Brucella agar

According to the technique described in the OIE Manual of Diagnostic Tests and Vaccines [28 , 29 ], the primary isolation of Brucella was performed by culturing the samples in Brucella broth supplemented with Farrell’s mix of antibiotics [30 (link)] and on Brucella agar (Oxoid, Basingstoke, Hampshire, UK) supplemented with 5% horse serum and antibiotics at the following amounts per 1 L of media: bacitracin (25 000 IU), polymyxin B (5000 IU), natamycin (50 mg), nalidixic acid (5 mg), nystatin (100 000 IU), and vancomycin (20 mg). The broth was incubated at 37°C ± 2°C in an atmosphere supplemented with 5% to 10% CO₂ (v/v) for up to 6 weeks. From the broth, two plates per sample were inoculated each week: one plate was incubated in aerobiotic conditions at 37°C ± 2°C and the other in an atmosphere supplemented with 5 to 10% CO₂ (v/v) at 37°C ± 2°C. The plates were observed after 3 days and then daily to identify Brucella-like colonies. The plates were discarded if no specific growth was evident after 7 to 10 days of incubation. Suspected colonies were subcultured onto serum dextrose agar from which subsequent growth was examined microscopically using Gram stain and biochemical (urease, oxidase, and catalase) and motility tests.

The minimal inhibitory concentrations (MIC) of metronidazole, vancomycin, clindamycin and ciprofloxacin were determined by gradient test with the M.I.C. Evaluator^™ strips (M.I.C.E.^™, Oxoid, USA). Briefly, a suspension of C. difficile was prepared in sterile 0.9% saline, from a pure culture after 24 hours’ growth in Brucella agar, using McFarland standard 1 as the reference. The test was performed on Brucella agar (Oxoid, USA) with 5% lysed blood, supplemented with hemin (Difco Laboratories, USA) and vitamin K (Sigma-Aldrich Co, USA). Plates were incubated at 37°C in an anaerobic atmosphere, and the MIC were measured after 48 hours of incubation. The MIC values were interpreted according to clinical breakpoints from the CLSI and EUCAST guidelines [29 –31 (link)].

To ensure IL-8 suppression did not result from the antagonism of C. difficile growth by soluble factors in LCM, C. difficile B2-CU-0001-54 was assayed for viability after co-incubation with LCM and HT-29 cells. Briefly, co-culture supernatants were serially diluted and cultured anaerobically on Brucella agar (Oxoid, England) at 37°C for 48 h. Counts of isolated C. difficile B2-CU-0001-54 colonies from co-culture assays with and without LCM treatment were compared.

Thirty-four Lactobacillus spp. isolated from infant feces were analyzed in this study (Additional file 1). All lactobacilli were routinely cultured in an anaerobic chamber (Concept Plus, Ruskinn Technology, UK) (10% CO₂, 10% H₂, and 80% N₂) for 24 h at 37°C in de Man, Rogosa, Sharpe (MRS) medium (Oxoid, England).
A C. difficile isolate, designated strain B2-CU-0001-54 was obtained from feces of an infected patient positive for C. difficile toxins A and B by VIDAS® Clostridium difficile A & B (Biomérieux, France) at the Department of Microbiology, Faculty of Medicine, Chulalongkorn University. This strain is positive for TcdA and TcdB as determined by PCR for toxin A and B genes [70 (link)] and the reactivity with mouse anti-TcdA and anti-TcdB monoclonal antibodies (Meridian Life Science, Inc.). C. difficile B2-CU-0001-54 was routinely cultured anaerobically on Brucella agar (Oxoid, England) at 37°C for 48 h. Cells were harvested, re-suspended in McCoy’s medium, and adjusted to a McFarland 6 standard (1.8×10⁹ cells/mL) prior to co-culture with HT-29 cells. This study was approved by the Ethics Committee of Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA no.617/2011, IRB no.246/54). Written informed parental consent for fecal samples was obtained from participants.

H. suis strain HS5bLP, isolated from the gastric mucosa of a sow [26 (link)], was grown on Brucella agar (Oxoid, Basingstoke, UK) supplemented with 20% fetal calf serum, 5 mg amphotericin B/l (Fungizone; Bristol-Myers Squibb, Epernon, France), Campylobacter selective supplement (Skirrow, Oxoid; containing 10 mg/l vancomycin, 5 mg/l trimethoprim lactate and 2500 U/l polymyxin B) and Vitox supplement (Oxoid). In addition, the pH of the agar was adjusted to 5 by adding HCl to a final concentration of approximately 0.05%. Brucella broth (Oxoid) with a pH of 5 was added on top of the agar to obtain biphasic culture conditions. After 3 days of incubation at 37°C under microaerobic conditions (85% N₂, 10% CO₂, 5% O₂), the Brucella broth, containing the bacteria, was harvested [18 (link)]. Bacteria were washed, concentrated by centrifugation (5000g, 10 min, 4°C) and suspended in phosphate buffered saline (PBS). The bacterial suspension was sonicated 8 times for 30 seconds, with a frequency of 20 kHz (Sonicator ultrasonic processor XL 2015; MISONIX, Farmingdale, USA), resulting in lysis of the bacteria [18 (link)]. After centrifugation (13,000g, 10 min, 4°C), the supernatant fluid was collected and stored at -70°C until further use.

Toxin production of the isolates was tested using an immunochromatographic method, detecting toxin A and toxin B (C. difficile QuikChek Complete, Techlab, Blacksburg, VA, USA). Samples with a GDH-positive, but toxin A/B negative results were further tested with the TC (toxigenic culture) method [44 (link)]. Strain typing was performed by PCR ribotyping method as described by Stubbs et al. [53 (link)].
Metronidazole (MET), vancomycin (VAN), clindamycin (CLI) and rifampicin (RIF) susceptibility of the isolates were tested using the E-test methodology (AB BIODISK, Solna, Sweden). A Cd suspension (of 0.5 McFarland turbidity) of each strain was swabbed on Brucella agar (Oxoid, UK) supplemented with 5% defibrinated sheep blood, 1 mg/L hemin and 5 mg/L Vitamin K₁ [50 (link)]. E-test strips of MET, VAN, CLI and RIF were applied onto the agar surface and the plates were incubated in an anaerobic atmosphere for 24–48 h. MICs were read at the point at which the zone of complete inhibition intersected the MIC scale. For the evaluation of the susceptibility of the tested isolates, EUCAST breakpoints and epidemiological cut-off values (in case of RIF) were used (http://www.eucast.org). C. difficile ATCC 9689, C. perfringens ATCC 13,124, B. fragilis ATCC 25,285 and C. acnes ATCC 11,827 were used as control strains.