Genotypic Identification of AmpC β-Lactamases Production in Gram-Negative Bacilli Isolates

Abstract

Background:AmpC type β-lactamases are commonly isolated from extended-spectrum Cephalosporin-resistant Gram-negative bacteria. Also, resistance appeared in bacterial species not naturally producing AmpC enzymes. Therefore, a standard test for the detection of the plasmid-mediated AmpC enzyme and new breakpoints for extended spectrum Cephalosporins are urgently necessary.Objectives:To detect plasmid and chromosomal mediated AmpC-β-lactamases in Gram negative bacteria in community and hospital acquired infections.Materials and Methods:1073 Gram negative clinical isolates were identified by the conventional methods and were screened for AmpC production using Cefoxitin discs. Confirmatory phenotypic identifications were done for the Cefoxitin-resistant isolates using Boronic Acid for combined and double disc synergy tests, Cloxacillin based double disc synergy test, and induction tests. The genotypic identification of plasmid-mediated AmpC was done using multiplex PCR. ESBL production was also screened by discs of Ceftazidime and Cefotaxime with and without Clavulanic Acid (10 μg).Results:The AmpC-producing isolates among all identified Gram negative bacilli were 5.8% (62/1073) as detected by screening disc diffusion methods, where 72% were positive for AmpC by combined disc method (Cefotetan and Boronic Acid), 56.5% were positive by each of Boronic Acid and Cloxacillin double disc synergy tests, 35.5% were positive by the induction test, and 25.8% were plasmid-mediated AmpC β-lactamase producers by the multiplex PCR. Plasmid-mediated AmpC genes retrieved, belonged to the families (MOX, FOX, EBC and CIT). ESBL producers were found in 26 (41.9%) isolates, 15 (57%) of which also produced AmpC. Isolates caused hospital acquired infections were (53/62); of which (39/62) were AmpC producers. While only (8/62) of the isolates caused community-acquired infections, were AmpC producers, and (1.6%) (1/62) were non AmpC producer.Conclusions:The AmpC β-lactamases detection tests had to be included in the routine microbiology workup of Gram negative bacteria, namely Cefoxitin as a screening test, combined Boronic Acid disc test with Cefotetan, followed by synergy tests and finally by the induction test for phenotypic identifications. Multiplex PCR can successfully detect the plasmid AmpC genes.

Comparison between Multiplex PCR and Phenotypic Detection Methods for Identifying AmpC B-lactamases Among Clinical Isolates of Enterobacteriaceae in Zagazig University Hospitals, Egypt

“…In the present study 14 of 49 (28.5%) cefoxitin resistant isolates were confirmed to possess PAmpC gene by multiplex PCR. Our result showed agreement with Egyptian studies previously conducted at Cairo University Hospital [12,23], where PAmpC prevalence were 26% and 28.3% respectively. This result came in agreement with another study Japoni-Nejad et al [19] that reported 19% prevalence of PAmpC genes.…”

“…In our study cefoxitin susceptibility was used as screen test for AmpC. This was agreed by Gupta et al and Wassef et al [11,12].…”

“…Taneja et al, [16] reported using both piperacillin disc and piperacillin-tazobactam disc as useful screening procedure for detection of AmpC production as AmpC betalactamase producers are more susceptible to tazobactam as compared to clavulanic acid. [12], where cefoxitin prevalence rate was 5.8%, but it was lower than [17], where prevalence rate was 18.2%. This difference in prevalence rate could be explained by difference in type and sample size of the screened population as their study was conducted on specific group of patients having urinary tract infections.…”

Detection and genotype analysis of AmpC β-lactamase in Klebsiella pneumoniae from tertiary hospitals

“…The knowledge of potential risk factors for that resistance may help to limit its impact by enabling the implementation of effective control measures and judicious antimicrobial therapy (3). AmpC β-lactamases are either plasmid-or chromosomal-mediated (4). With regard to plasmid-mediated AmpC (pAmpC), no single phenotypic method is satisfactory for its detection; the combined application of phenotypic tests is necessary for the screening and confirmation of the presence of pAmpC-mediated resistance (5).…”

Detection of Inducible and Non-inducible (constitutive) AmpC β-lactamase-producing Gram-Negative Bacteria among Family Enterobacteriaceae by Two Phenotypic Methods-Disk Antagonism Test (DAT) and AmpC disk Test at a Tertiary Care Hospital, Himachal Pradesh, India

“…(Nasir et al, 2015) In a study at Egypt by Wassef M and Behiry I 86.4% (19/22) were chromosomally-mediated and 13.6 %( 3/22) plasmid-mediated AmpC. (Wassef et al, 2014) Amongst the 320 urinary Enterobacteriaceae isolates the inducible AmpC production was maximally detected by the ceftazidime-imipenem disk antagonism test (3.1%) and the noninducible isolates by AmpC disk test (2.2%). (Shevade and Agarwal, 2013) Cefoxitin and imipenem are strong inducers of the AmpC β-lactamases, whereas cefotaxime and ceftazidime are weak inducers.…”