title: WGS of MDRO pathogens in two hospitals: Pseudomonas aeruginosa from a Room Environment Sample
design: Samples were processed and cultured for vancomycin-resistant enterococci (VRE), Clostridioides difficile, and third-generation cephalosporin- or carbapenem-resistant Enterobacteriaceae within 24-48 hours of collection. Sponges were processed as previously described,5 using 45 mL sterile phosphate buffered saline with 0.02% Tween 80 (PBST). Vortexed swabs transport media and PBST sponge eluate were plated to C. difficile CCFA-HT agar and CCMB-TAL broth for C. difficile, Chromagar VRE and 5 mL bile esculin broth + 30 ug vancomycin disk for VRE, and MacConkey agar with 0.5 g/mL cefotaxime and 5 mL tryptic soy broth with a 30 ug vancomycin disk and a 30 ug ceftriaxone disk for ESBL-producing Enterobacteriaceae or CRE. Positive broths were plated onto the corresponding MDRO agar for further isolation and identification. Morphologically distinct isolates were selected for species identification by MALDI-TOF (Bruker Biotyper CA System, Bellerica, MA). Aliquots of swab transport medium or sponge eluate was also plated on non-selective blood agar to determine the overall bacterial bioburden of the samples. Putative VRE isolates were tested for vancomycin resistance by the disk diffusion method using 30 ug vancomycin disks. Enterobacteriaceae isolates were tested for third generation cephalosporin resistance using cefotaxime, ceftazidime, and aztreonam. Enterobacteriaceae resistant to carbapenems was tested using 10 ug ertapenem disks. All susceptibility results were interpreted using the 2018 Clinical Laboratory Standards Institute (CLSI) 6. ESBL-producing Enterobacteriaceae were defined as Enterobacteriaceae exhibiting phenotypic resistance to third or fourth generation cephalosporin  antibiotics as defined by the 2018 CLSI breakpoints 6. Testing for Cdiff toxin genes was performed by GenXpert PCR (Cepheid, Sunnyvale, CA).