Cotrimoxazole

MIC was determined to check the effect of infection-related in vitro gut conditions, such as bile, osmotic, high and low iron, pH and temperature conditions, on MIC variation in resistant and sensitive isolate by using Ezy MIC™ strips of ampicillin, co-trimoxazole, imipenem, nalidixic acid, ciprofloxacin, tetracycline, nitrofurantoin and chloramphenicol (HiMedia Laboratories Pvt. Ltd., Maharashtra, India). Both the isolates were grown up to mid-exponential phase (MEP) under in vitro gut conditions and swabbed onto Muller Hinton agar (MHA) plates. MIC strips were placed onto plates using an applicator followed by incubation at 37 °C for 16–18 h. The result was read where the ellipse intersects the MIC scale on the strip for the tested antibiotics.

Antibiotic susceptibility test (AST) of both clinical isolates was performed using the modified Kirby-Bauer disk diffusion method on Mueller Hinton agar (Hi-Media Laboratories, India) following standard zone size interpretative criteria set by the Clinical and Laboratory Standards Institute (CLSI) [25 ]. The different antibiotic disks used in this study during AST were procured from HiMedia Laboratories, India, and include amoxicillin (30 μg), gentamicin (10 μg), cotrimoxazole (25 μg), ciprofloxacin (5 μg), imipenem (10 μg), amoxicillin/clavulanic acid (20/10 μg), cefotaxime (30 μg), ceftriaxone (30 μg), ceftazidime (30 μg), aztreonam (30 μg), and cefpodoxime (10 μg). The E. coli and K. pneumoniae isolates were regarded as MDR isolates if they were resistant to at least one agent of three different classes of antimicrobial disks [2 (link)].

Antibiotic susceptibility testing was done using disk diffusion method as described by Clinical and Laboratory Standards Institute [35 ]. 95 isolates were randomly selected for convenience from all bacteria isolated from the three counties. The isolates were tested for susceptibility to commonly used antibiotics including Ampicillin, Tetracycline, Streptomycin, Co-trimoxazole, Kanamycin, Gentamicin, Sulphamethoxazole, and Chloramphenicol (HiMedia).
Isolates were grown on blood agar for 24 hours; 5 colonies were picked from each plate and suspended in 5 ml of sterile normal saline which was then adjusted to a density approximately equal to McFarland Opacity Standard number 0.5. A dry sterile cotton swab was then placed inside the suspension; excess liquid from the swab was expressed against the wall of the tube and the swab used to spread the bacterial suspension evenly on the surface of Mueller-Hinton agar in order to get confluent growth. Antibiotic disks were then placed on the surface of the inoculum and incubated for 18–24 hours. Zones of inhibition were measured to the nearest millimeter and interpretation as to whether the bacterium was resistant or susceptible to the particular antibiotic was done according to specifications defined by Clinical and Laboratory Standards Institute (CLSI 2006).

Natural graphite flakes, concentrated sulfuric acid (98%), potassium permanganate, 30% hydrogen peroxide, concentrated hydrochloric acid (98%) and ultrapure water were purchased from Sigma-Aldrich, Malaysia. All of the aqueous solutions were prepared in deionized water. Analytical grade absolute ethanol, NaCl, peptone, yeast extract, beef extract, agar were purchased from Merck, Germany. Antibacterial discs: azithromycin (AZM), gentamycin (GEN), ciprofloxacin (CIP), cefixime (CFM), amoxicillin (AMX), cotrimoxazole (COT), imipenem (IPM) and ceftriaxone (CTR) were bought from Himedia Laboratories, India. 6X loading dye, 1 Kb plus DNA ladder and Presto Blue dye were purchased from Thermo Fisher Scientific. Human serum and bacterial strains collected from urine were obtained from North East Medical College Hospital, Sylhet, Bangladesh (https://www.nemc.edu.bd/pathology-department/) following all the ethical conditions and legislations approved by the ethical committee with the Ref. No. NEMC/Sylhet/287/2013 [24 ].

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).