Brain heart infusion agar plates

The bacterial strains used in this study are listed in Table S4 in the supplemental material. E. coli and Salmonella strains were cultured on LB medium or LB agar plates (Coolaber, China) at 37°C. C. jejuni strains were generally grown in brain heart infusion broth or on brain heart infusion agar plates (Oxoid, United Kingom) at 42°C under microaerophilic conditions with ~5% O₂, 10% CO₂, and 85% N₂.

The organism used was a Staphylococcus aureus strain (NCTC 12981) obtained from Public Health England (Salisbury, UK). Stock cultures were maintained on beads at − 80 °C. Working cultures were maintained on Brain–Heart Infusion agar plates (BHIA; Oxoid, Basingstoke, UK) and sub-cultured weekly for a maximum of four weeks to maintain viability and colony characteristics. For broth inoculum preparation, 2–3 colonies were taken from a BHIA plate into 10 ml Brain–Heart Infusion broth (BHIB; Oxoid, Basingstoke, UK) and incubated for 16–18 h at 37 °C with gentle shaking (120 RPM). The reason for using BHIB is that it is a good, rich, general-purpose medium that permits good growth of a variety of bacteria. Standards were made by adjusting overnight cultures in phosphate buffered saline (PBS) to an OD_625nm of 0.08–0.12 (0.5 McFarland standard; approx. 1.5 × 10⁷ CFU/ml) and further diluted in a 1% Petone/PBS broth to a final density of 1.5 × 10⁵ CFU/ml. This was used in all microbiological assays unless specified. Since Teic is active on the cell wall of proliferating cells, the bacteria need to be metabolically active, hence the rationale for using a minimal medium supplemented with 1% peptone.

Beta-lactamase production was assayed using Beta-Lactamase Identification Sticks (Oxoid). Since beta-lactamase production can be induced in some clostridia strains upon exposure to beta-lactam antimicrobials (Hedberg and Nord, 1996 (link)), the beta-lactamase activity of the WT and Δmp derivative strains was evaluated with and without induction. For induction, strains were sequentially transferred in TPGY broth containing increasing concentrations of penicillin G or ampicillin (Sigma–Aldrich), starting from 0.062 μg/ml. The broths with the highest concentrations of beta-lactams that could support growth were streaked on TPGY agar plates containing the same beta-lactams concentrations. Colonies from the agar plates were directly assayed for beta-lactamase production. Single colonies of the beta-lactamase producing Staphylococcus aureus strain ATCC 29213 were used as the positive control on Brain Heart Infusion agar plates (Oxoid).