Colistin sulphate

The susceptibility of bacterial isolates against different antibiotics was tested by the disk diffusion method [modified Kirby-Bauer method] on Mueller Hinton agar (Hi-Media, India) following standard procedures recommended by the Clinical and Laboratory Standards Institute (CLSI), Wayne, USA [16 ]. Antibiotics that were tested in our study include Ampicillin (AMP 25 μg), Amoxycillin clavulanate (AMC20/10 μg), Aztreonam (30 μg) Gentamycin (GEN10μg), Ciprofloxacin (CIP5μg), Levofloxacin (LEV5μg) trimethoprim sulfamethoxazole/cotrimoxazole (COT30μg), Cephalexin (CN30 μg), Cefixime (CFM5μg), Ceftriaxone (CTR30μg), Ceftazidime (CAZ30μg), Piperacillin tazobactam (PIT 100/10 μg), Imipenem (IMP 10 μg), Meropenem (MRP 10 μg) Tigecycline (TGC30μg), and Colistin sulphate (CT10μg) (HiMedia Laboratories, India). Interpretations of antibiotic susceptibility results were made according to the zone size interpretative standards of CLSI [16 ]. Escherichia coli ATCC 25922 was used as a control organism for antibiotic susceptibility testing.

The susceptibility of bacterial isolates against different antibiotics was determined by the disk diffusion method [modified Kirby-Bauer method] on Mueller Hinton agar (Hi-Media, India) following standard procedures recommended by the Clinical and Laboratory Standards Institute (CLSI), Wayne, USA [18 ]. For this purpose following antibiotics with specified concentrations were used; ampicillin (10 μg), ampicillin-sulbactam (10/10 μg), ceftazidime (30 μg), ceftriaxone (30 μg), cefepime (30 μg), cefoxitin (30 μg), piperacillin-tazobactam (100/10 μg, aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg), gentamycin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), levofloxacin (5 μg), trimethoprim-sulphamethoxazole/co-trimoxazole (25 μg), polymixin B (300unit), colistin sulphate (10 μg)] and tigecycline (30 μg) from HiMedia Laboratories, India. Interpretations of antibiotic susceptibility results were made according to the guidelines of interpretative zone diameters of CLSI [18 ]. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as the control organisms for antibiotic sensitivity.

Colistin susceptibility was tested by the broth microdilution method using colistin sulphate (Himedia labs) and cation-adjusted Mueller Hinton II broth (CAMHB, Himedia labs) without supplementation of polysorbate-80 in polystyrene microtiter plates as per the CLSI- European Committee on Antimicrobial Susceptibility Testing (EUCAST) joint Polymyxin Breakpoints Working Group guidelines [24 ]. Three twofold dilutions ranging from 2 ug/mL to 8 ug/mL were used. E. coli, ATCC25922 and Pseudomonas aeruginosa, ATCC27853 were used as controls. Those isolates that exhibited growth at ≥4 µg/mL of colistin after 16–18 h, as detected by OD₆₀₀, were considered to be resistant [25 ,26 (link)]. The assays were done in triplicate and were repeated three times to confirm the findings.