Probiotics have shown favourable properties in maintaining oral health. By interacting with oral microbial communities, these species could contribute to healthier microbial equilibrium. This study aimed to investigate in vitro the ability of probiotic Lactobacillus rhamnosus GG (L.GG) to integrate in oral biofilm and affect its species composition. Five oral strains, Streptococcus mutans, Streptococcus sanguinis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Candida albicans were involved. The group setup included 6 mono-species groups, 3 dual-species groups (L.GG + S. mutans/S. sanguinis/C. albicans), and 4 multi-species groups (4/5 species and 4/5 species + L.GG, 4 species were all the tested strains except S. mutans). Cell suspensions of six strains were pooled according to the group setup. Biofilms were grown on saliva-coated hydroxyapatite (HA) discs at 37 °C in anaerobic conditions for 64.5 h. Biofilm medium was added and refreshed at 0, 16.5, and 40.5 h. The pH of spent media was measured. Viable cells of the 16.5 h and 64.5 h biofilms were counted. 64.5 h biofilms were stained and scanned with confocal laser scanning microscopy. Our results showed that L.GG and S. mutans demonstrated stronger adhesion ability than the other strains to saliva-coated HA discs. L.GG, C. albicans, S. mutans and F. nucleatum, with poor ability to grow in mono-species biofilms demonstrated better abilities of adhesion and reproduction in dual- and/or multi-species biofilms. L.GG slightly suppressed the growth of C. albicans in all groups, markedly weakened the growth of S. sanguinis and F. nucleatum in 4sp + L.GG group, and slightly reduced the adhesion of S. mutans in L.GG+ S. mutans group. To conclude, in this in vitro model L.GG successfully integrated in all oral biofilms, and reduced the counts of S. sanguinis and C. albicans and lowered the biofilm-forming ability of F. nucleatum, but only slightly reduced the adhesion of S. mutans. C. albicans significantly promoted the growth of L.GG.

Mass mortality of farmed small abalone Haliotis diversicolor occurred in Fujian, China, from 2009 to 2011. Among isolates obtained from moribund abalones, the dominant species AP37 exhibited the strongest virulence. After immersion challenge with 106 CFU ml-1 of AP37, abalone mortalities of 0, 53 and 67% were induced at water temperatures of 20°C, 24°C, and 28°C, respectively. Following intramuscular injection, AP37 showed a low LD50 (median lethal concentration) value of 2.9 × 102 CFU g-1 (colony forming units per gram abalone wet body weight). The LT50 (median lethal time) values were 5.2 h for 1 × 106 CFU abalone-1, 8.4 h for 1 × 105 CFU abalone-1, and 21.5 h for 1 × 104 CFU abalone-1. For further analysis of virulence, AP37 was screened for the production of extracellular factors. The results showed that various factors including presence of flagella and production of extracellular enzymes, such as lipase, phospholipase and haemolysin, could be responsible for pathogenesis. Based on its 16S rRNA gene sequence, strain AP37 showed >98.8% similarity to Vibrio harveyi, V. campbellii, V. parahaemolyticus, V. alginolyticus, V. natriegens and V. rotiferianus, so it could not be identified by this method. However, multi-locus sequence analysis (MLSA) of concatenated sequences, including the rpoD, rctB, gyrB, toxR and pyrH genes, identified strain AP37 as V. harveyi. Phenotypic characters of AP37 were identified by API 20E. In antibiotic susceptibility tests, strain AP37 exhibited susceptibility to 7 antibiotics and resistance to 13. This is the first report of a V. harveyi-related species being linked with the mass mortality of adult abalone H. diversicolor in southern China.

Clinical studies have shown that probiotics positively affect oral health by decreasing gum bleeding and/or reducing salivary counts of certain oral pathogens. Our aim was to investigate the inhibitory effect of six probiotic lactobacilli against opportunistic oral Candida species. Sugar utilisation by both lactobacilli and Candida was also assessed. Agar overlay assay was utilised to study growth inhibition of Candida albicans, Candida glabrata and Candida krusei by Lactobacillus rhamnosus GG, Lactobacillus casei Shirota, Lactobacillus reuteri SD2112, Lactobacillus brevis CD2, Lactobacillus bulgaricus LB86 and L. bulgaricus LB Lact. The inhibitory effect was measured at pH 5.5, 6.4, and 7.2, respectively, and in the presence of five different carbohydrates in growth medium (glucose, fructose, lactose, sucrose, and sorbitol). Growth and final pH values were measured at two-hour time points to 24 h. L. rhamnosus GG showed the strongest inhibitory activity in fructose and glucose medium against C. albicans, followed by L. casei Shirota, L. reuteri SD2112 and L. brevis CD2. None of the lactobacilli tested affected the growth of C. krusei. Only L. rhamnosus GG produced slight inhibitory effect on C. glabrata. The lower pH values led to larger inhibition zones. Sugar fermentation profiles varied between the strains. L. casei Shirota grew in the presence of all sugars tested, whereas L. brevis CD2 could utilise only glucose and fructose. All Candida species metabolised the available sugars but the most rapid growth was observed with C. glabrata. The results suggest that commercially available probiotics differ in their inhibitory activity and carbohydrate utilisation; the above properties are modified by different pH values and sugars with more pronounced inhibition at lower pH.

In order to investigate the effects of mouthwashes on oral biofilms with probiotics, we compared in biofilms the susceptibility to mouthwashes of probiotic Lactobacillus rhamnosus GG (LGG) and oral pathogens Streptococcus mutans, Streptococcus sanguinis, and Candida albicans. We also evaluated these pathogens’ susceptibility to the mouthwashes and their recovery after mouthwash-rinsing in biofilms with/without LGG. First, 1-day-/3-day-old LGG-integrated multi-species biofilms were exposed for 1 min to mouthwashes containing chlorhexidine, essential oils, or amine fluoride/stannous fluoride. Cells were plate-counted and relative survival rates (RSRs) of LGG and pathogens calculated. Second, 1-day-/3-day-old multispecies biofilms with and without LGG were exposed for 1 min to mouthwashes; cells were plate-counted and the pathogens’ RSRs were calculated. Third, 1-day-old biofilms were treated for 1 min with mouthwashes. Cells were plate-counted immediately and after 2-day cultivation. Recovery rates of pathogens were calculated and compared between biofilms with/without LGG. Live/Dead^® staining served for structural analyses. Our results showed that RSRs of LGG were insignificantly smaller than those of pathogens in both 1-day and 3-day biofilms. No significant differences appeared in pathogens’ RSRs and recovery rates after treatment between biofilms with/without LGG. To conclude, biofilm LGG was susceptible to the mouthwashes; but biofilm LGG altered neither the mouthwash effects on oral pathogens nor affected their recovery.