Browsing by Author "Okyere, Dora"

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    Beta-lactam resistance mechanisms in Enterobacter species isolates from Tygerberg Hospital.
    (Stellenbosch : Stellenbosch University, 2018-10-31) Okyere, Dora; Newton-Foot, Mae; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
    Background Resistance to carbapenem antibiotics in Gram-negative bacteria is a major public health problem due to limited treatment options. In Enterobacter species, carbapenemase production and reduced membrane permeability, resulting from reduced expression of outer membrane proteins OmpF and OmpC, in combination with extended-spectrum β-lactamase (ESBL) production, hyper-production of chromosomal blaAmpC β-lactamases and / or over-expression of the AcrAB/TolC efflux pump have been speculated to promote carbapenem resistance. This study investigated the mechanisms that promote ertapenem resistance in clinical Enterobacter cloacae isolates from Tygerberg Hospital. Materials and methods Twenty ertapenem non-susceptible clinical E. cloacae isolates, four ertapenem susceptible controls and five wild-type controls were selected based on the VITEK2®AES. Ertapenem MICs were determined using broth microdilution (BMD) and gradient diffusion. Resistance mechanisms were characterized using the phenotypic assays VITEK2®AES, Mastdiscs D68C, D69C and D63C combination sets, Rapidec® Carba NP kit and a synergy assay using disc diffusion in the presence of an efflux pump inhibitor (EPI). Molecular assays included multiplex PCR to identify carbapenemases, multiplex PCR and sequencing to identify ESBLs, SDS-PAGE to characterize OmpC and OmpF abundance and RT-qPCR to quantify expression of blaAmpC, ompC, ompF and acrB. Results and Discussion Seventeen (85%) ertapenem non-susceptible isolates were confirmed non-susceptible by BMD and six (30%) by gradient diffusion, suggesting possible undercalling of ertapenem resistance by gradient diffusion and overcalling by VITEK2®AES. Seven ertapenem non-susceptible isolates were predicted to be carbapenemase producers by the VITEK2®AES and one by the Rapidec® Carba NP kit, however no carbapenemases were detected by PCR. Nineteen ESBL producers were identified by the VITEK2®AES, eight by the D68C combination set and eleven by the D63C combination set. ESBLs were detected in 12 (60%) isolates by PCR and sequencing; of which eight were blaCTX-M, three were blaSHV-12 and one isolate contained both genes. Twelve (60%) ertapenem non-susceptible isolates were predicted to be derepressed blaAmpC producers by the VITEK2®AES, thirteen (65%) by the D68C combination set and six (30%) by the blaAmpC RT-qPCR.