Clindamycin

The antimicrobial resistance of GBS was determined through the disk diffusion method (Kirby-Bauer), according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [40 ]. The clinical isolates were tested for susceptibility to seven different antibiotics, including penicillin (10 units), ampicillin (10 μg), cefditoren (5 μg), vancomycin (30 μg), levofloxacin (5 μg), clindamycin (2 μg), and erythromycin (15 μg) (Thermo Fisher Scientific Oxoid Ltd., Basingstoke, UK). Briefly, GBS colonies were suspended in 5 ml of sterile physiological saline, and the turbidity adjusted to a 0.5 McFarland standard, corresponding to a concentration of approximately 1.5 × 10⁸ CFU/ml. A sterile cotton swab was dipped in the bacterial suspension and swabbed over the surface of the Mueller-Hinton agar plates supplemented with 5% defibrinated sheep blood (MHAB) (bioMérieux SA, Marcy-l’Etoile, France). The antibiotic disks were placed on the plates and incubated for 20–24 h at 35 °C in a 5% CO₂ atmosphere. After incubation, the zone of inhibition around the disks was measured by a calibrated ruler and interpreted using a standard chart (Table S1).

The antibiotic resistance profiles of S. aureus strains (NCCP 16830, NCCP 14567, NCCP 14754, and KVCC-BA0500624) were investigated using disc diffusion assay according to the guidelines from the Clinical and Laboratory Standards Institute [53 ]. Briefly, each of the S. aureus strains was aerobically grown at 37 °C on the BHI agar plate for 18 h, and colonies were picked and suspended in sterilized saline to meet turbidity of 0.5 McFarland standard. Each bacterial suspension was spread on the Mueller–Hinton agar plate and incubated in the presence of antibiotic discs at 35 °C for 18 h. The antibiotic discs used were cefoxitin (30 μg), oxacillin (1 μg), penicillin (10 μg), erythromycin (15 μg), clindamycin (2 μg), and chloramphenicol (30 μg) purchased from Thermo Fisher Scientific (Waltham, MA, USA). After incubation, the diameters of the inhibition zones were measured to evaluate antibiotic resistance or susceptibility of S. aureus strains.

Double-layer disc diffusion was used to determine the susceptibility to metronidazole (5μg), clindamycin (2μg), penicillin (2μg) and amoxicillin (10μg; Davies Diagnostics, South Africa), as described previously [58 (link)]. These experiments were performed in duplicate. For rifampicin and rifabutin (Sigma-Aldrich, USA), minimal inhibitory concentrations (MICs) were determined using two-fold serial dilutions according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2019 guidelines, with concentrations ranging from 5–0.00488μg/mL for rifabutin and 25–0.024μg/mL for rifampicin. MICs below the lowest or above the highest concentration tested were assigned a MIC half of the lowest or twice the highest concentration tested, respectively. Experiments were performed in duplicate. For a subset of Lactobacillus strains (n = 20), broader antibiotic susceptibility profiles were determined using Sensititre GPALL1F plates (including ampicillin, cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, daptomycin, erythromycin, gentamicin, levofloxacin, linezolid, moxifloxacin, nitrofurantoin, oxacillin, penicillin, quinupristin/dalfopristin, rifampin, streptomycin, tetracycline, tigecycline, trimethoprim/sulamethoxazole and vancomycin; Thermo Fisher Scientific Inc., USA), according to the manufacturer’s instructions.

Antibiotic drug susceptibility testing was performed on Mueller–Hinton agar supplemented with 5 % sheep blood (bioMérieux, Rio de Janeiro, Brazil). The disc diffusion test was performed with clindamycin (2 µg), erythromycin (15 µg), penicillin (10U), vancomycin (30 µg) and tetracycline (30 µg) discs (Oxoid, Altrincham, UK). Isolates were investigated for inducible clindamycin resistance by double disc diffusion test to identify the cMLS (constitutive methylation), iMLS (inducible methylation) and M (efflux mechanism) resistance phenotypes. Streptococcus pneumoniae ATCC 49619 was used as a control. Tests were performed and interpreted according to Clinical and Laboratory Standards Institute (CLSI) standards (M100 S26) [15 ].

The antibiotic susceptibility of P. konkukensis was determined according to the Kirby–Bauer disc diffusion method. The antibiotics used in this study were gentamycin (2 µg), vancomycin (30 µg), ampicillin (2 µg), tetracycline (30 µg), oxacillin (1 µg), kanamycin (64 µg), erythromycin (1 µg), chloramphenicol (30 µg), and clindamycin (2 µg) (Oxoid Ltd., Basingstoke, Hampshire, UK). The freshly prepared P. konkukensis bacterial culture incubated at 37 °C for 24 h was swabbed onto R2A agar plate using sterilized cotton swabs. Each disc of antibiotics was placed on the agar plate. After incubation at 37 °C for 48 h, a clear zone around the disc was observed.

The determination of antibiotic susceptibility was achieved as previously described [31 (link),32 (link)]. Briefly, Bacteroides strains were grown on BHI + H and incubated in an Anaerobic jar (AnaeroGen, Oxoid) at 37 °C for 24 h. Bacterial suspensions were prepared in 0.9% saline to a density of McFarland 1. The disc diffusion assays were performed using Brucella blood agar plates supplemented with hemin (0.1%) and vitamin K1 (1%). The antibiotic discs (Oxoid) were amoxycillin–clavulanate (20/10 μg) (AMC), clindamycin (10 μg) (DA), imipinem (10 μg) (IMP), moxifloxacin (5 μg) (MXF), piperacillin-tazobactam (30/6 μg) (TZP) and metronidazole (5 μg) (MTZ). The plates were incubated in anaerobic conditions at 37 °C for 24 h. Six discs (three per plate) were used. The susceptibility zone diameter provisional breakpoints previously suggested [31 (link),32 (link)] were followed: amoxycillin–clavulanate (≥15 mm), clindamycin (≥25 mm), imipenem (≥29 mm), moxifloxacin (≥19 mm), piperacillin-tazobactam (≥25 mm) and metronidazole (≥24 mm). Next, the results for imipinem and Metronidazole were confirmed by Epsilon test according to manufacturer’s procedure (Biomérieux, Portugal). The breakpoints were established according to EUCAST [33 ].