Rapid Detection of Plasmid AmpC Beta-Lactamases by a Flow Cytometry Assay

Abstract

Plasmidic AmpC (pAmpC) enzymes are responsible for the hydrolysis of extended-spectrum cephalosporins but they are not routinely investigated in many clinical laboratories. Phenotypic assays, currently the reference methods, are cumbersome and culture dependent. These methods compare the activity of cephalosporins with and without class C inhibitors and the results are provided in 24–48 h. Detection by molecular methods is quicker, but several genes should be investigated. A new assay for the rapid phenotypic detection of pAmpC enzymes of the Enterobacterales group-I (not usually AmpC producers) based on flow cytometry technology was developed and validated. The technology was evaluated in two sites: FASTinov, a spin-off of Porto University (Portugal) where the technology was developed, and the Microbiology Department of Ramón y Cajal University Hospital in Madrid (Spain). A total of 100 strains were phenotypically screened by disk diffusion for the pAmpC with the new 2 h assay. Molecular detection of the pAmpC genes was also performed on discrepant results. Forty-two percent of the strains were phenotypically classified as pAmpC producers using disk diffusion. The percentage of agreement of the flow cytometric assay was 93.0%, with 95.5% sensitivity and 91.1% specificity. Our proposed rapid assay based on flow cytometry technology can, in two hours, accurately detect pAmpC enzymes.

Keywords

• pAmpC
• flow cytometry
• resistance mechanisms
• rapid antimicrobial susceptibility testing