Tryptic soy broth (tsb)

The experiment used the flow test system described in point 4.5. A suspension with a density of 3 × 10⁸ CFU/mL was made from a 24-h culture in a Tryptic Soy Broth (Becton Dickinson, Warsaw, Poland). The suspension was flowed through the urinary catheter fragments (GalMed, Bydgoszcz, Poland) for a period of 2 h at a speed corresponding to the glomerular filtration rate—0.5 mL/min. After 2 h, a sterile solution of 50% TSB (Becton Dickinson, Warsaw, Poland) in 0.9% NaCl (Stanlab, Lublin, Poland) was flowed through the tested catheter fragment for another 24 h. After this time, solutions of the tested substances or sterile water were flowed through the catheters for 2 min. In the next step, the catheters were cut into 1 cm pieces. The 3rd, 4th and 5th fragment of the catheter were assessed using the Richards’ method. A catheter, which had not been washed with any solution after the biofilm forming step, served as a control. The efficacy of 0.05% solutions of polyhexanide (PHMB) (Carbosynth, Ltd., Compton, Newbery, UK) and octenidine (OCT) (Dishman Pharmaceuticals & Chemicals Ltd., Ahmedabad, India) and 0.1% solutions of betaine (BET) (Merck, Darmstadt, Germany), saponin (SAP) (Merck, Darmstadt, Germany) and phenoxyethanol (P-ETOH) (Merck, Darmstadt, Germany) was tested.

Bacterial strains representing species commonly found in bovine milk were used to construct a mock bacterial community (Table 1). Each bacterial strain was grown in standard laboratory culture medium with negative controls for that species and harvested at early stationary phase by centrifugation at 13,000 × g for 2 min. The laboratory culture media were as follows: Bacillus subtilis, Pseudomonas fluorescens, and Escherichia coli, LB (Lennox broth; Thermo Fisher Scientific); Enterococcus faecalis and Streptococcus agalactiae, brain heart infusion broth (Thermo Fisher Scientific); Staphylococcus aureus, tryptic soy broth (Becton Dickinson); Corynebacterium bovis, tryptic soy broth (Becton Dickinson) with 0.1% Tween 80; Lactococcus lactis, M17 broth (Becton Dickinson) with 0.5% glucose; and Clostridium tyrobutyricum, reinforced clostridial broth (Becton Dickinson). All strains were incubated at 37°C, with the exception of B. subtilis, L. lactis, and P. fluorescens, which were incubated at 30°C. B. subtilis, C. bovis, E. faecalis, E. coli, and P. fluorescens were grown under aeration (250 rpm).

All clinical isolates used in this study were obtained from the microbiology department of Shriners Hospitals for Children–Cincinnati. Bioluminescent PA-Xen41, derived from parental strain PAO1, was purchased from PerkinElmer (Waltham, MA). Luria broth (LB) medium was composed of 10 g tryptone, 5 g yeast extract, and 5 g NaCl (and an additional 15 g Bacto-agar for LB agar) per liter (all chemicals were from Fisher Scientific). Tryptic soy broth (TSB; Becton, Dickinson) and TSB plus 1.5% agar (TSA) were prepared according to the manufacturer’s instructions. All strains listed in Table S1 were grown with appropriate medium plates or broth.

The phoP-phoQ regulon of Salmonellae regulates numerous genes that results in sensitivity to endogenous cationic antimicrobial peptides, including α-defensins, and phoP null strain of S. Typhimurium is sensitive to α-defensins^{7 (link),9 (link)}. We used a defensin-sensitive ΔphoP strain of S. Typhimurium to enhance the sensitivity of our determinations. S. Typhimurium ΔphoP was pre-cultured in 3 mL of tryptic soy broth (Becton Dickinson) for 16 h followed by growing to an OD₆₀₀ of 0.6–0.8 in 30 mL of tryptic soy broth at 37 °C with shaking at 180 rpm. Exponential-phase bacteria were deposited by centrifugation at 3,000 g at 4 °C for 10 min. Bacteria were washed once and resuspended in ice-cold PBS to a concentration of 1 × 10¹⁰ CFU/mL.

Bacillus subtilis DE111^® was cultivated at 37°C for 24 h in Tryptic Soy Broth (TSB; Becton, Dickinson and Company, Berkshire, England). Indicator strains employed to determine the antimicrobial activity of B. subtilis DE111^® were either purchased from the American Type Culture Collection (ATCC, Middlesex, United Kingdom) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). These are listed in Table 1. All of these strains except Cutibacterium acnes and Gardernella vaginalis were cultivated at 37°C for 24 h in TSB (Becton, Dickinson and Company, Berkshire, England). Cutibacterium acnes was grown in Brain heart infusion medium (BHI) (Merck, Darmstadt, Germany), and G. vaginalis was cultivated in New York City (NYC) medium, both were incubated at 37°C under anaerobic conditions. In addition, other control strains employed for comparative analysis were Lactobacillus rhamnosus (ATCC 53103) cultivated at 37°C for 24 h in TSB (Becton, Dickinson and Company, Berkshire, England), and Lactobacillus fermentum ME-3 cultivated at 37°C for 24 h in De Man, Rogosa and Sharpe broth (MRS; Merck, Darmstadt, Germany).