Phenotypic detection of AmpC β-lactamases, extended-spectrum β-lactamases and metallo-β-lactamases in Enterobacteriaceae using a resazurin microtitre assay with inhibitor-based methods

Abstract

Abstract:Dissemination of antibiotic resistance in Enterobacteriaceae mediated by AmpC, ESBL and MBL β-lactamases is clinically significant. A simple, rapid method for the detection of these resistance phenotypes would greatly improve chemotherapeutic recommendation. This technology would provide valuable input in our surveillance of resistance on a global stage, particularly if the methodology could be applicable to resource poor settings. A resazurin microtitre plate (RMP) assay incorporating cloxacillin (CX), clavulanic acid (CA), and EDTA for the rapid phenotypic identification of AmpC, ESBL, MBL and the co-existence of β-lactamases has been developed. A total of 44 molecularly characterised Enterobacteriaceae clinical isolates producing AmpCs, ESBLs, co-producers of ESBL and AmpC, MBLs, and co-producers of ESBL and MBL were phenotypically examined using the RMP assay. The ceftazidime (CAZ)-based and cefotaxime (CTX)-based RMP assay successfully detected all 15 AmpC, 12 ESBL, 9 MBL producers, 6 ESBL-AmpC co-producers, and 2 ESBL-MBL co-producers without false positive results. The CAZ-based assay was more reliable in detecting AmpC alone, while the CTX-based assay performed better in identifying co-producers of ESBL and AmpC. There was no difference in detection of ESBL and MBL producers. The findings of the present study suggest that use of the RMP assay with particular β-lactamase inhibitors explicitly detects three different β-lactamases, as well as coexistence of β-lactamases within 6 h. This assay is applicable to carry out in any laboratory, is cost-effective and easy to interpret. It could be implemented in screening patients, controlling infection and for surveillance purposes. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

A highly multiplexed melt-curve assay for detecting the most prevalent carbapenemase, ESBL and AmpC genes

“…A total of 111 well-documented 40,41 bacterial isolates from a well characterized collection of clinical isolates sourced in the UK between 2012-2017 were tested. The isolates included E. coli (n=37), K. pneumoniae (n=27), Enterobacter cloacae (n=15), Enterobacter aerogenes (n=12), Citrobacter freundii (n=12), Pseudomonas aeruginosa (n=3), Morganella morganii (n=3), K. oxytoca (n=1) and Acinetobacter haemolyticus (n=1).…”

Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay

“…A total of 111 bacterial isolates were obtained from a collection of clinical isolates sourced in the UK between 2012 and 2017 UK 35 , 36 . The isolates included E. coli (n = 37), K. pneumoniae (n = 27), Enterobacter aerogenes (n = 12), Enterobacter cloacae (n = 15), Citrobacter freundii (n = 12), P. aeruginosa (n = 3), Morganella morganii (n = 3), K. oxytoca (n = 1) and A. haemolyticus (n = 1).…”

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended‐spectrum‐β‐lactamases, AmpC β‐lactamases and co‐β‐lactamases in Enterobacteriaceae

“…In the present study, the performance of the RCA assay in rapid screening and discrimination of ESBL, AmpC, and co‐producers of ESBL and AmpC was evaluated in 86 β‐lactamase‐producing Enterobacteriaceae isolates. The organisms used in the present study are summarized in Table . The molecular types included 15 Ambler class A ESBL producers (three CTX‐M‐3, one CTX‐M‐15, one SHV‐27, one SHV‐18, one SHV, one TEM‐214, one TEM‐10, one TEM‐70, one CTX‐M‐15+SHV‐27, one SHV‐27+TEM‐53, one SHV‐27+TEM‐71, one SHV‐110+TEM‐84 and one CTX‐M+SHV+TEM), 32 Ambler class C AmpC producers (six DHA family, seven CIT family, two MOX family, 11 EBC family and six FOX family) and nine co‐producers of ESBL and AmpC (one TEM+ACT‐type, four CTX‐M+ACT‐types, one TEM+SHV+ACT‐type, one TEM+CTX‐M+ACT‐type, one SHV+ACT type and one SHV+CTX‐M‐ACT‐type).…”