Evaluation of the effectiveness of polymerase chain reaction (PCR) with flow cytometry in the fast detection of carbapenemas-producing enterobacteriacae

Infections due to carbapenem-resistant Enterobacterales, which have increased worldwide in recent years, are a cause for concern. Especially carbapenemase production is the most important mechanism of carbapenem resistance seen in the Enterobacterales family. Lots of phenotypic and genotypic methods are used in laboratories to detect carbapenemases. In this study, it was aimed to rapidly detect the presence of the carbapenemase gene using flow cytometry in Enterobacterales family isolates and to evaluate its efficacy and sensitivity by comparing it with polymerase chain reaction (PCR). 45 isolates belonging to Enterobacteriales family detected low susceptable or resistant at least one carbapenem group antibody and 14 carbapenem susceptible isolates belong to Enterobacteriales in automatized system from blood cultures of hospitalized patients in intensive care units of Gaziantep University Faculty of Medicine Hospital between July 2019 and July 2020. Escherichia coli ATCC 25922 used as negative control and 18 isolates (strains include KPC, NDM, IMP, VIM, OXA-48 gene regions) as positive control. Carbapenemase gene regions were investigated by PCR after their susceptiblity was determined by the disk diffusion method. Then in flow cytometry method; eight isolates of OXA-48 detected by PCR randomly selected from the patient group and 18 isolates from the control group (8 KPC, 7 NDM, one isolate each VIM, IMP and OXA-48) carbapenem susceptible 14 isolates were studied. Bacterial suspensions were treated with meropenem + specific carbapenemase inhibitors (EDTA or APBA) and temocillin, stained with thiazole orange (TO) and propidium iodide (PI) to demonstrate dead/alive separation, and the percentage of dead cells was calculated. In the ROC analysis of flow cytometry method, PI staining rate (%) for meropenem was found to have a cut-off value of 14.37%, specificity as 100% and sensitivity as 65%. It was found that the results of flow cytometry method well compatible with PCR in determining of the carbapenemase gene region. Flow cytometry will continue to be a method open to improvement in the detection of antimicrobial susceptibility and resistance due to its versatile rapid analysis of many cells and its high compatibility with PCR results