Sheep blood

L. monocytogenes (EGD, BUG 600) was cultivated in Luria-Bertani (LB) broth. B. cereus (ATCC 14579) was grown in nutrient broth. For E. faecium (ATCC 6057) columbia agar containing 5% sheep blood (BD BBL, Heidelberg, Germany) or brain-heart-infusion (BHI) medium (Carl Roth, Karlsruhe, Germany) were used. All bacteria strains (Table 1) were cultivated at 37°C. For protein extraction of bacteria grown in the logarithmic and stationary growth phase, bacteria were cultured in broth overnight (stationary phase). The next day, bacterial culture was 1:20 diluted in fresh medium and grown at 37°C to an OD₆₀₀ of approximately 0.8 (logarithmic phase). Bacterial cultures were centrifuged at 4500 × g for 20 min at 4°C. Pelleted bacteria were then harvested in PBS (Sigma, Vienna, Austria) or lysis buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 % Triton X-100, 0.5 % DOC, 0.1 % SDS, 0.5 % NP-40). Where indicated 5 μg/ml lysozyme (Lactan, Graz, Austria) was added for 1 h at 37°C. Bacteria suspended in PBS or lysis buffer were sonicated on ice 3 times for 45 sec with 50 % power. All bacterial lysates were centrifuged at 16000 × g for 20 min at 4°C. The protein content of the lysates was measured using Bradford protein assay (Carl Roth, Karlsruhe, Germany). All experiments were repeated at least four times.

H. pylori clinical isolates and quality control strain ATCC43504 were tested for antibiotic susceptibility to amoxicillin, clarithromycin, levofloxacin, tetracycline, metronidazole, rifampicin, and BuT-DADMe-ImmA (BTDIA). In brief, 3- to 4-day-old cultures of H. pylori were prepared in prior to inoculation onto Mueller–Hinton agar with 5% sheep blood (BD BBL).^{22 (link)} Etests (bioMérieux, France) and agar dilution methods were used to determine MIC values after 72–96 h of incubation under microaerophilic conditions (5% O₂, 10% CO₂, and 85% N₂) at 37 °C. European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint tables (version 11.0) and CLSI M45 third edition breakpoints were used for MIC value interpretation in this study.

Spn 23F strain, P2499, was used in experimental work unless otherwise indicated. P2499 is a streptomycin-resistant derivative of P1121, an ST33 strain obtained from a study of experimental human Spn colonization, and previously tested to colonize mice efficiently^{14 (link),24 (link),43 (link)–45 (link)}. A mutant lacking the fratricidal genes (ΔcibABΔcbpD, P2576; kanamycin-resistant) was also used in this work^{24 (link)}. A streptomycin-resistant derivative of TIGR4, P2406, was used for competition experiments^{14 (link)}. All bacterial strains used in this experimental work are listed in Table S1. Colonies were grown from frozen stocks by streaking on TSA-II agar plates supplemented with 5% sheep blood (BD BBL, NJ, USA). Unless otherwise stated, starter cultures were prepared by inoculating streaked colonies in tryptic soy (TS) broth statically at 37°C until they reached an optical density at 620 nm (OD₆₂₀) of 1.0. The cells were then pelleted, washed and resuspended in sterile phosphate-buffered saline (PBS) for mouse inoculations. Bacterial numbers were enumerated by plating serial dilutions on TSA plates supplemented with 100 μl of catalase (38,000 U/ml; Worthington Biochemical Corporation, NJ) and the desired antibiotic (250 μg/ml kanamycin, 200 μg/ml streptomycin, or 200 μg/ml spectinomycin) and incubated overnight at 37°C with 5% CO₂.