2.3. Antibiotic Susceptibility Testing using Broth Microdilution

The antibiotic susceptibility testing of all Enterococcus isolates was performed with the MICRONAUT system for Gram-positive bacteria (MICRONAUT-S MRSA/GP; Merlin, Bornheim, Germany) according to the manufacturer’s recommendations. It allowed the determination of minimum inhibitory concentrations (MICs) of 22 antimicrobial agents, including ampicillin (β-lactam), cefoxitin (β-lactam; cephamycin), ceftaroline (cephalosporin 5th generation), clindamycin (lincosamide), daptomycin (cyclic lipopeptide), erythromycin (macrolide), erythromycin/clindamycin, fosfomycin (epoxide antibiotic), fusidic acid (steroide antibiotic), gentamicin (aminoglycoside), linezolid (oxazolidinone), moxifloxacin (fluorchinolone 4th generation), mupirocin, oxacillin (β-lactam), penicillin G (β-lactam), rifampicin (ansamycine), synercid (streptogramine), teicoplanin (glycopeptide), tigecycline (glycylcycline), trimethoprim/sulphamethoxazole (trimethoxybenzyl pyrimidine/sulfonamide) and vancomycin (glycopeptide).

The MICRONAUT-S FLI MHK plates allowed the determination of MICs for E. coli against 14 antimicrobial agents including AMK (amikacin), AMC (amoxicillin/clavulanic acid), CAZ (ceftazidim), CMP (chloramphenicol), CIP (ciprofloxacin), ERY (erythromycin), GEN (gentamicin), IMP (imipenem), LEV (levofloxacin), PEN (penicillin G), RAM (rifampicin), STR (streptomycin), TET (tetracycline) and T/S (trimethoprim/sulfamethoxazole) in serial dilutions of the antibiotics.

Overnight grown bacteria were suspended in NaCl solution (0.9%) to obtain a turbidity corresponding to a McFarland standard of 0.5 (Dr. Lange, CADAS photometer 30, Berlin, Germany). Three hundred microliters of the suspension were diluted with 11 mL of Mueller–Hinton broth (Oxoid Deutschland GmbH), resulting in a concentration of approximately 10⁶–10⁷ colony forming units (cfu)/mL. In total, 100 µL of the inoculum were given in each well of the plate. After sealing the plates, they were incubated for 18 to 24 h at 37 °C. Reading of plates was done optically. Interpretation was carried out as recommended by the Clinical and Laboratory Standards Institute [14].