Pseudomona aeruginosa gram stain

Pseudomona aeruginosa gram stain DEFAULT

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterium belonging to the γ-proteobacteria. Like other members of the Pseudomonas genus, it is known for its metabolic versatility and its ability to colonize a wide range of ecological niches, such as rhizosphere, water environments and animal hosts, including humans where it can cause severe infections. Another particularity of P. aeruginosa is its high intrinsic resistance to antiseptics and antibiotics, which is partly due to its low outer membrane permeability. In contrast to Enterobacteria, pseudomonads do not possess general diffusion porins in their outer membrane, but rather express specific channel proteins for the uptake of different nutrients. The major outer membrane ‘porin’, OprF, has been extensively investigated, and displays structural, adhesion and signaling functions while its role in the diffusion of nutrients is still under discussion. Other porins include OprB and OprB2 for the diffusion of glucose, the two small outer membrane proteins OprG and OprH, and the two porins involved in phosphate/pyrophosphate uptake, OprP and OprO. The remaining nineteen porins belong to the so-called OprD (Occ) family, which is further split into two subfamilies termed OccD (8 members) and OccK (11 members). In the past years, a large amount of information concerning the structure, function and regulation of these porins has been published, justifying why an updated review is timely.

Pseudomonas aeruginosa, porins, OprF, regulation, virulence, outer membrane

INTRODUCTION

Pseudomonas aeruginosa is the best known and investigated member of the genus Pseudomonas, the representatives of which are known for their high metabolic versatility. Pseudomonas syringae pathovars are important plant pathogens while P. aeruginosa causes infections in immunocompromised individuals and in cystic fibrosis (CF) patients (Goldberg ; Lyczak, Cannon and Pier ). Being a Gram-negative bacterium, P. aeruginosa has a cytoplasmic membrane with a symmetric phospholipid bilayer and an asymmetric outer membrane with a phospholipid inner face and a lipopolysaccharide outer layer, which generates a permeability barrier. The outer membrane of P. aeruginosa contains numerous proteins, including lipoproteins and channels (Remans et al. ). Exchange of nutrients across the outer membrane is orchestrated by β-barrel proteins producing water-filled diffusion channels, which give these membranes a molecular sieve-like appearance. These channels were first termed porins, given their pore shape and function, and were characterized by electron microscopy and conductance measurements in planar lipid membranes (Nakae ; Hancock, Decad and Nikaido ). Porins fold in the outer membrane as β-barrels made of antiparallel β-sheets with hydrophobic amino acids facing outward and hydrophilic residues inside the barrel and lining the constricted pore (Fernandez and Hancock ). This definition is still nowadays used in case of members of the Pseudomonas genus. However, in the case of Enterobacteriaceae, the term ‘porin’ was exclusively affected to large non-specific outer membrane β-barrel channels such as OmpF or OmpC (Nikaido ). Thus, two classes of outer membrane diffusion channels can be distinguished in Gram-negative bacteria: non-specific large general porins and substrate-specific channels (van den Berg ). Since Pseudomonas members do not display such large general porins, applying strictly this definition would result in the absence of porins. However, since numerous studies focusing on Pseudomonas members still use the term porin for outer membrane channels, it will be used throughout this review. We will therefore stick to the definition by Henderson et al. () who proposes that ‘the outer membrane β-barrel proteins termed porins allow the passage of solutes or contribute to the envelope stability’. Pseudomonas aeruginosa is characterized by the very low permeability of its outer membrane, representing about 8% of that of Escherichia coli, which at least partly contributes to the high intrinsic and induced resistance to antibiotics (Hancock ). One of the reasons for this low permeability is the already mentioned absence of large general diffusion porins, such as OmpF and OmpC (Pratt et al. ). Another reason is that the OprD (Occ) family comprises 19 members (Hancock and Brinkman ; Tamber, Ochs and Hancock ; Liu et al. a,b), which together are involved in the specific uptake of a wide range of small molecules of typically Da or less (Eren et al. ). Crystal structures for 14 members of this family have been obtained, all of which show channels that are substantially narrower than those of the enterobacteria porins (Eren et al. ).

There are different families of porins, including the so-called structural porins of the OmpA family (Smith et al. ; Confer and Ayalew ), the small porins of the OmpW family (8 β-sheets) (Hong et al. ; Benz et al. ; Catel-Ferreira et al. ) and larger diffusion porins with 18 β-sheets (Hancock and Brinkman ). Still, larger channels with 22 anti-parallel β-sheets are present in the outer membrane, the TonB-dependent receptors for the uptake of siderophores, heme and organic sulfur molecules. They are energized by the TonB inner membrane protein which relays the proton motive force opening the gate to permit the passage of large molecules (Cornelis and Bodilis ). Other specialized channels are involved in the efflux of toxic molecules, including antibiotics, or participate in secretion systems, but are not going to be discussed in this review (Hancock and Brinkman ; Schweizer ). This review will therefore focus on the 26 porins of P. aeruginosa, including OprF, which is the major non-lipoprotein outer membrane protein, and the homolog of OmpA of E. coli. Given the many data that recently emerged on OprF, and in terms of its numerous important functions as well as the complex regulation of its expression, this review will put a particular emphasis on this amazing protein. The other porins described here are the two small OprG and OprH proteins, the two OprB glucose porins, the OprO and OprP phosphate porins and the members of the so-called OprD (Occ) family (Hancock and Brinkman ; Tamber, Ochs and Hancock ; Liu et al. a,b). The 19 members of the OprD family which have been renamed Occ (outer membrane carboxylate channel) are phylogenetically split into two subfamilies, OccD being involved in the uptake of basic amino acids and the OccK for the uptake of negatively charged cyclic molecules (Tamber, Ochs and Hancock ; Eren et al., ). However, despite the new nomenclature that takes into account the functions of these channels, most articles still use the old Opr- Opd- names. To help understanding, the double nomenclature will be used throughout this review.

GENOMIC CONTEXT AND EXPRESSION REGULATION

Most porin genes (16 out of 26) are transcribed as monocistronic mRNAs, which does not mean that they are not co-transcribed or co-regulated with other genes in the vicinity (Table 1). However, some outer membrane porin genes are clearly in an operonic structure as shown in Table 1 and in Fig. 1. Many regulators are involved in the control of porin genes expression, including (i) two-component systems where a sensor in the inner membrane detects a signal which is relayed by phosphorylation to a response regulator (Rodrigue et al. ; Zschiedrich, Keidel and Szurmant ); (ii) extracytoplasmic function (ECF) sigma factors, which are transcription factors that are involved in stress responses perceived outside the cytoplasm, and/or in the regulation of numerous genes encoding proteins having ECFs (Potvin, Sanschagrin and Levesque ; Llamas et al. ; Schulz et al. ). The involvement of the different types of regulators in the expression of porin genes will be developed further in this review.

Figure 1.

Porin genes contained in operons. Operons including porin-encoding genes from P. aeruginosa PAO1 (pseudomonas.com). Predicted localization of the encoded proteins is indicated by colors as in Pseudomonas.com (green: outer membrane; orange: cytoplasmic membrane; red: cytoplasmic; yellow: periplasmic; gray: unknown). ABC: ATP-binding cassette; PBP: probable binding protein component of ABC transporter; MFS: major facilitator superfamily transporter; NmoII: type II nitronate monooxygenase (Salvi et al. ).

Figure 1.

Porin genes contained in operons. Operons including porin-encoding genes from P. aeruginosa PAO1 (pseudomonas.com). Predicted localization of the encoded proteins is indicated by colors as in Pseudomonas.com (green: outer membrane; orange: cytoplasmic membrane; red: cytoplasmic; yellow: periplasmic; gray: unknown). ABC: ATP-binding cassette; PBP: probable binding protein component of ABC transporter; MFS: major facilitator superfamily transporter; NmoII: type II nitronate monooxygenase (Salvi et al. ).

Table 1.

List of porin genes and alternative porin name designation, PAO1 gene number, PA14 gene number, molecular mass, direction (Dir.) of transcription (>forward strand, <reverse strand), operonic structure, possible function, antibiotic (ATB) transport, regulators controlling the porin expression, number of orthologs, orthologs group and associated transporter genes (http://pseudomonas.com; Winsor et al. ).

Gene . PAO1 n° . PA14 n° . Mass (kDa) . Dir. . Operon . Function . ATB transport . Regulators . Sigma factorsaOrthologs . Ortholog group (*) . Transporter-associated . 
oprFPA PA14_  NobStructuralcAmpR AlgU1,3, SigX*1, RpoS148 POG () 
oprBPA PA14_  PAGlucose uptake Anr, GltR RpoS2,338 POG () PA (ABC) 
oprB2PA PA14_  No Glucose uptake (SigX) 37 POG () 
oprGPA PA14_  No Fe2+ uptake? Anr SigX1,2,3, FecI248 POG () 
oprHPA PA14_  oprH-phoP-PhoQOM stabilization PhoP-PhoQ, BrlR, BqsR/CarR RpoS1,2, FecI141 POG () 
oprPPA PA14_  No Phosphate PhoB, TctD RpoH1,2, RpoS1, (SigX) 28 POG () 
oprOPA PA14_  No Pyrophosphate PhoB, TctD RpoH1, RpoS1, (SigX) 25 POG () 
oprD (occD1)PA PA14_  No Arginine Imipenem/ meropenem ArgR, CzcR RpoN1,3, SigX1,3, FliA137 POG () 
opdC (occD2)PA PA14_  No Histidine, arginine SigX1,2,3, FliA129 POG () 
opdP (occD3)PA PA14_ 53 PAGly-Glu, arginine RpoN2,338 POG () ABC transporter 
opdT (occD4)PA PA14_  No Tyrosine (FecI) 49 POG () 
opdI (occD5)PA PA14_  No Arginine 14 POG () 
oprQ (occD6)PA PA14_  No Arginine MexT RpoS1, SigX148 POG () 
opdB (occD7)PA PA14_  PAProline, arginine 23 POG () MFS 
opdJ (occD8)PA PA14_  PAArginine 14 POG () PA is an hydrolase 
opdK (occK1)PA PA14_  No Vanillate, benzoate Carbenicillin, cefoxitin, tetracyclin, temocillin 26 POG () 
opdF (occK2)PA PA14_  PAGlucuronate Carbenicillin, cefoxitin, gentamycin, temocillin (FecI) 68 POG () MFS 
opdO (occK3)PA PA14_  PAPyroglutamate Cefotaxime 14 POG () PA is an MFS 
opdL (occK4)PA PA14_  No Phenylacetate 26 POG () 
opdH (occK5)PA PA14_ 47 PAtctCBACis-aconitate, tri-carboxylates TctD 39 POG () (tctABC)Tri-carboxylate transport 
opdQ (occK6)PA PA14_  No NarXL 28 POG () 
opdD (occK7)PA PA14_  PAMeropenem 24 POG () Acyl-CoA dehydrogenase, PA nitropropane dioxygenase 
oprE (occK8)PA PA14_  No IHF, OxyR SigX1, RpoN1,2, FecI2153 POG () 
opdG (occK9)PA PA14_  No PA 23 POG () 
opdN (occK10)PA PA14_  No 26 POG () 
opdR (occK11)PA PA14_  PA13 POG () Acyl-coA metabolism 
Gene . PAO1 n° . PA14 n° . Mass (kDa) . Dir. . Operon . Function . ATB transport . Regulators . Sigma factorsaOrthologs . Ortholog group (*) . Transporter-associated . 
oprFPA PA14_  NobStructuralcAmpR AlgU1,3, SigX*1, RpoS148 POG () 
oprBPA PA14_  PAGlucose uptake Anr, GltR RpoS2,338 POG () PA (ABC) 
oprB2PA PA14_  No Glucose uptake (SigX) 37 POG () 
oprGPA PA14_  No Fe2+ uptake? Anr SigX1,2,3, FecI248 POG () 
oprHPA PA14_  oprH-phoP-PhoQOM stabilization PhoP-PhoQ, BrlR, BqsR/CarR RpoS1,2, FecI141 POG () 
oprPPA PA14_  No Phosphate PhoB, TctD RpoH1,2, RpoS1, (SigX) 28 POG () 
oprOPA PA14_  No Pyrophosphate PhoB, TctD RpoH1, RpoS1, (SigX) 25 POG () 
oprD (occD1)PA PA14_  No Arginine Imipenem/ meropenem ArgR, CzcR RpoN1,3, SigX1,3, FliA137 POG () 
opdC (occD2)PA PA14_  No Histidine, arginine SigX1,2,3, FliA129 POG () 
opdP (occD3)PA PA14_ 53 PAGly-Glu, arginine RpoN2,338 POG () ABC transporter 
opdT (occD4)PA PA14_  No Tyrosine (FecI) 49 POG () 
opdI (occD5)PA PA14_  No Arginine 14 POG () 
oprQ (occD6)PA PA14_  No Arginine MexT RpoS1, SigX148 POG () 
opdB (occD7)PA PA14_  PAProline, arginine 23 POG () MFS 
opdJ (occD8)PA PA14_  PAArginine 14 POG () PA is an hydrolase 
opdK (occK1)PA PA14_  No Vanillate, benzoate Carbenicillin, cefoxitin, tetracyclin, temocillin 26 POG () 
opdF (occK2)PA PA14_  PAGlucuronate Carbenicillin, cefoxitin, gentamycin, temocillin (FecI) 68 POG () MFS 
opdO (occK3)PA PA14_  PAPyroglutamate Cefotaxime 14 POG () PA is an MFS 
opdL (occK4)PA PA14_  No Phenylacetate 26 POG () 
opdH (occK5)PA PA14_ 47 PAtctCBACis-aconitate, tri-carboxylates TctD 39 POG () (tctABC)Tri-carboxylate transport 
opdQ (occK6)PA PA14_  No NarXL 28 POG () 
opdD (occK7)PA PA14_  PAMeropenem 24 POG () Acyl-CoA dehydrogenase, PA nitropropane dioxygenase 
oprE (occK8)PA PA14_  No IHF, OxyR SigX1, RpoN1,2, FecI2153 POG () 
opdG (occK9)PA PA14_  No PA 23 POG () 
opdN (occK10)PA PA14_  No 26 POG () 
opdR (occK11)PA PA14_  PA13 POG () Acyl-coA metabolism 

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Table 1.

List of porin genes and alternative porin name designation, PAO1 gene number, PA14 gene number, molecular mass, direction (Dir.) of transcription (>forward strand, <reverse strand), operonic structure, possible function, antibiotic (ATB) transport, regulators controlling the porin expression, number of orthologs, orthologs group and associated transporter genes (http://pseudomonas.com; Winsor et al. ).

Gene . PAO1 n° . PA14 n° . Mass (kDa) . Dir. . Operon . Function . ATB transport . Regulators . Sigma factorsaOrthologs . Ortholog group (*) . Transporter-associated . 
oprFPA PA14_  NobStructuralcAmpR AlgU1,3, SigX*1, RpoS148 POG () 
oprBPA PA14_  PAGlucose uptake Anr, GltR RpoS2,338 POG () PA (ABC) 
oprB2PA PA14_  No Glucose uptake (SigX) 37 POG () 
oprGPA PA14_  No Fe2+ uptake? Anr SigX1,2,3, FecI248 POG () 
oprHPA PA14_  oprH-phoP-PhoQOM stabilization PhoP-PhoQ, BrlR, BqsR/CarR RpoS1,2, FecI141 POG () 
oprPPA PA14_  No Phosphate PhoB, TctD RpoH1,2, RpoS1, (SigX) 28 POG () 
oprOPA PA14_  No Pyrophosphate PhoB, TctD RpoH1, RpoS1, (SigX) 25 POG () 
oprD (occD1)PA PA14_  No Arginine Imipenem/ meropenem ArgR, CzcR RpoN1,3, SigX1,3, FliA137 POG () 
opdC (occD2)PA PA14_  No Histidine, arginine SigX1,2,3, FliA129 POG () 
opdP (occD3)PA PA14_ 53 PAGly-Glu, arginine RpoN2,338 POG () ABC transporter 
opdT (occD4)PA PA14_  No Tyrosine (FecI) 49 POG () 
opdI (occD5)PA PA14_  No Arginine 14 POG () 
oprQ (occD6)PA PA14_  No Arginine MexT RpoS1, SigX148 POG () 
opdB (occD7)PA PA14_  PAProline, arginine 23 POG () MFS 
opdJ (occD8)PA PA14_  PAArginine 14 POG () PA is an hydrolase 
opdK (occK1)PA PA14_  No Vanillate, benzoate Carbenicillin, cefoxitin, tetracyclin, temocillin 26 POG () 
opdF (occK2)PA PA14_  PAGlucuronate Carbenicillin, cefoxitin, gentamycin, temocillin (FecI) 68 POG () MFS 
opdO (occK3)PA PA14_  PAPyroglutamate Cefotaxime 14 POG () PA is an MFS 
opdL (occK4)PA PA14_  No Phenylacetate 26 POG () 
opdH (occK5)PA PA14_ 47 PAtctCBACis-aconitate, tri-carboxylates TctD 39 POG () (tctABC)Tri-carboxylate transport 
opdQ (occK6)PA PA14_  No NarXL 28 POG () 
opdD (occK7)PA PA14_  PAMeropenem 24 POG () Acyl-CoA dehydrogenase, PA nitropropane dioxygenase 
oprE (occK8)PA PA14_  No IHF, OxyR SigX1, RpoN1,2, FecI2153 POG () 
opdG (occK9)PA PA14_  No PA 23 POG () 
opdN (occK10)PA PA14_  No 26 POG () 
opdR (occK11)PA PA14_  PA13 POG () Acyl-coA metabolism 
Gene . PAO1 n° . PA14 n° . Mass (kDa) . Dir. . Operon . Function . ATB transport . Regulators . Sigma factorsaOrthologs . Ortholog group (*) . Transporter-associated . 
oprFPA PA14_  NobStructuralcAmpR AlgU1,3, SigX*1, RpoS148 POG () 
oprBPA PA14_  PAGlucose uptake Anr, GltR RpoS2,338 POG () PA (ABC) 
oprB2PA PA14_  No Glucose uptake (SigX) 37 POG () 
oprGPA PA14_  No Fe2+ uptake? Anr SigX1,2,3, FecI248 POG () 
oprHPA PA14_  oprH-phoP-PhoQOM stabilization PhoP-PhoQ, BrlR, BqsR/CarR RpoS1,2, FecI141 POG () 
oprPPA PA14_  No Phosphate PhoB, TctD RpoH1,2, RpoS1, (SigX) 28 POG () 
oprOPA PA14_  No Pyrophosphate PhoB, TctD RpoH1, RpoS1, (SigX) 25 POG () 
oprD (occD1)PA PA14_  No Arginine Imipenem/ meropenem ArgR, CzcR RpoN1,3, SigX1,3, FliA137 POG () 
opdC (occD2)PA PA14_  No Histidine, arginine SigX1,2,3, FliA129 POG () 
opdP (occD3)PA PA14_ 53 PAGly-Glu, arginine RpoN2,338 POG () ABC transporter 
opdT (occD4)PA PA14_  No Tyrosine (FecI) 49 POG () 
opdI (occD5)PA PA14_  No Arginine 14 POG () 
oprQ (occD6)PA PA14_  No Arginine MexT RpoS1, SigX148 POG () 
opdB (occD7)PA PA14_  PAProline, arginine 23 POG () MFS 
opdJ (occD8)PA PA14_  PAArginine 14 POG () PA is an hydrolase 
opdK (occK1)PA PA14_  No Vanillate, benzoate Carbenicillin, cefoxitin, tetracyclin, temocillin 26 POG () 
opdF (occK2)PA PA14_  PAGlucuronate Carbenicillin, cefoxitin, gentamycin, temocillin (FecI) 68 POG () MFS 
opdO (occK3)PA PA14_  PAPyroglutamate Cefotaxime 14 POG () PA is an MFS 
opdL (occK4)PA PA14_  No Phenylacetate 26 POG () 
opdH (occK5)PA PA14_ 47 PAtctCBACis-aconitate, tri-carboxylates TctD 39 POG () (tctABC)Tri-carboxylate transport 
opdQ (occK6)PA PA14_  No NarXL 28 POG () 
opdD (occK7)PA PA14_  PAMeropenem 24 POG () Acyl-CoA dehydrogenase, PA nitropropane dioxygenase 
oprE (occK8)PA PA14_  No IHF, OxyR SigX1, RpoN1,2, FecI2153 POG () 
opdG (occK9)PA PA14_  No PA 23 POG () 
opdN (occK10)PA PA14_  No 26 POG () 
opdR (occK11)PA PA14_  PA13 POG () Acyl-coA metabolism 

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STRUCTURAL CHARACTERISTICS OF PSEUDOMONAS AERUGINOSA OUTER MEMBRANE PORINS

Most porins from P. aeruginosa have a molecular mass ranging from kDa (OpdG/OccK9) to 53 kDa (OpdP/OccD3), except the two smaller OprG (kDa) and OprH ( kDa) and the OprF porin ( kDa) (Table 1). The structure of 16 porins has been determined (Fig. S1,Supporting Information; Table 1), including the small β-barrel porin OprG with only 8 β-sheets (Kucharska et al., ), while the porins belonging to the OprD/Occ family all have 18 β-sheets. The structure of the OprF porin is still a matter of debate, since full-length OprF resists crystallographic efforts and the link between its functions and tertiary or quaternary structures remains controversial and will be discussed further. The OprD/Occ family comprise 19 members divided into two subfamilies: the OccD (8 members) and the OccK (11 members) (Tamber, Ochs and Hancock ; Eren et al. ). The structures of OccD and OccK1-OccK6 have been determined (Fig. S1) (Eren et al., ). The porins of the OccD family have a smaller pore compared to those of the OccK subfamily, but all have a basic ladder (row of Arg and Lys residues) in the barrel wall, the side chains pointing to the lumen (Eren et al. , ). The OccD porins have a more dynamic channel which can be closed or open while the OccK porins have a more rigid channel (Eren et al., ).

OprF, THE MAJOR AND MULTIFARIOUS PORIN OF PSEUDOMONAS AERUGINOSA

As the most abundant non-lipoprotein outer membrane protein in P. aeruginosa, OprF has been the object of numerous research works since (Hancock and Carey ; Hancock, Decad and Nikaido ). The most recent reviews dealing with OprF focused on two specific aspects, folding pathways (Sugawara, Nagano and Nikaido ) and involvement in pathogenesis (Krishnan and Prasadarao ), but a comprehensive review on this protein is missing. We therefore devoted a large part of the present review on OprF, which is justified by the large amount of available data, the variety of functions in which OprF is involved, and recent new findings, for example in the regulation of the oprF gene.

The elusive structure of OprF

OprF of P. aeruginosa is homologous to the outer membrane protein A (OmpA) of Escherichia coli, and these two proteins are the best studied members of the OmpA protein family. The structures of OmpA and OprF were reviewed and compared in (Reusch ; Sugawara, Nagano and Nikaido ). Briefly, OprF ( residues) folds into three domains: the crystallized N-terminal eight-stranded β-barrel located in the outer membrane (Brinkman, Bains and Hancock ; Reusch ) (Fig. 2A, Fig. S1), a cysteine-rich linker that may be partly surface exposed (Hancock and Carey ; Hancock, Decad and Nikaido ; Bodilis et al. ) and the C-terminal part containing α-helixes and/or β-strands (Sugawara, Nagano and Nikaido ). As OmpA, OprF generates two distinct conformers corresponding to a closed channel (the most abundant form) and a rare (<5% of the conformers) open channel (Sugawara and Nikaido ). The closed conformer contains the two domains mentioned above (the N-terminal eight-stranded β-barrel and the C-terminal periplasmic globular domain), whereas the open form folds as a single domain protein with a larger number of transmembrane β-strands (14 to 16) (Sugawara et al. ; Sugawara, Nagano and Nikaido ). The OprF channels mainly exist in weakly conductive subconformations and switch to the fully open state for a short time only (Nestorovich et al. ), thereby contributing to the low permeability of P. aeruginosa outer membrane reported earlier (Bellido et al. ). Interestingly, the channel conductance can be modulated in function of the bacterial growth temperature, with 80 pS and pS measured when bacteria were grown at 17°C or 37°C, respectively (Jaouen et al. ). While the small channel size is consistent with the predicted structure of the crystalized N-terminal domain, the largest one remains controversial, and has been attributed either to a folding of OprF as a large single domain or to the oligomerization of three subunits folding into three small channels of about 80 pS each (Jaouen et al. ; Sugawara et al. ). Remarkably, the recent discovery of unfolded OmpA monomers and oligomers that mimic amyloid fibers structures sheds new light into OmpA, and possibly also into OprF structures and associated functions (Wang et al. ; Danoff and Fleming ). It is interesting to mention that the largest OprF conformer proportion in the outer membrane increases in a mutant that does not make the periplasmic chaperone Skp, suggesting that in the absence of this chaperone, OprF could be misfolded like in the case of OmpA (Sugawara, Nagano and Nikaido , ).

Figure 2.

oprF genomic context and C-terminal protein similarity with OprL. (A) Schematic representation (PyMOL Molecular Graphics System, Version Schrödinger, LLC) viewed from the side (top) and from the extracellular environment (bottom) of the N-terminal part of OprF based on the X-ray crystal structure (PDB 4RLC). (B) Sequence (amino-acids) conservation between OprF and OprL. Conserved residues between the two proteins are shaded in gray. Peptidoglycan-binding residues are indicated in red (Cascales and Lloubes ). (C) Genomic locus including oprF and the seven genes upstream genes and localization of oprF promoter regions. The transcriptional initiation sites of the three promoters lying upstream of the oprF gene (PSigX, Pσ70, PAlgU), corresponding to SigX-, σ and AlgU-dependent promoters, respectively, are indicated by arrows. Their positions are indicated relative to the translational initiation start of oprF (+1). Position of AmpR putative binding site is indicated (blue square).

Figure 2.

oprF genomic context and C-terminal protein similarity with OprL. (A) Schematic representation (PyMOL Molecular Graphics System, Version Schrödinger, LLC) viewed from the side (top) and from the extracellular environment (bottom) of the N-terminal part of OprF based on the X-ray crystal structure (PDB 4RLC). (B) Sequence (amino-acids) conservation between OprF and OprL. Conserved residues between the two proteins are shaded in gray. Peptidoglycan-binding residues are indicated in red (Cascales and Lloubes ). (C) Genomic locus including oprF and the seven genes upstream genes and localization of oprF promoter regions. The transcriptional initiation sites of the three promoters lying upstream of the oprF gene (PSigX, Pσ70, PAlgU), corresponding to SigX-, σ and AlgU-dependent promoters, respectively, are indicated by arrows. Their positions are indicated relative to the translational initiation start of oprF (+1). Position of AmpR putative binding site is indicated (blue square).

OprF as a channel

OprF was first considered to function as a non-specific aqueous channel, allowing the passage of ions and low molecular mass sugars (Bellido et al. ), but it has also been suggested to allow the passage of toluene since a mutant not expressing oprF is more tolerant to toluene (Li et al. ). OprF has also been suggested to allow the diffusion of iron-chelated non-cognate siderophores (Meyer ). An interesting observation is the increased levels of OprF in P. aeruginosa cells grown under anaerobic conditions in the presence of nitrate, suggesting a possible involvement of OprF in the diffusion of nitrates and nitrites (Yoon et al. ).

OprF involvement in maintaining the outer membrane integrity

OprF plays a structural role, contributing to maintenance of the cell shape, especially under low osmolarity conditions, since the C-terminal part contains a peptidoglycan binding domain, which anchors the outer membrane to the peptidoglycan layer (Gotoh et al. ; Rawling, Brinkman and Hancock ). OprF mutants lacking various portions of the C-terminal part confirmed that the N-terminal amino acids are sufficient for protein production and membrane insertion, while the C-terminal part is needed for stable interaction with peptidoglycan (Rawling, Brinkman and Hancock ). The cells expressing C-terminally truncated OprF were both sensitive to low osmolarity and their cell length was reduced (Rawling, Brinkman and Hancock ). A comparison with OprL, the peptidoglycan-associated lipoprotein (PAL) (Lim et al. ), reveals the presence of remarkably conserved residues in the C-terminal part of both proteins (Fig. 2B). These residues are also conserved in the different PAL homologs in Gram-negative bacteria and are involved in the association with peptidoglycan or with TolA, an inner membrane protein which, together with TolC, TolQ and TolR, forms the Tol-PAL complex that insures the outer membrane integrity (Journet et al. ; Cascales et al. ; Cascales and Lloubes ). In E. coli, the PAL lipoprotein can also dimerize and interact with OmpA (Cascales et al. ). Since OprF belongs to the OmpA family, and because the critical residues for the interactions with peptidoglycan and Tol proteins are present in its C-terminal part, it is reasonable to suggest that OprF could either dimerize or interact with OprL, TolA, TolB and peptidoglycan, in line with the published evidence of its role in maintaining cell integrity under some environmental stresses (Rawling, Brinkman and Hancock ). In a study aiming at probing the protein interaction network of P. aeruginosa PAO1 by in vivo covalently linking interacting protein partners, OprF was confirmed to form homodimers and to interact with OprL (Navare et al. ). OprF and OprL were found to interact via their C-terminal part containing the conserved residues mentioned above. The small and abundant OprI lipoprotein (Cornelis et al. ) was also shown to interact with both OprF and OprL, suggesting the existence of ternary OprF-OprL-OprI complexes involved in cell shape and outer membrane stability. Four other OprF partners were identified: the translation initiation factor IF-2 encoded by the infB gene and the PA, PA and PA proteins of unknown functions. Of these four proteins, only PA is predicted to be an OmpA-like outer membrane lipoprotein (Remans et al. ; Navare et al. ; Winsor et al. ), but the relevance of these interactions remains unknown. Finally, it is worth mentioning that these conserved PAL motifs are not present in the other P. aeruginosa porins (results not shown).

OprF and biofilm formation

Pseudomonas aeruginosa forms thick biofilms (defined as microbial communities of sessile cells embedded into a matrix of extracellular polymeric substances that they have produced) under anaerobic conditions in the presence of nitrate, which is accompanied by higher levels of OprF in the outer membrane (Hassett et al. ; Yoon et al. ). Furthermore, an oprF-negative mutant forms very poor biofilms under these anaerobic conditions and the cells lack nitrite reductase activity, making an interesting link with the previous suggestion that OprF could be involved in the diffusion of nitrates/nitrites (Hassett et al. ; Yoon et al. ). In line with these results, it was demonstrated that hypoxic conditions, such as those encountered in the CF lung, favor a higher expression of oprF (Hogardt and Heesemann ; Eichner et al. ). In artificial sputum medium (ASM), mimicking the conditions in the CF lung, Sriramulu et al. () found that OprF is needed for the formation of microcolonies and that the high levels of amino-acids present in this medium favor high OprF production levels, whereas low OprF levels were observed in ASM without added amino acids. However, in LB medium under microaerobic conditions, but in the absence of additional nitrate, Bouffartigues et al. () showed that an oprF-negative mutant forms aggregates in liquid medium accompanied by higher levels of extracellular Pel polysaccharides, and more strongly attached biofilm, in stark contrast to the anaerobic growth conditions mentioned above. These phenotypes could partly be the result of increased levels of cyclic-di-GMP intracellular levels, which are known to favor biofilm formation (Bouffartigues et al. ). These contrasting results could be explained by the different conditions used (anaerobic vs aerobic), different media (LB vs ASM) and the presence or absence of nitrate.

OprF function in binding and adhesion to mammalian cells

Adhesion of P. aeruginosa to human lung alveolar epithelial cells has been found to be, at least partly, mediated by OprF since an oprF-negative mutant had its binding capacity reduced by about 60% while pre-incubation of epithelial cells with purified OprF or with a monoclonal antibody against OprF also reduced attachment (Azghani et al. ). Likewise, OprF has been found to be involved in the binding to human middle ear epithelial cells, probably facilitating the invasion of P. aeruginosa in otitis media (Mittal et al. ). In chronic suppurative otitis media caused by P. aeruginosa, actin rearrangement occurs due to phosphorylation of protein kinase C (PKC) and OprF was found to be necessary for PKC activation (Mittal et al. ). In a separate study, an oprF mutant of PAO1 showed a drastic reduction of adherence to rat glial cells and to Caco2/TC7 cells while wild-type adhesion level was restored upon complementation with the oprF gene in trans (Fito-Boncompte et al. ). OprF has also been found to interact with the lectin LecB, which is exposed at the surface of P. aeruginosa cells and both proteins contribute to the hemaglutination of erythrocytes (Funken et al. ).

OprF involvement in outer membrane vesicle biogenesis and functions

Outer membrane vesicles (OMVs) are nanostructures (20– nm in diameter) produced by almost all Gram-negative bacteria. They are spherical vesicles delimited by a bilayer membrane originating from the bacterial outer membrane. OMV membrane is therefore made of an outer lipopolysaccharide (LPS) leaflet, an inner phospholipid leaflet and outer membrane proteins. OMVs were also shown to contain bacterial periplasmic components (proteins and cell wall components), proteins of the bacterial inner membrane, cytoplasmic proteins, DNA and RNA, ions, metabolites and signaling molecules (Kulp and Kuehn ; Kim et al. ; Pathirana and Kaparakis-Liaskos ). They are produced by planktonic bacteria, but also by bacteria in biofilms and can thus be considered as components of biofilm matrixes (Schooling and Beveridge ).

Several general models of events leading to outer membrane budding and OMV biogenesis were proposed, including the loss of links between the outer membrane and the peptidoglycan layer in regions where vesiculation will take place (Kulp and Kuehn ). Since OprF and the lipoproteins OprL and OprI tether the outer membrane to peptidoglycan, their roles in P. aeruginosa PA14 OMV biogenesis were investigated and the absence of OprF led to OMV amounts increased by about 8-fold (Wessel et al. ). The PQS quorum-sensing signaling molecule is hydrophobic and associates with LPS in the external leaflet of the outer membrane, causing the outer leaflet to expand relative to the inner leaflet, yielding OMV budding from the bacterial surface (Schertzer and Whiteley ). In the oprF mutant of PA14, the higher OMV biogenesis level was shown to result from an increase in PQS production rather than from a decrease in outer membrane–peptidoglycan linkage (Wessel et al. ). An oprI mutant produced a 3-fold higher OMV level than the wild-type strain, while its PQS production was unaffected, indicating that the absence of OprI stimulated OMV biogenesis by reducing the outer membrane tethering to peptidoglycan. Finally, the OMV amount was unaffected in an oprL mutant compared to the wild-type strain (Wessel et al. ). The different mechanisms by which OprF and OprI impact OMV formation and the lack of effect of OprL indicate that, although these three proteins likely form ternary complexes maintaining the outer membrane integrity (Navare et al. ), they can also act individually. To the best of our knowledge, it remains unknown if variations in OprF levels in wild-type cells are sufficient to modulate OMV formation.

Several proteome studies identified OprF as a constituent of OMVs produced by P. aeruginosa PAO1 in various conditions: liquid cultures, biofilms, antibiotic treatment (Choi et al. ; Maredia et al. ; Toyofuku et al. ; Couto et al. ; Park et al. ). OprF was found to be the second most abundant protein in OMVs from liquid cultures (Choi et al. ) and was among the 30 most abundant proteins in OMVs from biofilms (Couto et al. ). Park et al. () observed that some outer membrane proteins can be preferentially incorporated or omitted into OMVs, supporting the notion of a specific protein packaging during OMV biogenesis. OprF was over-represented in biofilm and planktonic OMVs at the h time point (Park et al. ), suggesting that OprF could play important roles in OMV biogenesis. OMVs allow the transport of biological material from the parental bacterium to distal sites. In various Gram-negative bacteria, they were shown or proposed to display a variety of functions including transfer of antibiotic resistance, competition with other bacteria, stress response and bacterial survival, virulence factor delivery, bacterial adhesion and biofilm formation, nutrient and iron acquisition, cell communication, host cell invasion and modulation, and immune evasion (Kulp and Kuehn ; Kim et al. ; Orench-Rivera and Kuehn ; Pathirana and Kaparakis-Liaskos ). OprF could contribute to one or several of these functions, for example, given its involvement in the binding of P. aeruginosa to human cells (see above paragraph), it is tempting to hypothesize that OprF contributes to the interaction of OMVs with host cells. To our knowledge, the contribution of OprF or other individual proteins in OMV functions has not been reported yet.

OprF involvement in quorum-sensing response

Quorum sensing (QS) is a cell density-dependent mechanism characterized by the production of diffusible extracellular signal molecules, which trigger cellular responses, such as the production of virulence factors in the case of P. aeruginosa (Venturi ; Williams and Camara ; Papenfort and Bassler ). Pseudomonas aeruginosa produces two types of N-acyl-homoserine lactones, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C HSL), and N-butyryl-L-homoserine lactone (C4-HSL) (Venturi ) and two 4-hydroxyalkylquinolines (HAQs), which includes 3,4-dihydroxyheptylquinoline, also known as the Pseudomonas quinolone signal (PQS), and its precursor, 4-hydroxyheptylquinoline (HHQ) (Diggle et al. ; Dubern and Diggle ). Another molecule, termed IQS, has been proposed as well as the product of the ambBCDE locus (Lee et al. ). However, more recent data show that IQS is in fact aeruginaldehyde, a by-product of the pyochelin siderophore biosynthetic pathway rather than the product of the ambABCDE locus as proposed by Lee et al. (Ye et al. ; Rojas Murcia et al. ). In the las system, the synthase LasI produces 3-oxo-C12 HSL and the LasR regulator binds the signal molecule, activating several target genes, while the rhl system involves RhlI, the C4-HSL synthase and the RhlR response regulator. These two systems are inter-connected and activate together the production of numerous virulence factors including elastase, staphylolytic protease, exotoxin A, rhamnolipids, pyocyanin, lectins and superoxide dismutases (Venturi ; Williams and Camara ). On the other hand, the HHQ and PQS signal molecules interact with the PqsR/MvfR regulator and together trigger the production of virulence factors, including the LecB lectin and the phenazine compound pyocyanin (Diggle et al. ). Recently, it was found that MvfR is a truly global regulator of QS system in P. aeruginosa, regulating both rhl and las systems as well as genes for the defense against oxidative stress, highlighting the central role of the HHQ-PQS/MvfR system in P. aeruginosa (Maura et al. ).

The production of several QS-controlled virulence factors (pyocyanin, elastase, LecA lectin, exotoxin A) is strongly reduced in an oprF mutant (Fito-Boncompte et al. ). Accordingly, the production of PQS by the oprF mutant was found to be decreased while the production of HHQ was considerably increased (Fito-Boncompte et al. ). The conversion of HHQ to PQS involves a hydroxylation reaction realized by the PqsH enzyme, the gene of which is under the control of the LasR (Diggle et al. ). In line with the accumulation of HHQ, and the decreased production of 3-oxo-CHSL in the oprF mutant, the activity of a pqsH-lacZ fusion was decreased in the oprF mutant (Fito-Boncompte et al. ). An impact of OprF on PQS production was also reported by Wessel et al. (). but it was opposite: the PQS level was increased in an oprF mutant. This discrepancy could result from the use of different parental strains (the PAO1-derivative H by Fito-Boncompte et al. and PA14 by Wessel et al.), different culture conditions and/or different PQS quantification methods (LC/MS by Fito-Boncompte et al. and TLC by Wessel et al.). In a P. aeruginosa PAO1 pqsA mutant unable to produce HHQ and PQS, the transcription of oprF and sigX, the sigma factor gene upstream of oprF (see below) was 4-fold higher than in a wild-type background, suggesting an involvement of these QS regulators on transcriptional control of sigX (Gicquel et al. ).

OprF involvement in the perception of environmental cues

Several reports strongly support a role of OprF as an outer membrane protein involved in the perception of environmental signals, including some produced by the infected host. The innate immune system complement cascade is a first line of defense against pathogens and it involves the conversion of C3 to C3a and C3b, which typically recognizes a component at the surface of the pathogen, activating the C5 convertase and the formation of C5b-9 membrane attack complex (Joiner, Fries and Frank ). OprF is probably a P. aeruginosa C3b receptor since an oprF-negative mutant showed reduced C3b binding compared to the wild type while expression of oprF in E. coli increased the deposition of C3b (Mishra et al. ). Wu et al. () also demonstrated that among all tested cytokines, OprF binds interferon-gamma (IFN-γ), inducing expression by P. aeruginosa of the LecA lectin, a virulence factor under the control of the Rhl QS system. In line with the increased lecA expression, the same authors found that IFN-γ also induces the rhlI gene encoding the C4-HSL synthase (Wu et al. ).

During an acute phase of inflammation, the serum amyloid protein (SAA) is produced by hepatocytes and it was found that it tightly binds to Gram-negative bacteria, including E. coli, Salmonella enterica Thyphimurium, Shigella flexneri, Klebsiella pneumoniae, Vibrio cholerae and P. aeruginosa (Hari-Dass et al. ). In E. coli, OmpA is bound by SAA, while OprF is likely to be the target in P. aeruginosa since an oprF mutant is unable to bind SAA (Hari-Dass et al. ).

OprF importance in acute and chronic infections

In one study, mice were infected with P. aeruginosa PAO1, either to cause sepsis (acute infection) or chronic wound infection, and total gene expression was compared with cells grown in mid-logarithmic phase in a MOPS-succinate medium under laboratory conditions (Turner et al. ). Under both infection conditions, oprF expression was found to be downregulated, especially in the case of chronic wound infection where the expression was decreased by a factor (Turner et al. ). This result seems to be counterintuitive given the importance of OprF for the expression of virulence factors (Fito-Boncompte et al. ), but one should take other factors into account than the degree of expression in order to evaluate the contribution of a given gene in fitness during infection processes (Turner et al. ). One way to address this issue is the use of massive random transposon insertions in a Tn-seq approach by comparing the number of independent transposon insertions in one given gene when bacteria are grown in laboratory conditions or in vivo in acute or chronic infections. In their study, Turner et al. () found that the number of insertions in the oprF gene was quasi null in the case of bacteria recovered from acute infections and zero in the case of bacteria from chronic wound infection, indicative of a high fitness cost caused by oprF inactivation, supporting the importance of OprF in the infection process. Another type of infection caused by P. aeruginosa is the CF lung chronic colonization (Folkesson et al. ). In a recent study, it was revealed that transposon insertions in the oprF gene were almost absent when P. aeruginosa PAO1 was grown in CF sputum medium indicating an important fitness cost as well, while in the case of PA14, no insertion was found suggesting that oprF is an essential gene in this strain, at least in this growth condition (Turner et al. ). Interestingly, the same study showed that sigX is essential in both strains for growth in sputum medium (Turner et al. ). In non-CF bronchiectasis, an important T-cell response to OprF protein and an immune-dominant HLA-restricted T-cell epitope of OprF was detected (Quigley et al. ).

Genomic environment and regulation of oprF transcription

A first transcriptional study suggested that oprF was constitutively transcribed as a single gene from a σ70-type promoter (Duchene et al

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Chapter 27Pseudomonas

General Concepts

Clinical Manifestations

Pseudomonas aeruginosa and P maltophilia account for 80 percent of opportunistic infections by pseudomonads. Pseudomonas aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns; the case fatality is 50 percent. Other infections caused by Pseudomonas species include endocarditis, pneumonia, and infections of the urinary tract, central nervous system, wounds, eyes, ears, skin, and musculoskeletal system.

Structure, Classification, and Antigenic Types

Pseudomonas species are Gram-negative, aerobic bacilli measuring to , ¼m by to ¼m. Motility is by a single polar flagellum. Species are distinguished by biochemical and DNA hybridization tests. Antisera to lipopolysaccharide and outer membrane proteins show cross-reactivity among serovars.

Pathogenesis

Neutropenia in cancer patients and others receiving immunosuppressive drugs contributes to infection. Pseudomonas aeruginosa has several virulence factors, but their roles in pathogenesis are unclear. An alginate is antiphagocytic, and most strains isolated produce toxin A, a diphtheria-toxin-like exotoxin. All strains have endotoxin, which is a major virulence factor in bacteremia and septic shock.

Host Defenses

Phagocytosis by polymorphonuclear leukocytes is important in resistance to Pseudomonas infections. Antibodies to somatic antigens and exotoxins also contribute to recovery. Humoral immunity is normally the primary immune mechanism against Pseudomonas infection but does not seem to resolve infection in cystic fibrosis patients despite high levels of circulating antibodies.

Epidemiology

Pseudomonas species normally inhabit soil, water, and vegetation and can be isolated from the skin, throat, and stool of healthy persons. They often colonize hospital food, sinks, taps, mops, and respiratory equipment. Spread is from patient to patient via contact with fomites or by ingestion of contaminated food and water.

Diagnosis

Pseudomonas can be cultured on most general-purpose media and identified with biochemical media.

Control

The spread of Pseudomonas is best controlled by cleaning and disinfecting medical equipment. In burn patients, topical therapy of the burn with antimicrobial agents such as silver sulfadiazine, coupled with surgical debridement, has markedly reduced sepsis. Antibiotic susceptibility testing of clinical isolates is mandatory because of multiple antibiotic resistance; however, the combination of gentamicin and carbenicillin can be very effective in patients with acute P aeruginosa infections.

Introduction

The genus Pseudomonas contains more than species, most of which are saprophytic. More than 25 species are associated with humans. Most pseudomonads known to cause disease in humans are associated with opportunistic infections. These include P aeruginosa, P fluorescens, P putida, P cepacia, P stutzeri, P maltophilia, and P putrefaciens. Only two species, P mallei and P pseudomallei, produce specific human diseases: glanders and melioidosis. Pseudomonas aeruginosa and P maltophilia account for approximately 80 percent of pseudomonads recovered from clinical specimens. Because of the frequency with which it is involved in human disease, P aeruginosa has received the most attention. It is a ubiquitous free-living bacterium and is found in most moist environments. Although it seldom causes disease in healthy individuals, it is a major threat to hospitalized patients, particularly those with serious underlying diseases such as cancer and burns. The high mortality associated with these infections is due to a combination of weakened host defenses, bacterial resistance to antibiotics, and the production of extracellular bacterial enzymes and toxins.

Clinical Manifestations

Pseudomonas aeruginosa causes various diseases (Fig. ). Localized infection following surgery or burns commonly results in a generalized and frequently fatal bacteremia. Urinary tract infections following introduction of P aeruginosa on catheters or in irrigating solutions are not uncommon. Furthermore, most cystic fibrosis patients are chronically colonized with P aeruginosa. Interestingly, cystic fibrosis patients rarely have P aeruginosa bacteremia, probably because of high levels of circulating P aeruginosa antibodies. However, most cystic fibrosis patients ultimately die of localized P aeruginosa infections. Necrotizing P aeruginosa pneumonia may occur in other patients following the use of contaminated respirators. Pseudomonas aeruginosa can cause severe corneal infections following eye surgery or injury. It is found in pure culture, especially in children with middle ear infections. It occasionally causes meningitis following lumbar puncture and endocarditis following cardiac surgery. It has been associated with some diarrheal disease episodes. Since the first reported case of P aeruginosa infection in , the organism has been increasingly associated with bacteremia and currently accounts for 15 percent of cases of Gram-negative bacteremia. The overall mortality associated with P aeruginosa bacteremia is about 50 percent. Some infections (e.g., eye and ear infections) remain localized; others, such as wound and burn infections and infections in leukemia and lymphoma patients, result in sepsis. The difference is most probably due to altered host defenses.

Figure Diverse sites of infection by P aeruginosa.

Figure

Diverse sites of infection by P aeruginosa. This opportunistic pathogen may infect virtually any tissue. Infection is facilitated by the presence of underlying disease (e.g., cancer, cystic fibrosis) or by a breakdown in nonspecific host defenses (as (more)

Pseudomonas maltophilia is the second most frequently isolated pseudomonad species in clinical laboratories. In nature, P maltophilia is found in water and in both raw and pasteurized milk. It has been associated with a variety of opportunistic infections in humans, including pneumonia, endocarditis, urinary tract infections, wound infections, septicemia, and meningitis. Pseudomonas cepacia, although primarily a plant pathogen (onion bulb rot), also is an opportunist. Most human infections caused by P cepacia are nosocomial and include endocarditis, necrotizing vasculitis, pneumonia, wound infections, and urinary tract infections. Pseudomonas cepacia causes chronic lung infections in cystic fibrosis patients. These infections differ from those caused by P aeruginosa in that P cepacia has become systemic in a number of cystic fibrosis patients, whereas P aeruginosa infections remain confined to the lungs. Pseudomonas cepacia is highly resistant to aminoglycosides and other antibiotics, making it very difficult to control.

Unlike most pseudomonads, P mallei and P pseudomallei can cause disease in otherwise healthy individuals. Pseudomonas mallei is the agent of glanders, a disease primarily of equines. Humans generally become infected by inhalation or by direct contract through abraded skin. These infections are frequently fatal within 2 weeks of onset, although chronic infections also have been reported. Today, P mallei infections of equines are controlled and are rarely encountered in the western world. Similarly, melioidosis, an endemic glanderslike disease of animals and a human pulmonary infection caused by P pseudomallei, is rare in the western hemisphere. Melioidosis is still found in Southeast Asia, and travelers returning from that area are sometimes infected.

Structure, Classification, and Antigenic Types

Pseudomonas aeruginosa is a Gram-negative rod measuring to ¼m by to ¼m. Almost all strains are motile by means of a single polar flagellum, and some strains have two or three flagella (Fig. ). The flagella yield heat-labile antigens (H antigen). The significance of antibody directed against these antigens, aside from its value in serologic classification, is unknown. Clinical isolates usually have pili, which may be antiphagocytic and probably aids in bacterial attachment, thereby promoting colonization.

Figure Structure and pathogenic mechanisms of P aeruginosa.

Figure

Structure and pathogenic mechanisms of P aeruginosa. The proposed role of other products is listed in Table

The cell envelope of P aeruginosa, which is similar to that of other Gram-negative bacteria, consists of three layers: the inner or cytoplasmic membrane, the peptidoglycan layer, and the outer membrane. The outer membrane is composed of phospholipid, protein, and lipopolysaccharide (LPS). The LPS of P aeruginosa is less toxic than that of other Gram-negative rods. The LPS of most strains of P aeruginosa contains heptose, 2-ketodeoxyoctonic acid, and hydroxy fatty acids, in addition to side-chain and core polysaccharides. Recent evidence suggests that the LPS of a large percentage of strains isolated from patients with cystic fibrosis may have little or no polysaccharide side chain (O antigen), and that this finding correlates with the polyagglutinability of these strains with typing sera.

Studies of isolated outer membranes suggest strong conservation of many of the outer membrane proteins of P aeruginosa. Although numerous serologic types exist (based on evaluations of O-specific antigens), many of the outer membrane proteins from these strains are antigenically crossreactive.

Pseudomonas aeruginosa is a nonfermentative aerobe that derives its energy from oxidation rather than fermentation of carbohydrates. Although able to use more than 75 different organic compounds, it can grow on media supplying only acetate for carbon and ammonium sulfate for nitrogen. Furthermore, although an aerobe, it can grow anaerobically, using nitrate as an electron acceptor. This organism grows well at 25° C to 37° C, but can grow slowly or at least survive at higher and lower temperatures. Indeed, the ability to grow at 42° C distinguishes it from many other Pseudomonas species. In addition to its nutritional versatility, P aeruginosa resists high concentrations of salt, dyes, weak antiseptics, and many commonly used antibiotics. These properties help explain its ubiquitous nature and contribute to its preeminence as a cause of nosocomial infections.

Pathogenesis

Pseudomonas aeruginosa produces many factors that may contribute to its virulence. Table lists some of them. Almost all strains of P aeruginosa are hemolytic on blood agar plates, and several different hemolysins have been described. A heat-stable hemolytic glycolipid consisting of two molecules each of L-rhamnose and 1-²-hydroxydecenoic acid has been purified. Although this hemolytic glycolipid is not very toxic to animals (5 mg injected intraperitoneally is required to kill a mouse), it is toxic to alveolar macrophages. Furthermore, P aeruginosa strains isolated from respiratory tract infections produce more hemolysin than do environmental strains, suggesting that this glycolipid hemolysin may play a role in P aeruginosa pulmonary infections. Correlation of hemolysin production with infections of other sites has not been reported.

Several heat-labile protein hemolysins also have been described. One of these hemolysins may be identical to phospholipase C, which is produced by approximately 70 percent of all clinical strains of P aeruginosa. Phospholipase C, which hydrolyzes lecithin, is of unknown toxicity, and its role in P aeruginosa infections also remains unknown. Some strains of P aeruginosa produce a thermolabile protein (leukocidin), which lyses leukocytes from many species including humans but is nonhemolytic. This leukocidin (also called cytotoxin) damages lymphocytes and various tissue culture cells and is very toxic to mice (minimum lethal dose is 1 ¼g). Despite its toxicity, the role of leukocidin remains unknown.

Some strains of P aeruginosa produce large amounts of extracellular polysaccharide. These mucoid strains usually are isolated only from patients with cystic fibrosis. The role of these polysaccharides in the pathogenesis of P aeruginosa chronic lung infections is unknown, but they may impede phagocytosis and impair diffusion of antibiotics and thus facilitate colonization and persistence. Interestingly, mucoid strains are frequently deficient in production of elastase, toxin A, and flagella, and their LPS lacks long polysaccharide side chains.

Most strains of P aeruginosa also produce one or more pigments, the most common being pyocyanin (a phenazine pigment) and fluorescein. These pigments are nontoxic in animals. Pyocyanin, however, retards the growth of some other bacteria and thus may facilitate colonization by P aeruginosa. One or more of these pigments appear to function in iron acquisition by P aeruginosa. Additional work is needed to clarify the role of these pigments in P aeruginosa infections.

Approximately 90 percent of P aeruginosa strains produce extracellular protease. Three separate proteases have been purified that differ in pH optimum, isoelectric point, and substrate specificity. Although all are capable of digesting casein, one of them, protease II, also digests elastin. When injected into the skin of animals, purified P aeruginosa proteases induce formation of hemorrhagic lesions, which become necrotic within 24 hours. These proteases also cause rapid tissue destruction when injected into the cornea of animal eyes or into rabbit lungs; they also probably contribute to the tissue destruction that accompanies P aeruginosa eye or lung infections and may aid bacteria in tissue invasion. Their effects, however, appear to be localized, and they are not highly toxic to animals (LD50 = approximately ¼g/mouse) (Table ).

Toxin A

Toxin A, the most toxic known extracellular protein of P aeruginosa, is produced by 90 percent of all strains. The median lethal dose of pure toxin A is about ¼g/mouse. Its toxicity has been attributed to its ability to inhibit protein synthesis in susceptible cells. It achieves this by catalyzing the transfer of the ADP-ribosyl moiety of nicotinamide adenine dinucleotide (NAD) onto elongation factor 2 (EF-2) according to the following reaction:

Image ch27e1.jpg

The resultant ADP-ribosyl-EF-2 complex is inactive in protein synthesis. This intracellular mechanism of action of toxin A is identical to that of diphtheria toxin fragment A (see Ch. 32). Also like diphtheria toxin, Pseudomonas toxin A is released by P aeruginosa as a proenzyme. Toxin A is toxic to animals and cultured cells, but the proenzyme has little or no enzymatic activity. Table shows the relationship between the various forms of toxin A and their enzymatic activity and mouse toxicity. Evidence suggesting that toxin A may be a major virulence factor of P aeruginosa includes observations that toxin A-deficient mutants are less virulent in several animal models than their toxin A-producing parental strains, as well as the observation that most patients surviving P aeruginosa sepsis have elevated levels of antitoxin A antibody or are infected with strains that produce little or no detectable toxin A in vitro. These studies need to be expanded before firm conclusions can be reached.

Table Comparison of the Structure and Function of Toxin A and Its Fragments.

Table

Comparison of the Structure and Function of Toxin A and Its Fragments.

Exoenzyme S

A second ADP-ribosyltransferase, exoenzyme S, has been described. Exoenzyme S catalyzes the transfer of ADP-ribose onto a number of GTP-binding proteins, including the product of the proto-oncogene c-H-ras (p2lC-H-ras); however, it does not modify elongation factor 2. Exoenzyme S is produced by about 90 percent of clinical isolates of P aeruginosa. Transposon-induced S-deficient mutants are less virulent in several animal models than is their S-producing parental strain; thus, exoenzyme S may be involved in the pathogenesis of some P aeruginosa infections.

Host Defenses

Although 85 percent of P aeruginosa isolates are resistant to serum alone, addition of polymorphonuclear leukocytes results in bacterial killing. Killing is most efficient in the presence of type-specific opsonizing antibodies, directed primarily at the antigenic determinants of LPS. This suggests that phagocytosis is an important defense and that opsonizing antibody is the principal functioning antibody in protecting from P aeruginosa infections; however, once a P aeruginosa infection is established, other antibodies, such as antitoxin, may be important in preventing death. Although evidence suggests interaction between P aeruginosa and the cellular immune system, patients with diseases characterized by impaired cellular immune responses (e.g., Hodgkin's disease) do not have an increased incidence of severe P aeruginosa infections. However, patients with diminished antibody responses caused by underlying disease or its associated therapy, have more serious P aeruginosa infections. This underscores the importance of the humoral response in controlling P aeruginosa infections. Cystic fibrosis is the exception. Most cystic fibrosis patients have high levels of circulating antibodies to many bacterial antigens, but are unable to clear P aeruginosa efficiently from their lungs.

Epidemiology

Pseudomonas aeruginosa commonly inhabits soil, water, and vegetation. It is found in the skin of some healthy persons and has been isolated from the throat (5 percent) and stool (3 percent) of nonhospitalized patients. The gastrointestinal carriage rates increase in hospitalized patients to 20 percent within 72 hours of admission. Within the hospital, P aeruginosa finds numerous reservoirs: disinfectants, respiratory equipment, food, sinks, taps, and mops. Furthermore, it is constantly reintroduced into the hospital environment on fruits, plants, vegetables, and patients transferred from other facilities. Spread occurs from patient to patient on the hands of hospital personnel, by direct patient contact with contaminated reservoirs, and by the ingestion of contaminated foods and water.

Several different typing systems are available for epidemiologic studies: serologic, phage, pyocin, and DNA fingerprinting. In the pyocin system, pyocins (bacteriocins or aeruginocins) produced by the test strain are assayed for bactericidal activity against a series of indicator strains. A number of different serologic typing systems are used. Some employ combinations of heat-stable and heat-labile antigens, whereas others use only heat-stable antigens. No system is universally accepted. Recently, DNA fingerprinting has identified probes that are useful in typing P aeruginosa strains.

Diagnosis

Diagnosis of P aeruginosa depends on its isolation and laboratory identification. It grows well on most laboratory media and commonly is isolated on blood agar plates or eosin-methylthionine blue agar. It is identified on the basis of its Gram morphology, inability to ferment lactose, a positive oxidase reaction, its fruity odor, and its ability to grow at 4 2° C . Fluorescence under ultraviolet radiation helps in early identification of P aeruginosa colonies and also is useful in suggesting its presence in wounds. Other pseudomonads are identified by specific laboratory tests.

Control

The spread of P aeruginosa can best be controlled by observing proper isolation procedures, aseptic technique, and careful cleaning and monitoring of respirators, catheters, and other instruments. Topical therapy of burn wounds with antibacterial agents such as mafenide or silver sulfadiazine, coupled with surgical debridement, has dramatically reduced the incidence of P aeruginosa sepsis in burn patients.

Pseudomonas aeruginosa is frequently resistant to many commonly used antibiotics. Although many strains are susceptible to gentamicin, tobramycin, colistin, and amikacin, resistant forms have developed, making susceptibility testing essential. The combination of gentamicin and carbenicillin is frequently used to treat severe Pseudomonas infections, especially in patients with leukopenia. Several types of vaccines are being tested, but none is currently available for general use.

References

  1. Brown MRW (ed): Resistance of a Pseudomonas aeruginosa. John Wiley &#x; Sons, New York, .

  2. Clarke PH, Richman MN (eds): Genetics and Biochemistry of Pseudomonas. John Wiley &#x; Sons, New York, .

  3. Coburn J, Wyatt RT, Iglewski BH, Gill DM. Several GTP-binding proteins, including o24 C-H-ras, are preferred substrates of Pseudomonas aeruginosa exoenzyme S. J Biol Chem. ; [PubMed: ]

  4. Cross AS, Sadoff JC, Iglewski BH, Sokol PA. Evidence for the role of toxin A in the pathogenesis of infections with Pseudomonas aeruginosa in humans. J Infect Dis. ; [PubMed: ]

  5. Dunn M, Wunderink RG: Ventilator-associated pnemonia caused by Pseudomonas infection. (Review) Clinics of Chest Medicine. (16), . [PubMed: ]

  6. Hancock REW, Mutharia LM, Chan L. et al. Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chain. Infect Immun. ; [PMC free article: PMC] [PubMed: ]

  7. Liu PV. Extracellular toxins of Pseudomonas aeruginosa. J Infect Dis. ;supplS

  8. Mutharia LM, Nicas TI, Hancock REW. Outer membrane proteins of Pseudomonas aeruginosa serotyping strains. J Infect Dis. ; [PubMed: ]

  9. Poole K. Bacterial multidrug resistanceemphasis on efflux mechanisms and Pseudomonas aeruginosa. (Review) J Antimicrobial Chemotherapy. ;34(4) [PubMed: ]

  10. Pritchard AE, Vasal ML. Possible insertion sequences in a mosaic genomeorganization upstream of the exotoxin A gene in Pseudomonas aeruginosa. J Bacteriol. ; [PMC free article: PMC] [PubMed: ]

  11. Woods DE, Iglewski BH. Toxins of Pseudomonas aeruginosa: new perspectives. Rev Infect Dis, suppl. ;5:S [PubMed: ]

Sours: https://www.ncbi.nlm.nih.gov/books/NBK/
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Pseudomonas Aeruginosa

Gram Stain, Culture Characteristics, Infection

Antibiotic Resistance and Antimicrobial Susceptibility Testing

Water Treatment


Introduction


Pseudomonas aeruginosa is a member of the genus Pseudomonas. They are Gram-negative bacteria commonly found in various moist environments.

While the bacterium is a pathogen that is responsible for various hospital-acquired infections, these infections are particularly severe among individuals with a compromised immune system. 


Culture Characteristics



As mentioned, Pseudomonas aeruginosa is ubiquitous in nature and can be found in various moist environments. This has been attributed to the fact that the bacterium is characterized by extensive metabolic diversity allowing it to live and thrive in various ecological niches. 

With regards to microbial culture, this is particularly important given that a variety of growth media can be used. While the bacterium grows well at 37 degrees C, it can also survive at the temperature range between 4 and 42 degrees C. 

To study various characteristics of Pseudomonas aeruginosa, some of the media used include Pseudomonas isolation agar, LB Broth, King A, and MOPS ([3-(N-morpholino) propane-sulfonic acid]).

To grow the bacteria in Pseudomonas Isolation Agar, the plates (clean and sterile plates) are first allowed to warm at room temperature. The agar is then poured into the plates and allowed to dry before inoculation. Once the agar has dried, the specimen has to be inoculated within the shortest time possible after it has been collected. 

Generally, inoculation involves streaking the specimen over the surface of the agar (covering about a third of the surface). Here, streaking must be done using a sterile loop in order to avoid contaminating the medium with other microorganisms.

Following inoculation, the plate or plates are incubated for one (1) day at 37 degrees C aerobically. After 24, Pseudomonas aeruginosa appear as blue-green colonies. If other Pseudomonas or non-fermenting bacteria are present, they are not blue-green in color.

It's worth noting that the pigments (water-soluble pigments) produced by Pseudomonas aeruginosa may vary depending on the medium or strain of bacteria.

Some of the most common pigments include; pyocyanin which is blue-green in color, pyoverdine which is yellow-green in color, and pyorubin which is red-brown in color. 

Apart from the pigmented colonies, Pseudomonas aeruginosa grown in media is also characterized by grape-like smell. Being a non-fermentor, the bacterium is also associated with acid production in culture rather than gases which are commonly associated with fermenting bacteria.

Studies have shown that in the presence of nitrate, Pseudomonas aeruginosa can grow slowly in an anaerobic environment at about 42 degrees C. 

Apart from the media mentioned above, Pseudomonas aeruginosa can also be grown in MacConkey agar (a bacterial culture medium commonly used to grow lactose fermenting bacteria). While the bacterium cannot use lactose present in this medium, it survives on peptone.

In MacConkey agar, Pseudomonas aeruginosa forms flat and smooth colonies that are between 2 and 3mm in diameter. Generally, these colonies have regular margins and have an alligator skin-like appearance when viewed from above. 

See more on Cell Culture.

Some of the other characteristics of Pseudomonas aeruginosa in culture include:

·       Colorless in MacConkey agar - This is attributed to the fact that the bacteria does not ferment lactose

·       In Centrimide agar - Pseudomonas aeruginosa colonies (greenish-blue in color) are medium-sized and characterized by an irregular growth

·       In Nutrient agar -  Pseudomonas aeruginosa are associated with several odors ranging from a sweet to earth smell


Gram Stain


Growing bacteria in culture is important in that it allows researchers to analyze and study various characteristics (smell, texture, and shape of colonies, the color of the colony, etc) of the organism.

Using Gram staining technique makes it possible for researchers to not only identify morphological characteristics of bacterial cells, but also differentiate them based on their cell wall components. 


Requirements for Gram stain include:

Sample - Pseudomonas aeruginosa grown in culture can be used.


Procedure

·       If the sample is obtained from a culture plate, then it's necessary to add a drop of water onto the glass slide before aseptically adding a small amount of the bacteria. This can be achieved by using a sterile wire loop to place the sample on to the drop of water

·       Using the wire loop or glass stir stick/rod, make a good smear of the sample at the middle of the glass slide

·       Allow the slide to air dry - If bacteria from the broth was used, then it's important to allow all the liquid to evaporate before fixing

·       Once the slide has dried, heat fix by passing over the flame - You can pass the slide over the flame about three times and avoid overheating 

·       Flood the slide with crystal violet for about 1 minute and then rinse with water 

·       Treat the slide with Gram's Iodine for about 1 minute and then gently rinse with water 

·       Add a few drops of alcohol (95 percent alcohol) on to the slide and then rinse with water 

·       Flood the slide with Safranin (counterstain) and then rinse with water

·       Add a drop of immersion oil onto the slide and observe under the microscope 

* Preparing several slides (about 3 slides) is always recommended 

See more on Cell staining.


Observation


When viewed under the microscope, Pseudomonas aeruginosa will appear as reddish/pink rods. This indicates that they are Gram-negative bacteria given that they are unable to retain the primary stain (crystal violet).

Under high magnification, studies have shown Pseudomonas aeruginosa to range from to um in diameter and to um in length (rod-like bacteria). They are also characterized by a single polar flagellum used for motility.

For some of the strains, studies have revealed the presence of two to three polar flagella used for movement. In addition to the flagella, Pseudomonas aeruginosa also possess pili on their surface which are used for adhesion and a form of motility known as twitching motility. 

Read more about Gram positive and Gram negative bacteria.


Infection


As mentioned, Pseudomonas aeruginosa is responsible for various hospital-acquired infections.

According to a report that was published by the European Center for Disease Prevention and Control in , about 9 percent of all health-care-associated infections are caused by Pseudomonas aeruginosa making it the fourth most common pathogen responsible for infections in European hospitals.

For the most part, the bacterium is an opportunistic pathogen of the mucosal tissues. However, infections of the cornea (the eye) and the urinary tract have been reported.

Depending on the patient, infections of the respiratory tract range from nosocomial pneumonia to lung infections among patients with cystic fibrosis. 


Nosocomial Infections


Essentially, nosocomial infections are hospital-acquired infections and thus occur post-admission.

Some of these infections include:

Burn wound infections - In addition to Staphylococcus aureus and Streptococcus pyrogens, Pseudomonas aeruginosa is one of the leading causes of invasive infections among burn patients. Here, the site of injury (from the burn) allows for the successive invasion of the bacterium.

Apart from entering the body through the injured skin, the bacterium has been shown to gain entry as a result of inhalation injury thus increasing the risk of respiratory infections.

Bacteremia - Pseudomonas aeruginosa is also one of the main causes of nosocomial bacteremia. Given that this particular organism has been shown to be resistant to various antimicrobials, these infections have been shown to result in higher mortality as compared to some of the other pathogens responsible for bacteremia.

Hospital and ventilator-associated pneumonia - Given that the respiratory tract provides favorable conditions for life, the bacterium is easily able to cause chronic and acute infections among patients with cystic fibrosis.

In addition, the pathogen has also been shown to be one of the main causes of ventilator-associated pneumonia (VAP) particularly in the case of increased duration of mechanical ventilation. 


Urinary Tract Infection 


Pseudomonas aeruginosa has been shown to be particularly effective at forming surface-associated biofilms. For patients who use catheters, the bacterium has been shown to form biofilm on the surface of these catheters (indwelling catheters) and ultimately cause an infection as they proliferate. 


Pathogenesis and Virulence Factors of Pseudomonas aeruginosa


Pathogenesis of Pseudomonas aeruginosa is made possible by several virulence factors that include:

Lipopolysaccharide - Lipopolysaccharide is one of the main components of the outer membrane of Pseudomonas aeruginosa. In addition to Lipid A, a hydrophobic domain, this component of the outer membrane also consists of O-antigen (distal polysaccharide) which not only determines the serotype of the organism but also activates the immune system of the host.

Eventually, the polysaccharide results in dysregulated inflammation which has been associated with morbidity and mortality. 

Flagellum - As mentioned, Pseudomonas aeruginosa contains a single polar flagellum used for swimming in moist environments. In addition to motility, this structure has also been shown to play an important role in attachment to the epithelium, invasion as well as biofilm formation. 

Type IV Pili - Type IV pili located on the surface of Pseudomonas aeruginosa play an important role in adhesion to various cells thus promoting infections. In addition to adhesion, the pili have also been shown to be involved in twitching motility which in turn promotes the formation of biofilms. 

Some of the other factors that promote the pathogenesis of Pseudomonas aeruginosa include:

·       Exotoxin A - associated with local tissue damage and gradual invasion 

·       Proteases - Pseudomonas aeruginosa produces a number of proteases including LasB and alkaline protease that destroy tissue 

·       Alginate -  is one of the main components of mucoid exopolysaccharide capsule and plays an important role in cell adherence 


Antibiotic Resistance


Following an infection, Pseudomonas aeruginosa has been shown to be resistant to a variety of antimicrobials.

There are several modes of resistance which include:

Intrinsic resistance to antibiotics - Essentially, intrinsic antibiotic resistance refers to the innate ability of bacteria to evade the impacts of antibiotics. This may be achieved through various structural and functional characteristics.

Some of the mechanisms through which Pseudomonas aeruginosa is able to diminish the efficacy of various antibiotics (intrinsically) include:

Permeability of the outer membrane - In Pseudomonas aeruginosa, the outer membrane is an asymmetric bilayer that consists of phospholipid and LPS (Lipopolysaccharides). It also consists of porins that are responsible for the beta-barrel protein channels.

The composition of this membrane makes it very restrictive and is responsible for limiting the penetration of antibiotics. However, the membrane does not completely prevent this penetration. Rather, slow uptake of these molecules contributes to intrinsic resistance. 

Efflux systems - Apart from the limiting outer membrane, Pseudomonas aeruginosa is also able to pump out toxic compounds. In particular, studies have shown proteins associated with the resistance-nodulation-division (RND) family to be largely involved in this activity in this bacterium.

Here, the proteins make up cytoplasmic membrane transporters and outer membrane porin channel proteins involved in expelling toxic compounds out of the cell. In cases where these pumps are overexpressed, the bacterium gradually develops resistance to a variety of drugs. 

Antibiotic-inactivating enzymes - One of the other factors that contribute to antibiotic resistance is the ability of the bacterium to produce enzymes capable of breaking down and modifying antibiotics.

In particular, Pseudomonas aeruginosa has been shown to produce such enzymes as hydrolytic enzyme β-lactamase which breaks the amide bond of certain antibiotics. In doing so, the drug is rendered ineffective against the pathogen. 

Also: How do antibiotics kill bacteria?


Acquired Antibiotic Resistance


Acquired antibiotic resistance is the second mechanism through which Pseudomonas aeruginosa have developed antibiotic resistance.

This is achieved through:

Mutational change - Mutational change is particularly beneficial for the pathogen as modification of antibiotic targets allows them to evade the intended actions of the drug. This may involve the overexpression of efflux pumps and thus the ability of the bacterium to remove toxic substances from the cell. 

Acquired resistance genes - Bacteria have been shown to be capable of acquiring genes through horizontal transfer. In the case of various P. aeruginosa strains, the acquisition of resistance genes allows the bacterium to develop resistance to various antibiotics. This transfer may occur through conjugation, transduction, or transformation. 


Adaptive Antibiotic Resistance


The last mechanism of resistance against antibiotics is through adaptive antibiotic resistance. Generally, this is achieved through the formation of a biofilm. A biofilm refers to adhesion or clustering of microorganisms on a given surface.

In the host, the biofilm formed by Pseudomonas aeruginosa is then covered by a matrix. As compared to other pathogenic cells, these cells tend to be less sensitive to antimicrobial agents. 


Antimicrobial Susceptibility Testing


As mentioned, Pseudomonas aeruginosa has been shown to be resistant to a number of antibiotics. This is due to a number of virulence factors associated with the organism. For this reason, Antimicrobial Susceptibility Testing is an important test to determine the most effective treatment to treat infections caused by the bacterium.

Essentially, Antimicrobial Susceptibility Testing involves placing a microorganism in contact with antibiotics in order to study whether or not the organism will grow in the presence of the antibiotics being used. 

Here, Mueller-Hinton agar using disc diffusion can be used for the test given that the technique is applicable for a wide range of non fastidious bacteria with little change of error. 

Agar preparation involves the following steps:

·       38 grams of the medium is suspended in a liter of purified water and mixed

·       The mixture is then heated for about 1 minute with frequent agitation in order to ensure that the contents mix properly 

·       The agar is autoclaved for 15 minutes at degrees C and then allowed to cool to 45 degrees C

·       The agar is then poured into Petri dishes to a depth of about 4mm

·       The plates are allowed to solidify at room temperature and investigated to ensure that the pH remains +11 at 25 degrees C

* Once the researcher is ready to test sensitivity/susceptibility of the pathogen the bacterium is inoculated and the antimicrobial disks placed on the culture (using sterile forceps). The plates are then inverted and incubated at 37 degrees C for between 16 to 18 hours. 

* Antibiotic disks contain the drug being tested. 

Some of the antibiotics used to test sensitivity/susceptibility of Pseudomonas aeruginosa include:

  • Ticarcillin
  • Aztreonam
  • Ciprofloxacin
  • Kanamycin
  • Cefepime


Water Treatment


While Pseudomonas aeruginosa is ubiquitous in nature, it's commonly found in moist environments. For this reason, it's also found in various water bodies including lakes and rivers, etc. In order to prevent possible infections, water treatment is necessary.

Antimicrobial susceptibility testing is used to determine the most effective water disinfectant. Here, this test also considers the impact that the disinfectants being tested can have on the health of those who use the water.

Based on previous studies, Pseudomonas aeruginosa has been shown to be susceptible to several disinfectants including ozone, iodine, chloramines, and chlorine. For this reason, they are commonly used to treat water in many developed and developing nations.

While UV (ultraviolet) disinfection has proven effective for a number of other microorganisms in water, this form of treatment has been shown to be less effective when it comes to Pseudomonas aeruginosa. As a result, the aforementioned water treatment options are often recommended. 


See also:Pseudomonas syringae

Return to Pseudomonas main page

Return to learning about Proteobacteria

Return to Bacteria under a microscope main page

Return to more on Bacteria - Size, Shape and Arrangement

Return from Pseudomonas aeruginosa to MicroscopeMaster home




References


Barbara H. Iglewski. (). Pseudomonas. Medical Microbiology. 4th edition.

Benie CKD et al. (). Prevalence and Antibiotic Resistance of

Pseudomonas aeruginosa Isolated from Bovine Meat, Fresh Fish and Smoked Fish.

Kristina D Mena and Charles P Gerba. (). Risk assessment of Pseudomonas aeruginosa in water. 

Patricia Ruiz-Garbajosa and Rafael Cantón. (). Epidemiology of antibiotic resistance in Pseudomonas aeruginosa. Implications for empiric and definitive therapy. 

Robert B. Fick, Jr. (). Pseudomonas Aeruginosa the Opportunist. 

Links

https://www.sciencedirect.com/science/article/pii/S#:~:text=Pseudomonas%20aeruginosa%20has%20been%20shown,et%20al.%2C%).

https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/PseudomonasIsolationAgar.htm

Sours: https://www.microscopemaster.com/pseudomonas-aeruginosa.html
Pseudomonas aureginosa(Clear overview).



Looking for the most current news, updates, and articles relating to microbiology, go to The American Society for Microbiology educational website Microbe World.







Kenneth Todar currently teaches Microbiology at the University of Wisconsin-Madison. His main teaching interest include general microbiology, bacterial diversity, microbial ecology and pathogenic bacteriology.

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Pseudomonas aeruginosa   (page 1)

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© Kenneth Todar, PhD



Gram stain of Pseudomonas aeruginosa cells

Pseudomonas aeruginosa is member of the Gamma Proteobacteria class of Bacteria. It is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. Since the revisionist taxonomy based on conserved macromolecules (e.g. 16S ribosomal RNA) the family includes only members of the genus Pseudomonas which are cleaved into eight groups. Pseudomonas aeruginosa is the type species of its group. which contains 12 other members.

Like other members of the genus, Pseudomonas aeruginosa is a free-living bacterium, commonly found in soil and water. However, it occurs regularly on the surfaces of plants and occasionally on the surfaces of animals. Members of the genus are well known to plant microbiologists because they are one of the few groups of bacteria that are true pathogens of plants. In fact, Pseudomonas aeruginosa is occasionally a pathogen of plants. However, Pseudomonas aeruginosa has become increasingly recognized as an emerging opportunistic pathogen of clinical relevance. Several different epidemiological studies track its occurrence as a nosocomial pathogen and indicate that antibiotic resistance is increasing in clinical isolates.

Pseudomonas aeruginosa is an opportunistic pathogen, meaning that it exploits some break in the host defenses to initiate an infection. In fact, Pseudomonas aeruginosa is the epitome of an opportunistic pathogen of humans. The bacterium almost never infects uncompromised tissues, yet there is hardly any tissue that it cannot infect if the tissue defenses are compromised in some manner. It causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns. The case fatality rate in these patients is near 50 percent.

Pseudomonas aeruginosa is primarily a nosocomial pathogen. According to the CDC, the overall incidence of P. aeruginosa infections in U.S. hospitals averages about percent (4 per discharges), and the bacterium is the fourth most commonly-isolated nosocomial pathogen accounting for percent of all hospital-acquired infections.

Characteristics

Pseudomonas aeruginosa is a Gram-negative rod measuring to µm by to µm. Almost all strains are motile by means of a single polar flagellum.

The bacterium is ubiquitous in soil and water, and on surfaces in contact with soil or water. Its metabolism is respiratory and never fermentative, but it will grow in the absence of O2 if NO3 is available as a respiratory electron acceptor.

The typical Pseudomonas bacterium in nature might be found in a biofilm, attached to some surface or substrate, or in a planktonic form, as a unicellular organism, actively swimming by means of its flagellum. Pseudomonas is one of the most vigorous, fast-swimming bacteria seen in hay infusions and pond water samples.

In its natural habitat Pseudomonas aeruginosa is not particularly distinctive as a pseudomonad, but it does have a combination of physiological traits that are noteworthy and may relate to its pathogenesis.

&#; Pseudomonas aeruginosa has very simple nutritional requirements. It is often observed "growing in distilled water", which is evidence of its minimal nutritional needs. In the laboratory, the simplest medium for growth of Pseudomonas aeruginosa consists of acetate as a source of carbon and ammonium sulfate as a source of nitrogen.

&#; P. aeruginosa possesses the metabolic versatility for which pseudomonads are so renowned. Organic growth factors are not required, and it can use more than seventy-five organic compounds for growth.

&#; Its optimum temperature for growth is 37 degrees, and it is able to grow at temperatures as high as 42 degrees.

&#; It is tolerant to a wide variety of physical conditions, including temperature. It is resistant to high concentrations of salts and dyes, weak antiseptics, and many commonly used antibiotics.

&#; Pseudomonas aeruginosa has a predilection for growth in moist environments, which is probably a reflection of its natural existence in soil and water.

These natural properties of the bacterium undoubtedly contribute to its ecological success as an opportunistic pathogen. They also help explain the ubiquitous nature of the organism and its prominence as a nosocomial pathogen.

P. aeruginosa isolates may produce three colony types. Natural isolates from soil or water typically produce a small, rough colony. Clinical samples, in general, yield one or another of two smooth colony types. One type has a fried-egg appearance which is large, smooth, with flat edges and an elevated appearance. Another type, frequently obtained from respiratory and urinary tract secretions, has a mucoid appearance, which is attributed to the production of alginate slime. The smooth and mucoid colonies are presumed to play a role in colonization and virulence.


Pseudomonas aeruginosa colonies on agar

P. aeruginosa strains produce two types of soluble pigments, the fluorescent pigment pyoverdin and the blue pigment pyocyanin. The latter is produced abundantly in media of low-iron content and functions in iron metabolism in the bacterium. Pyocyanin (from "pyocyaneus") refers to "blue pus", which is a characteristic of suppurative infections caused by Pseudomonas aeruginosa.


The soluble blue pigment pyocyanin is produced by many, but not all, strains of Pseudomonas aeruginosa



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Gram pseudomona stain aeruginosa

Pseudomonas aeruginosa

Species of bacterium

Pseudomonas aeruginosain Petri dish.

Pseudomonas aeruginosa is a common encapsulated, Gram-negative, strict aerobic (although can grow anaerobically in the presence of nitrate), rod-shapedbacterium that can cause disease in plants and animals, including humans.[1][2] A species of considerable medical importance, P. aeruginosa is a multidrug resistantpathogen recognized for its ubiquity, its intrinsically advanced antibiotic resistance mechanisms, and its association with serious illnesses – hospital-acquired infections such as ventilator-associated pneumonia and various sepsissyndromes.

The organism is considered opportunistic insofar as serious infection often occurs during existing diseases or conditions – most notably cystic fibrosis and traumatic burns. It generally affects the immunocompromised but can also infect the immunocompetent as in hot tub folliculitis. Treatment of P. aeruginosa infections can be difficult due to its natural resistance to antibiotics. When more advanced antibiotic drug regimens are needed adverse effects may result.

It is citrate, catalase, and oxidase positive. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in low-oxygen atmospheres, thus has colonized many natural and artificial environments. It uses a wide range of organic material for food; in animals, its versatility enables the organism to infect damaged tissues or those with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis. If such colonizations occur in critical body organs, such as the lungs, the urinary tract, and kidneys, the results can be fatal.[3] Because it thrives on moist surfaces, this bacterium is also found on and in medical equipment, including catheters, causing cross-infections in hospitals and clinics. It is also able to decompose hydrocarbons and has been used to break down tarballs and oil from oil spills.[4]P. aeruginosa is not extremely virulent in comparison with other major pathogenic bacterial species – for example Staphylococcus aureus and Streptococcus pyogenes – though P. aeruginosa is capable of extensive colonization, and can aggregate into enduring biofilms.[5]

Nomenclature[edit]

A culture dish with Pseudomonas

The word Pseudomonas means "false unit", from the Greek pseudēs (Greek: ψευδής, false) and (Latin: monas, from Greek: μονάς, a single unit). The stem word mon was used early in the history of microbiology to refer to germs, e.g., kingdom Monera.[6]

The species name aeruginosa is a Latin word meaning verdigris ("copper rust"), referring to the blue-green color of laboratory cultures of the species. This blue-green pigment is a combination of two metabolites of P. aeruginosa, pyocyanin (blue) and pyoverdine (green), which impart the blue-green characteristic color of cultures.[6] Another assertion from is that aeruginosa may be derived from the Greek prefix ae- meaning "old or aged", and the suffix ruginosa means wrinkled or bumpy.[7]

The names pyocyanin and pyoverdine are from the Greek, with pyo-, meaning "pus",[8]cyanin, meaning "blue", and verdine, meaning "green".[citation needed] Hence, the term "pyocyanic bacteria" refers specifically to the "blue pus" characteristic of a P. aeruginosa infection. Pyoverdine in the absence of pyocyanin is a fluorescent-yellow color.[citation needed]

Gram-stained P. aeruginosabacteria (pink-red rods)

Biology[edit]

Genome[edit]

The genome of P. aeruginosa consists of a relatively large circular chromosome (– Mb) that carries between 5, and 6, open reading frames, and sometimes plasmids of various sizes depending on the strain.[9] Comparison of genomes from different P. aeruginosa strains showed that just % is shared. This part of the genome is the P. aeruginosa core genome.[10]

strain:VRFPA04CPAO1PA14PACS2
Chromosome size (bp)6,,6,,6,,6,,6,,
ORFs5,5,5,5,5,

A comparative genomic study (in ) analyzed complete genomes from the Pseudomonas genus, of which were P. aeruginosa strains.[11] The study observed that their protein count and GC content ranged between – (average: ) and between –% (average: %), respectively.[11] This comparative analysis further identified aeruginosa-core proteins, which accounts for more than 30% of the proteome. The higher percentage of aeruginosa-core proteins in this latter analysis could partly be attributed to the use of complete genomes. Although P. aeruginosa is a very well-defined monophyletic species, phylogenomically and in terms of ANIm values, it is surprisingly diverse in terms of protein content, thus revealing a very dynamic accessory proteome, in accordance with several analyses.[11][12][13][14] It appears that, on average, industrial strains have the largest genomes, followed by environmental strains, and then clinical isolates.[11][15] The same comparative study ( Pseudomonas strains, of which are P. aeruginosa) identified that 41 of the P. aeruginosa core proteins were present only in this species and not in any other member of the genus, with 26 (of the 41) being annotated as hypothetical. Furthermore, another 19 orthologous protein groups are present in at least / P. aeruginosa strains and absent in all the other strains of the genus.[citation needed]

Population structure[edit]

The population of P. aeruginosa forms three main lineages, characterised by the finished genomes PAO1, PA14, and the highly divergent PA7.[16]

While P. aeruginosa is generally thought of as an opportunistic pathogen, several widespread clones appear to have become more specialised pathogens, particularly in cystic fibrosis patients, including the Liverpool epidemic strain (LES) which is found mainly in the UK,[17] DK2 in Denmark,[18] and AUST in Australia (also previously known as AES-2 and P2).[19] There is also a clone that is frequently found infecting the reproductive tracts of horses.[20][21]

Metabolism[edit]

P. aeruginosa is a facultative anaerobe, as it is well adapted to proliferate in conditions of partial or total oxygen depletion. This organism can achieve anaerobic growth with nitrate or nitrite as a terminal electron acceptor. When oxygen, nitrate, and nitrite are absent, it is able to ferment arginine and pyruvate by substrate-level phosphorylation.[22] Adaptation to microaerobic or anaerobic environments is essential for certain lifestyles of P. aeruginosa, for example, during lung infection in cystic fibrosis and primary ciliary dyskinesia, where thick layers of lung mucus and bacterially-produced alginate surrounding mucoid bacterial cells can limit the diffusion of oxygen. P. aeruginosa growth within the human body can be asymptomatic until the bacteria form a biofilm, which overwhelms the immune system. These biofilms are found in the lungs of people with cystic fibrosis and primary ciliary dyskinesia, and can prove fatal.[23][24][25][26][27][28]

Cellular cooperation[edit]

P. aeruginosa relies on iron as a nutrient source to grow. However, iron is not easily accessible because it is not commonly found in the environment. Iron is usually found in a largely insoluble ferric form.[29] Furthermore, excessively high levels of iron can be toxic to P. aeruginosa. To overcome this and regulate proper intake of iron, P. aeruginosa uses siderophores, which are secreted molecules that bind and transport iron.[30] These iron-siderophore complexes, however, are not specific. The bacterium that produced the siderophores does not necessarily receive the direct benefit of iron intake. Rather, all members of the cellular population are equally likely to access the iron-siderophore complexes. Members of the cellular population that can efficiently produce these siderophores are commonly referred to as cooperators; members that produce little to no siderophores are often referred to as cheaters. Research has shown when cooperators and cheaters are grown together, cooperators have a decrease in fitness, while cheaters have an increase in fitness.[31] The magnitude of change in fitness increases with increasing iron limitation.[32] With an increase in fitness, the cheaters can outcompete the cooperators; this leads to an overall decrease in fitness of the group, due to lack of sufficient siderophore production. These observations suggest that having a mix of cooperators and cheaters can reduce the virulent nature of P. aeruginosa.[31]

Enzymes[edit]

LigDs form a subfamily of the DNA ligases. These all have a LigDom/ligase domain, but many bacterial LigDs also have separate polymerase domains/PolDoms and nuclease domains/NucDoms. In P. aeruginosa's case the nuclease domains are N-terminus, and the polymerase domains are C-terminus, extensions of the single central ligase domain.[33]

Pathogenesis[edit]

Phagocytosis of P. aeruginosaby neutrophil in patient with bloodstream infection (Gram stain)

An opportunistic, nosocomial pathogen of immunocompromised individuals, P. aeruginosa typically infects the airway, urinary tract, burns, and wounds, and also causes other blood infections.[34]

It is the most common cause of infections of burn injuries and of the outer ear (otitis externa), and is the most frequent colonizer of medical devices (e.g., catheters). Pseudomonas can be spread by equipment that gets contaminated and is not properly cleaned or on the hands of healthcare workers.[35]Pseudomonas can, in rare circumstances, cause community-acquired pneumonias,[36] as well as ventilator-associated pneumonias, being one of the most common agents isolated in several studies.[37]Pyocyanin is a virulence factor of the bacteria and has been known to cause death in C. elegans by oxidative stress. However, salicylic acid can inhibit pyocyanin production.[38] One in ten hospital-acquired infections is from Pseudomonas. Cystic fibrosis patients are also predisposed to P. aeruginosa infection of the lungs due to a functional loss in chloride ion movement across cell membranes as a result of a mutation.[39]P. aeruginosa may also be a common cause of "hot-tub rash" (dermatitis), caused by lack of proper, periodic attention to water quality. Since these bacteria thrive in moist environments, such as hot tubs and swimming pools, they can cause skin rash or swimmer's ear.[35]Pseudomonas is also a common cause of postoperative infection in radial keratotomy surgery patients. The organism is also associated with the skin lesion ecthyma gangrenosum. P. aeruginosa is frequently associated with osteomyelitis involving puncture wounds of the foot, believed to result from direct inoculation with P. aeruginosa via the foam padding found in tennis shoes, with diabetic patients at a higher risk.

A comparative genomic analysis of compete Pseudomonas genomes, including complete P. aeruginosa genomes, identified several proteins that are shared by the vast majority of P. aeruginosa strains, but are not observed in other analyzed Pseudomonas genomes.[11] Intrigungly, these aeruginosa-specific core proteins, such as CntL, CntM, PlcB, Acp1, MucE, SrfA, Tse1, Tsi2, Tse3, and EsrC are known to play an important role in this species' pathogenicity.[11]

Toxins[edit]

P. aeruginosa uses the virulence factorexotoxin A to inactivate eukaryotic elongation factor 2 via ADP-ribosylation in the host cell, much as the diphtheria toxin does. Without elongation factor 2, eukaryotic cells cannot synthesize proteins and necrotise. The release of intracellular contents induces an immunologic response in immunocompetent patients. In addition P. aeruginosa uses an exoenzyme, ExoU, which degrades the plasma membrane of eukaryotic cells, leading to lysis. Increasingly, it is becoming recognized that the iron-acquiring siderophore, pyoverdine, also functions as a toxin by removing iron from mitochondria, inflicting damage on this organelle.[40][41]

Phenazines[edit]

Phenazines are redox-active pigments produced by P. aeruginosa. These pigments are involved in quorum sensing, virulence, and iron acquisition.[42]P. aeruginosa produces several pigments all produced by a biosynthetic pathway: pyocyanin, 1-hydroxyphenazine, phenazinecarboxamide, 5-methylphenazinecarboxylic acid betaine, and aeruginosin A. Two operons are involved in phenazine biosynthesis: phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2.[43][44] These operons convert a chorismic acid to the phenazines mentioned above. Three key genes, phzH, phzM, and phzS convert phenazinecarboxylic acid to the phenazines mentioned above. Though phenazine biosynthesis is well studied, questions remain as to the final structure of the brown phenazine pyomelanin.

When pyocyanin biosynthesis is inhibited, a decrease in P. aeruginosa pathogenicity is observed in vitro.[44] This suggests that pyocyanin is most responsible for the initial colonization of P. aeruginosain vivo.

Triggers[edit]

With low phosphate levels, P. aeruginosa has been found to activate from benign symbiont to express lethal toxins inside the intestinal tract and severely damage or kill the host, which can be mitigated by providing excess phosphate instead of antibiotics.[45]

Plants and invertebrates[edit]

In higher plants, P. aeruginosa induces soft rot, for example in Arabidopsis thaliana (Thale cress)[46] and Lactuca sativa (lettuce).[47][48] It is also pathogenic to invertebrate animals, including the nematode Caenorhabditis elegans,[49][50] the fruit fly Drosophila[51] and the moth Galleria mellonella.[52] The associations of virulence factors are the same for plant and animal infections.[47][53]

Quorum sensing[edit]

P. aeruginosa is an opportunistic pathogen with the ability to coordinate gene expression in order to compete against other species for nutrients or colonization. Regulation of gene expression can occur through cell-cell communication or quorum sensing (QS) via the production of small molecules called autoinducers that are released into the external environment. These signals, when reaching specific concentrations correlated with specific population cell densities, activate their respective regulators thus altering gene expression and coordinating behavior. P. aeruginosa employs five interconnected QS systems – las, rhl, pqs, iqs and pch – that each produce unique signaling molecules.[54] las and rhl systems are responsible for the activation of numerous QS-controlled genes, pqs system is involved in quinolone signaling and iqs system plays an important role in intercellular communication.[55] QS in P. aeruginosa is organized in a hierarchical manner. At the top of the signaling hierarchy is the las system, since las regulator initiate the QS regulatory system by activating the transcription of a number of other regulators, such as rhl. So, the las system defines a hierarchical QS cascade from the las to the rhl regulons.[56] Detection of these molecules indicates P. aeruginosa is growing as biofilm within the lungs of cystic fibrosis patients.[57] The impact of QS and especially las systems on the pathogenicity of P. aeruginosa is unclear, however. Studies have shown that lasR-deficient mutants are associated with more severe outcomes in cystic fibrosis patients[58] and are found in up to 63% of chronically infected cystic fibrosis patients[59] despite impaired QS activity.

QS is known to control expression of a number of virulence factors in a hierarchical manner, including the pigment pyocyanin. However, although las system initiates the regulation of the gene expression, its absence does not lead to loss of the virulence factors. Recently, it has been demonstrated that rhl system partially controls las-specific factors, such as proteolytic enzymes responsible for elastolytic and staphylolytic activities, but in a delayed manner. So, las is a direct and indirect regulator of QS-controlled genes.[55] Another form of gene regulation that allows the bacteria to rapidly adapt to surrounding changes is through environmental signaling. Recent studies have discovered anaerobiosis can significantly impact the major regulatory circuit of QS. This important link between QS and anaerobiosis has a significant impact on production of virulence factors of this organism.[60]Garlic experimentally blocks quorum sensing in P. aeruginosa.[61]

Biofilms formation and cyclic di-GMP[edit]

As in most Gram negative bacteria, P. aeruginosabiofilm formation is regulated by one single molecule: cyclic di-GMP. At low cyclic di-GMP concentration, P. aeruginosa has a free-swimming mode of life. But when cyclic di-GMP levels increase, P. aeruginosa start to establish sessile communities on surfaces. The intracellular concentration of cyclic di-GMP increases within seconds when P. aeruginosa touches a surface (e.g.: a rock, plastic, host tissues).[62] This activates the production of adhesive pili, that serve as "anchors" to stabilize the attachment of P. aeruginosa on the surface. At later stages, bacteria will start attaching irreversibly by producing a strongly adhesive matrix. At the same time, cyclic di-GMP represses the synthesis of the flagellar machinery, preventing P. aeruginosa from swimming. When suppressed, the biofilms are less adherent and easier to treat. The biofilm matrix of P. aeruginosa is composed of nucleic acids, amino acids, carbohydrates, and various ions. It mechanically and chemically protects P. aeruginosa from aggression by the immune system and some toxic compounds. P. aeruginosa biofilm's matrix is composed of 2 types of sugars (or "exopolysacharides") named PSL and PEL:

  • Polysaccharide synthesis locus (PSL) and cyclic di-GMP form a positive feedback loop. PSL stimulates cyclic di-GMP production, while high cyclic di-GMP turns on the operon and increases activity of the operon. This gene operon is responsible for the cell-cell and cell-surface interactions required for cell communication. It is also responsible for the sequestering of the extracellular polymeric substance matrix.[63]
  • PEL is a cationic exopolysaccharide that cross-links extracellular DNA in the P. aeruginosa biofilm matrix.[64]

Upon certain cues or stresses, P. aeruginosa revert the biofilm program and detach. Recent studies have shown that the dispersed cells from P. aeruginosa biofilms have lower cyclic di-GMP levels and different physiologies from those of planktonic and biofilm cells.[65][66] Such dispersed cells are found to be highly virulent against macrophages and C. elegans, but highly sensitive towards iron stress, as compared with planktonic cells.[65]

Biofilms and treatment resistance[edit]

Biofilms of P. aeruginosa can cause chronic opportunistic infections, which are a serious problem for medical care in industrialized societies, especially for immunocompromised patients and the elderly. They often cannot be treated effectively with traditional antibiotic therapy. Biofilms seem to protect these bacteria from adverse environmental factors. P. aeruginosa can cause nosocomial infections and is considered a model organism for the study of antibiotic-resistant bacteria. Researchers consider it important to learn more about the molecular mechanisms that cause the switch from planktonic growth to a biofilm phenotype and about the role of QS in treatment-resistant bacteria such as P. aeruginosa. This should contribute to better clinical management of chronically infected patients, and should lead to the development of new drugs.[60]

Recently, scientists have been examining the possible genetic basis for P. aeruginosa resistance to antibiotics such as tobramycin. One locus identified as being an important genetic determinant of the resistance in this species is ndvB, which encodes periplasmicglucans that may interact with antibiotics and cause them to become sequestered into the periplasm. These results suggest a genetic basis exists behind bacterial antibiotic resistance, rather than the biofilm simply acting as a diffusion barrier to the antibiotic.[67]

Diagnosis[edit]

Production of pyocyanin, water-soluble green pigment of P. aeruginosa(left tube)

Depending on the nature of infection, an appropriate specimen is collected and sent to a bacteriology laboratory for identification. As with most bacteriological specimens, a Gram stain is performed, which may show Gram-negative rods and/or white blood cells. P. aeruginosa produces colonies with a characteristic "grape-like" or "fresh-tortilla" odor on bacteriological media. In mixed cultures, it can be isolated as clear colonies on MacConkey agar (as it does not ferment lactose) which will test positive for oxidase. Confirmatory tests include production of the blue-green pigment pyocyanin on cetrimide agar and growth at 42&#;°C. A TSI slant is often used to distinguish nonfermenting Pseudomonas species from enteric pathogens in faecal specimens.[citation needed]

When P. aeruginosa is isolated from a normally sterile site (blood, bone, deep collections), it is generally considered dangerous, and almost always requires treatment.[68][69] However, P. aeruginosa is frequently isolated from nonsterile sites (mouth swabs, sputum, etc.), and, under these circumstances, it may represent colonization and not infection. The isolation of P. aeruginosa from nonsterile specimens should, therefore, be interpreted cautiously, and the advice of a microbiologist or infectious diseases physician/pharmacist should be sought prior to starting treatment. Often, no treatment is needed.[citation needed]

Identification[edit]

Test Results
Gram Stain -
Oxidase +
Indole Production -
Methyl Red -
Voges-Proskauer -
Citrate +
Hydrogen Sulfide Production -
Urea Hydrolysis -
Phenylalanine Deaminase -
Lysine Decarboxylase -
Motility +
Gelatin Hydrolysis +
acid from lactose -
acid from glucose +
acid from maltose -
acid from mannitol +
acid from sucrose -
nitrate reduction +
DNAse -
Lipase +
Pigment + (bluish green pigmentation)
Catalase +
Hemolysis Beta/variable

P. aeruginosa is a Gram-negative, aerobic (and at times facultatively anaerobic), rod-shaped bacterium with unipolar motility.[70] It has been identified as an opportunistic pathogen of both humans and plants.[71]P. aeruginosa is the type species of the genus Pseudomonas.[72]

Identification of P. aeruginosa can be complicated by the fact individual isolates often lack motility. Furthermore, mutations in the gene lasR drastically alter colony morphology and typically lead to failure to hydrolyze gelatin or hemolyze.[citation needed]

In certain conditions, P. aeruginosa can secrete a variety of pigments, including pyocyanin (blue), pyoverdine (yellow and fluorescent), pyorubin (red), and pyomelanin (brown). These can be used to identify the organism.[73]

Pseudomonas aeruginosafluorescence under UV illumination

Clinical identification of P. aeruginosa may include identifying the production of both pyocyanin and fluorescein, as well as its ability to grow at 42&#;°C. P. aeruginosa is capable of growth in diesel and jet fuels, where it is known as a hydrocarbon-using microorganism, causing microbial corrosion.[74] It creates dark, gellish mats sometimes improperly called "algae" because of their appearance.[citation needed]

Treatment[edit]

Many P. aeruginosa isolates are resistant to a large range of antibiotics and may demonstrate additional resistance after unsuccessful treatment. It should usually be possible to guide treatment according to laboratory sensitivities, rather than choosing an antibiotic empirically. If antibiotics are started empirically, then every effort should be made to obtain cultures (before administering the first dose of antibiotic), and the choice of antibiotic used should be reviewed when the culture results are available.

Due to widespread resistance to many common first-line antibiotics, carbapenems, polymyxins, and more recently tigecycline were considered to be the drugs of choice; however, resistance to these drugs has also been reported. Despite this, they are still being used in areas where resistance has not yet been reported. Use of β-lactamase inhibitors such as sulbactam has been advised in combination with antibiotics to enhance antimicrobial action even in the presence of a certain level of resistance. Combination therapy after rigorous antimicrobial susceptibility testing has been found to be the best course of action in the treatment of multidrug-resistant P. aeruginosa. Some next-generation antibiotics that are reported as being active against P. aeruginosa include doripenem, ceftobiprole, and ceftaroline. However, these require more clinical trials for standardization. Therefore, research for the discovery of new antibiotics and drugs against P. aeruginosa is very much needed. Antibiotics that may have activity against P. aeruginosa include:

  • aminoglycosides (gentamicin, amikacin, tobramycin, but notkanamycin)
  • quinolones (ciprofloxacin, levofloxacin, but not moxifloxacin)
  • cephalosporins (ceftazidime, cefepime, cefoperazone, cefpirome, ceftobiprole, but not cefuroxime, cefotaxime, or ceftriaxone)
  • antipseudomonal penicillins: carboxypenicillins (carbenicillin and ticarcillin), and ureidopenicillins (mezlocillin, azlocillin, and piperacillin). P. aeruginosa is intrinsically resistant to all other penicillins.
  • carbapenems (meropenem, imipenem, doripenem, but not ertapenem)
  • polymyxins (polymyxin B and colistin)[75]
  • monobactams (aztreonam)

As fluoroquinolones are one of the few antibiotic classes widely effective against P. aeruginosa, in some hospitals, their use is severely restricted to avoid the development of resistant strains. On the rare occasions where infection is superficial and limited (for example, ear infections or nail infections), topical gentamicin or colistin may be used.

For pseudomonal wound infections, acetic acid with concentrations from % to 5% can be an effective bacteriostatic agent in eliminating the bacteria from the wound. Usually a sterile gauze soaked with acetic acid is placed on the wound after irrigation with normal saline. Dressing would be done once per day. Pseudomonas is usually eliminated in 90% of the cases after 10 to 14 days of treatment.[76]

Antibiotic resistance[edit]

One of the most worrisome characteristics of P. aeruginosa is its low antibiotic susceptibility, which is attributable to a concerted action of multidrug efflux pumps with chromosomally encoded antibiotic resistance genes (e.g., mexAB, mexXY, etc.) and the low permeability of the bacterial cellular envelopes.[77] In addition to this intrinsic resistance, P. aeruginosa easily develops acquired resistance either by mutation in chromosomally encoded genes or by the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug resistance by P. aeruginosa isolates requires several different genetic events, including acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes. Hypermutation favours the selection of mutation-driven antibiotic resistance in P. aeruginosa strains producing chronic infections, whereas the clustering of several different antibiotic resistance genes in integrons favors the concerted acquisition of antibiotic resistance determinants. Some recent studies have shown phenotypic resistance associated to biofilm formation or to the emergence of small-colony variants may be important in the response of P. aeruginosa populations to antibiotics treatment.[60]

Mechanisms underlying antibiotic resistance have been found to include production of antibiotic-degrading or antibiotic-inactivating enzymes, outer membrane proteins to evict the antibiotics and mutations to change antibiotic targets. Presence of antibiotic-degrading enzymes such as extended-spectrum β-lactamases like PER-1, PER-2, VEB-1, AmpC cephalosporinases, carbapenemases like serine oxacillinases, metallo-b-lactamases, OXA-type carbapenemases, aminoglycoside-modifying enzymes, among others have been reported. P. aeruginosa can also modify the targets of antibiotic action, for example methylation of 16S rRNA to prevent aminoglycoside binding and modification of DNA, or topoisomerase to protect it from the action of quinolones. P. aeruginosa has also been reported to possess multidrug efflux pumps systems that confer resistance against a number of antibiotic classes and the MexAB-OprM (Resistance-nodulation-division (RND) family) is considered as the most important[78]. An important factor found to be associated with antibiotic resistance is the decrease in the virulence capabilities of the resistant strain. Such findings have been reported in the case of rifampicin-resistant and colistin-resistant strains, in which decrease in infective ability, quorum sensing and motility have been documented.[79]

Mutations in DNA gyrase are commonly associated with antibiotic resistance in P. aeruginosa. These mutations, when combined with others, confer high resistance without hindering survival. Additionally, genes involved in cyclic-di-GMP signaling may contribute to resistance. When grown in vitro conditions designed to mimic a cystic fibrosis patient's lungs, these genes mutate repeatedly.[80]

Two small RNAs&#;: Sr and ErsA were shown to interact with mRNA encoding the major porin OprD responsible for the uptake of carbapenem antibiotics into the periplasm. The sRNAs bind to the 5'UTR of oprD causing increase in bacterial resistance to meropenem. Another sRNA: Sr was suggested to positively regulate (post-transcriptionally) the expression of PagL, an enzyme responsible for deacylation of lipid A. This reduces the pro-inflammatory property of lipid A.[81] Furthermore, similarly to study in Salmonella[82] Sr regulation of PagL expression was suggested to aid in polymyxin B resistance.[81]

Prevention[edit]

Probiotic prophylaxis may prevent colonization and delay onset of Pseudomonas infection in an ICU setting.[83] Immunoprophylaxis against Pseudomonas is being investigated.[84] The risk of contracting P. aeruginosa can be reduced by avoiding pools, hot tubs, and other bodies of standing water; regularly disinfecting and/or replacing equipment that regularly encounters moisture (such as contact lens equipment and solutions); and washing one's hands often (which is protective against many other pathogens as well). However, even the best hygiene practices cannot totally protect an individual against P. aeruginosa, given how common P. aeruginosa is in the environment.[85]

Experimental therapies[edit]

Phage therapy against P. aeruginosa has been investigated as a possible effective treatment, which can be combined with antibiotics, has no contraindications and minimal adverse effects. Phages are produced as sterile liquid, suitable for intake, applications etc.[86] Phage therapy against ear infections caused by P. aeruginosa was reported in the journal Clinical Otolaryngology in August [87]

Research[edit]

In , João Xavier described an experiment in which P. aeruginosa, when subjected to repeated rounds of conditions in which it needed to swarm to acquire food, developed the ability to "hyperswarm" at speeds 25% faster than baseline organisms, by developing multiple flagella, whereas the baseline organism has a single flagellum.[88] This result was notable in the field of experimental evolution in that it was highly repeatable.[89]

P. aeruginosa has been studied for use in bioremediation and use in processing polyethylene in municipal solid waste.[90]

See also[edit]

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Sours: https://en.wikipedia.org/wiki/Pseudomonas_aeruginosa
Pseudomonas Aeruginosa for the USMLE Step 1

Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence

Introduction

Pseudomonas aeruginosa is a Gram-negative and ubiquitous environmental bacterium. It is an opportunistic human pathogen capable of causing a wide array of life-threatening acute and chronic infections, particularly in patients with compromised immune defense. It has been of particular importance since it is the main cause of morbidity and mortality in cystic fibrosis (CF) patients and one of the leading nosocomial pathogens affecting hospitalized patients while being intrinsically resistant to a wide range of antibiotics.

P. aeruginosa strains possess large genomes (5&#x;7 Mbp). Their metabolic capacity is extensive as exemplified by their ability to produce multiple secondary metabolites and polymers as well as their ability to use various carbon sources and electron acceptors. The repertoire of P. aeruginosa genes which are substantially conserved suggest the highest proportion of regulatory genes and networks observed in known bacterial genomes and is foundational to respond and adapt to diverse environments (Stover et al., ; Mathee et al., ; Frimmersdorf et al., ). The ubiquitous presence of P. aeruginosa as well as its prevalence and persistence in clinical settings including intrinsic resistance to therapeutics are attributed to its extraordinary capability of survival by recruiting an arsenal of responsive mechanisms.

In the present review, we have attempted to summarize the diversity of these mechanisms causing the versatility of P. aeruginosa to adapt and thrive in unfavorable conditions particularly during pathogenesis. To this end, we will describe the clinical importance of P. aeruginosa followed by the well-characterized and most recent findings about key strategic adaptation mechanisms including quorum sensing (QS), motility-sessility switch, biofilm formation, antibiotic resistance mechanisms, adaptive radiation for persistence, stringent response and persisters, and the CRISPR-Cas systems. Recent findings on adaptive mechanisms will be set into context of the overall physiology of P. aeruginosa by also depicting on future research needs.

Clinical Importance

The CF patients suffer from a multisystem disease due to inheritable genetic defects in the CF transmembrane conductance regulator (CFTR) gene. However, the recurrence of bacterial infections in the abnormal mucus layers is the main cause of morbidity and mortality of CF patients (Khan et al., ; Rosenfeld et al., ). The CFTR regulator is responsible for regulating the transport of electrolytes and chloride across epithelial cell membranes to maintain normal mucus properties and homeostasis. Therefore, the loss of function of the CFTR protein results in abnormally thick, dehydrated and sticky mucus layers in the lung (Flume and Van Devanter, ). Hence, the CF patients are largely susceptible to respiratory infections by P. aeruginosa from infancy. When they are under a year old, almost 30% of CF infants can acquire initial P. aeruginosa strains from the environment leading to acute infections. This rate increases to about 50% by the age of 3 years while mucoid phenotypes causing chronic infections have been reported emerging at the age of 3 to16 years (median of 13 years) (Rehm et al., ; da Silva et al., ; Jones et al., ). P. aeruginosa will adapt to CF airways and persist as overwhelming, predominant and ineradicable infections to the end of patients&#x; life in almost 70% of adults (Döring et al., ).

Furthermore, P. aeruginosa is also largely associated with hospital acquired infections including ventilator-associated pneumonia, central line-associated bloodstream infection, urinary catheter-related infection, and surgical/transplantation infections (Cardo et al., ; Nathwani et al., ; Trubiano and Padiglione, ). The International Nosocomial Infection Control Consortium reported that P. aeruginosa nosocomial infections have become a worldwide healthcare issue (Rosenthal et al., ). A cohort study reported that P. aeruginosa had the highest burden of healthcare-acquired infections in European intensive care units (Lambert et al., ). In the United States healthcare-associated P. aeruginosa infections were estimated to contribute to 51, cases each year (Eurosurveillance Editorial Team, ). P. aeruginosa is prevalent in healthcare settings because it is a common companion of patients under medical care and also it can survive on abiotic and biotic surfaces such as medical equipment resisting disinfection methods while being transmissible from patient-to-patient (Russotto et al., ).

P. aeruginosa infections are becoming more difficult to treat because this bacterium is naturally resistant to many antibiotics and the number of multidrug- and pan-drug-resistant strains is increasing worldwide. Strains have been reported which are resistant to almost all class of commonly used antibiotics including aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems (Hancock and Speert, ; Poole, ; Eurosurveillance Editorial Team, ). In the United States about 13% of P. aeruginosa infections are caused by multidrug resistant strains (Eurosurveillance Editorial Team, ).

P. aeruginosa utilizes sophisticated genotypic events to support various phenotypes and molecular mechanisms required for survival during pathogenesis and antibiotic treatment.

Therefore, at initial stages of CF lung colonization, a large number of virulence and intrinsic antibiotic resistance mechanisms mediate survival. After infection, bacteria are exposed to inflammatory responses including oxidative stress followed by treatment with antibiotics (Furukawa et al., ; Turner et al., ). These environmental stress factors induce the expression of different sets of genes enabling P. aeruginosa to adapt and switch to persisting and resistant phenotypes, while becoming less virulent, such as upon formation of mucoid biofilms (MacDougall et al., ; Poole, ; Gellatly and Hancock, ). Due to the existence of an arsenal of molecular mechanisms conferring resistance to multiple classes of antibiotics, therapeutic options are increasingly limited for treatment of infections, while the number of infection incidences and multi-drug resistance strains are increasing.

Central Regulatory Role of Quorum Sensing (QS) for Virulence and Adaptation

Communication between individual cells using specific chemical signals is a well-known capability of bacteria and is called quorum sensing. Indeed, QS controls social behavior of bacteria by multiple interconnected signaling pathways (LaSarre and Federle, ). It allows bacterial communities to regulate a variety of biological processes important for bacterial adaptation and survival. Basically, this phenomenon relies on regulating the expression of specific sets of genes in response to a critical threshold of signaling molecules known as autoinducers (AIs). QS will mediate population density dependent collective responses and is therefore beneficial for community survival. A study showed that cells&#x; responses to QS signals and the corresponding gene expression profile is heterogeneous within a given community leading to increasing fitness and chance of survival (Grote et al., ).

During pathogenesis P. aeruginosa QS plays a critical role for survival and colonization by coordinating phenotypic alterations at early stages of infection i.e., after attachment (González and Keshavan, ). The progress of acute to chronic infection is critically influenced by QS-dependent gene expression. More than 10% of P. aeruginosa genes are regulated by QS. These genes are mainly involved in virulence factor production, motility, motility-sessility switch and biofilm development, antibiotic resistance mechanisms and the adjustment of metabolic pathways for stress responses (Venturi, ; Williams and Camara, ; Barr et al., ). The role of QS in each physiological adaptation will be discussed below.

Molecular Mechanisms Underlying QS

As shown in Figure 1, four main pathways of QS dependent signaling exist in P. aeruginosa. These constitute a hierarchal network mediating integration of multiple signals via cross-talk between the QS signaling pathways. The most recently discovered IQS signaling pathway is less understood and its integration and impact on gene expression still needs to be unraveled. It was previously proposed that the IQS molecule (an aeruginaldehyde) is the product of enzymatic activity of proteins encoded by ambBCDE genes (Lee et al., ), while new findings showed the IQS molecule is a byproduct of the pyochelin biosynthesis pathway and AmbBCDE proteins are responsible for the biosynthesis of the toxin Laminomethoxy-transbutenoic acid (AMB) (Ye et al., ; Rojas Murcia et al., ; Sun et al., ).

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Figure 1. Hierarchical QS network in P. aeruginosa and regulation of virulence factors. (A) So far, four pathways including Las, Rhl, Pqs, and IQS have been understood as mediating QS responses in P. aeruginosa while LasR resides at the top of the cascade. In response to specific stimuli/stress, each pathway synthesizes cognate autoinducers (AIs) [HSL(3-oxo-Chomoserine lactone)], BHL (N-butyrylhomoserine lactone or C4-HSL), PQS (2-heptylhydroxyquinolone) and IQS [2-(2-hydroxyphenyl)-thiazolecarbaldehyde (aeruginaldehyde)]. Export and import of HSL, BHL, and PQS is mediated by the efflux pumps MexAB-OprM/MexEF-OprN, free diffusion and membrane vesicles, respectively. Question mark indicates unknown pathway of IQS transportation. As fine-tuned individual circuits, but interconnected (dashed lines), transcriptional factors (i.e., LasR, RhlR, and PqsR) are activated by AIs for upregulating expression of cognate AI synthases (respectively, LasI, RhlI, PqsABCDH) as well as others such as virulence factor genes. The IQS pathway remains unraveled and the IQS receptor is still unknown. Various secretion systems mainly type 1 and 2 secretion systems (T1SS/T2SS) and also PvdRT-OpmQ efflux pump mediate the secretion of virulence factors. (B) QS initiates upon cumulative production of AIs (small colorful circles) by increasing cell density and results in collective responses. AprA, alkaline protease; Pyd, pyoverdine; PLC, phospholipase C; Tox, toxin A; LasA, LasA elastase; LasB, LasB elastase; HCN, hydrogen cyanide; Pyo, pyocyanin; Rhld, rhamnolipids; Lec A, lectin A; CM, cytoplasmic membrane; OM, outer membrane.

In regard to the other three QS pathways, each system consists of at least two major functional elements; the first category of proteins (i.e., LasR, RhlR, or PqsR, respectively) is activated upon sensing specific autoinducers (AIs) and acts as transcriptional activator for genes encoding the second tier proteins, the cognate AI synthases (i.e., LasI, RhlI, and PqsABCDH, respectively). These activation steps constitute a fine-tuned circuit by which the synthesized AIs are exported outside the cells followed by being imported again (Figure 1). Transportation of these signals is not well understood, but it is proposed as being mediated by free diffusion, membrane transporters such as specific efflux systems or membrane vesicles (Mashburn and Whiteley, ; Martinez et al., ; Alcalde-Rico et al., ). Beyond this regulatory circuit the activated AI-sensing proteins act as transcriptional factors for activating the expression of other set of genes such as virulence genes in response to environmental stimuli (Figure 1). Transcriptional activation occurs via binding to conserved las/rhl boxes acting as operators residing upstream of these genes (González-Valdez et al., ; Lee and Zhang, ; Banerjee and Ray, ; Papenfort and Bassler, ). In the hierarchy of this network, LasR resides at the top of the cascade and along with RhlR mediates QS signaling at early stages of exponential growth phase while the PQS signaling pathway is active at late exponential growth phase (Choi et al., ). As abovementioned, cumulative and cell density-dependent production of AIs is required for reaching a specific threshold triggering collective responses by individual cells.

QS-Controlled Virulence Factors and Stress Responses

Production of virulence factors is a survival strategy for pathogens to evade the host immune defense resulting in progression of pathogenesis particularly at early stage of colonization and acute infection. A large number of virulence factors including cell-associated or secreted compounds, both low and high molecular weight compounds have been reported as important in colonization and establishment of infections by P. aeruginosa. Although they play a critical role in promoting bacterial growth and survival, they can cause devastating injuries to the host tissues and impair the immune responses. QS deficient mutants cause considerably less tissue damage and pathological changes during infections due to a significant decrease in the virulence and cytotoxicity (Nelson et al., ; Feng et al., ).

Production of many virulence factors is metabolically costly and requires community involvement. Hence, they are mainly under the regulatory control of the QS systems (Figure 1, Table 1; Whiteley et al., ; Diggle et al., ; Wagner et al., ; García-Contreras, ).

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Table 1. Key QS-dependent virulence factors produced by P. aeruginosa.

Analysis of bronchial secretions of CF patients during different stages of pulmonary exacerbations showed that QS upregulates the expression of genes involved in the production of some destructive virulence factors such as proteases (elastase, alkaline protease), phenazines (pyocyanin), toxins (exotoxin A), rhamnolipids and hydrogen cyanide (Jaffar-Bandjee et al., ; Lee and Zhang, ). Production of these toxic compounds is destructive to the host cells/tissues by impairing permeability barrier and by inhibiting protein production promoting cell death.

Recent findings suggested a correlation between systemic concentrations of some QS signaling molecules with the clinical status of pulmonary exacerbation and at least some QS signaling molecules were elevated at the start of either pulmonary exacerbation or antibiotic treatment when assessing different biofluids (Barr et al., ). In conclusion, virulence factors assist bacteria in colonization and survival aligned with worsened clinical course of infections. Thus, QS can determine the degree of pathological damages and clinical stages of infections in response to environmental factors.

Pathogenesis encompasses various stresses such as host immune factors, bacterial interspecies competition, phosphate/iron depletion and starvation. QS systems and production of some virulence factors mediate appropriate responses to these stresses to promote survival and adaptation (García-Contreras et al., ). Here, we provide some examples of stress responses mediated by the QS systems.

Interferon-³ (IFN-³) is a crucial cytokine of the human immune system during infection and it coordinates a wide array of immunological responses such as up-regulation of pathogen recognition and the activation of bactericidal effector functions (Schroder et al., ). IFN-³ produced by T-cells was shown to bind directly to P. aeruginosa OprF, an outer membrane protein. Upon formation of IFN-³-OprF complexes the rhl QS system was activated and resulted in up-regulation of the expression of lecA (or PA-I lectin) and synthesis of pyocyanin. The lecA gene encodes the virulence determinant, galactophilic lectin (or LecA) (Wu et al., ) which is cytotoxic and acts as adhesion factor mediating initiation of host recognition by P. aeruginosa (Chemani et al., ). It induces an increased permeability of the intestinal and respiratory epithelial cells enabling cytotoxic exoproducts such as exotoxin A (Laughlin et al., ) to enter host cells (Bajolet-Laudinat et al., ). In addition it also contributes to biofilm development (Diggle et al., ). Furthermore, the QS system has been reported to mediate a response to the host antimicrobial factor LL by increasing the production of pyocyanin, hydrogen cyanide, elastase and rhamnolipids (Strempel et al., ). The QS-dependent production of rhamnolipids has a crucial role in neutralizing the attack of neutrophils due to their necrotic property (Jensen et al., ; Van Gennip et al., ).

Recent findings indicated that the LasR and RhlR QS systems, but not the PQS system, play major roles in adaptation and response to environmental stresses such as oxidative, heat, heavy metal and salt stresses (García-Contreras et al., ).

The stress response of P. aeruginosa to the depletion of phosphate and iron was found to be linked (Slater et al., ). Different studies showed that acquisition of phosphate and iron are important for survival and pathogenesis of P. aeruginosa and the expression of cognate genes mediating acquisition of these elements are upregulated upon interaction with human respiratory epithelial cells (Frisk et al., ; Chugani and Greenberg, ). Various studies unraveled that phosphate- and iron-deficient conditions can trigger the activation of the QS system especially via the IQS- or PQS-dependent pathway leading to boosted activation of central QS and the production of virulence factors such as rhamnolipids, phenazines, cyanide, exotoxin A, LasA protease, elastase, and antimicrobials (Kim et al., ; Long et al., ; Zaborin et al., ; Bains et al., ; Lee et al., ; Nguyen et al., ). Production of such virulence factors can increase cytotoxic impact of bacteria on host tissue and promote pathogen survival.

The recently discovered IQS system which controls the expression of a large set of virulence factors was shown to be directly activated by phosphate limitation in P. aeruginosa (Lee et al., ; Lee and Zhang, ).

P. aeruginosa QS signaling molecules such as 2-heptylhydroxyquinoline (HHQ) and 2-heptylhydroxyquinoline-N-oxide (HQNO) can serve as antimicrobial agents against Staphylococcus aureus which is commonly present during early stages of pulmonary infections in CF patients. It is proposed that this antibacterial activity supports the dominance of P. aeruginosa during the course of infection. Interestingly, this inter-species competition is linked to the availability of iron as the depletion of iron potentiates the antistaphylococcal activity of these metabolites (Nguyen et al., ). Also, LasA is a staphylolytic protease produced by P. aeruginosa and it is under the regulation of the las QS system (Toder et al., ). Furthermore, when P. aeruginosa was grown together with the yeast Candida albicans in a mixed biofilm, the QS system upregulated the production of virulence factors such as pyoverdine, rhamnolipids and pyocyanin (Trejo-Hernández et al., ).

Oxygen depletion known as hypoxia is another stress factor for P. aeruginosa during pathogenesis. Hypoxia condition is influenced by various factors such as reduced ventilation through viscose layers, chronic inflammation, microbial population and biofilm formation (Hassett, ; Worlitzsch et al., ; Yoon et al., ; Alvarez-Ortega and Harwood, ; Hassett et al., ). However, P. aeruginosa can survive and grow under hypoxia to high cell densities. Under hypoxia stress P. aeruginosa retains the capability of microaerobic respiration, although occurrence of nitrate respiration was thought to be possible (Alvarez-Ortega and Harwood, ). The QS regulon expression occurs at low oxygen conditions. Hammond et al. () unraveled that the 4-hydroxyalkylquinolines (HAQ)-dependent QS pathway is active during hypoxia via the ANR protein as the master transcriptional regulator of anaerobic respiration while it is in the absence of LasR signaling (Hammond et al., ). Under low oxygen tension, the ANR protein positively regulated the production of the QS signaling molecule 4-hydroxyalkylquinolines and in turn the regulation of virulence-related genes could continue via PQS system (Hammond et al., ). Furthermore, under hypoxia stress the ANR protein and the QS systems cooperatively regulate hydrogen cyanide biosynthesis (Castric, , ; Pessi and Haas, ). This study provided further evidence that low oxygen-dependent QS inversely correlates with denitrification i.e., suppresses nitrate respiration (Hammond et al., ).

Overall, these examples have provided further insight into the versatility of P. aeruginosa to adapt to various environmental conditions by processing signals via integrated and cross-talking QS pathways resulting in enhanced survival i.e., in a medical context establishment of acute and chronic infections.

Persistence and Biofilm Formation

During acute infection the relationship between pathogen and host is reciprocally devastating as a variety of cytotoxic molecules produced by bacteria impair the host cellular processes while bacteria on the other hand encounter immune system responses such as production of antimicrobial compounds and reactive oxygen species, as well as enhanced phagocytosis. In this context, motile P. aeruginosa display a more virulent phenotype. Various modes of P. aeruginosa motility such as swimming and swarming involving flagella and twitching using type 4 pili are associated with virulent traits (Winstanley et al., ). A motile cell is readily detectable by the host immune system via flagellar and/or other motility components mediating recognition and induction of signaling pathways which trigger inflammatory responses and phagocytosis by murine or human macrophages (Amiel et al., ).

Switching to sessile lifestyle along with lower virulency is a survival advantage by which many pathogenic bacteria such as P. aeruginosa evade stresses and adverse conditions. They lose motility and attach to surfaces and form cellular aggregations or microcolonies which are embedded in extracellular polymeric substances (EPS) to protect bacteria from the surrounding environment. These structures are so called biofilms conferring an extreme capacity for persistence against phagocytosis, oxidative stresses, nutrient/oxygen restriction, metabolic waste accumulation, interspecies competitions, and conventional antimicrobial agents (Leid, ; Olsen, ).

Formation of mucoid biofilm by P. aeruginosa is the hallmark of chronic infections and indicative of disease progression and long-term persistence. As a consequence, P. aeruginosa dominates the microbial community of CF lungs in patients older than 24 years (McDaniel et al., ).

Other P. aeruginosa biofilm associated infections include chronic wound infection, chronic otitis media, chronic rhinosinusitis, catheter-associated urinary tract infection, and contact lens-related keratitis (Römling and Balsalobre, ).

Composition of the P. aeruginosa Biofilm

Formed on abiotic and biotic surfaces, the matrix of most biofilms embedding bacterial cells may account for over 90% of dry weight of whole biofilm mass. In fact, this matrix creates a niche rendering bacteria for intense cell-cell interaction and communication as well as a reservoir of metabolic substances, nutrients and energy for promoting growth while shielding cells from unfavorable conditions (Flemming and Wingender, ). The matrix is mainly formed by extracellular polymeric substances (EPS) which are mainly polysaccharides, proteins, extracellular DNA (eDNA) and lipids (Strempel et al., ). Major polymers and relevant characteristics are listed in Table 2.

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Table 2. Key polymeric substances in P. aeruginosa biofilm formation and development.

The exopolysaccharides Psl, Pel, and alginate are major constituents of the P. aeruginosa biofilm matrix involved in surface adhesion and together with eDNA determine the biofilm architecture. These EPS play an important role in resistance to immune responses and antibiotic treatments (Ghafoor et al., ; Gellatly and Hancock, ; Strempel et al., ). The differential role of each EPS has been analyzed at each stage of biofilm development. The various exopolysaccharides and eDNA were shown to interactively contribute to the biofilm architecture (Ghafoor et al., ). The presence of various EPS exhibiting different physiochemical properties confers a survival strategy for increasing the flexibility and stability of biofilms under various conditions (Jennings et al., ).

The Psl polysaccharide is a key element at early stage of biofilm formation when cells explore surfaces for adhesion (Overhage et al., ). It is anchored around cells in a helical arrangement initiating biofilm formation by enhancing cell migration, cell-cell interaction and cell-surface adhesion whereas in mature biofilms it is located to the periphery of mushroom shaped macrocolonies (Ma et al., ; Zhao et al., ). Psl can exist as a fiber-like matrix requiring type 4 pili-mediated migration of cells (Wang S. et al., ). It protects cells against phagocytosis and oxidative stress during infection (Mishra et al., ). Recent studies suggested that Psl can provide an instant protective role against anti-biofilm agents and a broad spectrum of antibiotics particularly at early stage of biofilm development (Zegans et al., ; Billings et al., ). Therefore, Psl provides a survival advantage during pathogenesis.

Similar to Psl, Pel is important for initiating and maintaining cell&#x;cell interaction in biofilms (Colvin et al., ). Pel and/or Psl are the primary matrix structural polysaccharides in non-mucoid P. aeruginosa strains as a predominant environmental phenotype. However, contribution of Psl and Pel to the structure of mature biofilms is strain-dependent while both unique and functionally redundant roles have been reported for these exopolysaccharides (Colvin et al., ). Recent studies elucidating the chemical structure and biological function of Pel demonstrated that it is a major structural component of the biofilm stalk where it cross-links eDNA and structurally compensates for the absence of Psl in the periphery of mature biofilm (Jennings et al., ). Furthermore, Pel was shown to protect bacteria against certain aminoglycoside antibiotics (Colvin et al., ).

Overproduction of the exopolysaccharide, alginate, is characteristic for mucoid phenotype of most clinical isolates from CF patients. During adaptation to the CF lung environment, alginate is overproduced and predominantly constitutes the matrix of mature biofilms conferring a slimy or mucoid phenotype. Indeed, it is greatly important in biofilm maturation, structural stability and protection as well as persistence by shielding P. aeruginosa cells against opsonophagocytosis, free radicals released from immune cells, and antibiotics used for treatment (Hay et al., a; Hay I. D. et al., ; Strempel et al., ). Some in vitro biofilm studies showed that the composition of the alginates can greatly influence biofilm characteristics such as viscoelastic property, bio-volume, cell density and architecture as well as cell-to-cell interaction, cell aggregation and surface attachment (Tielen et al., ; Moradali et al., ).

As abovementioned, eDNA is another important structural component for biofilm development and along with the Pel polysaccharide it can be detected within the stalks of mushroom-shaped macrocolonies. However, eDNA has multifaceted roles in biofilm formation such as contribution to forming cation gradients in the matrix via the chelating interaction of highly anionic DNA with cations such as Mg2+, Ca2+, Mn2+, and Zn2+, as a nutrient source during starvation, facilitating twitching motility and coordinating cell movements and conferring antibiotic resistance (Allesen-Holm et al., ; Mulcahy et al., ; Gloag et al., ).

Among the proteinaceous biofilm constituents, both flagella and the type 4 pili are important during maturation of the biofilm, however, these cell appendages are not commonly considered as classical matrix components of biofilms. Type 4 pili are important for adhesion and promote initial attachment of cells to surfaces at early stage of biofilm formation. Together with eDNA, flagella and the type 4 pili mediate migration required for the formation of the stalk and the cap in the mushroom-shaped microcolonies of the mature biofilm (Table 2; Barken et al., ; Mann and Wozniak, ).

Central Regulatory Network Governing the Motility-Sessility Switch

Transition from motility to sessility requires dynamic regulatory networks at transcriptional, post-transcriptional and post-translational levels resulting in coordinated timely expression of hundreds of genes. These events mainly arrest flagella based motility and the production of virulence factors such as exotoxins and proteases while positively regulating surface attachment, EPS production and biofilm maturation (Figure 2).

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Figure 2. Regulatory networks underlying biofilm formation by P. aeruginosa. (A) Elevation of the cyclic di-GMP molecule is a key determinant for the motility-sessility switch. Environmental cues are sensed by various proteins localized in the envelope of the cells where these proteins contribute to two-component systems (brown/green rectangles), chemoreceptor-like system (orange complex) and other receptor mediated signaling pathways (arranged in the left side of figure). Either triggered as phosphorylation cascades (small red circle) or protein-protein interactions, the signals induce diguanylate cyclases (containing GGDEF motif) (red rectangles) to synthesize cyclic di-GMP from two molecules of GTP (guanosine-5&#x;-triphosphate). Consequently, cyclic di-GMP sensing proteins act as receptor/effector for specific outputs such as induction of alginate and Pel polymerization, inhibition of motility and derepression of psl/pel expression via FleQ, induction of attachment and biofilm formation/maturation triggered by two component systems. The two-component systems are interconnected and the LadS/RetS/GacS/GacA/RsmA regulatory network (green rectangles) plays a key role in the phenotypic switch from motility to sessility and downregulation of QS and virulence factor production. (B) Various stages of biofilm formation and development were represented. Plus and minus signs represent positive and negative effect of transcriptional regulators, respectively. CM, cytoplasmic membrane; OM, outer membrane.

The small molecule cyclic-3&#x;5&#x;-diguanylic acid (cyclic di-GMP) is a key signal in post-transcriptional regulation of biofilm formation. It is an almost ubiquitous second messenger present in a wide range of bacteria that principally controls motility-sessility switch. The major determinant for this substantial phenotypic change is the cellular level of cyclic di-GMP, so that its elevation triggers biofilm formation while inhibiting motility. The cyclic di-GMP signaling system is very complex and two groups of proteins have been identified as main actors. The first group comprises cyclic di-GMP metabolizing enzymes including diguanylate cyclases (DGC) (containing GGDEF motif) and phosphodiesterases (containing EAL or HD-GYP motif) that respectively synthesize and degrade cyclic di-GMP in cells (Römling et al., ; Valentini and Filloux, ). At least 40 such proteins directly synthesize and/or degrade cyclic di-GMP in P. aeruginosa which controls cellular level of this molecule in response to perceived stimuli (Ryan et al., ). The second group is represented by cyclic di-GMP sensing proteins which also act directly as effectors or via protein-protein interactions to mediate the output response (Römling et al., ). For example, cyclic di-GMP is essential for the activation of alginate polysaccharide polymerization (Remminghorst and Rehm, ). Experimental evidence indicated that a pool of cyclic di-GMP is synthesized by MucR (a hybrid GGDEF/EAL domain-containing protein) in the proximity of the alginate biosynthesis/secretion multi-protein complex of P. aeruginosa (Hay et al., b; Wang et al., ). Cyclic di-GMP binds to PilZ domain of Alg44 protein and allosterically activates alginate polymerization via interaction with Alg8 glycosyltransferase (Hay et al., b; Moradali et al., ; Wang et al., ). Also, cyclic di-GMP binding to FleQ, a transcriptional master regulator represses flagella biosynthesis while it concomitantly derepresses the expression of pel and psl genes (Baraquet et al., ; Figure 2). Likewise, there are many other receptor/effector proteins which enhance required pathways for biofilm formation upon cyclic di-GMP binding while they inhibit motility and other virulence factor synthesis pathways.

In addition, the Wsp chemosensory system in P. aeruginosa is homologous to chemotaxis signaling pathways which regulate cyclic di-GMP synthesis via signal transduction (Figure 2). A cascade of phosphorylations is triggered upon surface attachment and possibly sensing mechanical stress or other environmental stimuli which then activate the cyclic di-GMP synthesizing protein WspR promoting biofilm formation (Hickman et al., ; Porter et al., ).

Furthermore, transduction of phosphorylation events via two-component regulatory systems controls biofilm formation in a stage-specific manner (Figure 2). This network consists of BfiRS, BfmRS, and MifRS and GacS/GacA regulatory components (Petrova and Sauer, ). The GacS/GacA two-component system is part of the global regulatory pathway comprising LadS/RetS/GacS/GacA/RsmA proteins (Figure 2). This pathway controls many physiological responses at post-transcriptional level and is involved in both motility-sessility and acute-chronic infection transitions. Of this regulatory pathway, the RNA-binding protein RsmA negatively controls biofilm formation pathways while it induces production of T3SS, type 4 pili and other virulence factors. RsmA binds to psl mRNA and inhibits the translation of required proteins for Psl polysaccharide biosynthesis (Irie et al., ; Jimenez et al., ). It also represses production of GGDEF/EAL encoding proteins; hence, it inhibits elevation of cyclic di-GMP levels. In P. aeruginosa, under stress conditions, this pathway generates non-coding RNAs (ncRNAs) known as RsmY and RsmZ which counteract RsmA translational repression activity, consequently derepressing biofilm formation mainly via cyclic di-GMP level increase resulting in exopolysaccharides production (Jimenez et al., ).

There are other regulatory pathways known to be involved in cyclic di-GMP turnover in response to external stimuli, but the further precise function still remains to be elucidated (Figure 2).

The Role of QS in Biofilm Development and Maturation

In addition to abovementioned regulatory networks, biofilm residents utilize QS systems for cell-to-cell communication and spatio-temporal regulation of expression of specific genes. During chronic infection, a major proportion of the colonizing population was thought to lose QS due to hypermutation events and phenotypic alterations. However, further investigations have now revealed that genes involved in the progress of biofilm maturation and persistence are positively regulated by QS in P. aeruginosa. Indeed QS-deficient mutants of P. aeruginosa (i.e., &#x;lasR&#x;rhlR and &#x;lasI&#x;rhlI) formed thin and much less developed biofilms which were more sensitive to antibiotic treatments and eradication (Shih and Huang, ; Nelson et al., ). Furthermore, Bjarnsholt et al. () demonstrated that at least a part of QS pathways i.e., rhl encoded system and the production of C4-HSL signals was retained in predominantly mucoid population at the end of chronic stages coinciding with overproduction of alginate and rhamnolipids (Bjarnsholt et al., ). The biosurfactants, rhamnolipids, have been suggested to play an active role in maintenance of the biofilm architecture by contributing to the formation of internal cavities within the mature biofilm, allowing proper flow of water and nutrients (Davey et al., ; Boles et al., ; Dusane et al., ; Chrzanowski et al., ). Additionally, the production of pel polysaccharide, eDNA and QS-controlled production of pyocyanin are critical for biofilm maturation. Pel cross-links eDNA in the biofilm stalk via ionic interactions and it serves as important structural components of the biofilm matrix of P. aeruginosa (Jennings et al., ). Furthermore, pyocyanin molecules can promote eDNA release by inducing bacterial cell lysis. Pyocyanin binds to eDNA increasing its solution viscosity which influences the physicochemical interactions of the biofilm matrix with environment as well as facilitates cellular aggregations (Das et al., , ). Collectively, such molecular and cellular interactions in combination with other polymeric substances lead to establishment of a robust and mature biofilm.

Antibiotic Resistance Mechanisms

Indeed, the emergence of antibiotic resistant bacteria is a global health issue. Among identified notoriously multi-drug resistant (MDR) bacteria, P. aeruginosa has been introduced as a major concern with a growing threat to global health resulting in dramatically increasing prevalence of nosocomial and chronic infections. This is due to the extraordinary capacity of these bacteria to develop resistance against a wide range of antimicrobials through various molecular mechanisms which are often simultaneously present in clinical isolates. Although each resistance mechanism is related to a specific class of antibiotics, multiple mechanisms mediate variably resistance to each class of antibiotics (Potron et al., ). Furthermore, the contribution of each mechanism varies from country to country. Loss or reduced copy numbers of OprD and overproduction of active efflux pumps, AmpC ²-lactamase and extended-spectrum ²-lactamases have been mainly reported as main contributors to multi-drug resistance phenotypes of P. aeruginosa isolates.

Recent reviews have described the prevalence and contribution of each resistance mechanism to each class of antibiotics in detail (Lister et al., ; Strateva and Yordanov, ; Sun et al., ; Hong et al., ; Potron et al., ). Here, we reviewed the most frequent and well-understood findings which are classified into intrinsic, acquired and adaptive mechanisms, and we provide an update on our understanding of how P. aeruginosa can survive antibiotic treatments.

Intrinsic Resistance Mechanisms

Like many Gram-negative bacteria, P. aeruginosa can be intrinsically resistant to particular antibiotics. Such intrinsic resistance mechanisms stem from the existence of genes in bacterial genome encoding inherent properties of cell structures and composition providing protection against toxic molecules and antimicrobials. It can also be contributed by the lack of susceptible sites which naturally exist in antibiotic sensitive species (e.g., resistance to triclosan) (Lambert et al., ; Blair et al., ; Figure 3).

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Figure 3. Intrinsic, acquired and adaptive mechanisms confer antibiotic resistance in P. aeruginosa. For each mechanism, various molecular strategies, which confer resistance to specific class of antipseudomonal antibiotics (Car., Carbapenems; Ceph., Cephalosporins; Pen., Penicillins; Ami., Aminoglycosides; Flu., Fluoroquinolones; Mac., Macrolides and Pol., Polymyxins), were presented at the top of the figure (underlined) Intrinsic mechanisms such as structural barriers [e.g., EPS (extracellular polymeric substances)], OprD reduction and basal production of AmpC ²-lactamase and MexAB/XY efflux pumps confer a basal resistance to some group of antibiotics. However, in acquired resistance, mutational changes in the oprD gene, transcriptional repressors causingupregulation of resistance genes and efflux pumps conferring resistance against a wider spectrum of antibiotics. Plasmid-mediated resistance is very potent as a variety of resistance genes can be exchanged among bacteria. Either mediated by mutational changes in the genome or in plasmids, resistance to polymyxins occurs via modification of LPS (lipopolysaccharide) components hindering binding of the antibiotic to this layer. Adaptive resistance occurs in the presence of antibiotics mainly via mutation in regulatory genes. This is a transient and reversible resistance, which will reverse upon removal of antibiotics. Stars represent antibiotics and dashed/wavy lines represent transcriptional levels of each gene product. CM, cytoplasmic membrane; OM, outer membrane.

However, hydrophilic antibiotics can enter cells by diffusing through membrane channels or porin proteins in a non-specific manner. As one of the intrinsic mechanisms, P. aeruginosa limits antibiotic entry by reducing the number of non-specific porin proteins and replacing them with specific or more-selective channels for taking up required nutrients resulting in lowered permeability to toxic chemicals (Tamber and Hancock, ; Figure 3). P. aeruginosa resistance to currently used broad-spectrum drugs such as carbapenems and cephalosporins is commonly caused by this adaptation (El Amin et al., ; Baumgart et al., ). Many of the clinical strains of P. aeruginosa displaying resistance to carbapenems such as imipenem are deficient in the OprD porin which specifically facilitates the diffusion of basic amino acids, small peptides as well as carbapenems into the cell (Trias and Nikaido, ; Strateva and Yordanov, ).

Active multidrug efflux pumps greatly contribute to antibiotic resistance observed in P. aeruginosa. The involved genes are ubiquitous in Gram-negative bacteria and they are located on the genome or plasmids. The multidrug efflux pumps are multi-protein complexes spanning the envelope of Gram-negative bacteria. They are responsible for expelling various toxic materials and a wide range of antimicrobials. Because of their broad substrate specificities they display resistance against different classes of antibiotics which are chemically unrelated (Blair et al., ; Venter et al., ).

P. aeruginosa possesses four well known active multidrug efflux pumps including MexAB-OprM, MexXY/OprM(OprA), MexCD-OprJ, and MexEF-OprN (Figure 3). The gene sets encoding these systems are under different regulatory factors; therefore, the expression levels of these systems significantly differ under various conditions. The MexAB-OprM and MexXY/OprM(OprA) are the most clinically important sets due to their large prevalence in clinical strains and significant contribution to a wide range of antibiotics (Avrain et al., ). The mexAB-oprM genes show a stable and constitutive expression in the cell guaranteeing a protective basal level production of the MexAB-OprM system to consistently expel a wide range of toxic molecules and antibiotics (Li et al., ). Hence it mainly contributes to natural resistance to antibiotics. The mexXY-(oprA) genes show lower basal expression levels and are mainly induced in response to protein synthesis inhibitors that target the ribosomal machinery (Matsuo et al., ; Hay T. et al., ). Both mexCD-oprJ and mexEF-oprN genes are not typically expressed in wild-type strains or their expression is very low and they have been proposed not to contribute significantly to natural antibiotic resistance (Llanes et al., ; Li et al., ).

There are other forms of multidrug efflux pumps such as MexJK, MexGHI-OpmD, MexVW, MexPQ-OpmE, MexMN, and TriABC. They are not expressed in wild-type strains but may contribute to adaptive resistance against antibiotic or biocide agents when expressed in resistant strains (Lister et al., ; Avrain et al., ).

On the other hand, they might play role in other physiological pathways as well. For example, The MexEF-OprN and MexGHI-OpmD sets can modulate QS systems by exporting the quinolone signaling molecule PQS reducing its cellular concentration resulting in the reduction of virulence factor production, which is presumably in favor of establishment of chronic infections (Köhler et al., ; Aendekerk et al., ; Lamarche and Déziel, ). However, many of these mechanisms remain still unclear with regard to their connection with other physiological pathways and their clinical relevance.

Another player of intrinsic resistance and basal lower level antibiotic susceptibility in P. aeruginosa is the gene encoding an inducible ²-lactamase (AmpC) (Figure 3). Particularly, chromosomal expression and production of AmpC confers low level resistance to aminopenicillins and most cephalosporins because these antibiotics strongly induce the production of AmpC which consequently hydrolyzes these substrates (Oliver et al., ). However, through adaptive or acquired resistance mechanisms AmpC can be overproduced, consequently conferring resistance to a wider range of antibiotics such as aminoglycosides and fluoroquinolones (Umadevi et al., ). These mechanisms will be further discussed later.

Acquired Resistance Mechanisms

P. aeruginosa can acquire resistance to antibiotics through mutation of intrinsic genes or horizontal acquisition from other bacteria through transferring plasmids carrying genetic materials encoding for antibiotic resistance (Davies, ; Davies and Davies, ). Contrary to intrinsic mechanisms, acquired resistance is related to antibiotic selection and this selective advantage occurs in the presence of antibiotic compounds leading to irreversible resistant population (Lee et al., ). Therefore, similar to intrinsic resistance, acquired resistance is stable too and it can be transmitted to progeny.

However, due to over-expression of resistance genes and transmissibility by plasmids, acquired resistance is a potent mechanism which confers resistance to a wide spectrum of antibiotics as well as leads to increased prevalence among clinical and environmental strains.

Boosted Antibiotic Resistance via Mutations

Intrinsic resistance genes are negatively or positively regulated by one or more regulatory mechanisms which confer a basal lower susceptibility of P. aeruginosa to a narrow spectrum of antibiotics. However, mutation in regulatory pathway could increase promoter activities resulting in unleashing gene expression and overproduction of protein products such as AmpC and multi-drug efflux pumps systems. Consequently, it causes higher level of resistance to antibiotics (Blair et al., ; Figure 3).

As a common mutational feature of P. aeruginosa isolates, resistant clinical mutants display a constitutive high level of AmpC production even in the absence of antibiotic inducers. This is mainly due to mutational inactivation of ampD (repressor of ampC) and specific point mutations of ampR, both encoding two regulatory proteins important in induction of the ampC gene (Juan et al., ; Figure 3). Consequently, it turns into a major cause of greater resistance to a wide range of antibiotics such as most of the ²-lactams (e.g., ticarcillin and piperacillin) as well as monobactams, third-generation and fourth-generation cephalosporins (Lister et al., ; Berrazeg et al., ). One study showed that 73% of tested clinical strains showed AmpC overproduction (Henrichfreise et al., ).

Several regulatory loci such as mexR, nalD, nalB, and nalC negatively control the expression of the mexAB-oprM operon in P. aeruginosa. On the other hand, various loss-of-function mutations in these loci derepress the expression of the mexAB-oprM operon leading to the overproduction of MexAB-OprM complex conferring a greater resistance to carbapenem antibiotics (Quale et al., ; Lister et al., ; Kao et al., ; Figure 3). Likewise, overproduction of other multidrug efflux pumps such as MexXY and MexCD-OprJ can occur via mutations in regulatory loci leading to unleashing gene expression and a greater resistance to a variety of antimicrobial agents (Lister et al., ; Figure 3).

Another clinically important and prevalent mutational alteration is attributed to OprD porin channel. This porin channel is localized in the outer membrane of P. aeruginosa and it is characterized as a carbapenem-specific porin (Figure 3). Therefore, loss or reduction of OprD can reduce permeability of the outer membrane to carbapenems (Epp et al., ; Gutiérrez et al., ; Kao et al., ). The emergence of resistance to imipenem and reduced susceptibility to meropenem has been reported upon the occurrence of oprD mutations. Genetic alteration in oprD can occur via nucleotide insertion or deletion and point mutations resulting in frameshift of the gene sequence, amino acid substitution, shortened putative loop L7 and premature stop codons (Kao et al., ). Furthermore, downregulation of oprD expression can be mediated by other regulatory factors such as MexT which itself concurrently upregulates mexEF-oprN expression (Köhler et al., ; Ochs et al., ).

Additionally, fluoroquinolone resistance among P. aeruginosa isolates can be mediated by either mutational changes within the fluoroquinolone targets i.e., DNA gyrase (gyrA and gyrB) and/or topoisomerase IV (parC and parE) or overproduction of active or inducible efflux pumps (Lee et al., ; Sun et al., ; Figure 3).

Plasmid-Mediated Resistance

Bacterial plasmids serve a central role as a potent vehicle for acquiring resistance genes and subsequent delivery to recipient host. This is so-called horizontal gene transfer whereby genetic elements can be transferred between bacterial cells particularly via conjugation. Some resistance plasmids are broad host range which can be transferred among various species via bacterial conjugation, while narrow host range plasmids are transferred among a small number of cells from similar bacterial species. For example, plasmid RP1 can transfer resistance genes to most Gram-negative bacteria (Kenward et al., ).

Plasmid-encoded antibiotic resistance confers resistance to different classes of antibiotics that are currently applied in frontline of clinical treatments such as ²-lactams, fluoroquinolones and aminoglycosides (Bennett, ; Figure 3). So far, P. aeruginosa resistance via horizontal gene transfer has been reported for the genes encoding ²-lactam-hydrolyzing enzymes known as the extended-spectrum ²-lactamases and the carbapenemases, aminoglycoside-modifying enzymes, 16S rRNA methylases resulting in high-level pan-aminoglycoside resistance (Poole, ).

The genes encoding extended-spectrum ²-lactamases and carbapenemase are clinically important not only due to their hydrolyzing activity on a wide range of ²-lactams such as carbapenems and extended-spectrum cephalosporins, but also for their worldwide prevalence (Paterson and Bonomo, ; Blair et al., ; Sullivan et al., ). The global epidemiology of carbapenem-resistant P. aeruginosa was recently analyzed by Hong et al (Hong et al., ). They reported that the geographical prevalence of these genes differs from country to country, whereas the genes encoding carbapenemases such as IMP, VIM, and NDM type metallo-²-lactamases have been found in all continents (Johnson and Woodford, ; Meletis and Bagkeri, ; Hong et al., ). Almost all types of transferable carbapenemases, except SIM-1, have been detected in P. aeruginosa, and the prevalence of carbapenem-resistant isolates of P. aeruginosa is gradually increasing (Meletis and Bagkeri, ; Hong et al., ).

It is of concern that transferable plasmids carrying some of the resistance genes are mobile among a wide range of unrelated Gram-negative bacteria which increases the antimicrobial resistance transfer rate causing increasing treatment complications (Hong et al., ). Recent findings about antibiotic resistance have been even more concerning and warning. Liu et al. reported the first evidence of plasmid-mediated colistin resistance from China (Liu et al., ; Figure 3). Colistin (or polymyxin E) belongs to the family of polymyxins. The members of this class of antibiotics such as polymyxin B and colistin have been the last resort for antibiotic treatment of carbapenem-resistant bacteria such as P. aeruginosa isolates and Enterobacteriaceae (Falagas and Kasiakou, ). Resistance to polymyxins was previously reported to occur via chromosomal mutations (Moskowitz et al., ; Gutu et al., ), however, new evidence suggests plasmid-mediated resistance through the mobilization of the mcr-1 gene which consequently confers resistance to colistin (Figure 3). This gene was discovered in E. coli strain SHP45 collected from agricultural products. It is more concerning that the plasmid carrying mcr-1 was mobilized into K. pneumoniae and P. aeruginosa via conjugation (Liu et al., ). This finding has triggered serious concerns about the emergence of pan-drug-resistant Gram-negative bacteria leading to almost untreatable infections. Recent findings provided some evidence of the spreading high-risk of clone ST of P. aeruginosa containing the genomic blaNDM&#x;1 resistance gene which also conferred resistance to colistin. It is likely that blaNDM&#x;1 was acquired via genetic exchange between P. aeruginosa and K. pneumoniae isolate in the same patient (Mataseje et al., ).

Adaptive Resistance Mechanisms

Compared to other types of resistance mechanisms, adaptive mechanisms are not really well understood. Adaptive resistance is an unstable and transient form of resistance, which is induced in the presence of specific antibiotics and other environmental stresses. This type of resistance mainly relies on induced alterations in gene expression and protein production or alterations in antibiotic targets and it is reversal upon removal of external stimuli leading to re-gaining susceptibility (Barclay et al., ; Xiong et al., ; Fernández et al., ). This mechanism has been seen mediating the resistance of P. aeruginosa isolates to ²-lactams, aminoglycosides, polymyxins and fluoroquinolones (Zhang et al., ; Poole, ; Fernández et al., ; Khaledi et al., ).

It has been seen that once strains encounters certain concentrations of antibiotics, they can tolerate higher concentrations in subsequent exposures, while cross-resistance to other antibiotics may occur as well (Mouneimné et al., ; Fujimura et al., ; Fernández et al., ; Pagedar et al., ). Furthermore, these alterations may link to other physiological events triggered by other stimuli and stresses as well as mutations in some specific genes (Xiong et al., ; Karlowsky et al., ; Fernández et al., ).

Using isolates from CF patients, it was shown that adaptive resistance of P. aeruginosa to fluoroquinolones such as ciprofloxacin is due to multiple mutations in the known-resistance genes including the gyrA, gyrB, nfxB, and orfN which were concomitant with mutations in the genes involved in cyclic di-GMP signaling (Figure 3). Mutations of nfxB were prevalent leading to loss of function of NfxB transcriptional repressor and consequently leading to the overproduction of MexCD-OprJ efflux pump (Wong et al., ; Figure 3). This efflux pump is an important determinant of resistance to fluoroquinolone antibiotics (Hirai et al., ). On the other hand, another study showed that expression of the mexCD-oprJ genes depends on the sigma factor AlgU and leads to resistance to the biocide chlorhexidine (Fraud et al., ). AlgU is well-known stress response sigma factor which positively regulates overproduction of alginate in mucoid isolates (Hershberger et al., ).

Another group showed that P. aeruginosa can acquire and lose resistance in the presence and absence of colistin, respectively. This occurred via adaptive multiple mutational mechanisms and genetic reversion (Lee et al., ). It was also demonstrated that resistance to certain polycationic antimicrobials such as aminoglycosides, polymyxins and cationic antimicrobial peptides can be mediated by altering the lipid A structure in LPS. This was caused by multiple mutations in cognate regulatory proteins such as the two-component systems PhoP-PhoQ, PmrA-PmrB, CprR-CprS, and ParR-ParS (Barrow and Kwon, ; Fernández et al., ; Figure 3). Other studies showed that further and complex genetic alterations affecting regulatory pathways including those causing amino acid substitutions in these cognate regulatory proteins such as PhoQ and PmrB are involved in polymyxin resistance. This is why the mechanism of resistance of P. aeruginosa to colistin was found to vary among isolates (Lee et al., ). Interestingly, this study showed that the acquisition of colistin resistance via many amino acid substitutions is reversible in colistin-susceptible revertants. However, even in the absence of colistin, resistance was preserved for some time and emergence of revertants may not occur so fast (Lee et al., ).

QS-Dependent Antibiotic Resistance

Some direct and indirect evidences have been found linking the QS systems with antibiotic resistance mechanisms in P. aeruginosa (Rasamiravaka and El Jaziri, ), but further exploration is needed for better understanding. Using clinical strains of P. aeruginosa, it was shown that the las system positively links to the expression of mexY gene encoding the inner-membrane drug/H+ antiporter protein MexY (Pourmand et al., ) which is a key subunit of the MexXY-oprM complex known as a major determinant of aminoglycoside resistance (Lau et al., ). On the other hand, some studies showed that CF-infecting strains with the common lasR loss-of-function mutations were more resistant to therapeutic antibiotics such as tobramycin, ciprofloxacin and ceftazidime. The reported antibiotic resistances in the lasR mutants were attributed to increased ²-lactamase activity, bacterial metabolic adaptation or metabolic shifts (D&#x;Argenio et al., ; Hoffman et al., ). However, the relationship of antibiotics susceptibility with the rhl encoded QS system and production of C4-HSL signals remains unclear (Bjarnsholt et al., ). Some supporting evidence was obtained by, treating P. aeruginosa biofilms with ciprofloxacin which upregulated the production and secretion of the virulent factor LasB, which is under the control of Rhl QS system (Oldak and Trafny, ; Figure 1).

Furthermore, two independent studies reported that the clinical strains of P. aeruginosa with QS-deficient phenotypes and negative for the production of QS-dependent virulence factors could cause infections and tend to be less susceptible to antimicrobial agents (Karatuna and Yagci, ; Wang H. et al., ). However, it was not shown how these mechanisms might link to each other while many of these clinical strains could also form biofilms with antibiotic resistance traits and many regulatory pathways for biofilm development are under the control of QS systems. Zhao et al. reported some supporting information showing the importance of QS systems in both biofilm formation and antimicrobials induced expression of ampC (Zhao et al., ). Earlier studies showed that by overexpressing the chromosomal type 1 ²-lactamase, QS-dependent virulence factors were reduced and strains were less virulent (Ramisse et al., ). Also, Kong et al. analyzed the dual role of the AmpR transcriptional regulator where it positively regulated ²-lactamases and negatively regulated the virulence factors through QS systems (Kong et al., ).

Balasubramanian et al. analyzed co-regulatory and transcriptional networks of three co-existing mechanisms involved in ²-lactam resistance, alginate production and modulation of virulence factor expression. They showed that while AmpR positively and negatively regulates ²-lactamases and QS-dependent proteases, respectively, there is an intimate crosstalk between the AmpR regulon and the master regulator AlgU which positively regulates alginate production (Balasubramanian et al., ). This gave more insight into the complexity of such co-existing networks. Recent findings also showed that high levels of cyclic di-GMP mediated by the SagS regulator contributes to elevated antibiotic resistance via BrlR regulon-dependent upregulation of cognate genes encoding MexAB-OprM and MexEF-OprN multidrug efflux pumps (Gupta et al., ).

The periplasmic TpbA tyrosine phosphatase was also reported as a regulatory candidate for linking QS signaling and biofilm formation. This protein was shown to be positively regulated by the las QS system at transcriptional level. Upon production of TpbA and its phosphatase activity in the periplasm, the cyclic di-GMP synthesizing protein TpbB is dephosphorylated at a tyrosine residue in periplasmic domain leading to inactivation of TpbB and a reduction in cyclic di-GMP levels and in turn Pel production, hence inhibiting biofilm development (Ueda and Wood, ). TpbA-dependent cyclic di-GMP reduction was also linked to increasing eDNA release by cell lysis (Ueda and Wood, ).

Overall, the reason for inconclusive information about the relation of the QS system and antibiotic resistance mechanisms is based on the fact that there are various layers of regulatory pathways associated with both QS systems and antibiotic resistance mechanisms in P. aeruginosa. Therefore, understanding the interplay between hierarchical QS systems and various antibiotic resistance mechanisms needs further exploration.

Adaptive Radiation for Persistence

Adaptation to the surrounding environments is an extraordinary capability of P. aeruginosa. It enables P. aeruginosa to inhabit diverse ecological niches such as colonization of various hosts as well as long term persisting infections. The adaptation process is designated as adaptive radiation by which initial clones would diversify into a variety of genotypes and phenotypes over time until the most favorable and adapted descendants are selected for long term persistence (Hogardt and Heesemann, ). A typical example is adaptation of P. aeruginosa isolates to the CF airways. Various studies have shown that initial colonization of CF lungs is caused by wild-type strains existing in the environment. For bacteria, the CF lungs encompass various stresses such as oxidative stresses and immune responses and inter-species competition followed by antibiotic treatment. Therefore, initial clones undergo substantial adaptation processes to survive such hostile environments.

Here, adaptive radiation is mainly due to intense genetic adaptations leading to the thousands of generations displaying diverse genotypes and phenotypes that emerge in vivo, while subjected to selection pressure imposed by the CF lung milieu (Figure 4). Therefore, selected variants display different genotypes when compared with initial wild-type colonizers and persist in the CF lungs leading to clonal expansion within patients and establishment of chronic infections (Mathee et al., ; Kong et al., ; Boles and Singh, ; Driffield et al., ; Workentine et al., ). By assessing a wide selection of phenotypes, Workentine et al. showed that the overall population structure in one chronically infected patient can be much more heterogeneous in phenotypes than what has been previously documented (Workentine et al., ). Furthermore, it has been reported that transmission of strains from patient to patient can result in the coexistence of highly divergent bacterial lineages (Winstanley et al., ).

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Figure 4. Remodeling of regulatory networks in P. aeruginosa during adaptive radiation and transition from acute to chronic infections. (A) During pathogenesis, adaptation to the CF lung environment occurs through adaptive radiation where intense genetic mutations lead to diverse genotypes and phenotypes (colorful ellipsoids) within bacterial populations followed by the selection of colonizers. Mutational adaptation and selection of generations drive bacterial transition from acute to chronic traits. (B) Remodeling of key regulatory networks between acute and chronic infections occurs mainly via mutational adaptation in cognate genes. Mutated lasR, ampR, and retS genes are key determinants in this process by which QS, virulence factor production and motility are downregulated, while synthesis of cyclic di-GMP, exopolysaccharides and various multidrug efflux pumps are upregulated. Mutation of mucA results in a defect in MucA (anti-sigma factor) releasing AlgU (positive regulator of alginate operon) that induces overproduction of alginate and the mucoid phenotype. Of important acute traits are flagellum, type 4 pili, T1SS, T2SS, T3SS (types 1 to 3 secretion systems), ExoT (exotoxins), Lip (lipases), AprA (alkaline proteases). The type 6 secretion system (biofilm-associated and toxin-delivering device to other bacteria) and efflux pumps and the production of EPS are part of chronic traits which confer antibiotic resistance and/or mediate biofilm formation. Plus and minus signs represent positive and negative effect of transcriptional regulators, respectively. Red cross indicates mutagenesis. CM, cytoplasmic membrane; OM, outer membrane.

Generally, phenotypic adaptation of these strains include slow growth, auxotrophy, virulence deficiency via downregulation of QS systems, loss of motility, biofilm formation, alginate overproduction and mucoid phenotype, antibiotic resistance, hypermutability, and lipopolysaccharide modifications. Downregulation of virulence factors such as flagella motility, T2SS/T3SS apparatus, and toxic components results in less inflammatory and phagocytic responses since the pathogen is less detectable for the immune system (Mahenthiralingam et al., ; Hogardt and Heesemann, ). Analysis of many clinical isolates showed these alterations represent convergent molecular evolution among many clinical isolates and mutation of 52 genes are mainly responsible for substantial phenotypic alterations associated with virulence traits and resistance (Diaz Caballero et al., ; Marvig et al., ). Of these genes, common adaptive mutations occur in regulatory genes including lasR, pvdS, rpoN, mucA, mexT, nfxB, mexR, nalD, retS, and ampR (Figure 4). Collectively, this leads to remodeling regulatory networks and developing a general adaptation pattern as explained below (Higgins et al., ; Hogardt and Heesemann, ; Rau et al., ; Winstanley et al., ).

Mutation of lasR, pvdS, and rpoN impairs central QS system signal processing leading to the deficiency in virulence traits. In wild-type strains, the LasR and PvdS regulators control the expression of a large number of genes including key virulence factors (Table 1, Figure 4) and pyoverdine for iron acquisition, respectively (Table 1; Hoffman et al., ; Imperi et al., ; Jiricny et al., ; LaFayette et al., ). The alternative sigma factor RpoN has been also found to regulate many cell functions such as motility and virulence factors production via QS system (Cai et al., ; Figure 4). Additionally, knocking out mutation of mucA locus encoding anti-sigma factor MucA results in releasing the RNA polymerase sigma factor Ã22 (AlgU) which itself positively regulates expression of the alginate biosynthesis operon and stress response mechanisms while it negatively regulates several virulence factors such as flagella, pili, T3SS, and Rhl quorum sensing signals (Folkesson et al., ). Development of the mucoid phenotype mediated by alginate overproduction as well as the formation of highly structured biofilms is the hallmark of chronic infections (Schurr et al., ; Hay I. D. et al., ; Figure 4).

Another common mutation has been reported in the mexT locus resulting in activation of MexT, the positive regulator of MexEF-OprN efflux pump, which in turn led to antibiotic resistance. In addition, the induction of MexEF-oprN production is linked to extruding QS signaling molecules and reduction of virulence factor production (Tian et al., ; Figure 4). The MexEF-OprN production is undetectable in wild-type strains due to the non-functionality of the mexT gene (Maseda et al., , ). Furthermore, mutations of repressor genes nfxB and mexR/nalD in clinical strains upregulated the production of the MexCD-OprJ and MexAB-OprM efflux pumps, respectively, conferring resistance to a wider range of antibiotics (Higgins et al., ; Sobel et al., ; Jeannot et al., ; Rau et al., ; Figure 4). Also, mutation of other genes such as gyrA/gyrB (DNA gyrase), mexZ (transcriptional regulator of the mexXY) and mexS (transcriptional regulator of the mexEF) are commonly attributed to antibiotic resistance during mutational adaptations (Marvig et al., ; Figure 4).

As part of RetS/GacS/GacA/RsmA regulatory pathway, the retS gene is important for phenotypic shifting from acute to chronic infections (Lapouge et al., ; Moscoso et al., ). The retS mutation repressed the production of virulence factors such as T3SS and swarming motility while it upregulated production of the T6SS (type 6 secretion system) and exopolysaccharides Pel/Psl required for biofilm formation (Moscoso et al., ; Figure 4). The T6SS is a puncturing device for delivery of proteins and toxins into the competing bacteria and the host cells and an important survival advantage for P. aeruginosa. It is also required for biofilm formation while being considered as a virulence factor (Chen et al., ). This transitional impact was shown to be mediated by high levels of cyclic di-GMP (Boehm et al., ; Paul et al., ; Moscoso et al., ).

Mutation of the ampR gene is another common mutational adaptation with a large impact on remodeling of physiological traits (Figure 4). The AmpR global regulator in P. aeruginosa regulates not only resistance to different classes of clinically relevant antibiotics, but also expression of hundreds of genes involved in diverse physiological processes such as virulence, QS systems and stress responses (Balasubramanian et al., ). It is understood that the ampR mutation induces adaptations leading to chronic infection including the downregulation of stress responses and virulence factors via downregulating QS systems, and boosting biofilm formation and alginate overproduction by causing elevation of cyclic di-GMP levels. Additionally, it induced AlgU activity, and resistance to fluoroquinolone through activation of MexT upregulating the MexEF-OprN efflux pump as well as increasing twitching motility and T6SS production (Balasubramanian et al., , , , ; Figure 4).

Other adaptive mutations of CF isolates have been commonly reported in anti-mutator genes including mutS, mutT, mutL, mutY, mutM, and uvrD conferring a hypermutability phenotype with elevated mutation rates due to the lack of DNA repair mechanisms. This phenotype has been described as being caused by later mutational events as they are understood to occur after mutation of the lasR and mucA genes known as earlier mutations. However, other reports postulated that mutation of anti-mutator loci may increase the rate of other adaptive mutations (Oliver and Mena, ).

Hypermutators are very prevalent in CF isolates and they are shown to have correlation with higher antibiotic resistance particularly in the late stage of chronic infections. However, hypermutators also display other phenotypes such as mucoidity, lack of motility and LPS production (Oliver et al., ; Ciofu et al., ; Varga et al., ).

Other distinct phenotypes correlated with adaptation to CF airways are the small-colony variants (SCVs). They are associated with prolonged persistence and chronic infections in CF lungs and obstructive pulmonary diseases (Malone, ). They have been characterized as variants forming rugose small colonies on solid media (1&#x;3 mm in diameter) with slow growing, autoaggregative and enhanced biofilm formation characteristics combined with enhanced surface attachment and hyperpiliation for twitching (Häussler et al., , ; Kirisits et al., ). In vitro analyses showed that the SCVs display increased resistance to a wide range of antibiotics (Wei et al., ). Different studies have demonstrated that the presence of SCVs in the CF lung is associated with poorer lung function and clinical outcomes (Häussler et al., , ; Schneider et al., ).

It has been understood that SCVs show high levels of cyclic di-GMP production aligned with increased production of Pel and Psl exopolysaccharides (Starkey et al., ). So far investigations regarding the molecular mechanisms underlying SCVs formation confirmed loss-of-function mutations in regulatory genes such as wspF, yfiR and rsmA and some other genes which alter regulatory networks in favor of enhanced cyclic di-GMP production (Irie et al., ; Malone et al., ; Blanka et al., ; Malone, ). On the other hand, an upregulating cyclic di-GMP synthesis pathway is a key determinant of exopolysaccharide production leading to highly developed biofilms. However, it still remains unclear how two distinct phenotypes i.e., cell within mucoid biofilm and SCV differ in regard to the cyclic di-GMP mediated signaling pathways.

Survival by Stringent Response and Persister Formation

Stringent response to environmental stresses such as nutritional starvation and response to antibiotics and oxidative stresses share a similar outcome of adaptation i.e., all are leading to dormancy and persister formation. In both responses, bacteria slow down their metabolism through downregulating the expression of genes participating in the biosynthesis of proteins, ribosomes, cell wall, nucleic acid metabolism, and virulence factors. These dramatic metabolic alterations result in arresting cell growth and cell division in favor of bacterial survival (Eymann et al., ; Hesketh et al., ; Durfee et al., ).

Persisters are defined as subpopulations of cells, occurring at very low frequency, which stochastically emerge in the presence of stress. They show very slow growth enhancing survival under stress while viability of the majority of the population is severely impaired. Upon stress removal, persisters turn back to normal growth to propagate, which coincides with regained sensitivity to stress. Such persistence was suggested to be based on the heterogeneity of population by means of epigenetic mechanisms, not genetic mutations (Fasani and Savageau, ).

Various studies have provided evidences showing the link between stringent response and persistence, but mostly using E. coli as a model which can be informative for P. aeruginosa as it possesses homologous signaling pathways (Fung et al., ; Maisonneuve et al., ; Amato et al., ; Ramisetty et al., ). There are only a few studies aiming to explain such responses, but they provided inconclusive explanations. Therefore, we summarized the general findings in order to propose the underlying molecular mechanisms in P. aeruginosa.

Molecular Mechanisms Underlying Stringent Responses and Persisters

Notably, increased levels of (p)ppGpp (collectively designated for guanosine pentaphosphate and guanosine tetraphosphate) molecules in the cells is a central triggering alarmone for both persistence and stringent response (Potrykus and Cashel, ; Wu et al., ; Amato et al., ). The cellular levels of (p)ppGpp are mediated by the activity of the (p)ppGpp-synthesizing and degrading enzymes such as RelA and SpoT in response to external stimuli (Bremer and Dennis, ).

In stringent response when E. coli encounters amino acid deprivation, the ribosome-associated RelA synthesizes ppGpp molecules to an upper level. In association with the transcriptional regulator DksA (global regulator of metabolism), ppGpp interacts with RNA polymerase and inhibits the transcription of ribosomal RNA promoters. This inhibitory impact is concomitant with activation and upregulation of pathways for amino acid biosynthesis and the transcription of stress response genes (Potrykus and Cashel, ; Dalebroux and Swanson, ; Amato et al., ). Amato et al. () found that stringent responses are linked to the emergence of persisters by involving ppGpp based regulatory events (Amato et al., ).

The persister state is typically based on the activity of genetically encoded toxin-antitoxin (TA) modules particularly in response to antibiotics, but proposed as being activated by the same acting elements i.e., RelA or SpoT and (p)ppGpp in E. coli (Maisonneuve et al., ). These TA system are widely distributed in genomes or plasmids of bacteria and archaea (Van Melderen, ). Basically, the toxin element is a stable protein while the cognate antitoxin element either is a protein or a small RNA molecule which are metabolically unstable under unfavorable conditions. A small RNA antitoxin directly inhibits the toxin translation by pairing with toxin mRNA or inactivate toxin by direct binding. A protein antitoxin on the other hand either degrades toxin mRNA or blocks the activity of cognate protein toxin via direct protein-protein interaction or protective interaction with toxin substrates. Impairment of antitoxin function under certain stresses such as antibiotics as well as nutritional stresses leads to the accumulation and activation of toxin proteins. Consequently, the toxin targets and interferes with key cellular processes such as DNA replication, and the synthesis of tRNA, membrane components, and ATP leading to the inhibition of cell growth and cell division to form a dormant or persister cell (Christensen-Dalsgaard et al., ; Wen et al., ). Presumably, bacteria switch off their active metabolism upon exposure to stress to evade starvation or antibiotic impact on their cellular targets. Christensen et al. demonstrated that E. coli encodes at least 10 TA loci and all of which could be induced by nutritional starvation and antibiotics (Christensen et al., ; Christensen-Dalsgaard et al., ).

So far, Maisonneuve et al. () have proposed the best model presenting the hierarchical molecular mechanisms for E. coli persistence with the involvement of the key players including RelA/SpoT enzymes, (p)ppGpp signaling, Lon protease (ATP-dependent protease), inorganic polyphosphate (PolyP), and toxin-antitoxins systems (Maisonneuve et al., ; Figure 5). This model explains that (p)ppGpp synthesized by RelA/SpoT inhibits PolyP degradation, while PolyP accumulation stimulates Lon protease to degrade the antitoxin leading to the activation of toxin for arresting cellular processes and growth. Previously, it was shown in E. coli that in response to nutritional stress and antibiotics, transcriptional activation of TA loci depended on protease activity of Lon protease on antitoxin which itself repressed the TA promoter (Christensen-Dalsgaard et al., ). Furthermore, it was suggested that the complex of Lon protease with PolyP could promote ribosomal protein degradation for supplying required amino acids during starvation (Kuroda et al., ; Figure 5).

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Figure 5. P. aeruginosa stringent response and persister formation. Stringent response is triggered by particular stresses such as amino acid and fatty acid starvation, iron/phosphor depletion and oxidative stress [e.g., reactive oxygen species (ROS)]. The (p)ppGpp alarmone is a key determinant for stringent response and it is elevated by RelA/SpoT enzymes. Generally, (p)ppGpp elevation and the PolyP (inorganic polyphosphate) and Lon protease complex interfere with normal biological processes in favor of bacterial survival via arresting metabolism, cell growth and cell division (dashed gray pathways are best understood for the E. coli model, but not or partially characterized in P. aeruginosa). In E.coli, (p)ppGpp signaling is linked to toxin (T)-antitoxin (A) system via activation of the Lon protease leading to the formation of persisters displaying dormant and antibiotic resistance phenotypes (dashed orange line). Generally, the TA complex is stable under normal conditions suppressing toxin activity and further expression of cognate genes. Upon antitoxin degradation, toxin becomes active to hinder biological processes. In the case of P. aeruginosa HigB/A, HicA/B, the toxin components perform endoribonuclease (RNase) activity on mRNA molecules. In P. aeruginosa, the (p)ppGpp alarmone is linked to the production of ROS scavengers probably via QS or RpoS regulators and Lon activity is required for biofilm formation, motility, virulence and antibiotic resistance. Furthermore, the TA system downregulates biofilm formation and virulence factor production while T3SS (type 3 secretion system) can be found upregulated. Although, the (p)ppGpp signaling, Lon protease activity and TA modules (i.e., HigB/A, HicA/B, and likely more complexes) are present in P. aeruginosa, their link to resistance to antibiotics and other stresses is poorly understood. AA, amino acids; QS, quorum sensing; RNAP, RNA polymerase. CM, cytoplasmic membrane; OM, outer membrane.

P. aeruginosa Stringent Response and Persisters

Required elements of stringent response and persistence including the ppGpp alarmone, SpoT, RelA, DksA, and the TA modules have been characterized in P. aeruginosa (Figure 5). Mutants deficient in relA and/or spoT genes showed increased sensitivity to heat shock, oxidative and osmotic stresses as well as antibiotics while becoming less virulent. Stringent response and RelA/SpoT activity for production of ppGpp were found to be crucial for regulation of virulence factor production (Erickson et al., ; Viducic et al., ; Boes et al., ; Nguyen et al., ; Vogt et al., ). Also, there is some experimental support that P. aeruginosa utilizes stringent response to protect cells from oxidative stresses generated by toxic reactive oxygen species (ROS) under aerobic conditions. The mutants deficient in relA and spoT genes were highly susceptible to multiple oxidants. This study showed that (p)ppGpp signaling is necessary for optimal expression of catalase and superoxide dismutase enzymes as major ROS scavengers, but it was assumed to be indirectly regulated through a complex regulatory network (Sampathkumar et al., ; Figure 5). Because, (p)ppGpp signaling is also required for full expression of other regulatory pathways controlling antioxidant response such as the stress response regulator RpoS as well as both Las and Rhl QS systems (van Delden et al., ; Kohanski et al., ; Schafhauser et al., ; Sampathkumar et al., ; Figure 5). The proposed E. coli model for the activation of the TA system via Lon protease has not been shown for P. aeruginosa, yet. However, different studies indicated that Lon protease activity was induced by aminoglycosides and lon mutants were highly susceptible to ciprofloxacin while lon was also required for biofilm formation, motility and virulence (Marr et al., ; Breidenstein et al., ). Molecular mechanism of these pathways have not been well characterized, but as proposed previously, interconnection of various regulatory and signaling pathways for appropriate responses leading to either as stringent/persistence, biofilm formation or virulence is anticipated (Kim et al., ).

To date five types (I-V) of TA systems have been described in bacteria based on the nature and mode of action of antitoxin (Wang et al., ). So far, HigB/HigA and HicA/HicB TA modules encoded by genomic loci have been experimentally demonstrated in P. aeruginosa, while other TA systems such as the relBE and parDE loci were predicted but they have not been characterized, yet (Pandey and Gerdes, ; Fernández-García et al., ; Figure 5). The HigB/HigA and HicA/HicB TA modules have been also widely reported for other bacteria (Pandey and Gerdes, ; Li G. et al., ; Wood and Wood, ).

These TA modules belong to the type II TA system where both toxin (i.e., HigB/HicA) and antitoxin (i.e., HigA/HicB) are proteins directly interacting with each other retaining the toxin inactivated, such as inhibiting the RNase activity of HigB or HicA (Christensen et al., ; Rocker and Meinhart, ; Figure 5). The HigB/HigA TA module was found to influence P. aeruginosa pathogenicity where toxin HigB was shown to reduce the production of the virulence factors pyochelin, pyocyanin, swarming, and impaired biofilm formation representing a novel function for a TA systems (Wood and Wood, ; Figure 5). Another study showed that the HigB/HigA TA module was necessary for the ciprofloxacin induced persister formation by P. aeruginosa. Concurrently, HigB overproduction upregulated the expression of T3SS genes leading to the accumulation of T3SS proteins in persisters as well as increasing bacterial cytotoxicity against host immune cells (Li M.et al., ; Figure 5).

Furthermore, these TA systems have been shown to be highly prevalent in the clinical strains (Pandey and Gerdes, ; Williams et al., ; Li G. et al., ). It is believed that persisters are one of the main reasons for recurring and chronic infections where persisters withstand antibiotic treatments and spawn new infecting population upon removal of antibiotic treatment (Lewis, ; Wang and Wood, ). They are abundant in P. aeruginosa biofilms which is the hallmark of long-term infections particularly in CF patients (Lewis, ; Mulcahy et al., ).

P. aeruginosa Resistance to Foreign DNA

Infection of bacteria with viruses or bacteriophages is a natural phenomenon which can lead to bacterial lysis. Bacteria harness various mechanisms to destroy such foreign DNAs leading to resistance. The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) systems form the only adaptive immune system in prokaryotic cells which also mediates P. aeruginosa survival during viral invasions (Cady et al., ; Bondy-Denomy and Davidson, ). A CRISPR region is an array of multiple repeated sequences on the bacterial genome or a plasmid ranging from 21 to 48 bp in length and separated by 26 to 72 bp hypervariable spacers (Bhaya et al., ; Cady et al., ). The cas locus encoding Cas proteins is located in the vicinity of the CRISPR region (Bhaya et al., ). In principle, the molecular mechanism is based on acquisition and integration of small fragments of foreign DNAs such as derived from viruses into the spacer regions between two adjacent repeats within the CRISPR locus mediated by Cas proteins with nuclease activity. Subsequently, the CRISPR region is transcribed resulting in pre-CRISPR RNA (pre-crRNA) which undergoes hydrolysis by endoribonucleases forming small CRISPR RNAs (crRNAs). The mature crRNAs in association with a multiprotein complex known as CASCADE (CRISPR-associated complex for antiviral defense) recognizes invasive DNAs upon complementarity which results in the initiation of the cleavage of the crRNA&#x;foreign DNA hybridization complex, mediating survival of bacteria after viral infections while protecting themselves from lysis (Brouns et al., ; Mojica et al., ; Deveau et al., ; Garneau et al., ; Bhaya et al., ). The CRISPR-Cas systems have been classified into three major types (I, II and III) and at least 11 subtypes (IA-F, IIA-C and IIIA-B) encoding distinct crRNA-guided surveillance complexes (Makarova et al., ).

A study showed that 36% of tested P. aeruginosa clinical isolates harbored CRISPR-Cas systems developing adaptive immunity against various mobile genetic elements such as temperate phages, prophages, pathogenicity island transposons which were integrated into the genome (Cady et al., ). Different studies demonstrated that the types I-F and I-E CRISPR-Cas systems are naturally active in P. aeruginosa isolates (Cady et al., ; Pawluk et al., ). A recent phylogenetic study revealed the existence of the type I-C CRISPR-Cas system in some isolates of P. aeruginosa (van Belkum et al., ).

On the other hand, the activity of CRISPR/Cas system can be inhibited by anti-CRISPR/Cas genes harbored by phages infecting P. aeruginosa which counteract the type I-F and I-E systems (Bondy-Denomy et al., ; Maxwell, ). Also it has been shown that phages producing anti-CRISPR activity are closely related to each other and with high sequence similarity to bacteriophage DMS3 (Bondy-Denomy et al., ). Bacteriophage DMS3 was isolated from clinical isolates of P. aeruginosa and it was shown to inhibit biofilm formation and swarming motility, and P. aeruginosa cannot develop immunity against it due to the lack of complementarity between crRNA and protospacers of DMS3 genome (Budzik et al., ; Zegans et al., ). Furthermore, the CRISPR-Cas systems show a strong correlation with antibiotic resistance/susceptibility (van Belkum et al., ). Additionally, the same study showed that the CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa strains. Accessory genome is referred to highly variable regions of the genome versus a relatively invariable core genome. P. aeruginosa accessory genome varies from strain to strain, ranging from to % of the total genome, and is mainly comprised of integrative and conjugative elements, replacement islands, prophages and phage-like elements, transposons, insertion sequences and integrons (Kung et al., ; Ozer et al., ). According to this finding the CRISPR typing with regard to the frequency of spacer integration and deletion between related strains can potentially be used for identifying the lineage of strains especially within outbreaks (van Belkum et al., ).

Overall, understanding of the CRISPR-Cas and anti-CRISPR-Cas systems is gradually becoming important in the context of pathogenesis and strain lineage identification. These links were highlighted by the discovery of the interaction of bacteriophage DMS3 and the type I-F CRISPR, and its impact on biofilms (Zegans et al., ; Palmer and Whiteley, ) as well as the role of different CRISPR/Cas systems on virulence and antibiotic resistance (Louwen et al., ). Overall, these findings suggest a more diverse function of CRISPR/Cas systems within the context of pathogenesis, requiring further in depth studies to elucidate the underlying molecular mechanisms.

Conclusions and Perspectives

For many years, P. aeruginosa has been a model organism and received much attention from scientific community to study the bacterial lifestyle and pathogenesis. It always has been of particular importance due to causing persistent infections in CF and immunocompromised patients. Nowadays, this ubiquitous bacterial pathogen is accepted worldwide as a public health risk due to its increasing prevalence in healthcare acquired infections combined with its ability to develop resistances to multiple classes of antibiotics. Over the past decade, extensive research studies have focused on these growing concerns aiming at deciphering the nature of P. aeruginosa capability and underlying molecular mechanisms applying different modes of persistence and antibiotic resistance.

In this review, we summarized several of the well characterized molecular mechanisms which enable P. aeruginosa to survive various hostile conditions such as during pathogenesis and antibiotic treatment. These mechanisms form multiple layers of physiological adaptations correlating with social behavior and lifestyle of bacteria while responding environmental stimuli. Such extraordinary adaptive capability relies on extensive numbers of regulatory or controlling factors within integrated and complex signal processing pathways. These enable bacteria perceive and process environmental cues in order orchestrate physiological changes to promote adaptation to unfavorable conditions. Many of these regulatory pathways, their cognate player, their signals and how they are integrated with global regulatory networks still remain poorly understood.

Furthermore, we highlighted key molecular pathways driving P. aeruginosa survival and persistence at different stages of pathogenesis such as QS elements for virulence traits, cyclic di-GMP signaling in biofilm formation and development of chronic traits, (p)ppGpp signaling/TA system in persister formation and various strategic adaptations for developing resistance to divers classes of antibiotics.

Recent technological advances in genomic characterization of pathogens have provided invaluable information about the dynamics of P. aeruginosa populations and their heterogeneity at different stages of pathogenesis. These results which were explained under adaptive radiation term emphasized that this species shows a stunning capability to become resilient during pathogenesis to withstand antibacterial treatment.

Available information indicated that sole therapy which only relies on bacteriostatic/bactericidal compounds can readily be defeated by bacterial resiliency and a management program is still required to combat infections. This program should be able to predict and evaluate physiological adaptations at each stage of infection for exerting appropriate treatments which could interfere with adaptation rather than increasing the chance of bacterial survival. A good example is improper and frequent application of antibiotics which must be avoided. Instead a comprehensive hygiene program must be applied in healthcare settings and among personnel to stop the spread of nosocomial infections specifically caused by multidrug resistance strains. Also, further research on identification of new drugs and developing new alternative prevention and treatment strategies for interfering with key regulatory pathways is needed.

At the end we suggest all efforts should consider international coordinated multidisciplinary programs with results of laboratory outputs being deposited in centralized accessible databases to expedite advances in control of infections and its implementation into clinical settings. The steadily growing concern of emerging antibiotic resistance strains in the world, would justify the set-up of such databases which then allow developing world-wide guidelines for monitoring and recording antibiotic resistance cases around the world. This should provide healthcare experts with appropriate guidelines for well managing bacterial infections and preventing the rate and spread of resistance strains.

Author Contributions

MM and BR conceived and wrote the majority of the manuscript. SG contributed biofilm and alginate related aspects to the manuscript.

Funding

The work was funded via the Massey University Research fund.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors are grateful to all past and present members of the BR Research group at the Institute of Fundamental Sciences (Massey University) for their contributions to the Pseudomonas aeruginosa research.

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Pseudomonas aeruginosa

A Microbial Biorealm page on the genus Pseudomonas aeruginosa

Classification

Higher order taxa

Domain Bacteria
Phylum Proteobacteria
Class Gamma proteobacteria
Order Pseudomonadales
Family Pseudomonadaceae
Genus Pseudomonas
Species Group Pseudomonas aeruginosa group

Species

Genus species Pseudomonas aeruginosa

Description and significance

Pseudomonas aeruginosa is a gram-negative, rod-shaped, asporogenous, and monoflagellated bacterium that has an incredible nutritional versatility. It is a rod about µm long and µm wide. P. aeruginosa is an obligate respirer, using aerobic respiration (with oxygen) as its optimal metabolism although can also respire anaerobically on nitrate or other alternative electron acceptors. P. aeruginosa can catabolize a wide range of organic molecules, including organic compounds such as benzoate. This, then, makes P. aeruginosa a very ubiquitous microorganism, for it has been found in environments such as soil, water, humans, animals, plants, sewage, and hospitals (1). In all oligotropic aquatic ecosystems, which contain high-dissolved oxygen content but low plant nutrients throughout, P.aeruginosa is the predominant inhabitant and this clearly makes it the most abundant organism on earth (2).

P.aeruginosa is an opportunistic human pathogen. It is “opportunistic” because it seldom infects healthy individuals. Instead, it often colonizes immunocompromised patients, like those with cystic fibrosis, cancer, or AIDS (3). It is such a potent pathogen that firstly, it attacks up two thirds of the critically-ill hospitalized patients, and this usually portends more invasive diseases. Secondly, P.aeruginosa is a leading Gram-negative opportunistic pathogen at most medical centers, carrying a % mortality rate. Thirdly, it complicates 90% of cystic fibrosis deaths; and lastly, it is always listed as one of the top three most frequent Gram-negative pathogens and is linked to the worst visual diseases (4). Furthermore, P.aeruginosa is a very important soil bacterium that is capable of breaking down polycyclic aromatic hydrocarbons and making rhamnolipids, quinolones, hydrogen cyanide, phenazines, and lectins (5). It also exhibits intrinsic resistance to a lot of different types of chemotherapeutic agents and antibiotics, making it a very hard pathogen to eliminate (1).

P. aeruginosa was first described as a distinct bacterial species at the end of the nineteenth century, after the development of sterile culture media by Pasteur. In , the first scientific study on P. aeruginosa, entitled “On the blue and green coloration of bandages,” was published by a pharmacist named Carle Gessard. This study showed P. aeruginosa’s characteristic pigmentation: P. aeruginosa produced water-soluble pigments, which, on exposure to ultraviolet light, fluoresced blue-green light. This was later attributed to pyocyanine, a derivative of phenazine, and it also reflected the organism’s old names: Bacillus pyocyaneus, Bakterium aeruginosa, Pseudomonas polycolor, and Pseudomonas pyocyaneus (3). P. aeruginosa has many strains, including Pseudomonas aeruginosa strain PA01, Pseudomonas aeruginosa PA7, Pseudomonas aeruginosa strain UCBPP-PA14, and Pseudomonas aeruginosa strain (5). Most of these were isolated based on their distinctive grapelike odor of aminoacetophenone, pyocyanin production, and the colonies’ structure on agar media (6).

Genome structure

P. aeruginosa has the genome size of about to 7 million base pairs (Mbp) with 65% Guanine + Cytosine content. It is a combination of variable accessory segments and a conserved core. The variable accessory genome is characterized by a set of genomic islands and islets from a primeval tRNA-integrated island type. The core genome consists of a low level of nucleotide divergence of % and a conserved synteny of genes, which means two or more genes, whether they are linked or not, are on the same chromosome (7).

P. aeruginosa has a single and supercoiled circular chromosome in the cytoplasm (4). It also carries a lot of chromosome-mobilizing plasmids that are very significant to the organism’s lifestyle as a pathogen. The plasmids, TEM, OXA, and PSE, for instance, are encoded for betalactamase production, which is necessary for its resistance to antibiotics, thus allowing P. aeruginosa to be a formidable pathogen (8).

The two strains that have the complete genome sequence are Pseudomonas aeruginosa PA01 and Pseudomonas aeruginosa PA14 (9):

--In , a group of volunteer "Pseudomonas scientists", including those from the Washington PathoGenesis Corportaion and the Department of Biology of the University of California, San Diego, worked under the Pseudomonas aeruginosa Community Annotation Project (PseudoCAP) to publish the complete genome sequence of Pseudomonas aeruginosa PA This was done because knowing the genomic sequence would provide new information about this bacterium as a pathogen and about its ecological versatility and genetic complexity. At 6,, base pairs, its bacterial genome is the largest to ever be sequenced. It also contains 5, predicted open reading frames (ORFs), and thus it almost has the genetic complexity of simple eukaryotes, such as Saccharomyces cerevisiae. Using whole-genome-shotgun sampling, the complete Mbp genome of Pseudomonas aeruginosa PA01 is very much similar to the P. aeruginosa’s physical map, with only one major exception, which is the inversion of about a quarter of the Pseudomonas aeruginosa PA01 genome. This inversion comes from the homologous recombination of the rrnA and rrnB loci, and earlier studies on genomic sequence inversions of ribosomal DNA loci in S. typhimurium and E. coli suggest that this inversion might have adaptive significance (10).

--The complete genome sequence of Pseudomonas aeruginosa PA14 is currently being done by Harvard Medical School scientists. The goal of this study is achieve a public data of Pseudomonas aeruginosa PA14 genome. The shotgun-sequencing phase of the project was finished in , yielding Mbp of PA14 sequence. It is currently being compared to the genome of Pseudomonas aeruginosa PA01 and preliminary results have shown that they are very similar but have several regions of marked differences, such as the insertion of the bp in PA14, which is absent in PA Approximately, there is % of the DNA sequence of PAO1 is in PA14, and % of PA14 DNA sequence is in PA01 (11).

Cell structure and metabolism

Protein F--Since P. aeruginosa is a Gram-negative microbe, it has an outer membrane which contains Protein F (OprF). OprF functions as a porin, allowing certain molecules and ions to come into the cells, and as a structural protein, maintaining the bacterial cell shape. Because OprF provides P. aeruginosa outer membrane with an exclusion limit of Da, it lowers the permeability of the outer membrane, a property that is desired because it would decrease the intake of harmful substances into the cell and give P. aeruginosa a high resistance to antibiotics (12).

Flagellum and Pili--P. aeruginosa uses its single and polar flagellum to move around and to display chemotaxis to useful molecules, like sugars. Its strains either have a-type or b-type of flagella, a classification that is based primarily on the size and antigenicity of the flagellin subunit. The flagellum is very important during the early stages of infection, for it can attach to and invade tissues of the hosts (13). Similarly to its flagellum, P. aeruginosa pili contribute greatly to its ability to adhere to mucosal surfaces and epithelial cells. Specifically, it is the pili’s tip that is responsible for the adherence to the host cell surface. P. aeruginosa have N-methyl-phenyl-alanine (NMePhe) or type IV pili (1). The pili are characterized as long polar filaments made up of homopolymers from the protein pilin, which is encoded by the pilA gene (4). Overall, P. aeruginosa flagellum and pili have similar functionality (for attachment) and structure (both are filamentous structures on the surface of the cell), and their motility is controlled by RpoN, especially during initial attachment to the human host and under low nutrient conditions (1).

When infecting its host, P. aeruginosa is starved for iron because iron deprivation of an infecting pathogen is the key part in the humans’ innate defense mechanism. To overcome this challenge, P. aeruginosa synthesizes two siderophores: pyochelin and pyoverdin. P. aeruginosa then secrets these sideophores to the exterior of the cell, where they bind tightly to iron and bring the iron back into the cell. Additionally, P. aeruginosa can also use iron from enterobactin, a special siderophore produced by E. coli for iron transport, to satisfy its iron need (14).

P. aeruginosa is a facultative aerobe; its preferred metabolism is respiration. It gains energy by transferring electrons from glucose, a reduced substrate, to oxygen, the final electron acceptor (15). The breakdown of glucose requires it to oxidize to gluconate in the periplasm, then it will be brought inside the inner membrane by a specific energy-dependent gluconate uptake system. Once inside, gluconate is phosphorylated to 6-P-gluconate, which will enter the central metabolism to produce energy for the cell (16). When P. aeruginosa is in anaerobic conditions, however, P. aeruginosa uses nitrate as a terminal electron acceptor(17). Under oxidative-stress conditions, P. aeruginosa synthesizes Fe- or Mn- containing superoxide dismutase (SOD) enzymes, which catalyze the very reactive O- to H2O2 and O2. It also detoxifies H2O2 to O2 and H2O by using catalase (1).

Ecology

Since P. aeruginosa can live in both inanimate and human environments, it has been characterized as a “ubiquitous” microorganism. This versatility is made possible by a large number of enzymes that allow P. aeruginosa to use a diversity of substances as nutrients. Most impressively, P. aeruginosa can switch from growing on nonmucoid to mucoid environments, which comes with a large synthesis of alginate. In inanimate environment, P. aeruginosa is usually detected in water-reservoirs polluted by animals and humans, such as sewage and sinks inside and outside of hospitals. It is also found in swimming pools and whirlpools because the warm temperatures are favorable to its growth (3). Because it thrived in warm conditions, however, it was determined to be the culprit of the Hot Tub Rash, in which direct contact between the skin and the infected water from the tub will make the infected skin itchy and turn it a bumpy red color (19). In addition, P. aeruginosa is an opportunistic human pathogen that causes chronic infections in patients with cystic fibrosis and is the leading cause of death by Gram-negative bacteria (more under pathology) (3).

Although most P. aeruginosa-plant interactions are detrimental to the plant, a recent study has found a P. aeruginosa strain that actually supports plant growth. This characteristic, along with the fact that P. aeruginosa can degrade polycyclic aromatic hydrocarbons, suggests the future uses of P. aeruginosa for environmental detoxification of synthetic chemicals and pesticides and for industrial purposes (3). Psuedomonas aeruginosa is unique due to its ability to infect both humans and plants, one of the few organisms that can infect both kingdoms.

P. aeruginosa groups tend to form biofilms, which are complex bacterial communities that adhere to a variety of surfaces, including metals, plastics, medical implant materials, and tissue. Biofilms are characterized by “attached for survival” because once they are formed, they are very difficult to destroy. Depending on their locations, biofilms can either be beneficial and detrimental to the environment. For instance, the biofilms found on rocks and pebbles underwater of lakes and ponds are an important food source for many aquatic organisms. On the contrary, those that developed on the interiors of water pipes might cause clogging and corrosions (19) (20).

Pathology

P. aeruginosa rarely causes disease in healthy humans. It is usually linked with patients whose immune system is compromised by diseases or trauma. It gains access to these patients’ tissues through the burns, for the burn victims, or through an underlying disease, like cystic fibrosis. First, P. aeruginosa adheres to tissue surfaces using its flagellum, pili, and exo-S; then, it replicates to create infectious critical mass; and lastly, it makes tissue damage using its virulence factors (21). Since the powerful exotoxins and endotoxins released by P. aeruginosa during bacteremias continue to infect the host even after P. aeruginosa has been killed off by antibiotics, acute diseases caused by P. aeruginosa tend to be chronic and life-threatening. Furthermore, with the exception of the cystic fibrosis strain, most P. aeruginosa strains that attack compromised patients tend to be nonmucoid (2). And even though a small amount of patients infected by P. aeruginosa developed severe sepsis with lesions with black centers, most patients exhibited no obvious pathological effects of the colonization (22).

Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasians. With a mutation on chromosome 7, a CF lung cannot transport chloride (Cl-), sodium (Na+), and water from the basolateral to the secretory epithelia. This disruption in the salt and water balance in the cell results in the production of a thick mucus, which becomes the ideal home for potential pathogens. P. aeruginosa attacks CF patients via airway and once it is in, it uses its flagellum to go to the hypoxic zone, an oxygen-depleted environment. At this location, P. aeruginosa undergoes a transition from an aerobic to an anaerobic microbe and starts forming biofilms anaerobically. Once this is formed, the P. aeruginosa in this community can sense their population via quorum sensing, where they secret low molecular weight pheromones that enable them to communicate with each other (23). This gives them the ability to resist many defenses, including anti-Pseudomonas antibiotics such as ticarcillin, ceftazidime, tobramycin, and ciprofloxacin, because once the bacteria sense that their outer layer of biofilm is being destroyed, the inner layers will grow stronger to reestablish the community (24). P. aeruginosa is also resistant to many antibiotics and chemotherapeutic agents due to their intrinsic resistance. This is caused by the low permeability to antibiotics of the outer membrane and by the production of β-lactamases against multidrug efflux pumps and β-lactam antibiotics (22).

P. aeruginosa communicates with other cells through quorum-sensing. This form of communication allows the cells to regulate gene production which results in control of certain cell functions. One of the enzymes responsible for quorum sensing is tyrosine phosphatase (TpbA). This enzyme relays extracellular quorum sensing signals to polysaccharide production and biofilm formation outside the cells (32). P. aeruginosa attaches to surfaces by way of biofilm production. Quorum-sensing can be a drug target to cure infections caused by P. aeruginosa. Quorum-quenching is used to blocks the signaling mechanism of quorum-sensing and prevents biofilm formation in P. aeruginosa. Yi-Hu Dong and his colleagues were able to prevent biofilm formation in mice under laboratory conditions (33).

P. aeruginosa secrets many virulent factors to colonize the cells of its host. For example, exotoxin A, the most toxic protein produced by P. aeruginosa, catalyzes the ADP-ribosylation to form ADP-ribosyl-EF-2, which inhibits the protein synthesis of the host’s cells. Moreover, elastase, an extracellular zinc protease, attacks eukaryotic proteins such as collagen and elastin and destroys the structural proteins of the cell. It also breaks down human immunoglobin and serum alpha proteins (1).

Furthermore, P. aeruginosa infects animals. In an experiment, intravenous injection of virulent P. aeruginosa was injected into mice and these animals usually died within hours. When a smaller dose was injected, characteristic signs of infection such as weight loss, focal lesions in liver, spleen, and kidneys, followed by death within days, would take place. P. aeruginosa has also been found to cause outbreaks of pneumonia in guinea pigs, and although it also attacks plants, not a lot of research has been done in this area (22).

Pseudomonas aeruginosa is an environmentally ubiquitous opportunistic pathogen. Epidermal infections often result from P. aeruginosa infiltrating through a human host’s first line of defenses, entering the body through the skin at the site of an open wound. P. aeruginosa is a common member of hospital bacterial communities where it can infect immunocompromised individuals including burn victims. P. aeruginosa is a source of bacteremia in burn victims [36]. Following severe skin damage, the prevalence of P. aeruginosa in the environment increases the probability of the organism accessing the bloodstream through the burn victim’s exposed deep epidermal tissue [36]. Previous research of antibody-mediated host defenses indicates that on the fifth day after the initial burn, Fc receptor expression is reduced in polymorphonuclear leukocytes (PMNs). Without the Fc receptor, PMN chemotaxis is greatly reduced and the PMNs become less effective at preventing infection [36].

P. aeruginosa can be transmitted to a host via fomites, vectors, and hospital workers who are potential carriers for multiply-antibiotic-resistant strains of the pathogen. Furthermore, any P. aeruginosa already present on a burn victim’s skin before the injury can transform from an innocuous organism on the surface of the skin to a source of infection in the bloodstream and body tissues of the same individual [36].

The pili and flagella of P. aeruginosa play a vital role in the infection of burns and wounds [36]. Controlled infection of burn wounds on animal and plant models with P. aeruginosa strains devoid of pili and flagella demonstrate a trend of decreased virulence. Without these morphological virulence factors, the bacteria exhibit a substantially decreased survival rate at the wound site and a decreased ability to disseminate within the host organism [36]. The spread of P. aeruginosa within host organisms is also dependent on the microorganism’s elastase production and other protease mechanisms. Bacterial elastase and other bacterial proteases degrade the host’s proteins, including the structural proteins within membranes, disrupting the host’s physical barriers against the spread of infection. Elastase also assists P. aeruginosa in avoiding phagocytotic antibody-mediated cytotoxicity at the site of the wound by inhibiting monocyte chemotaxis [36].

Identification:

Macro morphology (smell):

Large, flat and greenish colonies ( mm in diameter) with irregular edges and typical metallic luster. The color is most visible on for instance TS-agar. Sometimes, a clear hemolysis zone is obtained on blood agar. Has distinctive smell (caramel, strawberry or raspberry soda). Some strains produce a green fluorescent pigment, pyoverdine. Some strains can also produce a blue pigment, pyocyanin.

Micromorphology: Small motile rod ( x µm) with a monotrichous flagellum.

Gram -:

Fig. Gram staining of Pseudomonas aeruginosa, strain ATCC The field B is a partial magnification (3 times) of A. The length of the scale bar corresponds to 5 µm. Date: G-

Metabolism: Is often classified as aerobic, but can also exploit NO3- as final electron acceptor in the respiratory chain. Should, therefore, be classified as facultatively anaerobic!

Catalase/Oxidase: +/+ Tryptophanase - Citrate +, methyl red -, Voges-Proskauer -.

Spec. Char.: Temperature optimum: 37°C, but can also grow at 42°C. Hosts: Cattle, dog, horse, mink, poultry, sheep, reptiles etc. (including humans) Reservoir: The environment: soil, water etc. Disease (Swedish): Mastit, pneumoni, otit mm Disease (English): Mastitis, pneumonia, otitis, etc

Application to Biotechnology

P. aeruginosa, as well as many other Pseudomonas, can degrade aromatic hydrocarbons such as methylbenzenes, which are the by-products of petroleum industries and are commonly used as solvents for enamels and paints as well as in the production of drugs and chemicals. Methylbenzenes are considered as environmental contaminants that are present in the atmosphere, underground and soils, and in surface water (25). P. aeruginosa can break down toluene, the simplest form of methylbenzene. P. aeruginosa degrades toluene through the oxidation of the methyl group to aldehyde, alcohol, and an acid, which is then converted to catechol. Hence, P. aeruginosa can be used in pollution control (26).

Current Research

Effect of Spaceflight on Microbial Gene Expression and Virulence (Microbe)

--The National Aeronautics and Space Administration (NASA) and the Biodesign Institute at Arizona State University are currently carrying out a research project called the Microbe Experiment. In this experiment, three microbial pathogens Pseudomonas aeruginosa, Salmonella typhimurium, and Candida albicans are being brought into space to see how their genetic responses and virulence change. These three microbes have been viewed as potential threat to the health of the astronauts, for P. aeruginosa had contaminated the spacecraft’s water system and infected a crew member during the Apollo era. Thus, understanding their adaptation and virulence in microgravity will give scientists more information about the crew’s space environment and better prepare the astronauts for future space explorations. The microbes were placed inside self-contained culture chambers and upon landing back on earth, one thirds of the sample will be used for virulence studies while the remaining will be kept frozen at oC. Because this is an ongoing research project, there have not been any results but NASA scientists are very hopeful that this study will lead to novel discoveries of vaccines against these microbes here on Earth and during spaceflight (27).

The Combination of PCR and Serology Increases the Diagnosis of Pseudomonas aeruginosa Colonization/Infection in Cystic Fibrosis

--Microbiological culturing methods are often used for the early diagnosis of P. aeruginosa infection in cystic fibrosis (CF) patients. These methods, however, have some disadvantages because P. aeruginosa might not be detected since initial infection is usually in low density. It was then proposed that serology and polymerase chain reaction (PCR) might be better techniques in detecting the early stage of P. aeruginosa infection in children with CF. The experiment was carried out by collecting sputum and serum from 87 CF children with a mean age of years. Then, 1) PCR was performed on the sputum, targeting P. aeruginosa algD GDP mannose dehydrogenase gene. 2) Serology was done against P. aeruginosa antigens: exotoxin A, elastase, and alkaline protease. 3) A combination of PCR and serology was done. When looking at the results, using the PCR or serology method alone did not yield statistically significant difference from the microbiological culturing methods. The combination of PCR and serology, however, identified a lot more patients than any of the two methods alone. Hence, a combination method that includes PCR will be an accurate technique to use in early diagnosis of P. aeruginosa colonization in CF patients (28).

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial

--Comparison between the P. aeruginosa PA01 strain and the more virulent P. aeruginosa PA14 was done to identity new virulence genes. First, shotgun genome sequencing was done on PA14 using 65, plasmids with kb fragments of PA14 DNA. Then, a long-range PCR based method was implemented to determine if certain P. aeruginosa genomes are similar to PA01 or PA14 genomes. It was found that although PA14 gemone ( Mbp) is somewhat larger than that of PA01 ( Mbp), PA14 and PA01 genomes are very similar. There were 58 gene clusters from PA14 that were missing in PA01 and it was assumed that some of these genes are what make PA14 a lot more virulent than PA Microarray genomotyping of 18 diverse strains in the C. elegans model, however, showed that those 58 PA14 gene clusters did not correlate with these strains’ virulence. Thus a conclusion was drawn that the virulence in P. aeruginosa is both combinatorial and multifactorial and that the genes required for one strain to be pathogenic are not required for virulence in other strains (29).

UV light test of fluorescent yellow/green pigment in nutrient broth

Research also done in a microbiology lab at Loyola University Chicago has concluded that the Pseudomonas aeruginosa develops a greenish/yellow fluorescent pigment in nutrient broth and casein hydrolysis. After placing this fluorescent pigment under UV light, we observed a fluorescent blue/green pigment within the test tube. (35)

Fig. 6A Thr

Under iron deficiency, a yellowish-green fluorescent pigment develops as a result of pyoverdins, a term named by Turfreijer for a group of compounds having a (1S)amino-2,3-dihydro-8,9-dihydroxy-1H-pyrimido-[1,2a] chinolincarboxylic acid chromophore. Figure 6A shows the different types of Pyoverdin groups that can be made on variations of their peptide chain. These pigment compounds only grow under iron limitation in a growth medium. Figure 6A shows the three different sv subgroups of Pyoverdin of P. aeruginosa. Since these three pyoverdin structures (also known as ferri-pyoverdins) are not produced by any other species of Pseudomonas they can be a quick way to identify the specific bacteria, Pseudomonas aeruginos. (34)

References

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30) Edited by students: Vivek Brahmbhatt and Varun Garg of M Glogowski at Loyola University. http://web.ebscohost.com/ehost/pdfviewer/pdfviewer?vid=4&hid=&sid=bc6b-e73c-4b8cc0b9d6cedf%40sessionmgr

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Written by Chelsea Dao, a student of Rachel Larsen

Edited by KLB

Edited by students: Vivek Brahmbhatt and Varun Garg / Michelle Chua and Safi Khan of Mary Glogowski at Loyola University, Irina Rojas and Aaron Beguelin at Hamilton College

Sours: https://microbewiki.kenyon.edu/index.php/Pseudomonas_aeruginosa


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