Colistin

Antimicrobial susceptibility testing was done by the Kirby-Bauer standard disk diffusion method [11] according to CLSI guidelines [12] for different antimicrobial agents like: ceftazidime (30 µg), cefotaxime (30 µg), cefpodoxime (10 µg), ceftriaxone (30 µg), cefepime (30 µg), aztreonam (30 µg), ampicillin (10 µg), piperacillin (100 µg), cefoxitin (30 µg), gentamicin (120 µg), amikacin (30 µg), ciprofloxacin (5 µg), tetracycline (30 µg), minocycline (30 µg), chloramphenicol (30 µg), trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg), colistin (10 µg), ertapenem (10 µg) and meropenem (10 µg) (BD Diagnostics, Franklin Lakes, NJ, USA).
The MIC values (mg/L) of cefotaxime, ertapenem, meropenem, amikacin, gentamicin and tigecycline were determined using Etest method (AB Biodisk, Solna, Sweden) and were interpreted according to CLSI guidelines as modified in 2013. The clinical breakpoints for meropenem were as follows: susceptible (S) ≤1.0 mg/L, intermediate (I) 2.0–3.0 mg/L, and resistant (R) ≥4.0 mg/L. The same for ertapenem were as follows: S ≤0.5 mg/L, I: 1.0 mg/L, R ≥2 mg/L. MIC50 and MIC90 of meropenem were calculated as the MIC at which 50% and 90% of the isolates were inhibited.

Specimens of wound tissue were homogenized in 0.5 mL volumes of sterile phosphate buffered saline (PBS, Sigma Aldrich, St Louis, MO). After mechanical homogenization, the specimens were seeded in Columbia agar (BD, Sparks, MD), MacConkey agar (BD), Sabouraud dextrose agar (BD) and Columbia agar supplemented with nalidixic acid and colistin (BD) using a spiral plater workstation (Don Whitley Scientific, Shipley, UK). Quantitative and qualitative microbiological analyses were performed after incubation of plates at 37 °C for 24 h. Isolated microorganisms were identified by standard methods and susceptibility testing was performed in accordance with Clinical and Laboratory Standards by the disk diffusion method [24 ]. The results were expressed as CFU per gram of tissue (CFU/g) and the limit of detection was 10 colony-forming units (CFU).

All third-generation cephalosporin-resistant E. coli isolates were investigated for their antimicrobial resistance using the disc diffusion test with the following 19 discs (BD Biosciences): amikacin (30 μg), amoxicillin/clavulanate (20/10 μg), ampicillin (10 μg), cefazolin (30 μg), cefepime (30 μg), cefoxitin (30 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), colistin (10 μg), doxycycline (30 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), neomycin (30 μg), norfloxacin (10 μg), streptomycin (10 μg), tetracycline (30 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg). Results were interpreted according to the Clinical and Laboratory Standards Institute guidelines [15 , 16 ]. The minimum inhibitory concentrations (MICs) for cefazolin, cephalothin, cefoxitin, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, and cefepime were determined by standard broth microdilution methods with Mueller–Hinton broth (BD Biosciences) according to the recommendations of the CLSI [15 , 16 ]. Escherichia coli ATCC 25,922 strain was used the control organisms in the antimicrobial susceptibility tests. Multi-drug resistance (MDR) was defined as acquired non-susceptibility to at least 1 agent in 3 or more antimicrobial categories [17 (link)].

A detailed antimicrobial (IMP, meropenem, cefoxitin, ceftazidime, cefpodoxime, ceftriaxone, amikacin, gentamicin, trimethoprim–sulfamethoxazole, polymyxin B, colistin, tigecycline, and fluoroquinolones (Becton Dickinson, Sparks, MD, USA) susceptibility analysis was conducted using the disc diffusion method according to CLSI guidelines.

Sensitivity to many different antibiotics was assessed by the Kirby–Bauer disc diffusion test. Bacterial cell suspensions in saline were normalized at 0.5 McFarland Standard and swabbed onto MH agar plates, using disks containing ciprofloxacin (5 µg), novobiocin (30 µg), rifampicin (5 µg), erythromycin (15 µg), streptomycin (10 µg), tobramycin (10 µg), imipenem (10 µg), and colistin (10 µg) (Becton Dickinson). Growth inhibition halos were measured after 24 h of growth at 22, 30, or 37 °C.
colistin sensitivity was also assessed with minimum inhibitory concentration (MIC) assay using the broth microdilution method. Briefly, strains were cultured in MH at 37 °C for 8 h, and then refreshed at 5 × 10⁵ cells/ml in the same medium in the presence of increasing concentrations of colistin (up to 16 μg/mL). MIC was defined as the lowest concentration of antibiotics for which no visible growth was observed after 24 h at 37 °C. Each strain was tested in at least three independent experiments.

The Kirby–Bauer disc diffusion test was performed using Mueller–Hinton agar plates (Becton, Dickenson and Company) according to Clinical and Laboratory Standard Institute standards [23] using the following antimicrobials: ampicillin (10 µg), cefazolin (30 µg), kanamycin (30 µg), streptomycin (10 µg), tetracycline (30 µg), chloramphenicol (30 µg), fosfomycin (50 µg), colistin (10 µg), sulfamethizole (250 µg), and nalidixic acid (30 µg) (Becton, Dickinson and Company).

One swab was inoculated into Todd-Hewitt broth supplemented with gentamicin 8 μg/ml and nalidixic acid 15 μg/ml (THB, Becton-Dickinson, NJ, USA). A second swab was inoculated into LIM enrichment broth (Todd-Hewitt broth with 1% yeast extract, 15 μg/ml nalidixic acid, and 10 μg/ml colistin, Becton-Dickinson, New Jersey, USA). Finally, we submerged the third swab into a transport medium (ESwab, Copan Diagnostics, Brescia, Italy). The first two swabs were incubated 18–24 hours at 37 °C in 5% CO2, then those 2 tubes along with the swab inside the transport medium were subcultured onto Petri dishes with Columbia agar (OXOID LTD, Basingstoke, Hampshire, England) with 5% sheep erythrocytes and incubated under the same conditions. After overnight incubation, suggestive colonies with or without hemolysis were analyzed to confirm GBS identification [19 , 20 (link)]. GBS isolation procedure used is available in protocols.io at dx.doi.org/10.17504/protocols.io.b4ytqxwn.