Luria bertani broth

Cell culture reagents were procured from Gibco Laboratories (Grand Island, NY, USA). TX-100, Tween 80, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO, USA). Middlebrook growth media 7H9 broth, 7H10 and 7H11 agar, Mueller-Hinton broth, brain heart infusion broth, tryptic soy broth, and Luria-Bertani (LB) broth were procured from Becton, Dickinson (MD, USA).

A clinical isolate of B. pseudomallei from a melioidosis case in University Malaya Medical Center (UMMC) isolated as previously described was used in the study [47 (link)]. The isolate, when cultured on agar medium at 37°C, was found to differentiate into two colony morphotypes, OB (WT, INSDC: APLK00000000.1) and OS (SCV, INSDC: APLL00000000.1). OB and OS were characterized using a commercial analytical profile index API 20NE (BioMėrieux) test and an in house PCR assay specific for B. pseudomallei [48 (link)]. The two morphotypes were cultured on nutrient agar and a single colony of each morphotype was inoculated into Luria-Bertani (LB) broth (Becton Dickinson, Franklin Lakes, New Jersey, USA) at 37°C overnight in a shaker incubator at 200 revolutions per minute (rpm). Following culture, glycerol (Acros Organics, Geel, Belgium) with a final concentration of 30% (v/v) was added to the LB cultures and stored at -80°C as a stock culture for the entire duration of the study.

A rifampicin-resistant E. coli O157:H7 strain (EDL933) was used. This strain cannot produce the shiga-like toxin I or II (stx1 and stx2). In addition, there were no differences of survival between toxin-positive and toxin-negative E. coli O157:H7. Firstly, the frozen bacteria were activated in Luria-Bertani (LB) broth (Becton Dickinson, Franklin Lakes, NJ, USA) overnight at 37 °C, then 1 mL of bacterial suspension was further incubated in LB for 12 h (200 rpm) to achieve the early stationary phase of the bacteria. Cells were harvested by centrifugation, and then washed three times with phosphate buffer (10 mM, pH 7.2). The washed cells were suspended in sterile deionized water. Afterwards, the cell concentration of the washed cell suspension was determined by spread plate method on Sorbitol MacConkey Agar (SMAC).

GAS A20 (emm1 type) and its animal-passage covS mutant AP3 strains were used in the previous study and shown in Tablle 1 (Chiang-Ni et al., 2016 (link)). Briefly, strain AP3 was isolated from the spleen of A20-infected BALB/c mouse (subcutaneous infection) after 3 days of infection. Strain AP3 was genome sequenced. In addition to the frameshift 143T deletion in the covS was identified, another five SNPs and an Indel were found in the repeat sequence regions of transposases or rRNA genes (data not shown). To be noted, trans-complementation of covR/covS in AP3 restored the expression of CovR-controlled genes to the levels similar to its parental A20 strain (Chiang-Ni et al., 2016 (link), 2017 (link)). GAS strains were cultured in TSB supplemented with 5% yeast extract (Becton, Dickinson and Company, Sparks, MD, United States). Escherichia coli DH5α was purchased from Yeastern (Yeastern Biotech Co., Ltd., Taipei, Taiwan) and cultured in Luria-Bertani (LB) broth (Becton) at 37°C with vigorous aeration. When appropriate, the antibiotic chloramphenicol (25 and 3 μg/mL for E. coli and GAS, respectively) was used for selection. Human leukemic monocyte lymphoma cell line U937 was cultured in RPMI medium supplemented with 10% of fetal calf serum (FCS) (Invitrogen, Waltham, MA, United States) at 37°C with 5% CO₂.

E. coli (Migula) Castellani and Chalmers strain 25922 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Luria–Bertani (LB) broth (Becton, Dickinson and Company, Sparks, MD, USA) or LB agar (FocusBio, Miaoli, Taiwan). Bacteria were cultured at 37 °C in an aerobic condition. Human normal skin fibroblast WS1 (ATCC, Manassas, VA, USA; CRL-1502) was cultured in Minimum Essential Medium (MEM) (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (Biological Industries; BI, Kibbutz Beit-Haemek, Israel) at 37 °C in a humidified incubator with 5% CO₂.

The endophytic selenobacteria isolated from roots of wheat plants (Bacillus sp. E5 identified as Bacillus paranthracis Accession no. KF561868 and Enterobacter sp. EC5.2 identified as Enterobacter ludwigi Accession no. KF561858) were selected for Se tolerance [13 (link)]. Both isolates were routinely pre-cultured aerobically in Luria Bertani (LB) broth (Becton Dickinson and Company, Sparks, MD, USA) at 30 °C with orbital shaking at 120 rpm. When needed, LB medium was solidified by adding 15 g/L of agar (OxoidTM, Thermo Fisher Scientific, Waltham, MA, USA).

Frozen (−80 °C) aliquots of each of the Salmonella strain stocks were inoculated into 3-ml volumes of Luria-Bertani (LB) broth (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated with continuous shaking at 35 °C for about 18 h. The overnight bacterial cultures were then diluted with LB broth heated to 42 °C. The dilution volumes were determined by a preliminary dose-finding experiment (data not shown). Equal volumes of the three cultures of each serovar were mixed, and a 0.3-ml aliquot of the pooled cocktail of S. Typhimurium was administered into the crop of eight 1-day-old chicks using syringes with gavage needles. A 0.3-ml aliquot of S. Infantis cocktail was then immediately administered to the same chicks. Bacterial cell counts were carried out for each of the cocktails following administration, and showed that the 0.3-ml aliquots of S. Typhimurium and S. Infantis contained 2.7 × 10⁶ and 3.1 × 10⁶ colony-forming units, respectively.