Investigation of Metallo-Beta-Lactamases in Carbapenem Resistant Pseudomonas aeruginosa Strains by Phenotypic and Genotypic Methods

Introduction: Carbapenem resistant Pseudomonas aeruginosa strains cause serious problems in treatment. A large number of identified metallo-beta-lactamase (MBL) enzymes produced by P. aeruginosa are one of the most important mechanisms in resistance to carbapenems. MBL genes are located on the chromosome or plasmid, and they can easily spread between different bacterial strains. The activities of these enzymes are zinc-dependent, and they are inhibited by ethylenediaminetetraacetic acid (EDTA). Therefore, this advantage is used in MBL identification tests. In this study, it was aimed to determine MBL among P. aeruginosa strains. Materials and Methods: MBL existence was investigated in 35 P. aeruginosa strains accepted to be mildly susceptible/resistant to any of the carbapenem group of antibiotics through phenotypic and genotypic methods. Phenotypic tests were performed as double disk synergy test (DDST), combined disk diffusion tests (CDDT) by using 0.1 M and 0.5 M EDTA, MBL E-test, and modified Hodge test (MHT). blaIMP, blaVIM, blaGIM, blaSIM, blaSPM genes and blaNDM gene were investigated by multiplex polimerase chain reaction (PCR) and PCR, respectively. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 standard bacteria were used in tests. VIM-1, VIM-2, IMP-13, SPM-1, NDM-1 type MBL-producing P. aeruginosa strains were used as positive controls. Results: Among the carbapenems resistant P. aeruginosa isolates, positivity of MBL was found as 54.2% by MBL E-test, 42.8% by DDST, 94.2% and 37.1% by CDDT method using 0.5 M and 0.1 M EDTA, respectively. Modified Hodge test and genotypic method did not detect MBL. Conclusion: In order to correctly evaluate the results of the phenotypic method, the investigation of resistance genes by molecular methods is also required. The most common metallo-beta-lactamase enzymes responsible for resistance to carbapenem in Pseudomonas were not observed. It was thought that different mechanisms might be responsible for the identified carbapenem resistance.

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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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Detection of VIM and NDM-1 metallo-beta-lactamase genes in carbapenem-resistant Pseudomonas aeruginosa clinical strains in Bahrain

Journal of Laboratory Physicians ◽

10.4103/jlp.jlp_118_18 ◽

2019 ◽

Vol 11 (02) ◽

pp. 138-143 ◽

Cited By ~ 1

Author(s):

Ronni Mol Joji ◽

Nouf Al-Rashed ◽

Nermin Kamal Saeed ◽

Khalid Mubarak Bindayna

Keyword(s):

Pseudomonas Aeruginosa ◽

Multiple Drug Resistance ◽

Infectious Agent ◽

Carbapenem Resistance ◽

Conventional Polymerase Chain Reaction ◽

Control Measures ◽

Multiple Drug ◽

Beta Lactamase ◽

Infection Control Measures ◽

Carbapenem Resistant

Abstract INTRODUCTION: Carbapenem-resistant Pseudomonas aeruginosa has emerged as a life-threatening infectious agent worldwide. Carbapenemase genes are reported to be some of the most common mechanisms for carbapenem resistance in P. aeruginosa. No reports are available from the Kingdom of Bahrain about carbapenem resistance and the underlying cause. In this study, we determined to study the presence of the metallo-beta-lactamase (M β L) genes of VIM family and NDM-1 in carbapenem-resistant P. aeruginosa strains. METHODOLOGY: Fifty carbapenem-resistant P. aeruginosa isolates were obtained from three main hospitals of Bahrain. They were subjected to antimicrobial susceptibility testing by disc diffusion test. Subsequently, MβL was detected by imipenem-ethylene diamine tetraacetic acid (EDTA) combined disc test and conventional polymerase chain reaction. RESULTS: Among 50 P. aeruginosa strains, 40 (80%) were imipenem resistant. Among the 40 imipenem-resistant strains, 35 (87.5%) strains were positive for the imipenem-EDTA combined disc test, and 21 (52%) were carrying MβL genes. Nineteen (47.5%) strains were positive for the VIM gene; one (2.5%) strain was carrying the NDM-1 gene, while one strain was carrying both the VIM and NDM-1 genes. None of the imipenem sensitive strains carried the VIM or NDM-1 gene. CONCLUSION: This is the first study to report the presence of the VIM family gene and NDM-1 genes in imipenem-resistant P. aeruginosa isolates in the Kingdom of Bahrain. The study also confirms the multiple drug resistance by the MβL strains, attention should therefore from now on, be focused on prevention of further spread of such isolates by firm infection control measures, and to reduce its threat to public health.

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Evaluation of different phenotypic tests for detection of metallo-β-lactamases in imipenem-resistant Pseudomonas aeruginosa

Journal of Laboratory Physicians ◽

10.4103/jlp.jlp_118_16 ◽

2017 ◽

Vol 9 (04) ◽

pp. 249-253 ◽

Cited By ~ 5

Author(s):

Rohit Sachdeva ◽

Babita Sharma ◽

Rajni Sharma

Keyword(s):

Pseudomonas Aeruginosa ◽

Large Scale ◽

Wide Spectrum ◽

Carbapenem Resistance ◽

Resistance Mechanisms ◽

Clinical Samples ◽

Resistant Strains ◽

Synergy Test ◽

Phenotypic Tests ◽

Simple Screening

Abstract PURPOSE: Pseudomonas aeruginosa causes a wide spectrum of infections including bacteremia, pneumonia, urinary tract infection, etc., Metallo-beta-lactamase (MBL) producing P. aeruginosa is an emerging threat and cause of concern as they have emerged as one of the most feared resistance mechanisms. This study was designed to know the prevalence of MBL production in P. aeruginosa and to evaluate the four phenotypic tests for detection of MBL production in imipenem-resistant clinical isolates of P. aeruginosa. METHODS: Totally, 800 isolates of P. aeruginosa isolated from various clinical samples were evaluated for carbapenem resistance and MBL production. All imipenem-resistant strains were tested for carabapenemase production by modified Hodge test. Screening for MBL production was done by double-disc synergy test and combined disc test (CDT). Confirmation of MBL production was done by the E-test (Ab BioDisk, Solna, Sweden). RESULTS: Out of the 800 isolates of P. aeruginosa, 250 isolates were found resistant to imipenem. Based on the results of E-test, 147 (18.37%) isolates of P. aeruginosa were positive for MBL production. The CDT has the highest sensitivity and specificity for the detection of MBL production as compared to other tests. CONCLUSION: The results of this study are indicative that MBL production is an important mechanism of carbapenem resistance among P. aeruginosa. Use of simple screening test like CDT will be crucial step toward large-scale monitoring of these emerging resistant determinants. Phenotypic test for MBL production has to be standardized, and all the isolates should be routinely screened for MBL production.

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GPC-1, a novel class A carbapenemase detected in a clinical Pseudomonas aeruginosa isolate

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz536 ◽

2020 ◽

Vol 75 (4) ◽

pp. 911-916 ◽

Cited By ~ 1

Author(s):

Jennifer Schauer ◽

Sören G Gatermann ◽

Daniel Hoffmann ◽

Lars Hupfeld ◽

Niels Pfennigwerth

Keyword(s):

Pseudomonas Aeruginosa ◽

Heterologous Expression ◽

Biochemical Characterization ◽

Resistance Mechanism ◽

Carbapenem Resistance ◽

Amino Acid Identity ◽

The Novel ◽

Bacterial Strains ◽

Class A ◽

Carbapenem Resistant

Abstract Objectives To investigate the carbapenem resistance mechanism of a carbapenem-resistant clinical Pseudomonas aeruginosa isolate. Methods A carbapenem-resistant P. aeruginosa isolate was recovered from a tracheal swab from a patient of a general ward in central Germany. Various phenotypic tests confirmed production of a carbapenemase that could not be identified further by PCR. A novel bla gene was identified by WGS and its carbapenemase activity was verified by heterologous expression in an Escherichia coli cloning strain. Kinetic parameters of the novel β-lactamase were determined by spectrophotometric measurements using purified enzyme. Results WGS confirmed the presence of a novel class A carbapenemase. The novel bla gene was named GPC-1 (GPC standing for German Pseudomonas Carbapenemase) and exhibited 77% amino acid identity to BKC-1. WGS also showed that blaGPC-1 was located on the chromosome surrounded by multiple ISs as part of a 26 kb genetic island. Heterologous expression of GPC-1 in E. coli TOP10 led to increased MICs of penicillins, oxyimino-cephalosporins, aztreonam and imipenem, but not of meropenem or ertapenem. Spectrophotometric measurements supported the MIC studies, but detected a slight hydrolysis of ertapenem and meropenem when using high concentrations of purified enzyme. Conclusions The biochemical characterization of GPC-1 emphasizes the ongoing emergence of novel carbapenemases. Strains expressing a weak carbapenemase like GPC-1 might go unrecognized by routine diagnostics due to low MICs for the bacterial strains producing such enzymes.

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Detection of prevalence of metallo-beta lactamases in clinical isolates of imipenem resistant Pseudomonas aeruginosa from neonatal septicaemia cases in a tertiary hospital in Odisha, India

International Journal of Contemporary Pediatrics ◽

10.18203/2349-3291.ijcp20175540 ◽

2017 ◽

Vol 5 (1) ◽

pp. 61 ◽

Cited By ~ 1

Author(s):

Surya Narayan Mishra ◽

Seba Ranjan Biswal ◽

Basanta Kumar Behera ◽

Dipti Pattnaik

Keyword(s):

Pseudomonas Aeruginosa ◽

Clinical Isolates ◽

Beta Lactamase ◽

Strict Adherence ◽

Neonatal Septicemia ◽

Gram Negative ◽

Beta Lactamases ◽

Pseudomonas Spp ◽

Neonatal Septicaemia ◽

Synergy Test

Background: Pseudomonas aeruginosa is a clinically troublesome gram-negative pathogen that causes both opportunistic infections and nosocomial outbreaks. Metallo beta lactamase have recently emerged as a worrisome resistance mechanism. Carbapenems had been the drug of choice for the infections caused by most penicillin- or cephalosporin-resistant gram-negative bacteria due to its broad-spectrum activity and stability to hydrolysis by most beta-lactamases. This does not hold good anymore due to rapid uprise of MBL producing strains. The current research covered 163 hospitalized cases of neonatal septicaemia from which Pseudomonas aeruginosa is isolated in the Paediatric Department of KIMS, Bhubaneswar. The study aimed at detecting the prevalence of metallo-beta lactamases in clinical isolates of imipenem resistant Pseudomonas aeruginosa from neonatal septicemia cases and to establish the antibiogram of Imipenem-resistant P. aeruginosa these cases. Methods: Clinical samples obtained from suspected cases of neonatal septicemia were first cultured by conventional methods and then identification was done by VITEK-2 instrument. Metallo beta lactamase (MBL) production was done by combined disc synergy test (CDST) using imipenem and EDTA (CDST-IPM) and double disc synergy test (DDST) using IPM and EDTA (DDST-IPM). Results: Among 1510 processed clinical specimens from cases of neonatal septicaemia; 637 (42.18%) showed positive growth of various clinically significant pathogens. Out of them in 163 (25.58%) cases Pseudomonas spp. was isolated. Of these, a total of 95 (58.28%) Pseudomonas spp. was found resistant to imipenem. All imipenem-resistant Pseudomonas isolates were positive for MBL by CDST imipenem-EDTA (CDST-IPM) method, whereas 89 (93.68%) were positive by DDST-IPM method, respectively. Pseudomonas aeruginosa was mostly isolated from endotracheal tube aspirate (57.89%) followed by pus (56.41%). Out of the 95 cases of MBL-producing Pseudomonas; 46 (48.42%) isolates showed the maximum susceptibility to piperacillin-tazobactam combination. All MBL-producing Pseudomonas isolates were resistant to ceftriaxone.Conclusions: MBL-producing Pseudomonas is found to be highly prevalent in our hospital, which is one of the major causes of multidrug resistance and need regular surveillance and strict adherence to a robust antibiotic policy.

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Detection of carbapenemase-producing Pseudomonas aeruginosa by phenotypic and genotypic methods in a tertiary care hospital of East India

Journal of Laboratory Physicians ◽

10.4103/jlp.jlp_136_19 ◽

2019 ◽

Vol 11 (04) ◽

pp. 287-291 ◽

Cited By ~ 1

Author(s):

Nishu Verma ◽

Ashok Prahraj ◽

Baijayantimala Mishra ◽

Bijayini Behera ◽

Kavita Gupta

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Mechanisms ◽

Tertiary Care ◽

Carbapenem Resistance ◽

Hospital Infection ◽

High Rate ◽

Care Hospital ◽

Carbapenem Resistant ◽

Hospital Infection Control ◽

Synergy Test

Abstract BACKGROUND: Carbapenemase-producing Pseudomonas aeruginosa is a serious threat in hospital infection due to its multidrug resistance. AIM: The aim of the study was to determine the frequency of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa and detect the presence of carbapenemase enzymes in carbapenem-resistant P. aeruginosa (CRPA) isolates by phenotypic and genotypic methods. MATERIAL AND METHODS: Double-disk synergy test [DDST] and combined disk synergy test [CDST]) was performed in CRPA isolates and the prevalence ofblaKPC,blaNDM-1,blaIMP,blaVIM,blaSIM,blaSPM,blaGIM, andblaOXA-48 was determined. RESULTS: Of 559 isolates included in the study, a total of 102 isolates were resistant to carbapenem that accounted for overall 18.24% (102/559) prevalence. Of these 102 isolates, 89 (87.25%) isolates were positive by DDST and 95 (93.17%) isolates were positive by CDST. Of 102 CRPA isolates,blaVIM was detected in 30 isolates (30/102, 29.1%), followed byblaNDM-1 in 29 (29/102, 28.4%) isolates andblaSIM andblaGIM in 6 isolates each (6/102, 5.8%). A combination of two carbapenemase genes was detected in 12 isolates, with six (6/102, 5.88%) CRPA isolates harboring with bothblaVIM andblaNDM-1 genes. Four isolates were found to harbor a combination of three carbapenem-resistant genes. CONCLUSION: A high rate of carbapenemase production was observed in P. aeruginosa. Coproducers of multiple carbapenemases are also a cause of concern. An in-depth understanding of molecular mechanisms of resistance will be helpful in optimizing patient management and hospital infection control.

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Activity of Cefiderocol, Ceftazidime-Avibactam, and Eravacycline against Carbapenem-Resistant Escherichia coli Isolates from the United States and International Sites in Relation to Clonal Background, Resistance Genes, Coresistance, and Region

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00797-20 ◽

2020 ◽

Vol 64 (10) ◽

Cited By ~ 1

Author(s):

Brian D. Johnston ◽

Paul Thuras ◽

Stephen B. Porter ◽

Melissa Anacker ◽

Brittany VonBank ◽

...

Keyword(s):

Escherichia Coli ◽

Carbapenem Resistance ◽

The United States ◽

Asia Pacific ◽

Beta Lactamase ◽

Content Type ◽

Beta Lactamases ◽

E Coli ◽

Carbapenem Resistant ◽

Primary Agent

ABSTRACT Emerging carbapenem resistance in Escherichia coli, including sequence type 131 (ST131), the leading cause of extraintestinal E. coli infections globally, threatens therapeutic efficacy. Accordingly, we determined broth microdilution MICs for three distinctive newer agents, i.e., cefiderocol (CFDC), ceftazidime-avibactam (CZA), and eravacycline (ERV), plus 11 comparators, against 343 carbapenem-resistant (CR) clinical E. coli isolates, then compared susceptibility results with bacterial characteristics and region. The collection comprised 203 U.S. isolates (2002 to 2017) and 141 isolates from 17 countries in Europe, Latin America, and the Asia-West Pacific region (2003 to 2017). Isolates were characterized for phylogenetic group, resistance-associated sequence types (STs) and subsets thereof, and relevant beta-lactamase-encoding genes. CFDC, CZA, and ERV exhibited the highest percent susceptible (82% to 98%) after tigecycline (TGC) (99%); avibactam improved CZA's activity over that of CAZ (11% susceptible). Percent susceptible varied by phylogroup and ST for CFDC and CZA (greatest in phylogroups B2, D, and F, and in ST131, ST405, and ST648). Susceptibility also varied by resistance genotype, being higher with the Klebsiella pneumoniae carbapenemase (KPC) for CZA, lower with metallo-beta-lactamases for CFDC and CZA, and higher with the beta-lactamase CTX-M for ERV. Percent susceptible also varied by global region for CZA (lower in Asia-Pacific) and by U.S. region for ERV (lower in the South and Southeast). Although resistance to comparators often predicted reduced susceptibility to a primary agent (especially CFDC and CZA), even among comparator-resistant isolates the primary-agent-susceptible fraction usually exceeded 50%. These findings clarify the likely utility of CFDC, CZA, and ERV against CR E. coli in relation to multiple bacterial characteristics and geographical region.

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Comparative Study of Phenotypic Detection Methods of Metallo-beta-lactamase (MβL) Among Carbapenem Resistant Gram-Negative Bacilli (GNB) Isolated from Intensive Care Units (IUC) Patients

10.51429/ejmm29310 ◽

2020 ◽

Vol 29 (3) ◽

pp. 75-80

Author(s):

Raghda Hager ◽

Bassant M. Sayed

Keyword(s):

Intensive Care ◽

Intensive Care Units ◽

Carbapenem Resistance ◽

Detection Methods ◽

Diffusion Method ◽

Beta Lactamase ◽

Gram Negative ◽

Carbapenem Resistant ◽

High Prevalence ◽

Synergy Test

Background: Metallo-beta-lactamase (MβL) mediated resistance is an emergency threat in health care settings, and its identification is essential for treatment and infection control. Objectives: this study aimed to detect the MβL prevalence in Gram negative bacilli (GNB) and to compare its phenotypic detection methods. Methodology: Ninety six (96) isolates of GNB were isolated from different clinical specimens collected from patients admitted to intensive care units (ICU) of Ain Shams Hospitals, from February 2018 to June 2018. Isolates were screened for carbapenem resistance with imipenem 10 µg and meropenem 10 µg discs. The resistant isolates were tested for antibiotic susceptibility by disc diffusion method, and Meropenem minimum inhibitory concentration (MIC) were determinated, then the production of MβL was detected by imipenem-ethylene diamine tetra-acetic acid (EDTA) combined disc test (IPM-EDTA CDT), ceftazidime -EDTA combined disc test (CAZ-EDTA CDT) and Imipenem - EDTA double disc synergy test (IPM-EDTA DDST).Results: Forty three (43) isolates (44.7%) were resistant to carbapenem. Klebsiella pneumoniae (K.pneumoniae) was the most common isolated species; 29 (67.4%) isolates. Forty (40) isolates (93%) were positive for MβL by IPM-EDTA CDT method, whereas 36 (83.7%) were positive by CAZ -EDTA CDT method and 19 isolates (44.2%) were positive for MβL by IPM-EDTA DDST. Conclusion: High prevalence of MβL was detected among our isolates and IMP-EDTA CDT can be used as a phenotypic test in detection of MβL production.

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Phenotypic and Molecular Detection of IMP and SPM Metallo-Beta-Lactamases in Clinical Isolates of Carbapenem Resistant Pseudomonas aeruginosa

10.21203/rs.3.rs-60519/v1 ◽

2020 ◽

Author(s):

Zahra Norouzi Bazgir ◽

Mohammad Ahanjan ◽

Hamid Reza Goli ◽

Roya Ghasemian ◽

Mohammad Bagher Hashemi-Soteh

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Susceptibility ◽

Molecular Detection ◽

Clinical Isolates ◽

Beta Lactamase ◽

Antibiotic Susceptibility Pattern ◽

Beta Lactamases ◽

Phenotypic Identification ◽

Carbapenem Resistant ◽

High Prevalence

Abstract Objectives: Metallo-beta-lactamases play a major role in the resistance of Pseudomonas aeruginosa to carbapenems. The aim of this study was the phenotypic and molecular detection of IMP and SPM carbapenemase genes in 100 carbapenem-resistant clinical isolates of P. aeruginosa. The isolates identified using standard microbiological tests, and their antibiotic susceptibility pattern determined by disk agar diffusion (Kirby Bauer) method. Phenotypic identification of Metallo-beta-lactamase-producing strains assessed by the combined disk test (CDT). Then, PCR was used to detect the presence of IMP and SPM genes.Results: The highest and lowest levels of antibiotic resistance were observed against gentamicin (40%) and piperacillin-tazobactam (13%), respectively. Besides, 40 isolates (40%) had the Multi-drug Resistant (MDR) phenotype, while 5 (12.5%) MDR isolates were resistant to all antibiotics tested. The results of the CDT showed that among 43 carbapenem non-susceptible clinical isolates of P. aeruginosa, 33 (76.74%) isolates were Metallo-beta-lactamase-producing strains. Also, the frequency of the IMP gene was determined to be 9%, while none of these isolates carried the SPM gene. Due to the high prevalence of carbapenem-resistant and MDR P. aeruginosa in this study, routine antibiotic susceptibility testing and phenotypic identification of carbapenemase production by this bacterium are necessary for proper selection of antibiotics.

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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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