Detection of plasmid-mediated AmpC in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis.
OBJECTIVE
This study investigated the prevalence of plasmid-mediated AmpC production in selected clinical isolates of Escherichia coli, Klebsiella species and Proteus mirabilis, and compared the results of boronic acid disc screening with conventional susceptibility testing for the detection of AmpC-positive isolates.


METHODS
E coli, Klebsiella species and P mirabilis with reduced susceptibility to amoxycillin-clavulanate, cefuroxime and cephalexin, but without phenotypic evidence of extended-spectrum beta-lactamases were screened for AmpC activity using enzyme-extraction methods. The presence of plasmid-mediated ampC was determined by multiplex PCR. Antibiotic susceptibilities were determined using both disc and dilution-based methods. A disc-based screening method for detection of AmpC-producing strains was evaluated using boronic acid as an inhibitor of AmpC, and cefoxitin as the antibiotic substrate.


RESULTS
Plasmid-mediated ampC was present in 26% of study isolates, with CMY-like enzymes detected predominantly in E coli and DHA-like enzymes predominantly in Klebsiella pneumoniae. Current susceptibility methods failed to detect a significant proportion of plasmid-mediated AmpC-producing isolates, with 33% of such strains interpreted as susceptible to third-generation cephalosporins using current Clinical Laboratory Standards Institute breakpoints. The boronic acid disc method showed sensitivity and specificity of 90% and 98% respectively in detecting AmpC-positive isolates.


CONCLUSIONS
The prevalence of plasmid-mediated ampC was high in the study population, and may be missed by conventional susceptibility testing methods. Inhibitor-based screening methods would improve detection of this emerging resistance phenotype.
Division of Laboratory Medicine, Changi General Hospital, Singapore. thean_yen_tan@cgh.com.sg
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D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D001897:Boronic Acids / Q000494:pharmacology; D002440:Cefoxitin / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests / Q000379:methods; D010957:Plasmids; D011446:Prospective Studies; D011513:Proteus mirabilis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D012680:Sensitivity and Specificity; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001897:Boronic Acids; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D002440:Cefoxitin; C048795:AmpC beta-lactamases; D001618:beta-Lactamases