Mueller hinton plates

Antibiotic discs (Oxoid, Thermo Scientific, Leicestershire, UK) stored at −20 °C were removed from the freezer and placed at room temperature 1 h before use.
With a sterile swab, Mueller–Hinton plates (Oxoid, Thermo Scientific, Leicestershire, UK) were inoculated with the previously prepared inoculum so as to fill the entire surface of the Petri dish (diameter 90 mm).
The discs were applied on the surface of the Mueller–Hinton medium within 15 min after the inoculation of the plates with the aid of sterile forceps at least 1.5 cm apart from each other. The plates were placed on a dark non-reflective surface, and inhibition halos were measured after 18 ± 2 h of incubation at 35 °C and interpreted according to the EUCAST guide to characterise the antimicrobial resistance of the strains of S. aureus [22 ]. A total of 14 antibiotics were used, namely, fusidic acid (10 µg), cefoxitin (30 µg), ceftaroline (5 µg), ciprofloxacin (5 µg), clindamycin (2 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), linezolid (10 µg), quinupristin–dalfopristin (15 µg), rifampicin (5 µg), teicoplanin (30 µg), tetracycline (30 µg) and tigecycline (15 µg). Inducible resistance to clindamycin was tested in all erythromycin-resistant strains by using the D-test [23 (link)] on Mueller–Hinton agar.

Antimicrobial susceptibility testing was performed for Enterobacteriaceae in accordance with the Clinical and Laboratory Standards Institute (CLSI) performance standards [61 ] using the disk-diffusion method on Mueller Hinton plates (Oxoid, Hampshire, UK) and the antibiotics ampicillin (AM), amoxicillin with clavulanic acid (AMC), azithromycin (AZM), cefazolin (CZ), cefepime (FEP), cefotaxime (CTX), chloramphenicol (C), ciprofloxacin (CIP), fosfomycin (FOS), gentamicin (G), kanamycin (K), nalidixic acid (NA), nitrofurantoin (F/M), streptomycin (S), sulfamethoxazole trimethoprim (SXT) and tetracycline (TE) (Becton Dickinson, Allschwil, Switzerland). Results were interpreted according to CLSI standards [61 ]. For azithromycin, an inhibition zone of ≤12 mm was interpreted as resistant.

The antimicrobial susceptibility of B. melitensis and B. abortus isolates was performed against eight clinically relevant antimicrobial agents (chloramphenicol, ciprofloxacin, erythromycin, gentamicin, imipenem, rifampicin, streptomycin and tetracycline) using gradient strip method (E-test, bioMerieux, Marcy L’Etoile, France) as described previously [48 (link)]. Briefly, a suspension of bacteria adjusted to 0.5 McFarland standard units was inoculated on Mueller-Hinton plates (Oxoid GmbH, Wesel, Germany) supplemented with 5% sheep blood and the gradient strips were applied. The plates were incubated at 37 °C with 5% CO₂ for 48 h before reading. As MIC breakpoints for clinically used antimicrobials are not yet established for brucellae, the guidelines for slow-growing bacteria (Haemophilus influenzae) were used as an alternative [57 ]. Quality control assays were performed using E. coli (161008BR3642, DSM 1103, ATCC 25922). The susceptibility profiles of Brucella isolates are presented as resistant and susceptible using minimum inhibitory concentrations (MIC), MIC₅₀ and MIC₉₀. The interpretations were performed using CLSI (The Clinical and Laboratory Standards Institute) [57 ] and EUCAST (The European Committee on Antimicrobial Susceptibility Testing) [58 ] using the criteria for slow growing bacteria. For rifampin, the strains were also classified as intermediate (Table 2).