EVALUATION OF MULTIPLEX PCR TECHNIQUES FOR KLEBSIELLA PRODUCING AMPC-Β LACTAMASES IN CLINICALLY SIGNIFICANT KLEBSILLA ISOLATES

Iman M A El-Kholy,Mona A Rezk,M H Abul-Aziz,Atef M Diab

doi:10.17628/ecb.2018.7.254-259

Abstract

Multiplex PCR for the detection of AmpC genes has proved useful as a rapid screening tool to distinguish cefoxitin resistant non-AmpC producers from cefoxitin resistant AmpC producers. In addition to AmpC gene detection, the data generated from the multiplex PCR method can distinguish which family of AmpC gene is present in the resistant organism there by distinguishing possible inducible AmpC producers from non-inducible producers of AmpC. The present study was designed to evaluate these issues among cephalosporin-resistant isolates of Klebsiella spp. and to assess the performance characteristics of phenotypic tests, using different inhibitors, compared to the PCR, for their rapid and accurate detection. Fifty eight out of 100 isolates were AmpC producers by PCR. Fifty six out of 58 isolates that were positive by PCR test were resistant to FOX. Thirty out of 58 AmpC producers were ESBL positive by E- test and MDDST in detection of ESBL in the presence of AmpC. While 23 /58 were positive by DDST for detection of ESBL in presence of Amp. This study reveals high prevalence of pAmpC and ESBL enzymes among bacterial isolates from our hospital. ESBL production may mask the phenotypic detection of pAmpC enzymes. Modified 3 dimensional (M3D) is a simple and reliable method for detection of pAmpCs. MDDST serve as reliable confirmatory tests for detection of ESBLs in AmpC-positive isolates.

Highlights

Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (AmpCs) are important mechanisms of resistance among Enterobacteriaceae
AmpCs can interfere with ESBL detection when using the current CLSI ESBL confirmatory tests
There are no guidelines or standardized phenotypic methods recommended for detection of AmpCs, though several methods have been described, including disc synergy assays using Amp-C inhibitors, a specific E test format for AmpC testing and a three-dimensional test. These methods are expensive, tedious and have not been systematically compared.[6,7]. This prospective study was designed and conducted at in the Central Microbiology Laboratory of Ain Shams University Hospital in Egypt to (a) determine the prevalence of ESBLs and AmpC-producers among bacterial isolates with reduced susceptibility to extended spectrum cephalosporines and cephamycins of Klebsiellae, (b) to evaluate the efficacy of the modified double disc synergy test (MDDST) as confirmatory ESBL tests in AmpC–positive isolates and (c) to assess the performance characteristics of phenotypic tests compared to the PCR as a gold standard test for the rapid and accurate detection ofAmpCs in ESBL-positive and ESBL-negative isolates

Summary

EVALUATION OF MULTIPLEX PCR TECHNIQUES FOR

The present study was designed to evaluate these issues among cephalosporin-resistant isolates of Klebsiella spp. and to assess the performance characteristics of phenotypic tests, using different inhibitors, compared to the PCR, for their rapid and accurate detection. Fifty six out of 58 isolates that were positive by PCR test were resistant to FOX. Thirty out of 58 AmpC producers were ESBL positive by E- test and MDDST in detection of ESBL in the presence of AmpC. While 23 /58 were positive by DDST for detection of ESBL in presence of Amp. This study reveals high prevalence of pAmpC and ESBL enzymes among bacterial isolates from our hospital. Modified 3 dimensional(M3D) is a simple and reliable method for detection of pAmpCs. MDDST serve as reliable confirmatory tests for detection of ESBLs in AmpC-positive isolates

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