Yeast extract

G. vaginalis strain UM241 was isolated from a woman diagnosed with BV [13 (link)]. Fifteen more bacterial species associated with BV were included in this study, namely: A. neuii, A. vaginae, B. ravenspurgense, C. amycolatum, C. tuscaniense, E. faecalis, N. ampullae, P. acnes, S. hominis, S. saprohyticus, S. simulans, S. warnerii, S. anginosus, M. mulieris, and P.bivia. More details on the species used here are found in Supplementary Table S1. The selection of these species was based on their feasibility to growth in vitro and the existence of previous phenotypic evidence of some key characteristics, including adhesion to HeLa cells, biofilm formation, cytotoxic assays as well as the determination of antimicrobial tolerance [22 (link), 23 (link), 41 (link), 42 (link)]. Each inoculum was grown in sBHI [Brain-heart infusion (Liofilchem, Rosetodegli Abruzzi, Italy) supplemented with 2% (wt/wt) gelatin (Liofilchem), 0.5% (wt/wt) yeast extract (Liofilchem), and 0.1% (wt/wt) soluble starch (Panreac, Barcelona, Spain)] for 24 h at 37 °C with 10% CO₂ (Shel Lab, Cornelius, Oregon, USA) [23 (link)]. The exceptions were with consortia with the strict anaerobes A. vaginae, M. mulieris, and P. bivia [43 (link)], which were grown in sBHI and incubated at 37 °C, under strict anaerobic conditions (AnaeroGen Atmosphere Generation system; Oxoid, United Kingdom).

For both bioaugmentation (BAa and BAp) treatments tested in the bioremediation experiment, a combined sample of the respective triplicates was taken, ten-fold diluted in sterile saline solution (0.85%) and spread out onto three different agar media (MA, SW, and BH) to provide different nutritional conditions and to try to recover the bacterial strains used as inocula. Per litre, agar media were prepared with the following: MA with marine agar (55.2 g L⁻¹) (Pronadisa); SW with 10 g soluble starch (Biochem Chemopharma), 4 g yeast extract (Liofilchem), 2 g peptone (Sigma-Aldrich), 33.3 g artificial sea salts (Sigma-Aldrich), 20 g agar (Liofilchem); BH with 3.27 g Bushnell-Haas broth (Difco™), 1 g sodium acetate anhydrous 99% (Alfa Aesar ^®), 17.5 g agar (Liofilchem), 20 g NaCl (EMSURE ^®). After 3–4 days of incubation at 28 °C, morphologically different colonies were described and purified. Isolated strains were then preserved in 21% glycerol at −80 C and biomass of each strain was also collected for DNA extraction. The 16S rRNA gene sequences of the bacterial identified strains were deposited in GenBank (Table 2).

G. vaginalis UM137, G. piotii UM035, G. leopoldii UGent 09.48 and G. swidsinskii GS 9838-1 were grown on Columbia Agar Base medium (Liofilchem, Roseto degli Abruzz, Italy) supplemented with 5% (v/v) defibrinated horse blood (Oxoid Ltd., Hampshire, UK) for 48 h [7 (link)]. For each experiment, the bacterial species were grown in Brain Heart Infusion (BHI, Liofilchem) supplemented (sBHI) with 2% (w/v) gelatine (Liofilchem), 0.1% (w/v) starch (Panreac, Barcelona, Spain) and 0.5% (w/v) yeast extract (Liofilchem) and incubated for 24 h at 37 °C and 10% CO₂ (Panasonic MCO-18AC, Bracknell, UK).

Various strains from the genus Gardnerella were analyzed in this study. Gardnerella sp. UM241, previously recovered from patients diagnosed with BV^{93 (link)}, G. vaginalis UM137, G. piotii UM035, G. leopoldii UGent 09.48, and G. swidsinskii GS 9838-1, previously identified by MALDI-TOF^{15 (link),94 (link)}, were used herein. The strains were kept frozen in Brain Heart Infusion medium (BHI, Liofilchem, Roseto degli Abruzz, Italy) with 23% (v/v) of glycerol (Panreac, Barcelona, Spain) and plated in Columbia Blood Agar (CBA) [Columbia Base Agar medium (Liofilchem) supplemented with 5% (v/v) of defibrinated horse blood (Oxoid Ltd, Hampshire, UK)] and incubated for 48 h at 37 °C and 10% CO₂. For each experiment, planktonic cells were grown in BHI supplemented (sBHI) with 2% (w/v) gelatin (Liofilchem), 0.1% (w/v) starch (Panreac) and 0.5% (w/v) yeast extract (Liofilchem) and incubated for 24 h at 37 °C and 10% CO₂ (CO₂ Incubator MCO-18AC, Panasonic, Bracknell, UK).