Inactivation of Nitrite-Dependent Nitric Oxide Biosynthesis Is Responsible for Overlapped Antibiotic Resistance between Naturally and Artificially Evolved Pseudomonas aeruginosa

Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance.

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Fis Contributes to Resistance of Pseudomonas aeruginosa to Ciprofloxacin by Regulating Pyocin Synthesis

Journal of Bacteriology ◽

10.1128/jb.00064-20 ◽

2020 ◽

Vol 202 (11) ◽

Cited By ~ 1

Author(s):

Yuqing Long ◽

Weixin Fu ◽

Su Wang ◽

Xuan Deng ◽

Yongxin Jin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Bacterial Resistance ◽

Global Gene Expression ◽

Chronic Infections ◽

Mobility Shift ◽

Content Type ◽

Regulatory Pathways ◽

Opportunistic Pathogenic

ABSTRACT Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa. In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa. The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

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High-Quality Complete Genome Sequences of Three Pseudomonas aeruginosa Isolates Retrieved from Patients Hospitalized in Intensive Care Units

Microbiology Resource Announcements ◽

10.1128/mra.01624-18 ◽

2019 ◽

Vol 8 (9) ◽

Cited By ~ 3

Author(s):

Bárbara Magalhães ◽

Laurence Senn ◽

Dominique S. Blanc

Keyword(s):

Pseudomonas Aeruginosa ◽

Intensive Care ◽

Intensive Care Units ◽

Genome Sequences ◽

High Quality ◽

Gram Negative ◽

Content Type ◽

Hospital Acquired Infections ◽

Hospital Acquired ◽

High Quality Genome

Pseudomonas aeruginosa is one of the major Gram-negative pathogens responsible for hospital-acquired infections. Here, we present high-quality genome sequences of isolates from three P. aeruginosa genotypes retrieved from patients hospitalized in intensive care units.

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Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid

Journal of Bacteriology ◽

10.1128/jb.00300-20 ◽

2020 ◽

Vol 203 (2) ◽

pp. e00300-20

Author(s):

Katie V. Farrant ◽

Livia Spiga ◽

Jane C. Davies ◽

Huw D. Williams

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Immune System ◽

Hypochlorous Acid ◽

Secretion System ◽

Content Type ◽

Type 3 Secretion System ◽

Lung Infections ◽

Type 3

ABSTRACTPseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host’s immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

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Complete Genome Sequences of Bacteriophages Kaya, Guyu, Kopi, and TehO, Which Target Clinical Strains of Pseudomonas aeruginosa

Microbiology Resource Announcements ◽

10.1128/mra.01043-21 ◽

2021 ◽

Vol 10 (48) ◽

Author(s):

Belinda Loh ◽

Xiaoqing Wang ◽

Xiaoting Hua ◽

Junhan Luo ◽

Tanye Wen ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Complete Genome ◽

Multidrug Resistant ◽

Health Concern ◽

Genome Sequences ◽

Content Type ◽

Hospital Acquired Infections ◽

Resistant Strains ◽

Drug Resistant Strains ◽

Hospital Acquired

Pseudomonas aeruginosa is a major public health concern, as drug-resistant strains increase mortality in hospital-acquired infections. We report the isolation and complete genome sequences of four lytic bacteriophages that target clinical multidrug-resistant P. aeruginosa strains.

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Identification of Two Regulators of Virulence That Are Conserved inKlebsiella pneumoniaeClassical and Hypervirulent Strains

mBio ◽

10.1128/mbio.01443-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 18

Author(s):

Michelle Palacios ◽

Taryn A. Miner ◽

Daniel R. Frederick ◽

Victoria E. Sepulveda ◽

Joshua D. Quinn ◽

...

Keyword(s):

Gene Expression ◽

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Virulence Factors ◽

Content Type ◽

Hospital Acquired Infections ◽

Capsule Type ◽

Capsule Production ◽

Invasive Infections ◽

Hospital Acquired

ABSTRACTKlebsiella pneumoniaeis widely recognized as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of hospital-acquired infections (urinary tract infections [UTI], pneumonia, sepsis) and community-acquired invasive infections. The genetic heterogeneity ofK. pneumoniaeisolates complicates our ability to understand the virulence ofK. pneumoniae. Characterization of virulence factors conserved between strains as well as strain-specific factors will improve our understanding of this important pathogen. The MarR family of regulatory proteins is widely distributed in bacteria and regulates cellular processes such as antibiotic resistance and the expression of virulence factors.Klebsiellaencodes numerous MarR-like proteins, and they likely contribute to the ability ofK. pneumoniaeto respond to and survive under a wide variety of environmental conditions, including those present in the human body. We tested loss-of-function mutations in all themarRhomologues in a murine pneumonia model and found that two (kvrAandkvrB) significantly impacted the virulence of K1 and K2 capsule type hypervirulent (hv) strains and thatkvrAaffected the virulence of a sequence type 258 (ST258) classical strain. In thehvstrains,kvrAandkvrBmutants displayed phenotypes associated with reduced capsule production, mucoviscosity, and transcription fromgalFandmanCpromoters that drive expression of capsule synthesis genes. In contrast,kvrAandkvrBmutants in the ST258 strain had no effect on capsule gene expression or capsule-related phenotypes. Thus, KvrA and KvrB affect virulence in classical andhvstrains but the effect on virulence may not be exclusively due to effects on capsule production.IMPORTANCEIn addition to having a reputation as the causative agent for hospital-acquired infections as well as community-acquired invasive infections,Klebsiella pneumoniaehas gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. Due to the rapid emergence of carbapenem resistance amongK. pneumoniaestrains, a better understanding of virulence mechanisms and identification of new potential drug targets are needed. This study identified two novel regulators (KvrA and KvrB) of virulence inK. pneumoniaeand demonstrated that their effect on virulence in invasive strains is likely due in part to effects on capsule production (a major virulence determinant) and hypermucoviscosity. KvrA also impacts the virulence of classical strains but does not appear to affect capsule gene expression in this strain. KvrA and KvrB are conserved amongK. pneumoniaestrains and thus could regulate capsule expression and virulence in diverse strains regardless of capsule type.

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A Serendipitous Mutation Reveals the Severe Virulence Defect of a Klebsiella pneumoniae fepB Mutant

mSphere ◽

10.1128/msphere.00341-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 12

Author(s):

Michelle Palacios ◽

Christopher A. Broberg ◽

Kimberly A. Walker ◽

Virginia L. Miller

Keyword(s):

Antibiotic Resistance ◽

Urinary Tract ◽

Klebsiella Pneumoniae ◽

Drug Targets ◽

Carbapenem Resistance ◽

Content Type ◽

Hospital Acquired Infections ◽

Tract Infections ◽

Hospital Acquired ◽

Rapid Emergence

ABSTRACT In addition to having a reputation as the causative agent of several types of hospital-acquired infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of infections, including urinary tract infections, pneumonia, and sepsis. Because of the rapid emergence of carbapenem resistance among Klebsiella strains, there is a dire need for a better understanding of virulence mechanisms and identification of new drug targets. Here, we identify the periplasmic transporter FepB as one such potential target. Klebsiella pneumoniae is considered a significant public health threat because of the emergence of multidrug-resistant strains and the challenge associated with treating life-threatening infections. Capsule, siderophores, and adhesins have been implicated as virulence determinants of K. pneumoniae, yet we lack a clear understanding of how this pathogen causes disease. In a previous screen for virulence genes, we identified a potential new virulence locus and constructed a mutant (smr) with this locus deleted. In this study, we characterize the smr mutant and show that this mutation renders K. pneumoniae avirulent in a pneumonia model of infection. The smr mutant was expected to have a deletion of three genes, but subsequent genome sequencing indicated that a much larger deletion had occurred. Further analysis of the deleted region indicated that the virulence defect of the smr mutant could be attributed to the loss of FepB, a periplasmic protein required for import of the siderophore enterobactin. Interestingly, a ΔfepB mutant was more attenuated than a mutant unable to synthesize enterobactin, suggesting that additional processes are affected. As FepB is highly conserved among the members of the family Enterobacteriaceae, therapeutic targeting of FepB may be useful for the treatment of Klebsiella and other bacterial infections. IMPORTANCE In addition to having a reputation as the causative agent of several types of hospital-acquired infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of infections, including urinary tract infections, pneumonia, and sepsis. Because of the rapid emergence of carbapenem resistance among Klebsiella strains, there is a dire need for a better understanding of virulence mechanisms and identification of new drug targets. Here, we identify the periplasmic transporter FepB as one such potential target.

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Bacteriophages ϕMR299-2 and ϕNH-4 Can Eliminate Pseudomonas aeruginosa in the Murine Lung and on Cystic Fibrosis Lung Airway Cells

mBio ◽

10.1128/mbio.00029-12 ◽

2012 ◽

Vol 3 (2) ◽

Cited By ~ 126

Author(s):

Debebe Alemayehu ◽

Pat G. Casey ◽

Olivia McAuliffe ◽

Caitriona M. Guinane ◽

James G. Martin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Cell Line ◽

Treatment Plant ◽

Bacteriophage Therapy ◽

Exciting Field ◽

Content Type ◽

Murine Lung ◽

Human Lung Cells

ABSTRACTPseudomonas aeruginosais a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance ofPseudomonasare major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (ϕNH-4) and a podovirus (ϕMR299-2). Both phages were active against clinical isolates ofP. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killingP. aeruginosaNH57388A (mucoid) andP. aeruginosaMR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 107to 2 × 107P. aeruginosa.Pseudomonaswas effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinicalPseudomonasisolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistantPseudomonaslung infections in CF patients.IMPORTANCEGiven the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment ofPseudomonasinfection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can killPseudomonasgrowing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

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Hypermutator Pseudomonas aeruginosa Exploits Multiple Genetic Pathways To Develop Multidrug Resistance during Long-Term Infections in the Airways of Cystic Fibrosis Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02142-19 ◽

2020 ◽

Vol 64 (5) ◽

Cited By ~ 2

Author(s):

C. A. Colque ◽

A. G. Albarracín Orio ◽

S. Feliziani ◽

R. L. Marvig ◽

A. R. Tobares ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Multidrug Resistance ◽

Parallel Evolution ◽

Acquired Resistance ◽

Antibiotic Resistance Genes ◽

Purifying Selection ◽

Chronic Infections ◽

Content Type

ABSTRACT Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolates recovered from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic infections in CF patients. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations, with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to have been positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed toward the establishment of a population constituted by genotypically diversified coexisting sublineages, all of which converged to multidrug resistance. These sublineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and ftsI, encoding the β-lactamase and penicillin-binding protein 3, respectively, were found to be among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying β-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence.

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Antagonistic Donor Density Effect Conserved in Multiple Enterococcal Conjugative Plasmids

Applied and Environmental Microbiology ◽

10.1128/aem.00363-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4537-4545 ◽

Cited By ~ 13

Author(s):

Arpan Bandyopadhyay ◽

Sofie O'Brien ◽

Kristi L. Frank ◽

Gary M. Dunny ◽

Wei-Shou Hu

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Virulence Determinants ◽

Content Type ◽

Hospital Acquired Infections ◽

Donor Density ◽

Donor Cells ◽

Inhibitor Peptide ◽

Hospital Acquired ◽

High Donor

ABSTRACTEnterococcus faecalis, a common causative agent of hospital-acquired infections, is resistant to many known antibiotics. Its ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious concern for public health. In some cases, induction of transfer ofE. faecalisplasmids results from peptide pheromones produced by plasmid-free recipient cells, which are sensed by the plasmid-bearing donor cells. These plasmids generally encode an inhibitory peptide that competes with the pheromone and suppresses self-induction of donors. We recently demonstrated that the inhibitor peptide encoded on plasmid pCF10 is part of a unique quorum-sensing system in which it functions as a “self-sensing signal,” reducing the response to the pheromone in a density-dependent fashion. Based on the similarities between regulatory features controlling conjugation in pAD1 and pAM373 and those controlling conjugation in pCF10, we hypothesized that these plasmids are likely to exhibit similar quorum-sensing behaviors. Experimental findings indicate that for both pAD1 and pAM373, high donor densities indeed resulted in decreased induction of the conjugation operon and reduced conjugation frequencies. This effect was restored by the addition of exogenous inhibitor, confirming that the inhibitor serves as an indicator for donor density. Donor density also affects cross-species conjugative plasmid transfer. Based on our experimental results, we propose models for induction and shutdown of the conjugation operon in pAD1 and pAM373.IMPORTANCEEnterococcus faecalisis a leading cause of hospital-acquired infections. Its ability to transfer antibiotic resistance and virulence determinants by sharing its genetic material with other bacteria through direct cell-cell contact via conjugation poses a serious threat. Two antagonistic signaling peptides control the transfer of plasmids pAD1 and pAM373: a peptide pheromone produced by plasmid-free recipients triggers the conjugative transfer in plasmid-containing donors, and an inhibitor peptide encoded on the plasmid and produced by donor cells serves to modulate the donor response in accordance with the relative abundance of donors and recipients. We demonstrate that high donor density reduces the conjugation frequency of both of these plasmids, which is a consequence of increased inhibitor concentration in high-donor-density cultures. While most antibiotic strategies end up selecting resistant strains and disrupting the community balance, manipulating bacterial signaling mechanisms can serve as an alternate strategy to prevent the spread of antibiotic resistance.

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Phenotypic and Genotypic Determination of Resistance to Common Disinfectants Among Strains of Pseudomonas Aeruginosa Producing and Non-Producing Biofilm Isolated From Iran

10.21203/rs.3.rs-918248/v1 ◽

2021 ◽

Author(s):

Mehdi Bakht ◽

Safar Ali Alizadeh ◽

Sara Rahimi ◽

Raana Kazemzade anari ◽

Mohammad Rostamani ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Acetic Acid ◽

Sodium Hypochlorite ◽

Efflux Pump ◽

Antimicrobial Susceptibility Test ◽

Present Survey ◽

Hospital Acquired Infections ◽

Hospital Acquired

Abstract Background: One of the most important reasons for human mortality worldwide is Hospital-acquired infections, which can be controlled by efficient use of proper disinfectants for the Hospital settings. The main aim of the present survey was to assess the susceptibility of Pseudomonas aeruginosa producing and non-producing biofilm isolated to the five commonly used Hospital disinfectants, and evaluation of the synergistic effect of selective disinfectants and Ethylene-diamine-tetra acetic acid (EDTA), and the effect of exposure to sub-inhibitory concentrations of Sodium hypochlorite on antimicrobial susceptibility test.Results: The results showed that Sodium hypochlorite 5%, and Ethanol 70% is the most and less potent disinfectants against Pseudomonas aeruginosa, respectively. Clearly, the addition of EDTA increased the efficacy of selected disinfectants significantly. The changes in the antibiotic-resistance profiles after exposure to sub-inhibitory concentrations of disinfectants were observed for different classes of antibiotics. As well as near the all isolates harbored efflux pump genes and produced biofilm. Conclusion: For disinfection of Hospital surfaces and instruments, the mixture of disinfectant and EDTA were the most suitable selection in this study. In our study, it was clear that exposure to sub-inhibitory concentrations of disinfectants results in resistance to antibiotics. Also, strong and intermediate biofilm formers belonged to MDR/XDR strains.

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