Genomic surveillance of Pseudomonas aeruginosa in the Philippines, 2013–2014

Pseudomonas aeruginosa is an opportunistic pathogen that often causes nosocomial infections resistant to treatment. Rates of antimicrobial resistance (AMR) are increasing, as are rates of multidrug-resistant (MDR) and possible extensively drug-resistant (XDR) infections. Our objective was to characterize the molecular epidemiology and AMR mechanisms of this pathogen. We sequenced the whole genome for each of 176 P. aeruginosa isolates collected in the Philippines in 2013–2014; derived the multilocus sequence type (MLST), presence of AMR determinants and relatedness between isolates; and determined concordance between phenotypic and genotypic resistance. Carbapenem resistance was associated with loss of function of the OprD porin and acquisition of the metallo-β-lactamase (MBL) gene blaVIM. Concordance between phenotypic and genotypic resistance was 93.27% overall for six antibiotics in three classes, but varied among minoglycosides. The population of P. aeruginosa was diverse, with clonal expansions of XDR genomes belonging to MLSTs ST235, ST244, ST309 and ST773. We found evidence of persistence or reintroduction of the predominant clone ST235 in one hospital, and of transfer between hospitals. Most of the ST235 genomes formed a distinct lineage from global genomes, thus raising the possibility that they may be unique to the Philippines. In addition, long-read sequencing of one representative XDR ST235 isolate identified an integron carrying multiple resistance genes (including blaVIM-2), with differences in gene composition and synteny from the P. aeruginosa class 1 integrons described previously. The survey bridges the gap in genomic data from the Western Pacific Region and will be useful for ongoing surveillance; it also highlights the importance of curtailing the spread of ST235 within the Philippines.

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Genomic Surveillance of Pseudomonas aeruginosa in the Philippines from 2013-2014

10.1101/2020.03.19.998229 ◽

2020 ◽

Author(s):

Jeremiah Chilam ◽

Silvia Argimón ◽

Marilyn T. Limas ◽

Melissa L. Masim ◽

June M. Gayeta ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Resistance ◽

Sequence Data ◽

Acquired Resistance ◽

Carbapenem Resistance ◽

The Philippines ◽

Surveillance Program ◽

Drug Resistant ◽

Loss Of Function ◽

Genotypic Resistance

AbstractPseudomonas aeruginosa is an opportunistic pathogen often causing nosocomial infections that are resilient to treatment due to an extensive repertoire of intrinsic and acquired resistance mechanisms. In recent years, increasing resistance rates to antibiotics such as carbapenems and extended-spectrum cephalosporins have been reported, as well as multi-drug resistant and possible extremely drug-resistant rates of approximately 21% and 15%, respectively. However, the molecular epidemiology and AMR mechanisms of this pathogen remains largely uncharacterized.We sequenced the whole genomes of 176 P. aeruginosa isolates collected in 2013-2014 by the Antimicrobial Resistance Surveillance Program. The multi-locus sequence type, presence of antimicrobial resistance (AMR) determinants, and relatedness between the isolates were derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined.Carbapenem resistance was associated namely with loss-of function of the OprD porin, and acquisition of the metallo-β-lactamase VIM. The concordance between phenotypic and genotypic resistance was 93.27% overall for 6 antibiotics in 3 classes, but varied widely between aminoglycosides. The population of P. aeruginosa in the Philippines was diverse, with clonal expansions of XDR genomes belonging to multi-locus sequence types ST235, ST244, ST309, and ST773. We found evidence of persistence or reintroduction of the predominant clone ST235 in one hospital, as well as transfer between hospitals. Most of the ST235 genomes formed a distinct Philippine lineage when contextualized with international genomes, thus raising the possibility that this is a lineage unique to the Philippines. This was further supported by long-read sequencing of one representative XDR isolate, which revealed the presence of an integron carrying multiple resistance genes, including blaVIM-2, with differences in gene composition and synteny to other P. aeruginosa class 1 integrons described before.We produced the first comprehensive genomic survey of P. aeruginosa in the Philippines, which bridges the gap in genomic data from the Western Pacific region and will constitute the genetic background to contextualize ongoing prospective surveillance. Our results also highlight the importance of infection control interventions aimed to curtail the spread of international epidemic clone ST235 within the country.

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Carbapenem-resistance mechanisms of multidrug-resistant Pseudomonas aeruginosa

Journal of Medical Microbiology ◽

10.1099/jmm.0.058354-0 ◽

2013 ◽

Vol 62 (9) ◽

pp. 1317-1325 ◽

Cited By ~ 23

Author(s):

Ester Fusté ◽

Lídia López-Jiménez ◽

Concha Segura ◽

Eusebio Gainza ◽

Teresa Vinuesa ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Carbapenem Resistance ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Class 1 Integron ◽

Carbonyl Cyanide ◽

Class 1

Clonal dissemination of multidrug-resistant Pseudomonas aeruginosa (MDRPA) is a major concern worldwide. The aim of this study was to explore the mechanisms leading to the carbapenem resistance of an MDRPA clone. Isolates were obtained from a surgical wound, sputum, urine and a blood culture. Pulsed-field gel electrophoresis (PFGE) showed high genomic homogeneity of these isolates and confirmed the circulation of an endemic clone belonging to serotype O4. Outer membrane protein (OMP) bands were visualized by SDS-PAGE, meropenem accumulation was measured in a bioassay and integrons were detected by PCR. Efflux pumps were studied for several antimicrobial agents and synergic combinations thereof in the presence or absence of both carbonyl cyanide m-chlorophenylhydrazone (CCCP) and Phe-Arg-β-naphthylamide (PAβN) at final concentrations of 10 and 40 mg l−1, respectively. On OMP electrophoretic profiles, MDRPA showed a reduction of outer membrane porin D (OprD) and PCR demonstrated the presence of a class 1 integron with a cassette encoding aminoglycoside adenyltransferase B (aadB). Meropenem accumulation was slightly higher in bacilli than in the filamentous cells that formed in the presence of antibiotics. Overexpression of the efflux pump MexAB-OprM and a functional MexXY-OprM were detected in all isolates.

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Prevalence of metallo-β-lactamase genes among Pseudomonas aeruginosa isolated from various clinical samples in China

Journal of Laboratory Medicine ◽

10.1515/labmed-2019-0162 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Wei Wang ◽

Xiaoya Wang

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Ethylenediaminetetraacetic Acid ◽

Carbapenem Resistance ◽

Opportunistic Pathogen ◽

Detection Methods ◽

Clinical Samples ◽

Routine Practice ◽

Carbapenem Resistant ◽

High Prevalence

AbstractBackgroundPseudomonas aeruginosa is an opportunistic pathogen which is associated with nosocomial infections and causes various diseases including urinary tract infection, pneumonia, soft-tissue infection and sepsis. The emergence of P. aeruginosa-acquired metallo-β-lactamase (MBL) is most worrisome and poses a serious threat during treatment and infection control. The objective of this study was to identify antibiotic susceptibility, phenotypic detection of MBL production and to determine the prevalence of MBL genes in carbapenem-resistant P. aeruginosa isolated from different clinical samples.MethodsA total of 329 non-duplicate P. aeruginosa isolated from various clinical samples from two hospitals in China between September 2017 and March 2019 were included in this study. Phenotypic detection of MBL was performed by the combined detection method using imipenem and imipenem-ethylenediaminetetraacetic acid (EDTA) discs. MBL-encoding genes including blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-2, blaSPM-1, blaSIM, blaNDM-1 and blaGIM were detected by polymerase chain reaction (PCR).ResultsOf the 329 P. aeruginosa, majority of the isolates were resistant to imipenem (77.5%) followed by meropenem (64.7%). Of the 270 P. aeruginosa isolates tested, 149 (55.2%) isolates were found to be positive for MBL detection. Of the different samples, 57.8% (n = 26) of P. aeruginosa isolated from blood were found to be positive for MBL production. Of the various MBL genes, blaIMP-1 (28.2%) was the most predominant gene detected followed by blaVIM-2 (18.8%), blaVIM-1 (16.1%), blaNDM-1 (9.4%), blaIMP-2 (6.7%), blaSIM (6.0%), blaSPM-1 (4.0%) and blaGIM (1.3%) genes.ConclusionsThe high resistance of P. aeruginosa toward imipenem and meropenem and the high prevalence of blaIMP-1 and blaVIM-2 set the alarm on the increasing, perhaps the increased, carbapenem resistance. In addition to routine antibiotic susceptibility testings, our results emphasize the importance of both the phenotypic and genotypic MBL detection methods in routine practice for early detection of carbapenem resistance and to prevent further dissemination of this resistant pathogen.

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Complete Sequence of pOZ176, a 500-Kilobase IncP-2 Plasmid Encoding IMP-9-Mediated Carbapenem Resistance, from Outbreak Isolate Pseudomonas aeruginosa 96

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00423-13 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3775-3782 ◽

Cited By ~ 48

Author(s):

Jianhui Xiong ◽

David C. Alexander ◽

Jennifer H. Ma ◽

Maxime Déraspe ◽

Donald E. Low ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Carbapenem Resistance ◽

Complete Sequence ◽

Open Reading Frames ◽

Content Type ◽

Conserved Sequence ◽

Carbapenem Resistant ◽

Class 1 ◽

Usage Analysis ◽

Type 3

ABSTRACTPseudomonas aeruginosa96 (PA96) was isolated during a multicenter surveillance study in Guangzhou, China, in 2000. Whole-genome sequencing of this outbreak strain facilitated analysis of its IncP-2 carbapenem-resistant plasmid, pOZ176. The plasmid had a length of 500,839 bp and an average percent G+C content of 57%. Of the 618 predicted open reading frames, 65% encode hypothetical proteins. The pOZ176 backbone is not closely related to any plasmids thus far sequenced, but some similarity to pQBR103 ofPseudomonas fluorescensSBW25 was observed. Two multiresistant class 1 integrons and several insertion sequences were identified. TheblaIMP-9-carrying integron containedaacA4→blaIMP-9→aacA4, flanked upstream by Tn21 tnpMRAand downstream by a completetnioperon of Tn402and amermodule, named Tn6016. The second integron carriedaacA4→catB8a→blaOXA-10and was flanked by Tn1403-liketnpRAand asul1-type 3′ conserved sequence (3′-CS), named Tn6217. Other features include three resistance genes similar to those of Tn5, a tellurite resistance operon, and twopiloperons. The replication and maintenance systems exhibit similarity to a genomic island ofRalstonia solanacearumGM1000. Codon usage analysis suggests the recent acquisition ofblaIMP-9. The origins of the integrons on pOZ176 indicated separate horizontal gene transfer events driven by antibiotic selection. The novel mosaic structure of pOZ176 suggests that it is derived from environmental bacteria.

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The rapid in vivo evolution of Pseudomonas aeruginosa in ventilator-associated pneumonia patients leads to attenuated virulence

Open Biology ◽

10.1098/rsob.170029 ◽

2017 ◽

Vol 7 (9) ◽

pp. 170029 ◽

Cited By ~ 17

Author(s):

Ke Wang ◽

Yi-qiang Chen ◽

May M. Salido ◽

Gurjeet S. Kohli ◽

Jin-liang Kong ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Ventilator Associated Pneumonia ◽

Loss Of Function ◽

Short Term ◽

Airway Infections ◽

Evolutionary Trajectories ◽

Human Airways ◽

Late Stages

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe airway infections in humans. These infections are usually difficult to treat and associated with high mortality rates. While colonizing the human airways, P. aeruginosa could accumulate genetic mutations that often lead to its better adaptability to the host environment. Understanding these evolutionary traits may provide important clues for the development of effective therapies to treat P. aeruginosa infections. In this study, 25 P. aeruginosa isolates were longitudinally sampled from the airways of four ventilator-associated pneumonia (VAP) patients. Pacbio and Illumina sequencing were used to analyse the in vivo evolutionary trajectories of these isolates. Our analysis showed that positive selection dominantly shaped P. aeruginosa genomes during VAP infections and led to three convergent evolution events, including loss-of-function mutations of lasR and mpl , and a pyoverdine-deficient phenotype. Specifically, lasR encodes one of the major transcriptional regulators in quorum sensing, whereas mpl encodes an enzyme responsible for recycling cell wall peptidoglycan. We also found that P. aeruginosa isolated at late stages of VAP infections produce less elastase and are less virulent in vivo than their earlier isolated counterparts, suggesting the short-term in vivo evolution of P. aeruginosa leads to attenuated virulence.

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F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

10.1101/2021.02.16.431561 ◽

2021 ◽

Author(s):

Senjuti Saha ◽

Chidozie D. Ojobor ◽

Erik Mackinnon ◽

Olesia I. North ◽

Joseph Bondy-Denomy ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bactericidal Activity ◽

Pathogenic Bacteria ◽

Therapeutic Potential ◽

Experimental Studies ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Antibiotic Resistant ◽

Genes Encoding ◽

Intraspecies Competition

ABSTRACTMost Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3’-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

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Three Novel Class 1 Integrons Detected in Multidrug-Resistant Pseudomonas aeruginosa Hospital Strains

Molecular Genetics Microbiology and Virology ◽

10.3103/s0891416819010026 ◽

2019 ◽

Vol 34 (1) ◽

pp. 8-15

Author(s):

E. I. Astashkin ◽

A. I. Lev ◽

O. N. Ershova ◽

T. S. Novikova ◽

E. N. Ageeva ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Multidrug Resistant ◽

Class 1 Integrons ◽

Class 1

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GidA Posttranscriptionally Regulates rhl Quorum Sensing in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00335-09 ◽

2009 ◽

Vol 191 (18) ◽

pp. 5785-5792 ◽

Cited By ~ 31

Author(s):

Rashmi Gupta ◽

Timothy R. Gobble ◽

Martin Schuster

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Flavin Adenine Dinucleotide ◽

Skim Milk ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Trna Modification ◽

Loss Of Function ◽

Novel Genes ◽

Lasa Protease

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa utilizes two interconnected acyl-homoserine lactone quorum-sensing (acyl-HSL QS) systems, LasRI and RhlRI, to regulate the expression of hundreds of genes. The QS circuitry itself is integrated into a complex network of regulation by other factors. However, our understanding of this network is still unlikely to be complete, as a comprehensive, saturating approach to identifying regulatory components has never been attempted. Here, we utilized a nonredundant P. aeruginosa PA14 transposon library to identify additional genes that regulate QS at the level of LasRI/RhlRI. We initially screened all 5,459 mutants for loss of function in one QS-controlled trait (skim milk proteolysis) and then rescreened attenuated candidates for defects in other QS phenotypes (LasA protease, rhamnolipid, and pyocyanin production) to exclude mutants defective in functions other than QS. We identified several known and novel genes, but only two novel genes, gidA and pcnB, affected all of the traits assayed. We characterized gidA, which exhibited the most striking QS phenotypes, further. This gene is predicted to encode a conserved flavin adenine dinucleotide-binding protein involved in tRNA modification. Inactivation of the gene primarily affected rhlR-dependent QS phenotypes such as LasA, pyocyanin, and rhamnolipid production. GidA affected RhlR protein but not transcript levels and also had no impact on LasR and acyl-HSL production. Overexpression of rhlR in a gidA mutant partially restored QS-dependent phenotypes. Taken together, these results indicate that GidA selectively controls QS gene expression posttranscriptionally via RhlR-dependent and -independent pathways.

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Problematic clinical isolates of Pseudomonas aeruginosa from the university hospitals in Sofia, Bulgaria: current status of antimicrobial resistance and prevailing resistance mechanisms

Journal of Medical Microbiology ◽

10.1099/jmm.0.46986-0 ◽

2007 ◽

Vol 56 (7) ◽

pp. 956-963 ◽

Cited By ~ 17

Author(s):

Tanya Strateva ◽

Vessela Ouzounova-Raykova ◽

Boyka Markova ◽

Albena Todorova ◽

Yulia Marteva-Proevska ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

Carbapenem Resistance ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Current Status ◽

University Hospitals ◽

Active Efflux ◽

Genes Encoding

A total of 203 clinical isolates of Pseudomonas aeruginosa was collected during 2001–2006 from five university hospitals in Sofia, Bulgaria, to assess the current levels of antimicrobial susceptibility and to evaluate resistance mechanisms to antipseudomonal antimicrobial agents. The antibiotic resistance rates against the following antimicrobials were: carbenicillin 93.1 %, azlocillin 91.6 %, piperacillin 86.2 %, piperacillin/tazobactam 56.8 %, ceftazidime 45.8 %, cefepime 48.9 %, cefpirome 58.2 %, aztreonam 49.8 %, imipenem 42.3 %, meropenem 45.5 %, amikacin 59.1 %, gentamicin 79.7 %, tobramycin 89.6 %, netilmicin 69.6 % and ciprofloxacin 80.3 %. A total of 101 of the studied P. aeruginosa isolates (49.8 %) were multidrug resistant. Structural genes encoding class A and class D β-lactamases showed the following frequencies: bla VEB-1 33.1 %, bla PSE-1 22.5 %, bla PER-1 0 %, bla OXA-groupI 41.3 % and bla OXA-groupII 8.8 %. IMP- and VIM-type carbapenemases were not detected. In conclusion, the studied clinical strains of P. aeruginosa were problematic nosocomial pathogens. VEB-1 extended-spectrum β-lactamases appear to have a significant presence among clinical P. aeruginosa isolates from Sofia. Carbapenem resistance was related to non-enzymic mechanisms such as a deficiency of OprD proteins and active efflux.

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