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Characterization of a novel AmpC ?-lactamase produced by a carbapenem-resistant Cedecea davisae clinical isolate.

ABSTRACT: A Cedecea davisae isolate, which was intermediate or resistant to third-generation cephalosporins and carbapenems, was recovered from a urine sample. Susceptibility testing, isoelectric focusing, and analysis of outer membrane proteins showed that AmpC ?-lactamase expression combined with porin deficiency accounted for the carbapenem resistance. A cloning experiment followed by phenotypic and enzymatic characterization identified a novel class C enzyme that was phylogenetically and biochemically close to the chromosome-borne ?-lactamases of the genera Enterobacter and Citrobacter.

SUBMITTER: Ammenouche N

PROVIDER: S-EPMC4249424 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Characterization of a novel AmpC β-lactamase produced by a carbapenem-resistant Cedecea davisae clinical isolate.

Ammenouche Nacim N Dupont Hervé H Mammeri Hedi H

Antimicrobial agents and chemotherapy 20140818 11

A Cedecea davisae isolate, which was intermediate or resistant to third-generation cephalosporins and carbapenems, was recovered from a urine sample. Susceptibility testing, isoelectric focusing, and analysis of outer membrane proteins showed that AmpC β-lactamase expression combined with porin deficiency accounted for the carbapenem resistance. A cloning experiment followed by phenotypic and enzymatic characterization identified a novel class C enzyme that was phylogenetically and biochemically ...[more]

PMID: 25136020

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Project description:BackgroundAcinetobacter baumannii is a nosocomial pathogen which is establishing as a major cause of morbidity and mortality within the healthcare community. The success of this pathogen is largely due to its ability to rapidly gain resistance to antimicrobial therapies and its capability to persist in an abiotic environment through the production of a biofilm. Our tertiary-care hospital has showed high incidence of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates.MethodsIn this study we explore both genotypic and phenotypic properties of 26 CRAB isolates: 16 isolates were collected from January 2010 to March 2011, and 10 were collected between February and May 2015.ResultsWe determined that all 26 CRAB isolates possessed multiple β-lactamase genes, including genes from Groups A, C, and D. Specifically, 42% of the isolates possesses the potentially plasmid-borne genes of OXA-23-like or OXA-40-like β-lactamase. The presence of mobile gene element integron cassettes and/or integrases in 88% of the isolates suggests a possible mechanism of dissemination of antibiotic resistance genes. Additionally, the location of insertion sequence (IS) ISAba1 in promotor region of of the OXA-51-like, ADC-7, and ampC genes was confirmed. Multilocus sequence typing (MLST) demonstrated that all 26 CRAB isolates were either sequence type (ST)-229 or ST-2. Interestingly, ST-2 went from being the minority CRAB strain in the 2010-2011 isolates to the predominant strain in the 2015 isolates (from 32 to 90%). We show that the ST-2 strains have an enhanced ability to produce biofilms in comparison to the ST-229 strains, and this fact has potentially led to more successful colonization of the clinical environment over time.ConclusionsThis study provides a longitudinal genetic and phenotypic survey of two CRAB sequence types, and suggests how their differing phenotypes may interact with the selective pressures of a hospital setting effecting strain dominance over a 5-year period.

Project description:WCK 5222 (cefepime/zidebactam) is a β-lactam/β-lactamase inhibitor combination that is effective against a broad range of highly drug-resistant bacterial pathogens, including those producing metallo-β-lactamase. In this study, we isolated a multidrug-resistant Pseudomonas aeruginosa clinical strain that is resistant to a variety of β-lactam antibiotics and the ceftazidime-avibactam combination. A metallo-β-lactamase gene blaDIM-2 was identified on a self-transmissible megaplasmid in the strain, which confers the resistance to β-lactam antibiotics, leaving WCK 5222 potentially one of the last treatment resorts. In vitro passaging assay combined with whole-genome sequencing revealed mutations in the pbpA gene (encoding the zidebactam target protein PBP2) in the evolved resistant mutants. Among the mutations, a V516M mutation increased the bacterial virulence in a murine acute pneumonia model. Reconstitution of the mutations in the reference strain PAO1 verified their roles in the resistance to zidebactam and revealed their influences on cell morphology in the absence and presence of zidebactam. Microscale thermophoresis (MST) assays demonstrated that the mutations reduced the affinity between PBP2 and zidebactam to various extents. Overall, our results revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings. IMPORTANCE Antibiotic resistance imposes a severe threat on human health. WCK 5222 is a β-lactam/β-lactamase inhibitor combination that is composed of cefepime and zidebactam. It is one of the few antibiotics in clinical trials that are effective against multidrug-resistant Pseudomonas aeruginosa, including those producing metallo-β-lactamases. Understanding the mechanisms and development of bacterial resistance to WCK 5222 may provide clues for the development of strategies to suppress resistant evolvement. In this study, we performed an in vitro passaging assay by using a multidrug-resistant P. aeruginosa clinical isolate. Our results revealed that mutations in the zidebactam target protein PBP2 play a major role in the bacterial resistance to WCK 5222. We further demonstrated that the mutations reduced the affinities between PBP2 and zidebactam and resulted in functional resistance of PBP2 to zidebactam.

Dataset Information

Characterization of a novel AmpC ?-lactamase produced by a carbapenem-resistant Cedecea davisae clinical isolate.

Publications

Characterization of a novel AmpC β-lactamase produced by a carbapenem-resistant Cedecea davisae clinical isolate.

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