Brucella broth

H. pylori strains ATCC700392, KS0048, and KS0145 were used in this study. The KS strains were clinically isolated and maintained at −80°C in Brucella broth (Becton–Dickinson, Franklin Lakes, NJ, USA) containing 25% (vol/vol) glycerol. The KS0048 and KS0145 strains were used as the Mtz-resistant strains with SodB overexpression owing to Fur amino acid mutations. ATCC700392 pHel3::sodB strain was used as the SodB-overexpressing strain of H. pylori ATCC700392, and ATCC700392 pHel3 ctrl strain was used as the control strain of ATCC700392 pHel3::sodB strain [10 (link)]. It was confirmed that the SodB activity in ATCC700392 pHel3::sodB was higher as compared with that of ATCC700392 pHel3 ctrl strain [10 (link)]. ATCC700392ΔfecA1, KS0048ΔfecA1, and KS0145ΔfecA1 strains were used as fecA1-deletion mutant strains of H. pylori ATCC700392, KS0048, and KS0145, respectively [8 (link)]. It was confirmed that the SodB activities in fecA1-deletion mutant strains were significantly decreased as compared with wild-type strains [8 (link)]. These strains were maintained at −80°C in Brucella broth (Becton–Dickinson, Franklin Lakes, NJ, USA) containing 25% (vol/vol) glycerol. The bacteria were cultured on Brucella agar containing 7% sheep blood and 7% fetal bovine serum for 2 days at 37°C under microaerobic conditions maintained with AnaeroPack MicroAero (Mitsubishi Gas, Tokyo, Japan).

The C. hepaticus strains HV10^T, 19L^{11 (link)}, VICOCT18, WESTERN3, NSWJUNE19, SAJULY18 and DALE3^{51 (link)} were used in the study. These strains are representative isolates from independent SLD outbreaks from widely separated geographical locations in Australia. All C. hepaticus strains were stored at − 80 °C in 70% Brucella broth (BD BBL™) and 30% glycerol. C. hepaticus was routinely cultured on Brucella agar plates (Brucella broth (BD BBL™) + 1.5% agar (BD BBL™)) supplemented with 5% defibrinated horse blood (Equicel) (HBA) and cultured at 37 °C under microaerobic conditions (created using Campygen 3.5L gas generation packs (Oxoid)) in an anaerobic jar, for 96 h to recover C. hepaticus cells from − 80 °C stock or for 72 h if they were subcultured from HBA plates.

Media were prepared according to guidelines from CLSI. The test medium used for the anaerobic bacterial was supplemented Brucella broth (BD, Lot No. 6278735) containing 5 μg/mL hemin (Sigma (St. Louis, MO. USA), Lot No. SLBC4685V), 1 mg/mL Vitamin K1 (Sigma, Lot No. MKBN5958V), and 5% (v/v) laked horse blood (LHB, Cleveland Scientific (Bath, OH, USA), Lot No. 385663). Brucella broth (BD) supplemented with 10% fetal bovine serum (FBS, Gibco, Lot No. 1709261) was used for testing of H. pylori. Cation-adjusted Mueller Hinton Broth (CAMHB) media was used for the spectrum determination against the spectrum panel. Difco Middlebrook (Waltham, MA, USA) 7H9 Broth (Catalog No. 271310) supplemented with 0.2% (v/v) glycerol, 0.05% Tween 80, and 10% (v/v) albumin-dextrosecatalase (BBL Middlebrook (Waltham, MA, USA) ADC Enrichment, Catalog No. 212352) (7H9-ADC-T) was used for the MIC assay against the M. tuberculosis isogenic resistant mutant panel.

For the in vitro studies, we used two CagA+ H. pylori carcinogenic strains, “J166” a clinical isolate of human-derived H. pylori, and “7.13” a rodent adapted strain derived from B128 H. pylori, which have been described previously [24 (link), 44 (link)–47 (link)]. In the in vivo study, we used the wild-type rodent-adapted cagA+ H. pylori strain (PMSS1), a clinical isolate from a duodenal ulcer patient and the parental strain of the mouse-derivative Sydney strain 1 (SS1) [48 (link)]. All H. pylori plate cultures were performed on Brucella agar (BBL/Becton Dickinson, Sparks, MD) supplemented with 5% heat-inactivated newborn calf serum (Invitrogen) and ABPNV (amphotericin B, 20 mg/liter; bacitracin, 200 mg/liter; polymyxin B, 3.3 mg/liter; nalidixic acid, 10.7 mg/liter; vancomycin, 100 mg/liter) antibiotics (all from Sigma-Aldrich, Milwaukee, WI). H. pylori MPSS1 strain liquid cultures for mouse inoculation were grown in Brucella broth (BD Biosciences) with 5% NCS and antibiotic supplementation for approximately 24 h, pelleted by centrifugation, and suspended in Brucella broth.

B. melitensis attenuated strain B115 was provided by the Veterinary Laboratories Agency (VLA) of Weybridge (U.K.) and cultured to prepare the bacterial extract according to previously described protocols [21 (link), 22 (link)].
Briefly, the bacteria were cultured in 1 l of Brucella broth (Becton Dickinson, France) at 37 °C in aerobic conditions under stirring for 3 days. When the culture reached an optical density (OD) of 2.080, the broth was centrifuged at 5000 g (ALC PK131R centrifuge, Milan, Italy) for 20 min. The pellet was washed with saline solution, inactivated at 100 °C for 10 min, sonicated, centrifuged and the supernatant was dialysed against distilled water before measurement of the protein content as previously described by Corrente et al. 2015.

Susceptibility to clindamycin, metronidazole, and vancomycin was evaluated using E-test strips (bioMérieux, Saint-Laurent, QC) on BBL Brucella agar with 5% horse blood, hemin, and vitamin K₁ (Becton, Dickinson and Co.) as previously described [37 (link)]. Briefly, bacteria were suspended in Brucella broth (Becton, Dickinson and Co.) prior to agar inoculation, and MICs were read after 48 hours of growth with Bacteroides fragilis ATCC 25285 and Staphylococcus aureus ATCC 29213 as controls. MIC interpretation for clindamycin and metronidazole was based on Clinical Laboratory Standards Institute (CLSI) breakpoints [38 ], while susceptibility to vancomycin was determined with European Committee on Antimicrobial Susceptibility Testing breakpoints [39 ] in the absence of established CLSI criteria.