Porphyromonas gingivalis

A total of 35 clinical isolates were used. Included were Porphyromonas saccharolytica (n=9), Porphyromonas gingivalis (n=13), Prevotella intermedia (n=9), Prevotella melaninogenica (n=4). All isolates belonged to a collection of the microbiology laboratory at UHS. For quality control, two reference strains, namely Porphyromonas gingivalis (ATCC 33277) and Bacteroides fragilis (ATCC 25285), were included in the study obtained from reference center (MicroBioLogics, USA) and identified by morphological, cultural, and biochemical profile (API -20A, bio Merieux, France). These reference strains were included in every cycle of culture and susceptibility testing to monitor the consistency of the procedure.

Porphyromonas gingivalis (Pg) (ATCC 33,277), Staphylococcus aureus (ATCC 29,213), and Prevotella intermedius (Pi) (ATCC 25,611) were purchased from ATCC. Cultures of Pg and Pi were grown overnight in brain heart infusion broth (BHI) (Difco, USA) under anaerobic conditions (80% N₂, 10% CO₂, and 10% H₂) at 37 °C in an anaerobic chamber. S. aureus was grown overnight in BHI in a microaerophilic environment (5% CO₂). Then, the bacterial suspensions were centrifuged at 2000 rpm/min for 5 min, and the final concentrations of the cultures were adjusted to 0.5 × 10⁵ CFU/mL using a spectrophotometer.

Streptococcus mutans Clarke (strain designation NCTC 10449), Pseudomonas aeruginosa (Schroeter) Migula (strain designation PAO1), Fusobacterium nucleatum, subspecies nucleatum (strain VPI 4355), and Porphyromonas gingivalis (strain 2561) were purchased from ATCC (LGC Standards Srl, Milan, Italy). Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis were obtained from clinical isolates and characterized by morphological, biochemical, and molecular typing. S. mutans, S. mitis, S. oralis, and S. sanguinis were maintained in Brain Heart Infusion (BHI) broth or agar under microaerophilic conditions. P. aeruginosa was maintained in trypticase soy (TS) agar or broth. F. nucleatum was maintained in TS agar or broth with defibrinated sheep blood under anaerobic conditions. P. gingivalis was maintained in ATCC Medium 2722 supplemented with TS broth or agar under anaerobic conditions. Before the experiments, bacterial cultures were inoculated in fresh media (dilution 1:100) and grown for 16 h at 37 °C.

Porphyromonas gingivalis ATCC 33277, Veillonella parvula ATCC 17745, Streptococcus mitis ATCC 49456, Neisseria flavescens ATCC 13120, Hemophilus parainfluenzae NCTC 10665, Rothia mucilaginosa ATCC 49041, and Lautropia mirabilis ATCC 51599 were obtained from the American Tissue Culture Collection (ATCC) or Public Health England. V. parvula was cultivated in Brain Heart Infusion (BHI) supplemented with 1.5 of 60% sodium lactate. All other bacteria were cultivated in BHI supplemented with 0.5% hemin, 0.1% Vitamin K and 1% Isovitalex. P. gingivalis and V. parvula were grown in anaerobic conditions (10% hydrogen, 10% CO₂, and 80% nitrogen), L. mirabilis was grown aerobically, and the other five bacteria were grown in 5% CO_2. All strains were grown at 37°C.

Prevotella copri DSMZ 18205, Staphylococcus epidermidis ATCC 14990, Akkermansia muciniphila ATCC BAA-835, Porphyromonas gingivalis ATCC 33278, and Bacteroides ovatus ATCC 8483 were obtained from DSMZ (Braunschweig, Germany) or ATCC (Manassas, VA) and grown in Schaedler Broth, Nutrient Broth, Brain Heart Infusion Broth, Supplemented Tryptic Soy Broth, or Modified Chopped Meat Medium, respectively. P. copri, A. muciniphila, and B. ovatus were grown in an anaerobic glove box containing approximately 2.5% CO₂, 2.5% H₂, and 95% N₂. All strains were grown at 37°C until the mid- to late-log phase growth. 250 ml of the culture was spun down at 3000 g for 10 min, the supernatant was removed, the pellet was resuspended in 4 ml of PBS, and sonicated using a probe sonicator (30 s followed by 1 min rest, repeated 3 times) for the preparation of bacterial lysates. The bacterial lysates were clarified by centrifugation at 10,000 rpm for 1 min at 4°C. The clarified bacterial lysates were then treated with Polymyxin beads (Sigma) to remove endotoxin content followed by estimation of total protein content in the lysates through BCA protein assay method (ThermoFisher). Fecal lysates were generated by diluting 100 mg of fecal sample in 1 ml PBS and sonicated 3 times for 1 min at 4°C with 30 s of rest in between. The fecal lysates were clarified by centrifugation at 15000 rpm for 5 min before use.

The following oral pathogens were studied: Candida albicans CBS 562 from “Centraalbureau voor Schimmelcultures” and bacteria Streptococcus sanguis ATCC 10556, Streptococcus mitis ATCC 903, Porphyromonas gingivalis ATCC 33277 and Fusobacterium nucleatum ATCC 25586 from American Type Culture Collection. The microorganisms were stored at −70°C in Sabouraud Dextrose Broth (SDB, Merck® - C. albicans) and Mueller-Hinton Broth (Difco® - bacteria) with 15% glycerol. It was considered the oxygen exigencies of each microorganism (C. albicans - aerobiosis, S. mitis and S. sanguis microaerophilie and F. nucleatum and P. gingivalis anaerobiosis) to choose bacteria growth conditions.