Heart infusion broth

The biofilm sample was collected from a patient by the assistance of an experienced dentist. The dental plaque samples were collected from the surfaces of the teeth and placed in Eppendorf tubes containing 2.0 mL phosphate buffered solution (PBS). Informed consent was obtained from patients in accordance with ethical approval from the ethics committee of Abasyn University. Six different dental plaque bacterial species, including Proteus spp., Escherichia coli, Pseudomonas spp., Salmonella spp., Streptococcus spp., and Staphylococcus aureus as previously isolated and identified were used for the biofilm formation. Heart infusion broth (Oxoid, UK) was used to grow and maintain Streptococcus spp., and all other bacterial spp. and maintain in tryptic soya broth and agar (Oxoid, UK). All the bacteria were preserved at 4 °C and by sub-culturing regularly [13 (link),16 (link)].

For a full list of bacterial strains and plasmids used in this work, please refer to Table S2 andS3 in the supplemental material, respectively. Unless stated otherwise, C. sordellii isolates were grown in HIS broth (37 g/liter heart infusion broth [Oxoid], 5 g/liter yeast extract, 1 g/liter l-cysteine, 0.375% [wt/vol] glucose) or on HIS agar (HIS broth with 15 g/liter agar) at 37°C in an anaerobic chamber (Coy Laboratory Products, Inc.) in an atmosphere of 10% H₂, 10% CO₂, and 80% N₂. Escherichia coli strains were grown in 2YT broth (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl) or on 2YT agar (2YT broth with 15 g/liter agar) at 37°C. When required after sterilization of media, the following antibiotics were included at the indicated concentrations (unless otherwise specified): erythromycin (Erm; 10 μg/ml), tetracycline (Tet; 10 μg/ml), chloramphenicol (Cm; 25 μg/ml), thiamphenicol (10 μg/ml), streptomycin (Str; 200 μg/ml), rifampin (Rif; 20 μg/ml), anhydrous tetracycline (AnTet; 20 ng/ml), and d-cycloserine (DCy; 250 μg/ml).

S. capitis isolate AYP1020 was originally isolated from blood and confirmed to be S. capitis by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) and confirmed to be subsp. capitis based on 16S rRNA sequence. After isolation, AYP1020 was cultured on Horse Blood Agar, grown in Heart Infusion Broth (Oxoid) then stored at −80°C in 25% (v/v) Glycerol. S. epidermidis RP62a is a biofilm-producing, methicillin-resistant S. epidermidis (MRSE) strain isolated from a patient with intravascular catheter-associated sepsis (Christensen et al., 1985 (link)). It has a fully sequenced genome (accession number NC_002976) (Gill et al., 2005 (link)) and was used in this study for comparative genomic analysis. Antibiotic susceptibility testing was performed using broth dilution for penicillin G, gentamicin, methicillin, erythromycin, and streptomycin according to Clinical Laboratory Standards Institute guidelines (C.L.S.I, 2013 ).

We performed whole genome sequencing (WGS) on a representative subset of bacterial isolates, stratified by bacterial species and month of isolation. All bacterial isolates were routinely grown on Heart Infusion agar (Oxoid) for 16 h at 37°C, inoculated into 3 mL Heart Infusion broth (Oxoid), and grown for a further 16 h at 37°C with orbital shaking at 150 rpm. Genomic DNA was extracted from 300 µL of liquid bacterial culture using the GenFind V3 Reagent Kit (Beckman Coulter) as per manufacturer's instructions. Libraries were prepared using the Nextera Flex DNA Library Prep Kit (Illumina), and 150 bp paired-end sequencing was performed on the NovaSeq 6000 system (Illumina). Genomes were assembled using Unicycler (v.0.4.9) [14] (link). Multi-locus sequence types (STs) were identified using mlst (https://github.com/tseemann/mlst).
For the genomes of the most prevalent ST, we determined relatedness by calculating pairwise single nucleotide variation (SNV) distances between all isolates. Genomes were assembled using SKESA v.2.3 [15] (link) using default parameters. Each pair of genome assemblies was compared using Catpac (https://github.com/rrwick/Catpac) to determine the number of SNVs between pairs. Pairs with ≤5 SNVs per mbp between them were considered likely candidates for strain transmission, as defined by Gorrie et al. [16] (link).

Table S1 lists bacterial strains used in this analysis. Unless stated otherwise, clostridial species were cultured in HIS broth (37 g/L heart infusion broth (Oxoid), 5 g/L yeast extract, 1 g/L L-cysteine, 0.375% (wt/vol) glucose) or on HIS agar (HIS broth with 15 g/L agar) at 37 °C in an anaerobic chamber (Coy Laboratory Products, Inc., Grass Lake, MI, USA) in an atmosphere of 10% H2, 10% CO2, and 80% N2. E. coli strains were grown with shaking at 200 rpm in 2YT broth (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) or on 2YT agar (2YT broth with 15 g/L agar) at 37 °C. When required, the following antibiotics were included for selection: erythromycin (10 mg/L), chloramphenicol (25 mg/L), thiamphenicol (10 mg/L), and lincomycin (50 mg/L).