Evaluation of Screening Methods To Detect Plasmid-Mediated AmpC in Escherichia coli , Klebsiella pneumoniae , and Proteus mirabilis

Abstract

There are currently no standardized phenotypic methods for the screening and detection of AmpC enzymes. This study aimed to evaluate different methods to detect AmpC enzymes in Escherichia coli, Klebsiella spp., and Proteus spp., comparing the results from two disk-based methods and an agar dilution method. AmpC activity was determined for 255 clinical isolates by use of a three-dimensional enzyme assay combined with a multiplex PCR assay for plasmid-borne ampC genes. These results were compared against a disk-based inhibitor assay using various combinations of cefpodoxime and cefoxitin as antibiotic substrates and boronic acid or cloxacillin as an AmpC inhibitor. The presence of enzyme induction by disk approximation was evaluated using imipenem, cefoxitin, and amoxicillin-clavulanate as inducing agents against ceftazidime. Finally, an agar dilution assay was performed, using cefoxitin with and without added cloxacillin. AmpC activity was present in 49.8% of test isolates, 93.7% of which were positive for plasmid-borne ampC genes. CIT-like enzymes were predominant in E. coli, and DHA-like enzymes were predominant in Klebsiella spp. The disk-based inhibitor tests performed better than the agar dilution assay, while detection of AmpC by disk induction had a poor sensitivity. The cefoxitin-cloxacillin disk combination provided the best overall performance, with a sensitivity and specificity of 95%. This study confirmed the accuracy of disk-based inhibitor screening for AmpC enzymes, which proved reliable at detecting CIT-and DHA-like plasmid-borne ampC genes. The methods are simple enough for introduction into clinical microbiology laboratories.

Dissemination of IncF plasmids carrying beta-lactamase genes in Gram-negative bacteria from Nigerian hospitals

“…Multiplex PCR was used for bla AmpC genes [19]. Amplimers resulting from these PCR reactions were sequenced to confirm the identity and specific variant of each gene identified, and sequences were aligned to known reference sequences using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html).…”

AmpC and Extended Spectrum Beta Lactamase Producing Isolates of E. coli, Klebsiella spp. and P. mirabilis in a Tertiary Care Center and their Sensitivity to Other Antibiotics

“…Despite this fact, coproduction of AmpC and ESBLs has been documented in many studies (Grover et al, 2013;Doi and Paterson, 2007;Bakthavatchalu et al, 2013;Mohanty et al, 2010). Prevalence of AmpC producers have been reported to be from 8% to as high as 41.3% in various studies from India and from 1.15% to as high as 46.7% in various studies from other countries (Peter-Getzlaff et al, 2011;Grover et al, 2013;Bakthavatchalu et al, 2013;Polsfuss et al, 2011;Li et al, 2003;Singhal et al, 2005;Manoharan et al, 2012;Maraskolhr et al, 2014;Rudresh and Nagarathnamma, 2011;Tan et al, 2009;Helmy and Wasfi, 2014;Ingram et al, 2011;Yilmaz et al, 2013;Coudron, 2005).…”

Evaluation of Phenotypic Methods for Detection of Plasmid-Mediated AmpC β-Lactamases (PMABLs) among Klebsiella pneumoniae

“…However, Tan et al, (2009) demonstrated that using of CC-DD test accurately detected the majority of AmpC-positive isolates with best sensitivity and specificity; both (95%) when using a ≥ 4mm increase in zone diameter as a cutoff and 200µg as cloxacillin concentration on 255 clinical isolates of Enterobacteriaceae. Similarly, found that the sensitivity for CC-DD test for detection of AmpC was 97.2% and the specificity was 100% when using the same concentration and the same cutoff compared to both Modified three dimensional test and PCR.…”

“…However, they are expensive and not available in all institutions as routine tests. So, various phenotypic methods have been developed such as AmpC disk test (Mohamudha et al, 2012), modified double disk test (Singhal et al, 2005), cefoxitin-cloxacillin double-disk synergy test (CC-DDS) , modified three dimensional test (Shahid et al, 2004), cefoxtin agar medium method (Nasim et al, 2004), agar dilution method using cefoxitin with and without added cloxacillin (Tan et al, 2009) and Etest AmpC .…”