Comprehensive Microbial Detection and Characterization Protocol

At least two samples of blood cultures were taken and inoculated in aerobic and anaerobic bottles (BD BACTEC™ Plus Aerobic/F and Plus Anaerobic/F) and monitored in the automatic system BD BACTEC (Becton Dickinson, Sparks, Maryland, USA) for a minimum incubation period of five days. Bacteremia was defined as the isolation of pathogenic bacteria in at least one bottle of blood culture. MDR-GNB was defined as a GNB resistant to three or more of the following categories of antibiotics: carbapenems, piperacillin/tazobactam, third and fourth-generation cephalosporins, aztreonam, fluoroquinolones, or aminoglycosides [33 (link),34 (link)]. Microbiological identifications were made with MALDI-TOF (BD Bruker Microflex MALDI Biotyper, Bruker Daltonics, Bremen, Germany). Antibiotic susceptibility testing was performed by disk diffusion, epsilometric tests, and/or the BD Phoenix automated system (Becton Dickinson). Breakpoints and interpretation were according to the CLSI recommendations. In carbapenem-resistant bacteria, carbapenemase production was investigated by the Blue-Carba assay and/or the double disk synergy tests (with carbapenems disks placed close to a boronic acid disk for KPC and an EDTA disk for identification of metallo-β-lactamases). The presence of genes coding for major carbapenemases (i.e., bla_VIM, bla_NDM, bla_IMP, bla_KPC, and bla_OXA-48-group) was investigated by a multiplex polymerase chain reaction (PCR) using specific primers [35 ]. In order to detect colonization with carbapenemase-producing Enterobacterales, ESBL-producing Enterobacterales, and multidrug-resistant Pseudomonas aeruginosa, rectal swabs were routinely collected (once a week and in every pre-transplant evaluation) and seeded in appropriate chromogenic media (CHROMAgar, Paris, France). Additionally, a multiplex PCR was performed directly from rectal swabs in order to detect bla_KPC and bla_OXA-48-group.
Clostridioides difficile was investigated in every patient with diarrhea by immunochromatography for the presence of glutamate dehydrogenase (GDH) antigen and toxins A and B using a commercial kit (C. Diff Quick Check Complete TECHLAB Inc, Blacksburg, Virginia, USA). Those samples with positive GDH and negative toxins were analyzed by real-time PCR (RT-PCR), using the LightMix^® Kit Clostridium difficile EC in the Light Cycler 2.0 equipment (LC, Roche Diagnostics, Mannheim, Germany) [36 (link),37 (link)].