Muller hinton agar

Seventy-six V.cholerae isolates were identified from different geographic locations during 2007-2010 representing multiple cholera outbreaks. Of the 76 isolates, 56 were from multiple outbreaks at distinct geographic locations during 2009 and the other 20 isolates were from 2007, 2008 and 2010. The V. cholerae isolates were preserved at -80°C and revived by inoculation into alkaline peptone water at 37°C for 6 hrs and then subcultured on thiosulfate citrate bile salts sucrose agar (TCBS, EiKen Tokyo, Japan) and incubated at 37°C for 18-24 hrs. Suspected V. cholerae colonies were subcultured onto Muller-Hinton agar (Becton, Dickson (BD) and company, Sparks, MD, USA) plates and incubated at 37°C for 18-24 hrs [7 ]. All the isolates were confirmed by biochemical reactions based on sugar fermentation, oxidase test and serology using polyvalent O1, and monovalent Ogawa, and Inaba antisera. Of the 76 V. cholerae isolates, 44 representative ones were randomly selected for antimicrobial susceptibility testing and PFGE using NotI, based on their period of isolation and the area of the outbreak (3 isolates from 2007, 4 from 2008, 31 from 2009 and 6 from 2010). For ribotyping by BglI digestion, 26 strains were selected to represent cholera cases from various affected areas at different periods.

To determine the MICs of CLR, CLSI agar dilution method was used [43 ]. Briefly, two-fold dilutions of CLR were prepared in Muller-Hinton agar (Becton Dickinson, MD) with 5% sheep blood. Following the preparation of H. pylori suspension in physiological saline adjusted to a 2.0 McFarland standard, a 1 to 3 μl inoculum was spotted on agar plate. Following incubation at 37°C for 72 h under microaerophilic atomosphere, the MICs of CLR were established as the drug concentration showing no growth.

In vitro antibacterial activities were determined using the broth microdilution method [22 (link)]. Assays were performed as described in Clinical and Laboratory Standards Institute protocols in triplicate using cation-adjusted Muller–Hinton broth (Becton Dickinson and Company, Franklin Lakes, NJ, USA) in 96-well plates with a total assay volume of 100 µL. Two-fold serial dilutions of test compounds were prepared over the concentration range of 1–64 µg/mL. The bacterial concentration was adjusted to OD₆₀₀ = 0.06. MW2 was incubated with the test compounds at 37 °C for 18 h, OD₆₀₀ was measured and the lowest concentration of each compound that visually inhibited bacterial growth was reported as the minimal inhibitory concentration (MIC) [23 (link)]. A 10 µL aliquot from each well in the MIC assays was plated onto Muller–Hinton agar (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and incubated overnight at 37 °C. The lowest concentration of compound for which no growth was observed on agar plates was reported as the minimal bactericidal concentration (MBC).

The clinical isolate of strains 119 (S. aureus MSSA) and 3 (E. coli) was used for antimicrobial assay. A suspension of S. aureus or E. coli was prepared by the colony suspension method: three to five colonies from a nonselective nutrient agar medium incubated at 37 °C for 18 h were taken by a loop and moved to a sterile 0.9% sodium chloride solution. The suspension was adjusted to produce turbidity equivalent to 0.5 McFarland standard, corresponding to approximately 1.5 × 10⁸ CFU × mL^–1 of bacteria. Then, the obtained suspension was diluted to produce bacterial density equal to 1.5 × 10⁶ CFU × mL^–1. The tested sample in calculated concentration was suspended in 1.0 mL of Mueller–Hinton Broth (Becton Dickinson, Franklin Lakes, NJ, USA). The resulting concentration of the sample in the wells varied from 63 to 1000 μg/mL. After 100 μL of the suspension was added to the microplate wells and then the wells were inoculated with 100 μL of the bacterial suspension. Microplates were incubated at 37 °C for 18 h. After incubation, the material from each well was seeded on Muller Hinton Agar (Becton Dickinson, Franklin Lakes, NJ, USA) for the quantitative counting of surviving cells using 10-fold dilution series. Petri dishes were incubated at 37 °C for 18 h After incubation, the grown colonies were counted, and the number of CFUs was calculated.

Conjugation experiments were performed with strain SL12517 used as a donor and rifampin-resistant Escherichia coli EC600 as a recipient (38 (link), 39 (link)). Donor and recipient strains (3 mL each) were cultured overnight at 37°C and mixed together. The mixed cells were harvested by centrifugation for 3 min at 1,200 × g, washed with 3 mL of Luria-Bertani (LB) broth and resuspended in 150 μL of LB broth. The mixture was spotted on a 1-cm² hydrophilic nylon membrane filter with a 0.45-μm pore size (Millipore), which was placed on an LB agar plate and then incubated for mating at 37°C for 6 h. The cells were recovered from the filter membrane and spotted on Muller-Hinton (MH) agar (BD Biosciences) plates containing 1,500 μg/mL rifampin and 4 μg/mL colistin for selecting an mcr-10.1-carrying transconjugant.