Porphyromonas gingivalis

Streptococcus oralis ATCC® 9811 was obtained from the American Type Culture Collection (ATCC®, Manassas, VA, USA). Actinomyces naeslundii DSM 43013 and Porphyromonas gingivalis DSM 20709 were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). Aggregatibacter actinomycetemcomitans MCCM 2474 strain was obtained from the Microbial Culture Collection Marburg.
All bacterial strains were routinely stored as glycerol stocks at −80 °C. As culture medium Todd-Hewitt Broth (Oxoid Limited, Hampshire, UK) supplemented with 10% yeast extract (THBy, Carl Roth GmbH + Co. KG, Karlsruhe, Germany), Brain–Heart Infusion (BHI, Oxoid Limited) supplemented with 10 μg mL⁻¹ vitamin K (Oxoid Limited) and Fastidious Anaerobe Broth (FAB, Oxoid Limited) were used as specified in Table 1. For the bacteria culture, 20 μL (S. oralis, A. naeslundii, Aac) or 30 μl (P. gingivalis) of the glycerol stocks were added into 10 mL of the corresponding medium in a 50 mL centrifugal tube and incubated under the conditions shown in Table 1. Then, bacterial cells were washed three times with autoclaved deionized water by centrifuging at 2000 RCF for 5 min.

Streptococcus oralis ATCC^® 9811^™, Actinomyces naeslundii DSM 43013, Veillonella dispar DSM 20735 and Porphyromonas gingivalis DSM 20709 were acquired from the German Collection of Microorganisms and Cell Cultures (DSMZ) and the American Type Culture Collection (ATCC). The bacteria were routinely cultured in brain heart infusion medium (BHl; Oxoid, Wesel, Germany), supplemented with 10 μg/mL vitamin K (Roth, Karlsruhe, Germany) under anaerobic conditions (80% N₂, 10% H₂, 10% CO₂) at 37°C.

Probiotic strains, namely L. rhamnosus SP1, L. helveticus SP27, and L. paracasei CBA-L87, key bioactive components of this mucoadhesive gel, in lyophilized form, were evaluated individually. Five pathogenic bacteria, from the “red complex” consortium, were selected for this competition test. Pathogenic strains (Prevotella melaninogenica DSM 7089, Tannerella forsythia DSM 102835, Porphyromonas gingivalis DSM 20709, Treponema denticola DSM 14222, and Aggregatibacter actinomycetemcomitans DSM 8324) were purchased from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany) and propagated in Columbia blood agar base (Oxoid, Thermo Fisher Scientific, Altrincham, UK), supplemented with 5% defibrinated horse blood to check their growth and purity in anaerobic chamber (atmosphere composed by 80% N₂, 10% CO₂, and 10% H₂).
T. forsythia DSM 102835 required N-Acetylmuramic acid NAMA (Merck KGaA, Darmstadt, Germany) for its growth, and for this reason, for all the experiment, NAMA was added to the medium at a final concentration of 10 µg/mL. Tested items are indicated in Table 16. The agar well diffusion test on the above-described strain was performed using the same approach followed in our previous work [19 (link)].