Dataset Information

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and Microbroth Dilution Methods for Detecting mecA-Mediated ?-Lactam Resistance in Contemporary Staphylococcus epidermidis Isolates.

ABSTRACT: Methicillin (?-lactam) resistance in Staphylococcus epidermidis is mediated by the mecA gene, with resistance reported to be as high as 90%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and broth microdilution (BMD) methods for the detection of mecA-mediated ?-lactam resistance in 100 human isolates of S. epidermidis (48 mecA-positive isolates and 52 mecA negative isolates). Oxacillin DD tests using the Clinical and Laboratory Standards Institute (CLSI) M100-S28 breakpoints for S. pseudintermedius/S. schleiferi accurately differentiated mecA-positive and -negative S. epidermidis isolates, with categorical agreement (CA) of 100% and no very major errors (VMEs) or major errors (MEs) identified. Likewise, oxacillin BMD and cefoxitin DD tests using the coagulase-negative Staphylococcus species (CoNS) breakpoints were highly reliable for detecting mecA-mediated ?-lactam resistance in S. epidermidis isolates. For cefoxitin DD and BMD results interpreted using S. aureus/S. lugdunensis breakpoints, the CA was 97.6% and 96.2%, respectively. There were 4.9% VMEs for cefoxitin DD with 0% MEs, and 3.6% VMEs and 3.9% MEs for cefoxitin BMD. Oxacillin BMD using S. aureus/S. lugdunensis breakpoints yielded the highest VMEs at 17.4% and 90% CA. Our findings demonstrate that oxacillin DD tests using the CLSI M100-S28 breakpoints for S. pseudintermedius/S. schleiferi and oxacillin BMD and cefoxitin DD tests using the CoNS breakpoints reliably identified mecA-mediated ?-lactam resistance in S. epidermidis Using mecA PCR as the gold standard, the PBP2a SA culture colony test (Abbott Diagnostics) exhibited 100% sensitivity and specificity whereas 2 false negatives were identified using the PBP2' latex agglutination test kit (Thermo Fisher Scientific) with sensitivity and specificity of 95.8% and 100%, respectively.

SUBMITTER: Naccache SN

PROVIDER: S-EPMC6879287 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Evaluation of Oxacillin and Cefoxitin Disk Diffusion and Microbroth Dilution Methods for Detecting mecA-Mediated β-Lactam Resistance in Contemporary Staphylococcus epidermidis Isolates.

Naccache Samia N SN Callan Katrina K Burnham C A CA Wallace Meghan A MA Westblade Lars F LF Dien Bard Jennifer J

Journal of clinical microbiology 20191122 12

Methicillin (β-lactam) resistance in Staphylococcus epidermidis is mediated by the mecA gene, with resistance reported to be as high as 90%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and broth microdilution (BMD) methods for the detection of mecA-mediated β-lactam resistance in 100 human isolates of S. epidermidis (48 mecA-positive isolates and 52 mecA negative isolates). Oxacillin DD tests using the Clinical and ...[more]

PMID: 31462553

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Project description:The oxacillin- and cefoxitin-susceptible mecA-positive Staphylococcus aureus is a novel "stealth" methicillin-resistant S. aureus (MRSA) type. Here, we sequenced the whole genome of two oxacillin- and cefoxitin-susceptible mecA-positive MRSA isolates from breast abscesses in a lactating woman and a nasal swab of a healthy student in Guangzhou for investigating the mechanism underlying its occurrence. The reversion of these isolates was selected by exposure to sub-MICs of cefoxitin with or without mupirocin. The mecA expression of both parental strains and their revertants was determined, and the whole genome of the revertants was sequenced. Comparative whole-genome analyses performed for both strains revealed that mecA of the clinical strain was mutated by a single-bp insertion at the 262nd position in the tandem repeat region of the gene, and this mutation that led to the formation of a premature stop codon. The colonizing strain was mutated by a novel G-to-A base substitution in the second promoter region (-35 bp) of mecA. The mecA expression level of strain 697 revertant was 37 times higher than that of the parental strain. Although the mecA expression level was even higher for parental strain 199 compared with that for its revertant, its cDNA sequence contained a single-bp insertion. Collectively, both the missense and single substitution mutations of the second promoter of mecA could render MRSA isolates as "stealth" MRSA, thereby emphasizing the importance of combining phenotype tests with mecA or penicillin-binding protein 2a detection for the identification of MRSA. IMPORTANCE The oxacillin- and cefoxitin-susceptible mecA-positive Staphylococcus aureus is a novel type of "stealth" methicillin-resistant S. aureus (MRSA), which is difficult to be detected using conventional methods. To investigate the genomic basis of their occurrence, we sequenced the whole genome of two previously recovered oxacillin- and cefoxitin-susceptible mecA-positive MRSA isolates from breast abscesses in a lactating woman and a nasal swab of a healthy student in Guangzhou. Complete SCCmec structure was absent except for mecA in clinical isolate 199. Additionally, a novel single-base pair insertion was observed in the clinical strain, which resulted in premature termination and a frameshift mutation. The colonizing isolate 697 had a Scc-mec-type IVa, and the second promoter region (-35 bp) of mecA was mutated by a novel G-to-A base substitution. The reversion of oxacillin- and cefoxitin-susceptible mecA-positive S. aureus to resistant MRSA isolates was selected by exposure to subminimum inhibitory cefoxitin with or without mupirocin.

Dataset Information

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and Microbroth Dilution Methods for Detecting mecA-Mediated ?-Lactam Resistance in Contemporary Staphylococcus epidermidis Isolates.

Publications

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and Microbroth Dilution Methods for Detecting <i>mecA</i>-Mediated β-Lactam Resistance in Contemporary Staphylococcus epidermidis Isolates.

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