Clostridium (Clostridioides) difficile infection (CDI) is recognized worldwide as a public health concern, related mainly with hypervirulent strains. In Brazil there are few studies about molecular epidemiology of C. difficile, for this reason, we aimed to characterize C. difficile isolates from a large cohort study of three different Brazilian states to identify virulence and resistance genes, specifically genes related to metronidazole and vancomycin resistance. All 153 fecal samples were submitted to C. difficile culture in CM0601 broth. Identification of suspected colonies was confirmed by matrix-assisted laser desorption/ionization (MALDI-TOF/MS, Brucker Daltonics, Germany). The tcdA and tcdB toxin were searched by PCR. The sequence type (ST) was determinate by multilocus sequencing typing (MLST) and susceptibility profile was performed by agar dilution method. Among the 16 isolates, we identified fourteen different STs, five belonging to Clade 1, one to Clade 2 and eight news STs with high similarity levels. Resistance (ermB, tetM, VanW and nimB) and virulence genes (cwp84, cwp66, cwp2, fbpA and secA) were found in toxigenic strains. Differently from other studies, we found high levels of resistance to vancomycin. These results suggest that the main circulating strains in Brazil belong to Clade 1 and have high pathogenicity and resistance profile.

Detecting carbapenemase-associated carbapenem resistance is a subject of major clinical and epidemiological concern as it influences therapeutic choice. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a means to assess bacterial resistance mechanisms. We aimed to detect the KPC enzyme directly from positive blood cultures using MALDI-TOF MS. To do so, 102 clinical Enterobacteria were evaluated, including 59 bla_KPC positives. Proteins were extracted using formic acid, isopropyl alcohol, and water (17:33:50) and spotted onto a steel target plate using the double-layer sinapinic acid technique. Two parameters were considered: (i) the visual detection of a clear peak with the expected KPC m/z and (ii) the evaluation of the relative intensity of the ions in the peak. A peak was observed in 56/59 bla_KPC-positive isolates (94.9% sensitivity), with no false-positive results (100% specificity). When considering intensity, with a cut-off ≥120 (a.u.), sensitivity was 94.9% and specificity was 95.3%. We proposed a “buffer” zone, with intermediate values of intensity (115 to 125) reaching 100% sensitivity and specificity. The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid, which may improve appropriate patient therapy and antimicrobial stewardship.