Background: The probiotic organisms Lactobacillus reuteri and Streptococcus oligofermentans are known for their inhibitory effect on cariogenic bacteria. Recently, they were shown to reduce Streptococcus mutans counts and lesion depth in a three-species model (S. mutans, Lactobacillus rhamnosus and Actinomyces naeslundii) of dental biofilm [Chen et al.: Microorganisms 2020;8:1272]. Here, we aimed to design and validate specific fluorescence in situ hybridization (FISH) probes for L. reuteri and S. oligofermentans and to investigate how both organisms contribute to biofilm architecture in the model.
Materials and methods: Biofilms (n=10) were grown for 10 days on bovine enamel slabs and rinsed daily with either L. reuteri (LR) or S. oligofermentans (SO). Probe sequences (L. reuteri: LAROI1 5’-GAGAGAGAGAGAG-3’; S. oligofermentans: SCRI1 5’- GAGAGAGAGAGAG -3’) were selected in the hypervariable segment V1 of the 16S rRNA gene and checked for duplex and hairpin formation using Oligo 7. Probe specificity and hybridization conditions were tested in silico using Probe Match and mathFISH. Hybridizations were performed at 30% formamide and 46°C. Fixed cells of L. reuteri, S. oligofermentans, S. mutans, L. rhamnosus and A. naeslundii served as controls. After FISH (n=5), samples were analyzed by confocal microscopy. Total and strain-specific bacterial numbers were assessed by quantitative polymerase chain reaction (n=5).
Results: LAROI1 and SCRI1 specifically targeted L. reuteri and S. oligofermentans, respectively, and both organisms constituted an important part of the biofilms. LAROI1, however, competed with EUB338 for binding and could not be used concomitantly. LR biofilms showed significantly reduced numbers of L. rhamnosus (82%) and S. mutans (90%), and S. mutans (94%) and A. naeslundii (58%) were significantly reduced in the SO group (p<0.05).
Conclusion: L. reuteri and S. oligofermentans are firmly integrated in multi-species biofilms.