Mrs broth

The multi-strain probiotic preparation consisted of Lactiplantibacillus plantarum and L. pentosus from the Department of Agricultural, Forest and Food Sciences collection previously characterized for its in vitro probiotic ability [62 (link)]. Strains were cultured in MRS broth (VWR, Milan, Italy) overnight at 37 °C. One milliliter of fresh pure culture was subsequently cultured into 100 mL MRS broth and incubated for 24 h at 37 °C. Ten milliliter of fresh pure culture was subsequently cultured into 1000 mL MRS broth and incubated for 24 h at 37 °C. Cells were then harvested after centrifugation (14,000 × g, 30 min), washed twice with sterile Ringer solution and resuspended in Ringer. Viable cells per gram culture were determined by plating onto MRS Agar (VWR). Plates were incubated at 37 °C for 24 h under aerobic conditions. The two strains were combined to yield a total cell count of around 10⁹ CFU/g.

Media used were the following: modified API50CH medium: Lactose 2%; Tryptone 1%; Yeast Extract 0,5%; K₂HPO₄ 250 ppm; MnSO₄ 50 ppm, Bromocresol purple 170 ppm; YEL: Sodium lactate 1,25%; Tryptone 1%; Yeast Extract 1%; K₂HPO₄ 328 ppm; MnSO₄ 56 ppm (agar 2% when needed); YEL_w/oYE: as YEL without Yeast Extract; YELactose: Lactose 2%; Tryptone 1%; Yeast Extract 1%; K₂HPO₄ 328 ppm; MnSO₄ 56 ppm (agar 2% when needed); MRS broth (deMan Rogosa and Sharpe medium, VWR, Italy), Iso-Sensitest Agar (Oxoid, Basingstoke, UK). Scotta composition was as follows: Scotta 1: Lactose 4.5%; ash 0.52%; fat 12.5%; protein 0.89%; dry weight 6.38%; pH 6.06. Scotta 2: Lactose 4.0%; ash 2.26%; fat 0.14%; protein 2.15%; dry weight 8.08%; pH 6.23.

The probiotic Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 were provided by Dr Gregor Reid, Lawson Health Research Institute, London, Ontario [35 (link)]. Both strains were grown anaerobically in MRS broth (VWR, Mississauga, ON, Canada) at 37 °C. Prior to the addition of bacteria to GECs, the RC-14 and GR-1 were washed to remove the MRS medium. The bacteria were then re-suspended in primary cell culture medium and added to the GEC monolayers at a concentration of 100 CFU/cell. Following 2 h of incubation, unattached bacteria was removed, and the GEC monolayers were washed with PBS and fresh medium was added.
HIV-1 virus strain ADA (M-tropic) was propagated by infection of adherent monocyte derived macrophages isolated from human PBMCs, followed by concentration of the virus using the Amicon Ultra-15 filtration system (Millipore, Billerica, MA, USA). Viral stocks were titered for infectious viral units per milliliter using the TZM-bl indicator cell assay as previously described [38 (link)]. HIV-1 was added as 10⁵ infectious units in 100 µL quantity on apical side of confluent GEC cultures. GECs were incubated with virus for 24 h.

The multi-strain probiotic preparation consisted of Lactiplantibacillus plantarum and L. pentosus from the Department of Agricultural, Forest and Food Sciences collection previously characterized for its in vitro probiotic ability [62 (link)]. Strains were cultured in MRS broth (VWR, Milan, Italy) overnight at 37 °C. One milliliter of fresh pure culture was subsequently cultured into 100 mL MRS broth and incubated for 24 h at 37 °C. Ten milliliter of fresh pure culture was subsequently cultured into 1000 mL MRS broth and incubated for 24 h at 37 °C. Cells were then harvested after centrifugation (14,000 × g, 30 min), washed twice with sterile Ringer solution and resuspended in Ringer. Viable cells per gram culture were determined by plating onto MRS Agar (VWR). Plates were incubated at 37 °C for 24 h under aerobic conditions. The two strains were combined to yield a total cell count of around 10⁹ CFU/g.

Lactobacillus reuteri CCM 3625 was purchased from the Czech Collection of Microorganisms (Brno, Czech Republic). The bacterial strain was cultivated in MRS broth (VWR, USA) at 37°C in anaerobic conditions for 18 h.

Lactic acid bacteria strains (Lactiplantibacillus plantarum subsp. plantarum, LMG6907, Lacticaseibacillus rhamnosus LMG25859, Lacticaseibacillus casei LMG6904, Levilactobacillus brevis LMG11437, and Pediococcus pentosaceus LMG10740) were purchased from the Belgian Coordinated Collections of Microorganisms-Laboratory of Microbiology (BCCM-LMG, Ghent, Belgium). The dried cultures were grown in sterile de Man, Rogosa, and Sharper (MRS) broth (Basingstoke, Hampshire, England) and stored in cryovials with 20% v/v glycerol (VWR International, Leuven, Belgium) at −20 °C.
Before use, each strain was activated twice in MRS broth at 30 °C (Lactiplantibacillus plantarum subsp. plantarum, Levilactobacillus brevis, and Pediococcus pentosaceus) and 37 °C (Lacticaseibacillus casei and Lacticaseibacillus rhamnosus) for 24 h (stationary growth phase). The cultures were centrifuged (Hermle Z300K, Hermle Labortechnik GmbH, Wehingen, Germany) at 2540× g (4000 rpm) for 15 min at 4 °C, the biomass washed twice in saline diluent (0.85% w/v sodium chloride, VWR International, Belgium) and then re-suspended in the same diluent [17 (link)].