Proteose peptone

Bacteria were standardized (1 × 10⁷ cfu/mL) in artificial saliva (AS), which contained the following constituents, as described previously
[11 (link)]. This included porcine stomach mucins (0.25% w/v), sodium chloride (0.35 w/v), potassium chloride (0.02 w/v), calcium chloride dihydrate (0.02 w/v), yeast extract (0.2 w/v), lab lemco powder (0.1 w/v), proteose peptone (0.5 w/v) in ddH₂O (Sigma, Poole, UK). Urea was then added to independently to a final concentration of 0.05% (v/v). To initiate multispecies biofilm development the pioneer species S. mitis biofilm were first formed for 24 h in 5% CO₂ on 13 mm diameter Thermanox™ coverslips within 24 well plates (Corning, NY, USA). The supernatant was then removed and F. nucleatum added, which was incubated anaerobically at 37°C for a further 24 h. The supernatant was removed and P. gingivalis and A. actinomycetemcomitans added to the dual species biofilm, which was incubated anaerobically at 37°C for a further 4 days, replacing the AS daily to produce a mixed four species biofilm (Figure 
1A).

Gardnerella isolates of different species (52 (link)) were obtained from the Laboratory of Bacteriology, University of Ghent, Belgium. These isolates include strains purchased from culture collections and fresh isolates from BV patients obtained from the University Clinic Bruges. Gardnerella isolates were grown on chocolate (Choc) agar plates (Becton, Dickinson) under anaerobic conditions in an anaerobic chamber equipped with anaerobic atmosphere generation bags (Sigma-Aldrich) for 48 h. All isolates were cultured in New York City broth III (NYCB), consisting of 10 mM HEPES (Sigma-Aldrich), 15 g/L proteose peptone (Sigma-Aldrich), 3.8 g/L yeast extract (Thermo Fisher Scientific), 86 mM sodium chloride (Carl Roth), and 28 mM α-d-glucose (Sigma-Aldrich), supplemented with 10% horse serum (HS) (Thermo Fisher Scientific). Table S1 lists all Gardnerella and Lactobacillus isolates studied.

E. coli profiled with PCR were screened for clonal relatedness using a PhP-RE plate (PhP-RE, PhPlate AB, Microplate Techniques, Stockholm, Sweden) following the manufacturer's instructions [37 ]. The system consisted of a 96-well plate coated with 11 carbon sources: cellobiose, lactose, rhamnose, deoxyribose, sucrose, sorbose, tagatose, D-arabitol, raffinose, gal-Lacton, and ornithine. Three hundred µL of media containing a pH indicator (bromothymol blue 0.11% w/v) and proteose peptone (Sigma-Aldrich: St. Louis, MO) was combined with ∼1 mg of each bacterial isolate. After 1.5 hours, 12 µL of inoculum was transferred into each substrate well. One row of substrate was not inoculated and served as a negative control. As an internal check for reproducibility, three replicate isolate pairs were grown on separate 96-well plates and were determined to have correlation values of 0.94 and above. Plates were incubated at 37°C, covered with light-sensitive material, and read at 620 nm on a BioTek Cytation™3 (BioTek Instruments: Winooski, VT) at 8 h, 24 h, and 48 h. Clonal relatedness was estimated using PhenePlate™ software (PhPlate AB), which examined variability in absorbance across each substrate and presented relatedness as a dendrogram. A cutoff value of <97.5% similarity in functional profiles defined isolates as clonally distinct from each other.