Detection of AmpC Beta-Lactamase in Escherichia coli: Comparison of Three Phenotypic Confirmation Assays and Genetic Analysis

Abstract

Two mechanisms account for AmpC activity in Escherichia coli, namely, mutations in the ampC promoter and attenuator regions resulting in ampC overexpression and acquisition of plasmid-carried ampC genes. In this study, we analyzed 51 clinical E. coli isolates with reduced susceptibility to amoxicillin-clavulanic acid, piperacillin-tazobactam, or extended-spectrum cephalosporins for the presence of AmpC production. Three phenotypic AmpC confirmation assays (cefoxitin-cloxacillin disk diffusion test, cefoxitin-EDTA disk diffusion test, and AmpC Etest) were compared for the detection of AmpC activity. All 51 isolates were characterized genetically by mutational analysis of the chromosomal ampC promoter/attenuator region and by PCR detection of plasmid-carried ampC genes. Altogether, 21/51 (41%) E. coli isolates were considered true AmpC producers. AmpC activity due to chromosomal ampC promoter/attenuator mutations was found in 12/21 strains, and plasmid-carried ampC genes were detected in 8/21 isolates. One strain contained both ampC promoter mutations and a plasmid-carried ampC gene. All three phenotypic tests were able to detect the majority (>90%) of AmpC-positive strains correctly. Cefoxitin resistance was found to be a discriminative parameter, detecting 20/21 AmpC-producing strains. Susceptibility to extended-spectrum cephalosporins, e.g., ceftriaxone, ceftazidime, and cefotaxime, was found in 9 of the 21 AmpC-positive strains. Considering the elevated zone diameter breakpoints of the 2010 CLSI guidelines, 2/21 AmpC-positive strains were categorized as susceptible to extended-spectrum cephalosporins.

Detection and epidemiology of plasmid-mediated AmpC β-lactamase producing Escherichia coli in two Irish tertiary care hospitals

“…Nucleotide sequencing of a representative group of these isolates (n = 7) revealed the bla CMY-2 gene. This observation is consistent with many other studies worldwide in which CMY-2 is the most commonly acquired plasmid and widely geographically distributed AmpC enzyme [5,[15][16][17][18][19][20][21][22].…”

“…Other studies carried out in hospital laboratories and similar in design to this study have revealed various prevalence rates. For example, a study from Denmark reported a similar prevalence to our data, in which 17% (4/24) of E. coli carried a plasmid ampC gene [14], whilst a study in Switzerland reported a higher prevalence at 38% (8/21) [15]. The predominant ampC gene detected in this study was bla CIT , identified in 14 E. coli isolates.…”

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

“…In our study, 6/26 (23.1%) PCR-positive isolates were carrying plasmid encoded AmpC β-lactamases of the family ACC rendering these strains appearing cefoxitin susceptible; as Peter-Getzlaff et al, (2011) stated that ACC-1 is inhibited by cefoxitin; so, FOX may appear susceptible.…”

“…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. Also, Peter-Getzlaff et al, (2011) demonstrated that CC-DD test correctly detected 20/21 (95.2%) AmpC (chromosomal and plasmid-mediated) positive E. coli strains by PCR and did not give false-positive results when using the same concentration and the same cutoff. Ingram et al, (2011) demonstrated that the sensitivity and specificity of CC-DD test when using cloxacillin (100µg) were 53% and 99% respectively for detection of AmpC production.…”

“…On the other hand, Peter-Getzlaff et al, (2011) demonstrated that 21/51 (41%) E. coli isolates were considered true AmpC producers by PCR. Cefoxitin resistance was found to be a discriminative parameter, detecting 20/21 (95.2%) AmpC-producing strains.…”