Rifampin

Tryptic soy broth (TSB) was purchased from MilliporeSigma (Burlington, MA), Petri dishes, agar, and general supplies/reagents from Fisher Scientific (Hampton, NH), and brain heart infusion (BHI) broth from Research Products International (Mt Prospect, IL). CDC biofilm reactors and titanium (Ti) coupons were purchased from Biosurface Technologies (Bozeman, MT). Levofloxacin was acquired from Chem-Impex International (Wood Dale, IL) and rifampin from ThermoFisher Scientific (Waltham, MA).

The chemicals and reagents used in this study were obtained as follows: Sigma-Aldrich (St. Louis, MO, USA) for bull serum albumin (BSA), Evans blue, Triton X-100, Tween-20, 4′,6-diamidino-2-phenylindole (DAPI), isopropyl-β-d-thiogalactoside (IPTG), Coomassie brilliant blue G 250, rifampin, kanamycin, gentamicin, and methyllycaconitine citrate (MLA); Thermo Fisher Scientific (Waltham, MA, USA) for fluorescent α-bungarotoxin conjugates; Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for penicillin G, streptomycin, glutamine, and pyruvate; Abcam (Cambridge, UK) for antibodies against SLURP1, α7 nAChR or β-actin; Proteintech (Proteintech Group, Chicago, IL, USA) for enzyme-linked immunosorbent assay (ELISA) kits. The rest reagents were purchased from Beyotime Institute of Biotechnology, Shanghai, China.

L. delbrueckii subsp. bulgaricus LBB.B5 was obtained from the LBB Culture Collection (LB Bulgaricum Plc, Sofia, Bulgaria). A spontaneous rifampin-resistant mutant of L. delbrueckii subsp. bulgaricus LBB.B5 (LBB.B5-R) was used for this study by selecting a single-colony isolate grown on deMan, Rogosa, and Sharpe agar (MRS) (BD, Franklin Lakes, NJ) containing 50 μg/ml rifampin (Thermo Fisher Scientific, Waltham, MA). When indicated, the strain was incubated in ultrahigh temperature (UHT)-processed 2% reduced fat milk (Gossner Foods, Logan, UT). To measure cell growth in response to exogenous nucleobases, purine (adenine and guanine) or pyrimidine (uracil) bases were added to the milk to a final concentration of 20 μg/ml. Bacterial cultures were serially diluted, plated onto MRS agar containing 50 μg/ml rifampin, and incubated at 37°C for 2 days prior to colony enumeration.
Equal quantities of L. delbrueckii subsp. bulgaricus LBB.B5-R (approximately 10⁶ CFU/ml) were inoculated into either MRS (n = 3) or milk (n = 6) and incubated at 37°C without aeration for 24 h. After 16 h at 37°C, three of the cultures in milk were transferred to 4°C and incubated for the subsequent 5 days. For all cultures, pH was periodically measured with the S20 SevenEasy (Mettler-Toledo LLC, Columbus, OH), and numbers of viable cells were estimated on MRS plates containing 50 μg/ml RIF.