Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae

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Background: Multidrug resistance and, in particular, carbapenem resistance is spreading worldwide at an alarming 10 rate, comprehending a variety of bacterial species and causing both nosocomial and community acquired 11 outbursts. Early and efficient detection of infected patients or colonized carriers are mandatory steps in infection 12 control and prevention of multidrug resistance diffusion. The latest EUCAST guidelines for detection of 13 carbapenemase-producing Enterobacteriaceae have set low clinical breakpoints to ensure the maximum detection 14 sensitivity of positive samples. Current workflows involve an initial screening step for species and resistance pattern 15 detection followed by phenotypic and/or genotypic confirmation. The aim of the present study was to assess the 16 efficiency of six widely used and validated phenotypic assays for the detection of carbapenemases/AmpC in 17 Enterobacteriaceae, to estimate the best workflow in the routine characterization of Enterobacteriaceae isolates. 18 Methods: A panel of 108 non-repetitive Enterobacteriaceae isolates with reduced susceptibility to carbapenems was 19 analyzed by means of 1) Modified Hodge Test, 2) Metallo Beta Lactamase Etest, 3) Double disk test with EDTA, 4) Rosco 20 Diagnostica KPC and MBL confirm kit (RDCK™), 5) AmpC Etest and 6) Cloxacillin inhibition test. Confirmation and 21 validation of results was achieved by genotypic analysis. 22 Results: The most accurate identification of resistance determinants was obtained with the combined disc test (Rosco 23 Diagnostica KPC and MBL confirm kit) which had to be coupled with the cloxacillin inhibition test for correct detection 24 of AmpC enzymes. However, in general phenotypic tests failed to characterize isolates harboring multiple carbapenem 25 resistance determinants, which were successfully assessed only by PCR-based analysis. 26 Conclusions: To detect and control the spread of pathogens with complicated resistance patterns, both optimized 27 phenotypic analysis (i.e. Rosco Diagnostica KPC and MBL confirm kit coupled with the cloxacillin inhibition test) and 28 genotypic assays are recommended in the routine diagnostic of clinical laboratories.