Metataxonomic and metabolomic evidence of biofilm homeostasis disruption related to caries: An in vitro study

Abstract

The ecological dysbiosis of a biofilm includes not only bacterial changes but also changes in their metabolism. Related to oral biofilms, changes in metabolic activity are crucial endpoint, linked directly to the pathogenicity of oral diseases. Despite the advances in caries research, detailed microbial and metabolomic etiology is yet to be fully clarified. To advance this knowledge, a meta-taxonomic approach based on 16S rRNA gene sequencing and an untargeted metabolomic approach based on an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis (UHPLC/Q-TOF-MS) were conducted. To this end, an in vitro biofilm model derived from the saliva of healthy participants were developed, under commensal and cariogenic conditions by adding sucrose as the disease trigger. The cariogenic biofilms showed a significant increase of Firmicutes phyla (p = 0.019), due to the significant increase in the genus Streptococcus (p = 0.010), and Fusobacter (p < 0.001), by increase Fusobacterium (p < 0.001) and Sphingomonas (p = 0.024), while suffered a decrease in Actinobacteria (p < 0.001). As a consequence of the shift in microbiota composition, significant extracellular metabolomics changes were detected, showed 59 metabolites of the 120 identified significantly different in terms of relative abundance between the cariogenic/commensal biofilms (Rate of change > 2 and FDR < 0.05). Forty-two metabolites were significantly higher in abundance in the cariogenic biofilms, whereas 17 metabolites were associated significantly with the commensal biofilms, principally related protein metabolism, with peptides and amino acids as protagonists, latter represented by histidine, arginine, L-methionine, glutamic acid, and phenylalanine derivatives.

Strategies to Combat Caries by Maintaining the Integrity of Biofilm and Homeostasis during the Rapid Phase of Supragingival Plaque Formation

“…When there is a high and constant consumption of carbohydrates and fermentable sugars, the pH of the medium alters from an alkaline condition (6.0–7.0)—favorable for commensal bacteria—to a more acidic 5.5, which decreases the flow and buffering capacity of saliva [ 24 , 59 ], resulting in changes in the bacterial composition. These changes favor the rapid growth of Streptococcus , Actinomyces , and Lactobacillus , which metabolize carbohydrates more easily, degrading glucose into pyruvate, lactate, and acetate, generating an anaerobic environment [ 9 , 59 ]. In this scenario, the growth of certain species of P. gingivalis , Treponema denticola , Tannerella forsythia , and Aggregatibacter actinomycetemcomitans also produce other acids, such as lactic and formic acid [ 9 , 59 , 60 ], which complicate the situation.…”

“…These changes favor the rapid growth of Streptococcus , Actinomyces , and Lactobacillus , which metabolize carbohydrates more easily, degrading glucose into pyruvate, lactate, and acetate, generating an anaerobic environment [ 9 , 59 ]. In this scenario, the growth of certain species of P. gingivalis , Treponema denticola , Tannerella forsythia , and Aggregatibacter actinomycetemcomitans also produce other acids, such as lactic and formic acid [ 9 , 59 , 60 ], which complicate the situation. The accumulation of more diverse microbiota tolerant to an acidic environment on the supragingival surface of the tooth, along with elevated carbohydrate fermentation, disrupts the demineralization–remineralization balance of dental tissues, facilitating the onset of carious lesions [ 23 , 40 , 60 , 61 ].…”

“…The amino acid proline has also been postulated as a prebiotic. In vitro assays have indicated that proline affects host–pathogen interactions by modulating cell signaling and osmotic stress either as an antibacterial molecule or as a prebiotic [ 9 , 103 ]. Studies have also reported that a proline-containing peptide (tripeptide Ile-Pro-Ile (described as diprotin A)) led to increased resistance in biofilms to sucrose-induced decreases in pH [ 95 , 96 ].…”

“… Illustration exemplifying the possible development of multispecies biofilms. Scanning electron microscopy corresponds to an in vitro supragingival biofilm model of ( a ) 3, ( b ) 12, ( c ) 24, and ( d ) 48 h of evolution (to methodology [ 9 ]): ( a ) initial colonization of a substratum covered in an acquired film composed of polysaccharides and proteins. In vitro, various bacteria of coccoid and bacillary morphology can be seen on the surface after 3 h of biofilm process initiation; cell division can be appreciated mainly in coccoid bacteria (green arrows; scale bar = 9 µm); ( b ) rapid growth and division of initial colonizers and production of extracellular polysaccharide (EPS) leading to the development of microcolonies from several bacterial species—green arrows point to EPS surrounding bacteria in a microcolony after 12 h of biofilm development in vitro, which is identifiable in the image as a compact mass of greater brightness—(scale bar = 5 µm); ( c ) coadhesion and coaggregation of bacteria into a young multispecies biofilm—after 24 h of in vitro evolution, the surface appeared to be covered by bacteria consisting primarily of a larger colonies—outward-growing masses of bacterial cells alternating with flat homogenous layers of cells (scale bar = 50 µm); and ( d ) maturation of the multispecies biofilm—after 48 h of in vitro incubation, biofilm demonstrated the characteristic organization of these communities: covering the surface with bacteria clusters, forming stacks, and showing channels inside the structure—(scale bar = 20 µm).…”

“…The main ethological component of dental caries consists of bacterial species (mainly streptococci, lactobacilli, and bifidobacteria) organized in sessile and highly specialized communities (the supragingival dental plaque) located in the proximal spaces and occlusal surfaces of the gingival margin [ 2 , 3 , 4 , 5 , 6 , 7 ]. Advances in the various omics techniques applied to the field of dental plaque have revealed that this oral ecosystem is inhabited by hundreds of bacterial species, most of which are considered commensal but include low levels of opportunistic pathogens that become the dominant species in caries-affected sites at the expense of health-associated taxa [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ].…”