Microscan autoscan 4 system

Secondary infections were identified by clinical assessment. Nutrient agar, blood agar, MacConkey agar and Sabouraud dextrose agar were used for bacterial and fungal culture. Microscopy and biochemical assays were carried out to identify microbial species. Antibiotic susceptibility testing from identified species were performed using the semi-automatic analyser Microscan Autoscan-4 system (Beckman Coulter, Indianapolis, IN, USA). The antibiotics tested were ampicillin, azithromycin, cefoxitin, ciprofloxacin, erythromycin, fosfomycin, fusidic acid and gentamicin. All the laboratory analyses for microbial identification were carried out at King Abdulaziz Centre for Science and Technology (KACST).

Kirby–Bauer disk diffusion was performed using the 0.5 McFarland standard of bacteria suspension seeded into Mueller–Hinton agar plates. The antibiotics tested included ampicillin (10 µg), cefotaxime (30 µg), norfloxacin (10 µg), ceftazidime (30 µg), ertapenem (10 µg), cefoxitin (30 µg) and cefuroxime (30 µg). The measured zone sizes were interpreted according to the Clinical and Laboratory Standard (CLSI, M100, 26th ed, 2018) guidelines (CLSI 2018). P. aeruginosa was tested against gentamicin (10 µg), cefepime (30 µg), ceftazidime (30 µg), ciprofloxacin (5 µg), piperacillin tazobactam (110 µg), amikacin (30 µg) and meropenem (10 µg). Pseudomonas stutzeri and Acinetobacter spp. were tested against 11 antibiotics in a microbroth dilution using the MicroScan autoSCAN-4-System with the NC 66 panel (Beckman Coulter Life Sciences, Indianapolis, IN, USA) following the manufacturer’s instructions. K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as control strains in the evaluation of the performance of the tests. An MDR phenotype was defined as non-susceptibility to ≥1 agent in ≥3 antimicrobial categories [46 (link)]. Isolates with non-susceptibility to 3rd-generation cephalosporins were examined for the possible presence of AmpCs and ESBLs according to the CLSI guideline.

For all 19 ESBL-producing Enterobacteriaceae from irrigation water, resistance to 32 clinically relevant antibiotics was determined in disk diffusion assays as described previously [15 (link)]. Additionally, all ESBL-producing donor and transconjugant strains obtained from filter conjugation experiments were screened for resistance against 31 microbial agents by broth dilution using the MicroScan autoSCAN-4 System (Beckman Coulter Life Sciences, Indianapolis, IN, USA) with the Neg MIC 44 panel. Susceptibility testing was performed according to the manufacturer’s guidelines.