P gingivalis

Bacterial strains were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Three bacterial strains were used: S. mutans (ATCC 25175), Aggregatibacter actinomycetemcomitans (ATCC 33384), and P. gingivalis (ATCC 33277). S. mutans and A. actinomycetemcomitans were grown on Bacto™ Brain Heart Infusion agar, and P. gingivalis on Bacto™ Tryptic soy agar with 5% defibrinated sheep blood. All bacteria were grown at 37°C with: S. mutans in aerobic conditions, A. actinomycetemcomitans in microaerophilic condition (5% CO₂), and P. gingivalis in anaerobic conditions. Colonies were isolated using the quadrant streak plate method (in triplicates) and incubated: S. mutans—24 hours—and A. actinomycetemcomitans and P. gingivalis—72 hours. Single colonies were selected from each plate. The bacteria were cultured in liquid broth and serially diluted to an optical density of 600 nm, which resulted in a concentration of 10⁶ CFU/mL.

P. gingivalis (strain 381) was purchased from ATCC (Manassas, VA, USA) and cultured anaerobically in Gifu anaerobic medium (GAM; Nissui, Tokyo, Japan) containing 5 mg/mL hemin and 0.5 mg/mL 3-phytyl-menadione (vitamin K) in an 80% nitrogen, 10% hydrogen, and 10% CO₂ anaerobic system (Anoxomat; Advanced instruments, Norwood, MA, USA). P. gingivalis was collected by centrifugation at 5000 rpm for 5 min and resuspended in PBS to infect cells at the indicated multiplicity of infection (MOI) for 24 h.

Anaerobic bacteria T. denticola (ATCC 35405), P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) were purchased from ATCC. T. denticola, P. gingivalis and F. nucleatum were grown as described previously at 37°C under oxygen deprived anaerobic conditions in New Oral Spirochete Medium or in Brain-heart Infusion (BHI) broth supplemented with hemin (5 μg ml^-1) and vitamin K (1 μg ml^-1) [143 , 144 (link)]. Purity of spirochete cultures was confirmed by dark field microscopy prior to use in experiments. Oral commensal bacteria S. gordonii (ATCC 10558), S. salivarius (ATCC 13419) and V. parvula (ATCC 10790) were grown overnight at 37°C under anaerobic conditions in BHI broth then harvested for subsequent assays. Gram staining followed by microscopic evaluation and colony morphology on Mitis Salivarius plates were used to confirm purity of cultures. T.denticola lipoologosaccharide (Td-LOS) was a gift from Daniel Grenier, Universite Laval, Quebec, Canada, P.gingivalis lipopolysaccharide (Pg-LPS) and F.nucleatum lipopolysaccharide (Fn-LPS) were provided by Richard P. Darveau, University of Washington, Seattle and V. parvula lipopolysaccharide (Vp-LPS) was provided by Christopher Fenno, University of Michigan, Ann Arbor, MI.

OSCC cell lines CAL27, SCC4 and SCC25 were acquired from the American Tissue Culture Collection (ATCC), certified as mycoplasma-free and STR-authenticated. The cell lines were grown in 5% CO₂ at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2.5 mM L-glutamine (CAL27) or a 1:1 mixture of DMEM and Ham’s F12 medium containing 2.5 mM L-glutamine, 400 ng/ml hydrocortisone and 10% FBS (SCC4 and SCC25).
Bacterial strains S. mitis ATCC 49456, N. flavescens ATCC 13120 and P. gingivalis ATCC 33277 were obtained from ATCC whereas H. parainfluenzae NCTC 10665 was obtained from Public Health England. P. gingivalis was included as a pathogenic control given existing evidence on its carcinogencity [2 ,3 (link)]. Brain Heart Infusion (BHI) supplemented with 0.5% hemin, 0.1% Vitamin K and 1% Isovitalex was used as a culture medium for all bacteria. The health-associated strains were grown at 37°C in 5% CO₂; P. gingivalis was grown at 37°C in anaerobic conditions (10% hydrogen, 10% CO₂, and 80% nitrogen).

The following strains commonly present in the oral microbiome (Aas et al., 2005 (link); Maddi and Scannapieco, 2013 (link); Loozen et al., 2014 (link)) were obtained from the American Type Culture Collection (ATCC): A. actinomycetemcomitans (ATCC 43718), F. nucleatum (ATCC 10953), P. gingivalis (ATCC 33277), P. intermedia (ATCC 25611), S. mutans (ATCC 25175), S. sobrinus (ATCC 33478), T. forsythia (ATCC 43037), Actinomyces naeslundii (ATCC 51655), Capnocytophaga sputigena (ATCC 33612), Streptococcus gordonii (ATCC 49818), Actinomyces viscosus (ATCC 15987), and S. mitis (ATCC 49456). Veillonella parvula was obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSM 2007); S. sanguinis was acquired from the BCCM/LMG Bacteria Collection (LMG 14657). S. salivarius strain TOVE-R (Tanzer et al., 1985 (link)) was utilized. Bacterial strains were classified as either commensal (indigenous), potentially health-associated or disease-associated, as indicated in Table 1.