Trimethoprim

Antibiotic susceptibility profile for A. hydrophila Ah17 was determined by the Kirby‐Bauer disk diffusion method (Bauer, Kirby, Sherris, & Turck, 1966). The following antibiotics were tested: Amikacin (AK: 30 μg), Amoxicillin (AMC: 30 μg), Ampicillin (AMP: 10 μg), Azithromycin (AZM: 15 μg), Cefixime (CFM: 5 μg), Cefoperazone (CPZ: 75 μg), Cefpodoxime (CPD: 10 μg), Ciprofloxacin (CIP: 5 μg), Chlorompenicol (C: 30 μg), Clarithromycin (CLR: 15 μg), Co‐Trimoxazole (COT: 25 μg), Doxycycline hydrochloride (DO: 30 μg), Fosfomycin (FO: 200 μg), Fusidic acid (FC: 10 μg), Gentamicin (GEN: 10 μg), Imipenem (IPM: 10 μg), Kanamycin (K: 30 μg), Linezolid (LZ: 30 μg), Methicillin (MET: 5 μg), Minocycline (MI: 30 μg), Nalidixic acid (NA: 30 μg), Nitrofurantoin (NIT: 300 μg), Norfloxacin (NX: 10 μg), Penicillin G (P: 10 μg), Pristinomycin (RP: 15 μg), Rifampicin (RIF: 5 μg), Streptomycin (S: 10 μg), Teicoplanin (TEI: 30 μg), Tetracycline (TE: 30 μg), Trimethoprim (TR: 5 μg), Tobramycin (TOB: 10 μg), and Vancomycin (VA: 30 μg) (HiMedia). 200 μl of A. hydrophila Ah17 suspension was swabbed on Mueller‐Hinton agar (MHA) medium (HiMedia) and kept for drying. Antibiotic discs were placed on the MHA medium and incubated at 37°C for 24–48 hr. The diameter of the zone of inhibition was measured, and susceptibility was categorized according to the manufacturer's protocol.

LAB strains of approximately 10⁸ CFU were subjected to eight antibiotics in MRS agar plates and incubated at 37 °C for 24 h. The antibiotics used were Ampicillin(AMP)-10 mcg/disc, Methicillin(MET)-5 mcg/disc, Trimethoprim(TR)-5 mcg/disc, Amoxyclav(AMC)-30 mcg/disc, Polymyxin B(PB)-300 units/disc, Penicillin G(P)-10 units/disc, Erythromycin(E)-10 mcg/disc, and Rifampicin(RIF)-5 mcg/disc obtained from Hi-Media, Mumbai, India. The zone diameter inhibition (ZDI) values were measured and interpreted according to the CLSI criteria following the method adopted from [37 (link),38 ].

Antibiotic susceptibility testing was implemented in accordance with the Kirby–Bauer disk diffusion method following the Clinical and Laboratory Standards Institute (CLSI) guidelines [47 (link)]. In brief, 150 µL of overnight Vibrio culture was spread aseptically on LB agar plates. E. coli DH5α was used as the negative control (usually E. coli strain K12 MG1655 is used as a negative control). The antibiotics which are most commonly used for the treatment of cholera were chosen for antibiotic susceptibility testing [15 (link),33 (link)]. Different antibiotics discs [ampicillin (AMP, 10 μg), chloramphenicol (C, 30 μg), cefotaxime (Ce, 30 μg), ciprofloxacin (Cf, 5 μg), co-trimoxazole (Co, 25 μg), polymyxin-B (PB, 300 unit), furazolidone (FR, 100 μg), gentamycin (GEN, 10 μg), neomycin (N, 30 μg), norfloxacin (Nx, 10 μg), nalidixic acid (Na, 30 μg), tetracycline (T, 30 μg), cephalexin (CN, 30 μg), streptomycin (S, 10 μg), trimethoprim (Tr, 5 μg)] (HiMedia, Mumbai, India) were placed at certain distances on the surface of the agar plate. The plates were incubated at 37 °C for 24 h and then the diameter of the zone of inhibition of each antibiotic disc was measured.

To determine the effect of myrtenol in enhancing the susceptibility of AB to antibiotics, MIC of antibiotics (amikacin, ciprofloxacin, gentamycin and trimethoprim, HiMedia, India) was determined by performing microbroth dilution assay.
Further, combinatorial efficacy of myrtenol with the antibiotics was assessed by growing AB strains in the absence and presence of antibiotics at 1×, 0.5× and 0.25× of MIC. Briefly, 200 μL of TSBSY broth was added to each wells of 96 well plate and 1% overnight culture of AB strains was added and treated without and with 200 μg/mL of myrtenol and various concentrations of antibiotics. Plates were further incubated at 37 °C for 24 h and read at 600 nm.
The disc diffusion assay was also performed as per the Clinical and Laboratory Standards Institute guidelines to determine the antibiotic sensitivity of AB in the presence of myrtenol⁵⁴ . Briefly, TSA plates were prepared without and with myrtenol (200 μg/mL). Overnight culture of AB strains were swabbed on the TSA plates and sterile discs were placed on the centre of plates and loaded with MIC of antibiotics such as amikacin, ciprofloxacin, gentamycin and trimethoprim were. Then, plates were incubated at 37 °C for 24 h and the zone of inhibition was measured.

Transport medium containing biopsy specimens was vortexed prior to plating 100 µl of the solution on brain heart infusion agar (BHIA) supplemented with 5% (vol/vol) horse serum (Gibco, New Zealand), 0.5% (vol/vol) BBL IsoVitaleX enrichment medium (Becton, Dickinson and Company, USA), trimethoprim (5 µg/ml) and vancomycin (6 µg/ml) (HiMedia Ltd., India). The plates were then incubated in a microaerobic atmosphere (5% O₂, 10% CO₂ and 85% N₂) at 37 °C for a period of 3 to 7 days. Successful H. pylori isolate was identified based on its distinctive colony morphology, appearance of Gram-negative bacillus under the microscope, positive urease, oxidase and catalase reactions, and PCR detection of H. pylori-specific glmM gene.