Cephalothin

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)].

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.