Mueller hinton agar

Six microorganisms known for their pathogenic ability to cause skin diseases in humans were selected for the study. These American Type Culture Collection (ATCC) strains were purchased from Thermofisher Scientific, Johannesburg, South Africa. Two bacterial strains: Gram-negative Shigella flexneri (ATCC 12022) and Gram-positive Bacillus cereus (ATCC 10876), as well as four fungi: Trichophyton rubrium (ATCC 28188), Trichophyton tonsurans (ATCC 28942), Candida glabrata (ATCC 15126) and Candida krusei (ATCC 14243) were used for the in vitro antibacterial and antifungal evaluation. Sabouraud dextrose (SD) agar was used to culture Candida krusei and Candida glabrata while Yeast Malt (YM) agar (Becton Dickinson, Franklin Lakes, NJ, USA) was used to culture Trichophyton tonsurans and Trichophyton rubrium. Sabouraud dextrose (SD) broth and Yeast Malt (YM) broth were used to prepare overnight cultures for the respective fungal strains. Mueller Hinton (MH) agar and broth (Merck, Modderfontein, South Africa) were used to culture the bacteria and re-suspend the bacteria cultures overnight.

Copper sulfate was obtained from Sigma Aldrich, USA. Cation adjusted Mueller–Hinton broth was purchased from Thermo Fisher Scientific, Waltham, MA. Mueller–Hinton (MH) agar was obtained from Merck, Darmstadt, Germany, and the Malt extract (ME) agar was from WINLAB, Leicestershire, UK. The chromatographic gels, diaion HP-20, MCI-gel CHP-20P (Mitsubishi Chemical, Tokyo, Japan), Toyopearl HW-40F (TOSOH, Tokyo, Japan), Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden), and ODS-gel (YMC, Tokyo, Japan) were used. Dimethyl sulfoxide (DMSO) was procured from Sigma-Aldrich Inc., St. Louis, MO, USA.

The chemicals and media utilized in the current study were: DeMan-Rogosa agar (MRS agar) (OXOID), LB broth (Sigma Aldrich), Mueller Hinton (MH) agar (Sigma Aldrich), Trypticase Soy Broth (TSB) (Sigma Aldrich), Plate Count agar (PCA) (Sigma Aldrich), Rose Bengal chloramphenicol (RBCA), (Himedia), Violet Red Bile Agar (VRBA) (Sigma Aldrich), m-Enterococcus agar (Sigma Aldrich), Cu (NO₃)₂ (Sigma Aldrich) and C₁₂H₂₈O₄Ti (Sigma Aldrich).

The susceptibility to antibiotics of Lm strains was assessed by measuring the MICs through the E-test method (Table 3). Bacteria in the stationary phase were inoculated at a high density on Mueller Hinton (MH) agar (Sigma-Aldrich) plates with a sterile cotton swab. ETEST® strips (BioMérieux) of each antibiotic (Table 3) were applied on the inoculated plates before overnight incubation at 37 °C. The day after, the susceptibility halos were analyzed as recommended.

Lennox broth (LB) contained 10 g·L⁻¹ BBL^TM phytone peptone (BD), 5 g·L⁻¹ Bacto^TM yeast extract (BD) and 5 g·L⁻¹ NaCl in deionised water. Terrific broth (TB) was prepared by dissolving 47 g·L⁻¹ of terrific broth powder (Thermo Fisher Scientific) and 4 mL·L⁻¹ of glycerol in deionised water. Lennox agar contained 10 g·L⁻¹ BBL^TM phytone peptone, 5 g·L⁻¹ Bacto^TM yeast extract, 5 g·L⁻¹ NaCl and 15 g·L⁻¹ extra pure agar. Mueller-Hinton (M-H) agar (Sigma-Aldrich) contained 38 g·L⁻¹ of premade Mueller-Hinton agar powder in deionised water. Kanamycin (Sigma-Aldrich) was routinely used at a concentration of 50 µg·mL⁻¹ and ampicillin (Sigma-Aldrich) at 100 µg·mL⁻¹. Phosphate Buffered Saline (PBS) contained 8 g·L⁻¹ NaCl, 0.2 g·L⁻¹ KCl, 2.16 Na₂HPO₄·7H₂O and 0.2 g·L⁻¹ KH₂PO₄ in deionised water, adjusted to pH 7.2–7.4.

C. coli DH161 was isolated from a duck slaughterhouse in Shandong Province, China, in 2009. Additionally, a total of 2,002 Campylobacter isolates (1,458 of C. coli and 544 of C. jejuni) were tested for the presence of RE-cmeABC (Table 2). These Campylobacter strains were isolated from cecal contents, carcasses, and feces of swine and chickens from Shandong, Henan, Ningxia, Guangdong, and Shanghai under the surveillance program for antimicrobial resistance in Campylobacter of animal origin during 2012 to 2014 (16 (link), 18 (link)). All of the Campylobacter strains were grown on Mueller-Hinton (MH) agar (Sigma-Aldrich, St. Louis, MO) at 42°C under microaerobic conditions (5% O₂, 10% CO₂, 85% N₂).
Antimicrobial susceptibility testing was conducted by the standard agar dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (34 ). C. jejuni ATCC 33560 was used for quality control. All of the experiments described above were repeated three times.