Sensititre antimicrobial susceptibility testing system

Antimicrobial susceptibility was determined through the agar dilution method as per Clinical Laboratory Standards Institute (CLSI) recommendations (CLSI, 2019 ). Susceptibility to CAZ/AVI, ceftolozane/tazobactam and aztreonam were determined using the broth microdilution method using SensiTitre Antimicrobial Susceptibility Testing System according to manufacturer`s instructions (Thermo Fisher Scientific, United States). Susceptibility categorization was performed using the breakpoints proposed by the CLSI guidelines (CLSI, 2019 ).

Identification and antimicrobial susceptibility testing of K. pneumoniae isolates were routinely performed using two commercial microdilution methods: MicroScan Walk-Away Gram-negative NC53 pane (Beckman Coulter, USA) and/or Sensititre Antimicrobial Susceptibility Testing System (Thermo Fisher, USA). For gentamicin and fosfomycin, a broth microdilution method was performed using E. coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) as control strains. Susceptibility and resistance categories were assigned according to EUCAST breakpoints (35 ). In some cases, the identification was confirmed by MALDI-TOF (MALDI Biotyper, Bruker Daltonics GmbH, USA). KPC production was confirmed by immunochromatography with NG-test CARBA 5 (NG Biotech, France) and presence of the bla_KPC gene was confirmed by qPCR analysis.

The antibiotic susceptibility profiles of the S. aureus isolates were determined using the Sensititre^® Antimicrobial Susceptibility Testing System (Thermo Scientific, UK), following the manufacturer’s instructions. The minimum inhibitory concentration (MIC) to 15 antibiotics (cefazolin, ceftriaxone, cefuroxime, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, fusidic acid, gentamicin, mupirocin, penicillin, rifampicin, tetracycline, TRISUL, and vancomycin) were detected using the broth microdilution method (Novy et al., 2014 (link)). S. aureus ATCC 29213 and ATCC 35601 were used for quality control. The antimicrobial sensitivity breakpoints were interpreted according to the current Clinical and Laboratory Standards Institute (CLSI) breakpoints for S. aureus (CLSI, 2019 ), while sensitivity to cefazolin, ceftriaxone, and cefuroxime was interpreted according to a previous version of CLSI (Clinical and Laboratory Standards Institute/NCCLS, 2005 ). An E-test ((bioMérieux, Marcy-L’Étoile, France) was further performed on the isolates classified as mupirocin-resistant through broth microdilution.