Characterizing Escherichia coli carrying plasmid-mediated AmpC β-lactamases to optimize detection in a diagnostic laboratory setting.

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Plasmid-mediated AmpC β-lactamases are a cause of acquired cephalosporin resistance in Gram-negative bacteria. However, consensus regarding the optimal detection method is yet to be achieved and varies depending on local epidemiology and laboratory capacity. We determined the acquired genotypic resistance mechanisms of 250 Escherichia coli isolates with a positive AmpC screen, defined as cefoxitin MIC ≥ 8 mg/L and a positive AmpC double- disc diffusion test, using in-house designed high-resolution melt PCR, detecting plasmid-acquired genes from the CIT and DHA families. A proportion of these isolates (n = 170, 68%) underwent further genotypic characterization using whole- genome sequencing (WGS). Of 250 isolates with a positive screening test, 72 (28.8%) were determined to carry an acquired AmpC gene. There was 100% concordance between PCR and WGS in the identification of acquired AmpC genes. The phenotypic criteria were then assessed to determine their utility in predicting acquired AmpC gene carriage. Criteria 1 (cefoxitin MIC > 8 mg/L plus ceftriaxone MIC > 1 mg/L and/or ceftazidime MIC > 1 mg/L) yielded a sensitivity of 93.1% and a specificity of 47.8%. Criteria 2 (cefoxitin MIC > 16 mg/L plus ceftriaxone MIC > 4 mg/L) had a sensitivity of 33.3% and a specificity of 98.9%. DHA genes, whose expression may be induced following antibiotic exposure, were present in 19% of isolates testing susceptible to ceftriaxone (MIC ≤ 1 mg/L) and were significantly more likely than CIT genes to be detected in susceptible isolates (P < 0.0001). These findings highlight the importance of using genotypic methods to detect acquired AmpC resistance in E. coli isolates that meet phenotypic screening criteria.IMPORTANCEDetection of transmissible AmpC resistance remains a challenging problem for diagnostic laboratories, especially in Escherichia coli where the expression of its intrinsic AmpC gene can result in phenotypic resistance patterns indistinguishable from plasmid-mediated resistance. In conjunction with whole- genome sequencing (WGS), we describe the development and performance of a novel melt-curve PCR to identify the two most prevalent plasmid-mediated AmpC gene families: CIT and DHA. We then describe phenotypic testing algorithms that incorporate this PCR and can differentiate these from non-acquired resistance in E. coli. It is important to distinguish these, not only to spare patients from unnecessarily being treated with infection control precautions, but also to identify plasmid-mediated genes, especially of the DHA family, that have been associated with inducible drug resistance to third- generation cephalosporins.