Anaerogen pak jar

Limosilactobacillus reuteri DSM 17938 (Rosander et al., 2008 (link)), provided by BioGaia AB (Stockholm, Sweden), was used in the study. The bacteria were plated on DeMan, Rogosa, and Sharpe agar (MRSA; Oxoid Limited, Hampshire, United Kingdom), and incubated at 37°C for 24 h in an anaerobic atmosphere (Anaerogen Pak Jar, Oxoid Ltd.). The CFS and its fractions, SurE 10 K and 30 K, were obtained following the procedure described by Maccelli et al. (2020) (link). Regarding the other bacterial strains used, different media and growth conditions were used on the basis of the species.

In this study, the reference bacterial strain Pg ATCC 33277 (LGC Standards S.r.l., Sesto San Giovanni, Milano, Italy) was used and cultivated as previously reported [42 (link)]. Briefly, the bacterium was cultivated in Fastidious Anaerobe Agar (Neogen, Lansing, MI, USA) supplemented with 5% of defibrinated horse sterile blood (Oxoid Limited, Hampshire, UK) for 48 h in anaerobiosis (Anaerogen Pak Jar, Oxoid, UK). After incubation, all the colonies were collected, washed in PBS, and centrifugated to obtain a pellet corresponding to 6 × 10¹² CFU. Molecular analyses were performed to quantify the Pg in the oral cavities of all the participants. The total genomic DNA was isolated from the samples and the reference bacterial strain using a Quick DNA miniPrep Plus KIT (Zymo Research, Irvine, CA, USA). StepOne™ 2.0 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was used in a qPCR analysis to quantify the Pg abundance in each sample. A TaqMan-based assay that recognizes Pg 16S rRNA, the gene encoding the small subunit of 16S ribosomal RNA, was used, as previously reported [43 (link)]. A standard curve passing through 5 points was constructed, indicating the cycle threshold values versus the Pg 16S rRNA gene. This method allowed us to estimate the bacterial quantity based on the amount of total DNA isolated from the oral samples [43 (link)].

The bacterial strains used in this study were Fusobacterium nucleatum (Fn) ATCC 25586 and Porphyromonas gingivalis (Pg) ATCC 33277 (LGC Standards S.r.l., Sesto San Giovanni, Milano, Italy). The strains, stored at −80 °C in a glycerol stock, were thawed at room temperature, plated rapidly on Fastidious Anaerobe Agar (FAA) (Lab M, Heywood, UK) plus 5% of defibrinated horse sterile blood (Oxoid Limited, Hampshire, UK) and incubated at 37 °C for 48 h in an anaerobic atmosphere (Anaerogen Pak Jar, Oxoid Ltd.). After 48 h of incubation, the colonies of Fn and Pg were washed twice in sterile 0.01-M PBS and centrifuged at 4000× g to obtain a visible pellet that was used for the extraction of DNA. Colony-forming unit (CFU) enumeration was carried out by resuspending the samples in 1 mL of PBS; subsequently, serial dilutions of the stock were performed in PBS and plated on FAA. The plates were then incubated at 37 °C for 48 h in an anaerobic atmosphere. Bacterial DNAs were isolated from 4.7 × 10¹² CFU/mL for Fn and 6 × 10¹² CFU/mL for Pg by using a Quick DNA miniPrep Plus kit (Zymo Research, Irvine, USA), obtaining a DNA concentration of 63 ng/μL and 59 ng/μL, respectively.