Luria bertani broth lb

Bacterial strains and plasmids used in this study are listed in Table 1. Unless indicated, E. coli strains were grown at 37°C in Luria Bertani Broth (LB, Becton Dickinson), P. aeruginosa stains at 37°C in LB without sodium chloride (LBNS) or Jensen's, a chemically defined medium (Jensen, Fecycz, & Campbell, 1980). L‐arabinose (Sigma) was used as inducer for genes transcribed from P_BAD promoter in P. aeruginosa. Antibiotics for P. aeruginosa were added at the following concentrations: gentamicin 30 μg/ml; ampicillin 100 μg/ml; carbenicillin 300 μg/ml; chloramphenicol 25 μg/ml; tetracycline 12.5 μg/ml. Gentamicin at 15 μg/ml was used for E. coli. For Pseudomonas selection media, Irgasan at 25 μg/ml was used.

UTI89, a clinical isolate of uropathogenic E. coli (UPEC) (58 (link)), was grown statically at 37°C in Luria-Bertani broth (LB; Becton Dickinson, Sparks, MD). Overnight cultures were centrifuged at 7,500 × g at 4°C, and the resulting pellet was resuspended in sterile phosphate-buffered saline (PBS) to a final density of ~4 × 10⁸ colony-forming units (CFU)/mL. UTI was initiated in the morning by transurethral inoculation of 50 µL of prepared bacterial suspension, delivering an inoculum of 1–2 × 10⁷ CFU. For ex vivo experiments, the chromosomally GFP-expressing strain UTI89 att_HK022::COM-GFP was used (59 (link)), and heat killing was performed at 60°C for 30 min.

The nalidixic acid-resistant E. coli O157:H7 (EHEC) strain NCTC 12 900, a well-characterized Shiga-toxin negative strain of human origin [34 (link)], was used to test the uptake and intracellular localization of bLF by rectal epithelial cells in the presence and absence of EHEC. Bacteria were grown overnight at 37 °C in 10 mL Luria–Bertani broth (LB) (Becton–Dickinson, Claix, France) while shaking (200 rpm), harvested by centrifugation (4000 × g, 10 min, 4 °C) and re-suspended in DMEM to a concentration of 10⁷ CFU/mL.

Fifteen EHEC and five Salmonella strains were used in this study including five strains of each EHEC serogroup: O45 (TW09183, TW10121, TW14003, TW07947, TW00965), O121 (TW08980, TW07927, TW08039, I2016000899, I2016012950), and O145 (GS G5578620, 4865/96, TW08087, TW09153, TW05149). EHEC strains I2016000899, and I2016012950 were obtained from the Minnesota Department of Health¹. All other strains were obtained from the Michigan State University STEC Center². Four Salmonella strains (S. Typhimurium 2009K-0300, S. Agona SLR141, S. enteritidis 2415, S. Anatum 6802) were provided by our culture collection at the Center for Food Safety. The last strain, Salmonella Typhimurium (ATCC 14028) was purchased from the American Type Culture Collection³.
Strains stock cultures were stored at −70°C in tryptic soy broth (TSB; Difco Laboratories, Sparks, MD, United States) supplemented with 20% (vol/vol) glycerol. All bacterial cultures were subjected to two consecutive transfers (24 h at 37°C) before use. Working stocks of each strain were prepared using Luria-Bertani broth (LB; Difco Laboratories, Sparks, MD, United States), stored at 4°C and refreshed on a monthly basis. Inoculation cultures were prepared using these stocks.