Levofloxacin

The susceptibilities of Gram-negative bacteria (GNB) to the antimicrobial agents ampicillin (10 µg) amoxicillin/clavulanate (30 µg), cefuroxime (30 µg), cefotaxime (30 µg), chloramphenicol (30 µg), cotrimoxazole (25 µg), gentamicin (10 µg), amikacin (30 µg), ciprofloxacin (5 µg), tetracycline (30 µg) and levofloxacin (5 µg) (HiMedia Laboratories, India) were determined with Kirby-Bauer disc diffusion method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [16 ]. Erythromycin (30 µg), oxacillin (30 µg), and vancomycin (30 µg) were included for Gram positive bacteria (GPB). The reference strain E. coli ATCC 25922 and Staphylococcus aureus ATCC 10221 were included as quality controls in the susceptibility assays. Relative to the panel of antibiotics tested for each isolate, and according to the international standard definitions for acquired resistance, multidrug resistant (MDR) phenotype was defined as in vitro non-susceptibility to ≥1 agent in ≥3 antimicrobial categories [17 (link)]: penicillins, cephalosporins, beta-lactamase inhibitor combinations, fluoroquinolones, aminoglycosides, chloramphenicol, folate pathway inhibitors, tetracyclines, macrolides and glycopeptides.

Antibiotic sensitivity testing of isolated E. coli and Salmonella spp. was carried out using the disk diffusion assay as previously described [63 (link)]. Antibiotic classes included fluoroquinolones (levofloxacin, LEV—5 μg; ciprofloxacin, CIP—5 μg), aminoglycosides (gentamicin, GEN—10 μg; streptomycin, S—10 μg), carbapenems (Meropenem, MEM—10 μg; imipenem, IMP—10 μg), amphenicols (chloramphenicol, C—10 μg), macrolides (erythromycin, E—15 μg), and tetracyclines (tetracycline, TE—30 μg) purchased from Hi Media (India). Sensitivity tests were performed on freshly grown isolates having a concentration equivalent to 0.5 McFarland standard using Mueller-Hinton agar media (Hi Media, India). All results were interpreted according to the guidelines provided by Clinical and Laboratory Standards Institute [64 ]. Furthermore, isolates showing resistance against three or more different classes of antibiotics were defined as MDR [65 (link)].

The susceptibility of E. marmotae M-12 to antibiotics was determined by the standard disc-diffusion method. Testing was performed following EUCAST 2022 guidance on the Mueller–Hinton agar (Himedia). The following discs with antibiotics were used (20 antibiotics in total): amoxicillin-clavulanic acid (20–10 µg), gentamicin (10 µg), trimetoprim-sulfametoxazol (1.25–23.75 µg) (NICF, Russia); aztreonam (30 µg), piperacillin (30 µg), piperacillin-tazobactam (100–10 µg), amikacin (30 µg), tobramycin (10 µg), ticarcillin-clavulanic acid (75–10 µg), ticarcillin (75 µg) (Bioanalyse, Turkey); ampicillin (10 µg), meropenem (10 µg), imipenem (10 µg), cefotaxime (5 µg), cefepime (30 µg), ceftazidime (10 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), chloramphenicol (30 µg), trimethoprim (5 µg) (HiMedia, India).

Kirby-Bauer disk diffusion method was also performed using 10-cm diameter MHA plates (HiMedia Laboratories, India) and ampicillin (10 µg), amoxicillin-clavulanate (20/10 µg), piperacillin-tazobactam (100/10 µg), cefazolin (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), cefepime (30 µg), imipenem (10 µg), meropenem (10 µg), gentamicin (10 µg), amikacin (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), and trimethoprim-sulfamethoxazole (1.25/23.75 µg) disks (HiMedia Laboratories, India) with the same bacterial inoculum from protocol B and incubated at 35°C ± 2°C in ambient air for 16–18 hours. AST results were interpreted as per the performance standards for AST by CLSI.
The workflow of standard inoculation and direct inoculation protocols of AST is depicted in Fig. 1.

For establishing the MIC, the following antibiotics were used; amikacin, meropenem, levofloxacin, chloramphenicol, gentamycin, tobramycin, ceftriaxone and piperacillin (HiMedia, India). Quercetin (Cat No: Q4951) purchased from Sigma Aldrich (USA) was used.
The MIC of antibiotics and Quercetin against the strains were tested by broth micro-dilution method using 96-well plates, using Muller Hinton broth (MHB) (HiMedia, India) according to Clinical & Laboratory Standards Institute (CLSI) guidelines for MIC breakpoints [18 (link)]. Briefly, the antibiotics stocks (10 mg/mL) were prepared using sterile distilled water and Quercetin stock was prepared in dimethyl sulfoxide (DMSO) (10 mg/mL). Fresh MHB containing antibiotics (amikacin, tobramycin, levofloxacin, gentamycin and ceftriaxone) in a broad range between 0.5 to 128 μg/mL was inoculated with overnight cultures of the bacteria at a cell density of 1×10⁵ CFU/mL and incubated at 37°C for 24 h. The antibiotics showing difference of more than 4 μg/mL between visible growth and no visible growth, were further tested in a narrow range between the two concentrations with an increment of 1 μg/mL to establish the exact MIC value. After incubation, the plates were observed for visible growth and MIC was interpreted as the lowest concentration of the antibiotic at which no visible growth was observed.

The AMR profile of Campylobacter isolates was determined using standard Kirby-Bauer disc diffusion method as described by Taremi et al. [16 (link)]. A total of 38 revived isolates were tested against a panel of eight antibiotics that included ampicillin (AMP, 10 µg), gentamicin (GEN, 10 µg), ERY (15 µg), levofloxacin (LE, 5 µg), CIP (5 µg), nalidixic acid (NA, 30 µg), ceftriaxone (CTR, 30 µg), and co-trimoxazole (COT, 25 µg) (HiMedia). The isolates were revived on mCCDA plates supplemented with FD009 supplement. The growth suspension prepared in Tryptic soy broth and compared with 0.5 McFarland standard was spread on Mueller-Hinton agar plates supplemented with 7% sheep blood and incubated at 42°C in a CO₂ incubator at 5% CO₂ tension for 24 h. Zone diameter was measured and breakpoints were interpreted based on the recommendations of the Clinical and Laboratory Standards Institute standards for disc diffusion assay (CLSI 2016).

Antibiotic susceptibility testing (AST) was performed following the modified Kirby Bauer disc diffusion method using CLSI guidelines (2016) as a reference [17 ]. A total of 13 different commonly prescribed antibiotics (tetracycline (30 µg), imipenem (10 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), azithromycin (15 µg), methicillin (5 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), amikacin (30 µg), aztreonam (30 µg), and levofloxacin (5 µg)) procured from Hi-Media, India, were used for susceptibility testing.