Ciprofloxacin

The samples on nutrient agar slant were cultured under aerobic conditions at 37 °C for 24–48 h. The cultures that showed growth were transferred to the tubes with broth. Furthermore, the samples were stored at −20 °C with the addition of 20% glycerol.Viability and antibiotic blind testing of bacterial cells stored frozen in glycerol were observed by seeding 50 μL on nutrient agar and MacConkey (BBL, Edwardsburgh/Cardinal, ON, Canada) medium with antibiotics—cephalothin (30 μg/mL), ciprofloxacin (5 μg/mL), gentamicin (10 μg/mL), tetracycline (30 μg/mL), and vancomycin (30 μg/mL) (Laborclin, RJ, Brazil) according to CLSI’s guidelines [39 ]. These antimicrobials were used for each sample. After the growth of colony-forming units (CFUs), five CFUs with different morphology and pigmentation were selected, and the CFU were placed in nutrient agar or MacConkey agar with antibiotics, respecting the colony’s medium, for isolation at 37 °C for 24 to 48 h (meta-phenotyping methodology described in detail in Figure 1). Once the colonies were isolated, identification could be made. Disk diffusion of antimicrobials into the solid culture medium was used for E. faecalis to test for multi-resistance using tetracycline, azithromycin, ampicillin, ciprofloxacin, rifampicin, and vancomycin [58 (link)].

From recent overnight cultures, microbial suspensions with turbidity equivalent to 0.5 tube of the McFarland scale (1.5 × 10⁸ CFU/mL) were prepared. Each bacterial strain was seeded on Mueller-Hilton agar (MHA) plates (DIFCO-Becton Dickinson Microbiology Systems, Bergen, NJ, USA). Next, wells (6 mm diameter) prepared in MHA plates were filled with 50 μL of 200,000 µg/mL extract/fractions solutions (resulting in 10,000 µg of each sample in each well). Dimethylsufoxide (DMSO; 50 μL of a 1% solution; Sigma-Aldrich^®, St. Louis, MO, USA) and ciprofloxacin (50 μg per well; Laborclin, Paraná, Brazil) were used as negative and positive controls, respectively. The plates were incubated at 35 °C. After 24 h, the formation of inhibition diameter zone (IDZ) around the wells was recorded (CLSI, 2015). The experiments were performed in triplicate.

To evaluate the antimicrobial resistance profiles of each isolate, the disk diffusion method, proposed by the Clinical and Laboratory Standards Institute (CLSI) [18 ], was used. A total of 25 antibiotics were tested: Amoxicillin-clavulanate (20-10 μg), ampicillin (10 μg), amikacin (30 μg), aztreonam (30 μg), cephalexin (30 μg), ciprofloxacin (5 μg), enrofloxacin (5 μg), chloramphenicol (30 μg), streptomycin (10 μg), gentamicin (10 μg), trimethoprim/sulfamethoxazole (25 μg), tetracycline (30 μg), polymyxin (300 μg), cephalothin (30 μg), cefepime (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg), imipenem (10 μg), azithromycin, (15 μg), nalidixic acid (30 μg), meropenem (10 μg), cefaclor (30 μg), tobramycin (10 μg), and cefoxitin (30 μg) (Laborclin, Brazil). For quality control, the American Type Culture Collection (ATCC) standard strain, E. coli no. 25922 was used.