title: 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: Dissemination of antibiotic resistance in Enterobacteriaceae mediated by AmpC β-lactamase, extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) is clinically significant. A simple and relatively quick 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, clavulanic acid and EDTA for the rapid phenotypic identification of AmpC, ESBL and MBL and the co-existence of β-lactamases has been developed. A total of 47 molecularly characterized 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- and cefotaxime-based RMP assays successfully detected all 16 AmpC, 14 ESBL and 9 MBL producers, 6 ESBL-AmpC co-producers and 2 ESBL-MBL co-producers without false-positive results. The ceftazidime-based assay was more reliable in detecting AmpC alone, while the cefotaxime-based assay performed better in identifying co-producers of ESBL and AmpC. There was no difference in the 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 co-existence of β-lactamases, within 6 h of initial isolation of the pathogen. This assay is applicable to carry out in any laboratory, is cost-effective and is easy to interpret. It could be implemented in screening patients and controlling infection and for surveillance purposes.
authors.affiliation: School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK.|School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Yothin
authors.initials: Y
authors.lastname: Teethaisong
authors.affiliation: School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Griangsak
authors.initials: G
authors.lastname: Eumkeb
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Sakesit
authors.initials: S
authors.lastname: Chumnarnsilpa
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.|Faculty of Medical Science, Nakhon Ratchasima College, Nakhon Ratchasima 30000, Thailand.
authors.forename: Nongluk
authors.initials: N
authors.lastname: Autarkool
authors.affiliation: Mast Group Ltd, Bootle, Liverpool L20 1EA, UK.
authors.forename: Jon
authors.initials: J
authors.lastname: Hobson
authors.affiliation: School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK.
authors.forename: Ismini
authors.initials: I
authors.lastname: Nakouti
authors.affiliation: School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK.
authors.forename: Glyn
authors.initials: G
authors.lastname: Hobbs
authors.affiliation: School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK.
authors.forename: Katie
authors.initials: K
authors.lastname: Evans
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002439:Cefotaxime / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology; D004396:Coloring Agents / Q000737:chemistry; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D004756:Enterobacteriaceae Infections / Q000382:microbiology*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D008826:Microbial Sensitivity Tests; D010078:Oxazines / Q000737:chemistry*; D010641:Phenotype; D014966:Xanthenes / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D002442:Ceftazidime; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D002439:Cefotaxime