β-lactam Resistance in Pseudomonas aeruginosa: Current Status, Future Prospects

Pseudomonas aeruginosa is a major opportunistic pathogen, causing a wide range of acute and chronic infections. β-lactam antibiotics including penicillins, carbapenems, monobactams, and cephalosporins play a key role in the treatment of P. aeruginosa infections. However, a significant number of isolates of these bacteria are resistant to β-lactams, complicating treatment of infections and leading to worse outcomes for patients. In this review, we summarize studies demonstrating the health and economic impacts associated with β-lactam-resistant P. aeruginosa. We then describe how β-lactams bind to and inhibit P. aeruginosa penicillin-binding proteins that are required for synthesis and remodelling of peptidoglycan. Resistance to β-lactams is multifactorial and can involve changes to a key target protein, penicillin-binding protein 3, that is essential for cell division; reduced uptake or increased efflux of β-lactams; degradation of β-lactam antibiotics by increased expression or altered substrate specificity of an AmpC β-lactamase, or by the acquisition of β-lactamases through horizontal gene transfer; and changes to biofilm formation and metabolism. The current understanding of these mechanisms is discussed. Lastly, important knowledge gaps are identified, and possible strategies for enhancing the effectiveness of β-lactam antibiotics in treating P. aeruginosa infections are considered.

Download Full-text

Mutational Analysis of RetS, an Unusual Sensor Kinase-Response Regulator Hybrid Required for Pseudomonas aeruginosa Virulence

Infection and Immunity ◽

10.1128/iai.00575-06 ◽

2006 ◽

Vol 74 (8) ◽

pp. 4462-4473 ◽

Cited By ~ 53

Author(s):

Michelle A. Laskowski ◽

Barbara I. Kazmierczak

Keyword(s):

Pseudomonas Aeruginosa ◽

Mutational Analysis ◽

Response Regulator ◽

Acute Infection ◽

Opportunistic Pathogen ◽

Sensor Kinase ◽

Chronic Infections ◽

Reciprocal Regulation ◽

Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in a wide range of hosts. Expression of the type III secretion system (T3SS) proteins is correlated with virulence in models of acute infection, while downregulation of the T3SS and upregulation of genes important for biofilm formation are observed during chronic infections. RetS, a hybrid sensor kinase-response regulator protein of P. aeruginosa, plays a key role in the reciprocal regulation of virulence factors required for acute versus chronic infection and is postulated to act in concert with two other sensor kinase-response regulator hybrids, GacS and LadS. This work examines the roles of the putative sensing and signal transduction domains of RetS in induction of the T3SS in vitro and in a murine model of acute pneumonia. We identify distinct signaling roles for the tandem receiver domains of RetS and present evidence suggesting that RetS may serve as a substrate for another sensor kinase. Phenotypes associated with RetS alleles lacking periplasmic and/or transmembrane domains further indicate that the periplasmic domain of RetS may transmit a signal that inhibits RetS activity during acute infections.

Download Full-text

A Large-Scale Whole-Genome Comparison Shows that Experimental Evolution in Response to Antibiotics Predicts Changes in Naturally Evolved Clinical Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01619-19 ◽

2019 ◽

Vol 63 (12) ◽

Cited By ~ 6

Author(s):

Samuel J. T. Wardell ◽

Attika Rehman ◽

Lois W. Martin ◽

Craig Winstanley ◽

Wayne M. Patrick ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Experimental Evolution ◽

Large Scale ◽

Opportunistic Pathogen ◽

Clinical Isolates ◽

Genome Comparison ◽

Chronic Infections ◽

Content Type ◽

Large Deletions ◽

Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations, we experimentally evolved the antibiotic-sensitive strain P. aeruginosa PAO1 to become resistant to three widely used antipseudomonal antibiotics, namely, ciprofloxacin, meropenem, and tobramycin. Mutants could tolerate up to 2,048-fold higher concentrations of antibiotics than strain PAO1. Genome sequences were determined for 13 mutants for each antibiotic. Each mutant had between 2 and 8 mutations. For each antibiotic, at least 8 genes were mutated in multiple mutants, demonstrating the genetic complexity of resistance. For all three antibiotics, mutations arose in genes known to be associated with resistance but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in this study, we analyzed the corresponding genes in 558 isolates of P. aeruginosa from patients with chronic lung disease and in 172 isolates from the general environment. Many genes identified through experimental evolution had predicted function-altering changes in clinical isolates but not in environmental isolates, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. Additionally, large deletions of up to 479 kb arose in experimentally evolved meropenem-resistant mutants, and large deletions were present in 87 of the clinical isolates. These findings significantly advance understanding of antibiotic resistance in P. aeruginosa and demonstrate the validity of experimental evolution in identifying clinically relevant resistance-associated mutations.

Download Full-text

Roles of Two-Component Systems in Pseudomonas aeruginosa Virulence

International Journal of Molecular Sciences ◽

10.3390/ijms222212152 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12152

Author(s):

Maria Sultan ◽

Rekha Arya ◽

Kyeong Kyu Kim

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Opportunistic Pathogen ◽

Secretion Systems ◽

Chronic Infections ◽

Potential Threat ◽

Wide Range ◽

Component Systems ◽

Two Component ◽

Two Component Systems

Pseudomonas aeruginosa is an opportunistic pathogen that synthesizes and secretes a wide range of virulence factors. P. aeruginosa poses a potential threat to human health worldwide due to its omnipresent nature, robust host accumulation, high virulence, and significant resistance to multiple antibiotics. The pathogenicity of P. aeruginosa, which is associated with acute and chronic infections, is linked with multiple virulence factors and associated secretion systems, such as the ability to form and utilize a biofilm, pili, flagella, alginate, pyocyanin, proteases, and toxins. Two-component systems (TCSs) of P. aeruginosa perform an essential role in controlling virulence factors in response to internal and external stimuli. Therefore, understanding the mechanism of TCSs to perceive and respond to signals from the environment and control the production of virulence factors during infection is essential to understanding the diseases caused by P. aeruginosa infection and further develop new antibiotics to treat this pathogen. This review discusses the important virulence factors of P. aeruginosa and the understanding of their regulation through TCSs by focusing on biofilm, motility, pyocyanin, and cytotoxins.

Download Full-text

Signal Sensing and Transduction Are Conserved between the Periplasmic Sensory Domains of BifA and SagS

mSphere ◽

10.1128/msphere.00442-19 ◽

2019 ◽

Vol 4 (4) ◽

Author(s):

Jozef Dingemans ◽

Rebecca E. Al-Feghali ◽

Holger Sondermann ◽

Karin Sauer

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Structural Similarity ◽

Opportunistic Pathogen ◽

Sequence Conservation ◽

Sensor Kinase ◽

Antibiotic Tolerance ◽

Bacterial Cells ◽

Chronic Infections ◽

Content Type

ABSTRACT The hybrid sensor kinase SagS of Pseudomonas aeruginosa plays a key role in the transition from the planktonic to the biofilm mode of growth. Recently, we have shown that distinct sets of residues in its periplasmic HmsP sensory domain are involved in the regulation of biofilm formation or antibiotic tolerance. Interestingly, the HmsP domain of the phosphodiesterase BifA shows great predicted structural similarity to that of SagS, despite moderate sequence conservation and only a number of residues involved in SagS signaling being conserved between both proteins. Based on this observation, we hypothesized that BifA and SagS may use similar mechanisms to sense and transduce signals perceived at their periplasmic HmsP domains and, therefore, may be interchangeable. To test this hypothesis, we constructed SagS hybrids in which the HmsP domain of SagS was replaced by that of BifA (and vice versa) or by the DISMED2 sensory domain of NicD. The SagS-BifA hybrid restored attachment and biofilm formation by the ΔbifA mutant. Likewise, while the NicD-SagS hybrid was nonfunctional, the BifA-SagS hybrid partially restored pathways leading to biofilm formation and antibiotic tolerance in a ΔsagS mutant background. Furthermore, alanine substitution of key residues previously associated with the biofilm formation and antibiotic tolerance pathways of SagS impaired signal transduction by the BifA-SagS hybrid in a similar way to SagS. In conclusion, our data indicate that the nature of the sensory domain is important for proper functionality of the cytoplasmic effector domains and that signal sensing and transduction are likely conserved in SagS and BifA. IMPORTANCE Biofilms have been associated with more than 60% of all recalcitrant and chronic infections and can render bacterial cells up to a thousand times more resistant to antibiotics than planktonic cells. Although it is known that the transition from the planktonic to the biofilm mode of growth involves two-component regulatory systems, increased c-di-GMP levels, and quorum sensing systems among others, the exact signaling events that lead to biofilm formation remain unknown. In the opportunistic pathogen Pseudomonas aeruginosa, the hybrid sensor kinase SagS regulates biofilm formation and antibiotic tolerance through two independent pathways via distinct residues in its periplasmic sensory domain. Interestingly, the sensory domains of SagS and BifA show great predicted structural similarity despite moderate sequence conservation. Here we show that the sensory domains of BifA and SagS are functionally interchangeable and that they use a similar mechanism of signal sensing and transduction, which broadens our understanding of how bacteria perceive and transduce signals when transitioning to the biofilm mode of growth.

Download Full-text

σ Factor and Anti-σ Factor That Control Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00784-15 ◽

2015 ◽

Vol 198 (5) ◽

pp. 755-765 ◽

Cited By ~ 9

Author(s):

Bryan A. McGuffie ◽

Isabelle Vallet-Gely ◽

Simon L. Dove

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Cell Envelope ◽

Opportunistic Pathogen ◽

Chronic Infections ◽

Swarming Motility ◽

Content Type ◽

Σ Factor ◽

Stress Response System

ABSTRACTPseudomonas aeruginosais capable of causing a variety of acute and chronic infections. Here, we provide evidence thatsbrR(PA2895), a gene previously identified as required during chronicP. aeruginosarespiratory infection, encodes an anti-σ factor that inhibits the activity of its cognate extracytoplasmic-function σ factor, SbrI (PA2896). Bacterial two-hybrid analysis identified an N-terminal region of SbrR that interacts directly with SbrI and that was sufficient for inhibition of SbrI-dependent gene expression. We show that SbrI associates with RNA polymerasein vivoand identify the SbrIR regulon. In cells lacking SbrR, the SbrI-dependent expression ofmuiAwas found to inhibit swarming motility and promote biofilm formation. Our findings reveal SbrR and SbrI as a novel set of regulators of swarming motility and biofilm formation inP. aeruginosathat mediate their effects throughmuiA, a gene not previously known to influence surface-associated behaviors in this organism.IMPORTANCEThis study characterizes a σ factor/anti-σ factor system that reciprocally regulates the surface-associated behaviors of swarming motility and biofilm formation in the opportunistic pathogenPseudomonas aeruginosa. We present evidence that SbrR is an anti-σ factor specific for its cognate σ factor, SbrI, and identify the SbrIR regulon inP. aeruginosa. We find that cells lacking SbrR are severely defective in swarming motility and exhibit enhanced biofilm formation. Moreover, we identifymuiA(PA1494) as the SbrI-dependent gene responsible for mediating these effects. SbrIR have been implicated in virulence and in responding to antimicrobial and cell envelope stress. SbrIR may therefore represent a stress response system that influences the surface behaviors ofP. aeruginosaduring infection.

Download Full-text

Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection

BMC Microbiology ◽

10.1186/s12866-021-02427-4 ◽

2022 ◽

Vol 22 (1) ◽

Author(s):

Shamima Nasrin ◽

Nicolas Hegerle ◽

Shaichi Sen ◽

Joseph Nkeze ◽

Sunil Sen ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Vaccine Development ◽

Opportunistic Pathogen ◽

Diffusion Method ◽

Chronic Infections ◽

Disk Diffusion Method ◽

Agglutination Assay ◽

O Antigen ◽

Wide Range

Abstract Background Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005–2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method. Results Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04). Conclusions Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections.

Download Full-text

Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽

10.1128/mbio.00183-10 ◽

2010 ◽

Vol 1 (4) ◽

Cited By ~ 106

Author(s):

Judith H. Merritt ◽

Dae-Gon Ha ◽

Kimberly N. Cowles ◽

Wenyun Lu ◽

Diana K. Morales ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Extracellular Polysaccharide ◽

Diguanylate Cyclase ◽

Flagellar Motility ◽

Critical Question ◽

Cyclic Diguanylate ◽

Critical Signal ◽

Content Type ◽

Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

Download Full-text

Pseudomonas aeruginosa las and rhl quorum-sensing systems are important for infection and inflammation in a rat prostatitis model

Microbiology ◽

10.1099/mic.0.028464-0 ◽

2009 ◽

Vol 155 (8) ◽

pp. 2612-2619 ◽

Cited By ~ 21

Author(s):

Lisa K. Nelson ◽

Genevieve H. D'Amours ◽

Kimberley M. Sproule-Willoughby ◽

Douglas W. Morck ◽

Howard Ceri

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Tissue Damage ◽

Inflammatory Markers ◽

Bacterial Colonization ◽

Opportunistic Pathogen ◽

Infection Model ◽

Chronic Infections ◽

Strain Pao1 ◽

Rat Prostate

Pseudomonas aeruginosa frequently acts as an opportunistic pathogen of mucosal surfaces; yet, despite causing aggressive prostatitis in some men, its role as a pathogen in the prostate has not been investigated. Consequently, we developed a Ps. aeruginosa infection model in the rat prostate by instilling wild-type (WT) Ps. aeruginosa strain PAO1 into the rat prostate. It was found that Ps. aeruginosa produced acute and chronic infections in this mucosal tissue as determined by bacterial colonization, gross morphology, tissue damage and inflammatory markers. WT strain PAO1 and its isogenic mutant PAO-JP2, in which both the lasI and rhlI quorum-sensing signal systems have been silenced, were compared during both acute and chronic prostate infections. In acute infections, bacterial numbers and inflammatory markers were comparable between WT PA01 and PAO-JP2; however, considerably less tissue damage occurred in infections with PAO-JP2. Chronic infections with PAO-JP2 resulted in reduced bacterial colonization, tissue damage and inflammation as compared to WT PAO1 infections. Therefore, the quorum-sensing lasI and rhlI genes in Ps. aeruginosa affect acute prostate infections, but play a considerably more important role in maintaining chronic infections. We have thus developed a highly reproducible model for the study of Ps. aeruginosa virulence in the prostate.

Download Full-text

Putrescine and its metabolic precursor arginine promote biofilm and c-di-GMP synthesis in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00297-21 ◽

2021 ◽

Author(s):

Zhexian Liu ◽

Sarzana S. Hossain ◽

Zayda Morales Moreira ◽

Cara H. Haney

Keyword(s):

Pseudomonas Aeruginosa ◽

Immune Responses ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Bacterial Pathogen ◽

Guanosine Monophosphate ◽

Genetic Approach ◽

Chronic Infections ◽

Host Immune Responses ◽

Primary Mechanism

Pseudomonas aeruginosa , an opportunistic bacterial pathogen can synthesize and catabolize a number of small cationic molecules known as polyamines. In several clades of bacteria polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, L-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or through a metabolic derivative. Here we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa . Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that L-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation, but via a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and L-arginine induced a significant increase in the intracellular level of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor arginine promote biofilm and c-di-GMP synthesis in P. aeruginosa . Importance: Biofilm formation allows bacteria to physically attach to a surface, confers tolerance to antimicrobial agents, and promotes resistance to host immune responses. As a result, regulation of biofilm is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switching that favors chronic infection.

Download Full-text

PmtA Regulates Pyocyanin Expression and Biofilm Formation in Pseudomonas aeruginosa

Frontiers in Microbiology ◽

10.3389/fmicb.2021.789765 ◽

2021 ◽

Vol 12 ◽

Author(s):

Amy V. Thees ◽

Kathryn M. Pietrosimone ◽

Clare K. Melchiorre ◽

Jeremiah N. Marden ◽

Joerg Graf ◽

...

Keyword(s):

Oxidative Stress ◽

Pseudomonas Aeruginosa ◽

Metal Binding ◽

Biofilm Formation ◽

Binding Capacity ◽

Opportunistic Pathogen ◽

Small Molecular Weight ◽

Heavy Metal Binding ◽

The Impact

The opportunistic pathogen Pseudomonas aeruginosa expresses a small molecular weight, cysteine-rich protein (PmtA), identified as a metallothionein (MT) protein family member. The MT family proteins have been well-characterized in eukaryotes as essential for zinc and copper homeostasis, protection against oxidative stress, and the ability to modify a variety of immune activities. Bacterial MTs share sequence homology, antioxidant chemistry, and heavy metal-binding capacity with eukaryotic MTs, however, the impact of bacterial MTs on virulence and infection have not been well-studied. In the present study, we investigated the role of PmtA in P. aeruginosa PAO1 using a PmtA-deficient strain (ΔpmtA). Here we demonstrated the virulence factor, pyocyanin, relies on the expression of PmtA. We showed that PmtA may be protective against oxidative stress, as an alternative antioxidant, glutathione, can rescue pyocyanin expression. Furthermore, the expression of phzM, which encodes a pyocyanin precursor enzyme, was decreased in the ΔpmtA mutant during early stationary phase. Upregulated pmtA expression was previously detected in confluent biofilms, which are essential for chronic infection, and we observed that the ΔpmtA mutant was disrupted for biofilm formation. As biofilms also modulate antibiotic susceptibility, we examined the ΔpmtA mutant susceptibility to antibiotics and found that the ΔpmtA mutant is more susceptible to cefepime and ciprofloxacin than the wild-type strain. Finally, we observed that the deletion of pmtA results in decreased virulence in a waxworm model. Taken together, our results support the conclusion that PmtA is necessary for the full virulence of P. aeruginosa and may represent a potential target for therapeutic intervention.

Download Full-text