Insight into Oral Biofilm: Primary, Secondary and Residual Caries and Phyto-Challenged Solutions

Oral Biofilms and Orodental Infections

One of the preeminent examples of a biofilm that is structurally as well as functionally organized is dental plaque, which is a multi species biofilm comprising of hundreds of bacterial species, salivary polymers, and bacterial extracellular products. The microbial species colonize the teeth, hard palate, tongue, carious lesions, oral mucosa, and periodontal pockets. Supra-gingival and sub-gingival plaques are the most significant biofilms in oral cavity contributing to various orodental manifestations. The distribution of the microbial species in these plaque biofilms varies depending on the anatomical locations and environmental factors [3]. The bacterial plaque adheres resolutely to tooth surfaces as well as restorations and prosthetic appliances. Many molecular and biochemical interactions occur among the bacterial species within biofilm leading to either gradients of carbon dioxide and hydrogen or a strictly anaerobic environment [23].

Of fundamental importance to oral health maintenance and preventative measures to inhibit dental caries, gingivitis, periodontitis, endodontitis, apical periodontitis, and peri-implantitis is the control of oral biofilm formation as these are the major outcomes of oral biofilm [24]. These commensal microbes in the oral cavity function as a principal source of the formation of biofilm in the root canal. The anatomical complexities found within the root canal favor the sustenance and proliferation of biofilm and bacteria. MMPs secreted by the inflammatory cells digests the biofilm components thereby helps to slough of bacterial biofilm. But in a preformed biofilm inside the dentinal tubules and canal, diffusion barrier property to antibiofilm agents, antibacterial drugs and inter canal medicaments like CHX, tetracycline and its derivatives, chemically modified tetracyclines (CMTs) (frequently used chemical MMP inhibitors in dentistry), will enhance MMPs and cysteine cathepsins leading to further tissue destruction [25]. Various surveillance data indicates that approximately 90 percent of carious lesions occur in the pits and fissures of permanent posterior and molar teeth [26]. An increase in biofilm porosity is aided by extracellular polysaccharides enabling penetration of metabolites to the core of biofilm resulting in pH drop [27]. Following this, plaque formation proceeds at an expense of host defenses (saliva inflow rate and frequency and composition of diet) resulting in extensive demineralization of the tooth enamel eventually leading to caries [3]. The inhibition of oral biofilms, as well as the initial microbial colonization, can effectively prevent orodental infections.

Architecture and Formation of Biofilm

Initiation of biofilm formation begins with the planktonic forms, which are converted into replicating sessile forms on a surface. The cells colonize and irreversibly attach to the surface to be embedded in a self-assembled exopolymeric matrix. Biofilms consist of clusters of bacterial cells, either single species or mixed population. The base unit of biofilm is a cluster or micro colony and is characterized as a discrete group of bacterial cells, from one species or several, enclosed in the matrix [28]. Bacteria and bacterial biofilms are reported in 81.2% of the cementum and root canals as well as 46.8% of the apical delta. When compared to the apical third of the root canal, it was found that a higher bacterial load resides in the cervical and middle thirds [29]. Electron microscopic morphology of biofilms appears as tower or mushroom shaped micro-colony with water channels and interstitial voids within the matrix. These water channels and interstitial voids separate the micro colonies from the external environment through which fluids containing nutrients, waste metabolites, and oxygen move via convection (Lawrence et al. 1991). Bacteria found at the edges of these fluid channels typically exist in an aerobic environment, while those found within the center of a micro colony are more tightly packed and may live in a strictly anaerobic environment [30].

The assembly and structural organization of such micro colonies can vary depending on bacterial species, the substrate, and the surrounding media. The system of nutrients and metabolic end products distribution functions only in the peripheral regions of biofilms, while differences in oxygen and nutrient availability within the structure of the biofilm can affect the different metabolic activity amongst the cells. In addition, the cells within biofilms secrete signaling molecules serving to activate the formation of micro colonies with complex architecture and diverse functions [31]. This structural complexity of the biofilm provides an effective barrier that limits the penetration of antimicrobial agents throughout the biological layer [32]. The organization of biofilm is displayed in (Fig. 1).

Plaque Biofilm: The Precursors to Caries Formation

A dental plaque originates by acquired pellicle formation. A pellicle is the salivary component composing of glycoproteins coating, mucins, proline rich proteins, alpha-amylase and other proteins that form on the tooth surface immediately after cleaning. A number of interactions facilitate this bacterial adhesion such as hydrogen bonds, calcium bridges, van der Waals forces, acid-base interactions, hydrophobic interactions and electrostatic interactions which occur between various glycoproteins, salivary components as well as the tooth surface that induce conformational changes to the proteins which form the pellicle [33, 34]. This adhesion to living tissue is mediated through particular molecular components like lectin ligand or adhesins [35]. Result of these interactions is favorable for the colonization of the primary colonizers by a reversible adhesion process aided by the secretion of extracellular polysaccharide matrix (EPS) which assists the bacteria in staying bound together and attaching to the pellicle. The events in biofilm formation are displayed in (Figs. 2 and 3).

Key Players of Plaque Biofilm Organization

Streptococcus mutans, Streptococcus sanguis, Streptococcus oralis, Streptococcus gordonii, and Lactobacillus acidophilus are major pioneer organisms in the plaque formation, which are competitive at the low pH (due to anaerobic metabolism). Oral streptococci belongs to four main groups namely mitis, salivaris, mutans and anginosus. According to Love et al., the streptococcal antigen I/II family of polypeptides mediates the attachment of oral streptococci to collagen and determines their ability to invade dentinal tubules found in human teeth. These microbes penetrate the dentinal tubules as co-aggregates as their adaptive response to extreme environmental change makes their survival easy. The pathogenicity of Streptococcus anginosus in root canal is attributable to its attachment and co-aggregation. Streptococcus gordonii recruits Porphyromonas gingivalis to dental plaque and promotes its invasion into the dentinal tubules. Biofilm formation has also been shown to be dependent on the intracellular transport of Mn²⁺ in Streptococcus gordonii. While the role of S. parasanguis and S. oralis in endodontic infections is not known. Streptococcus oralis possesses surface-associated protein which aids in its survival while Streptococcus parasanguis have fimbriae which assist their spreading in the bloodstream and survival during adverse conditions.

Streptococcus mutans shows significant aggregation with Candida albicans [36]. Candida albicans co-aggregates with Streptococcus gordonii, Streptococcus sanguis and other α and non-haemolytic Streptococcus species and activates biofilm formation. Candida species have various glycoproteins located on the exterior of the cell wall, which help in attachment to the mucosal tissue and abiotic surfaces [37]. Candida possesses virulence factors such as adherence, thigmotropism for penetration, hyphae formation, protease secretion and phenotypic switching. They survive in a nutritionally deprived environment via the secretion of aspartyl protease and enzymes which degrade dentinal collagen [38]. Moreover, the Candida species can resist the effect of calcium hydroxide during dental restoration therapies. These features of Candida albicans indicate their higher rate of survival (10%) within the canal, contributing to root canal and periapical infections [39, 40]. Yeast such as; Geotrichium candidum, Candida glabrata, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida kefyr and Candida parapsilosis. are also common in the root canals [41]. They comprise less than 1% of the endodontic flora and are predominantly seen in persistent apical periodontitis [42].

Tahmourespour and Kermanshahi found that Lactobacillus acidophilus causes substantial reduction in the adherence of Streptococcus mutans where the interaction with non-mutan streptococci was less significant [43]. Such antagonistic interaction between these species forms the reason for the relative reduced number of mutan streptococci in infections of the root canal compared to non-mutans. The acidic environment is advantageous for the cariogens as it inhibits the growth as well as the metabolism of noncariogenic species in biofilm [44]. Other oral microbes like Actinomyces sp., Veillonella sp., Rothia mucilaginosa, Gemella sp., Capnocytophagae sp., Prevotella sp., Niesseria sp., are also frequently found during the early stages of plaque formation [45]. The members of secondary colonizers include Fusobacterium nucleatum, Treponema sp., Tannerella forsythensis, Porphyromonas gingivalis, Aggregat-ibacter actinomycetemcomitans [46]. Fusobacterium nucleatum is a well known co-aggregation bridge species that serves as facilitator of streptococcal and obligate anaerobes aggregation.

Likewise, Enterococcus faecalis and Enterococcus faecium harbor in root canals of the carious tooth and elicit persistent asymptomatic infections paving ways to endodontitis [47]. Deng et al. reported that the presence of a streptococcal-biofilm significantly raises the formation of biofilm in Enterococcus faecalis strains, but co-aggregation has not yet been reported for Enterococcus faecalis and Streptococcus mutans [48]. streptococci have been isolated in majority of cases with primary endodontic infection, during endodontic treatment and in retreatment, which constitutes approximately 20% of the endodontic mileu in post treatment cases.

Similarly, several ecological factors also regulate the survival of micro-flora found in the root canal. The bacterial synergy in which, the nutritional requirement of one bacterium is met by the metabolic product of the other microbial flora is also responsible for the coexistence of several root canal microorganisms. The majority of the endodontic micro-flora are given in Table (1) [49]. Microbial ecology is further influenced by nutritional status found in the coronal parts of the exposed root canal possessing exogenous nutrients (carbohydrates) and the body of the root canal containing endogenous nutrients (proteins and glycoproteins).

Factors Affecting Biofilm Formation

Proliferation of resident microbes within the biofilm increases the biomass production within the matrix. Various factors including pH, oxygen availability, temperature, and organic metabolites determine the maturation and growth of the biofilm. Moreover, the signaling molecules for cell-cell communication are also integral to population control. During the maturation process of the biofilm, the secondary colonizers or the late colonizers recognize the polysaccharides or protein receptors on the bacterial cell surface that facilitates co-aggregation. These interactions pave the way for the formation of morphologically distinct biofilms like corn cob forms, bristle brush forms, or mesh [68]. Mature biofilms typically contain many porous layers and water channels which provide essential nutrient/metabolite traffic to the residing bacteria [69].

Flushing action of saliva and associated first line of defense, mechanical action of oral cavity during nutrient consumption and fluid sheer force of saliva limit biofilm development. This results in the dispersion of the biofilm cells by erosion, sloughing, and seeding [70]. Also, EPS influences ion exchange within biofilms and controls the hydrophilic/hydrophobic characteristics of biofilm. Oxygen metabolism and exchange of metabolites in the biofilm between the resident aerobes and obligate anaerobes facilitate survival of the latter. Oxygen consumption by the aerobes limits the oxygen content and induces a local reduction potential that provides anaerobic micro-environment for the survival of obligate anaerobes.

The initial attachment of bacteria on to a surface can often result in the upregulation of EPS synthesis. Among the plaque bacteria, mutan streptococci and Lactobacilli, being acidophilic and aciduric, respectively, have been shown to be capable of demineralizing the dental enamel. Gradually, the bacterial surface structures for adhesion, such as fimbrea, fibrils, and other membrane proteins/glycoproteins, act as cues to interact with the pellicle [71,72]. Biofilm formation by mutan streptococci, particularly Streptococcus mutans and Streptococcus sorbinus follows two pathways: sucrose dependent adherence and sucrose independent adherence. In the later route, oral streptococcal surface protein antigen (a cell wall associated adhesin P1 and a member of antigen I/II (AgI/II) encoded by spaP gene) is involved [73, 69]. A 29 kDa surface protein known as wall associated protein A (WapA, antigen A or antigen III) also plays a vital structural role at the cell surface for the formation of biofilm and cell to cell interaction [70].

Sucrose dependent adherence is mediated by glucan binding proteins namely GbpA, GbpB, and GbpC of which GbpC was reported to be involved with rapid dextran dependent aggregation [74]. Furthermore, water insoluble glucans produced from sucrose by glucosyl transferase enzymes Gtf-B, Gtf C, and Gtf D also activate microbes for the formation of the biofilm formation as well as acting as a universal glue that forms the plaque matrix [75-78]. Such insoluble extracellular polysaccharides are an essential factor for Streptococcus mutans biofilm formation. However, glucan-binding proteins (GBPs) present within the salivary pellicle mediate initial adherence to the tooth [79]. Reports show that Streptococcus mutans colonizes the dentinal tubule to a greater extend and the depth of penetration is strain dependent [80] and hence also play a significant role in endodontic infections.

Oral streptococci produce lactic acid as an end-product of their carbohydrate fermentation process which can lead to a rapid-rate drop in environmental pH. Extracellular pH can modulate hyphal growth of Candida and thus biofilm formation as Candida albicans grows in the form of yeast at acidic levels of pH and in the hyphal form at alkaline pH levels [81]. For communication among the interspecies to occur between the plaque microbial communities, cell-cell signaling and cell-cell association is required. Co-aggregation is the leading mechanism behind microbial succession by secondary colonizers or late colonizers via cell surface adhesins [3]. Cell-cell association may decrease the space between the bacteria and enhance the cell-cell signaling and communication [82]. The polymers found on the surface of cells belonging to the same or different species enables cohesion between the organisms with the rigid alpha-1,3 linked glucans being particularly suited for cohesion [83].

Quorum sensing (QS) controls both intra and inter species communication as well as plays a prominent role in allowing bacteria to architect complex biofilm formation [84]. Streptococcus mutans mediates QS by means of Competence Stimulating Peptides (CSP) and a ComD/ComE two-component signal transduction system. The CSP-mediated QS system found in Streptococcus mutans also affects its aciduricity, acidogenicity, and genetic transformation along with bacteriocin production. Along with QS, autoinducer-2 (AI-2) also favors bacterial interactions [85].

The cell wall specific polysaccharide of streptococci and type 2 fimbrea of Actinomyces sp. are well reported for highly selective surface interactions [23]. An inducible high-affinity Mn²⁺ transporter in Streptococcus gordonii mediates its co-adhesion with Actinomyces naeslundii [23]. A lectin like receptor (ScaA) participates explicitly in the interactions of streptococci with Actinomyces, while receptors ScbA, PsaA and SsaB resembling the fimbrial adhesins favor co-aggregation [86]. Glucosyl transferase B (GtfB) produced by Streptococcus mutans binds effectively to both hyphal and yeast forms of Candida albicans in an enzymatically active form.

The resultant glucans formed in situ enhance the binding ability of Streptococcus mutans cells to the Candida albicans cells and promote the colonization of C. albicans on the surface of the tooth [87]. Lactobacilli cell surfaces have an S-layer (macromolecular paracrystalline arrays of proteins/glycoproteins) which determines the surface hydrophobicity. The S-layer is thought to act as an active mediator for bacterial adhesion to the host cells/ECM and also have protective and catalytic functions [88]. Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus plantarum isolated from the tongue, gums, dental plaque, and the saliva showed high hydrophobic properties [89].

The most potent virulence factors of Enterococcus faecalis are aggregation substances (AS), enterococcal surface protein (Esp), extracellular superoxide production, the lytic enzymes gelatinase and hyaluronidase, the toxin cytolysins, and capsular polysaccharides. Each of them may be associated with various stages of an endodontic infection and periapical inflammation [90]. It also act as cues for biofilm formation and microbial interactions. Quorum sensing in Enterococcus faecalis is mediated by auto inducing peptide (AIP) which interacts with the cell surface receptors for co-aggregation and biofilm formation [91].

Multivalent adhesins are present on the cell surface of Fusobacterium nucleatum are thus considered as a middle colonizer since they act as a link between the early colonizers and late colonizers. Specifically, an arginine-inhibitable adhesin (RadD) is responsible for its adherence to Streptococcus species. Some galactose-specific, lectin-like adhesin are involved in binding to the sugar moiety present within the capsule and lipopolysaccharide of the periodontal pathogens like Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Treponema denticola [83]. Tannerella forsythia associated with severe and chronic periodontitis has cell surface glycoproteins in S-layer acting as adhesins in coadhesion with Fusobacterium nucleatum [92]. A leucine-rich repeat in proteins from Treponema denticola and Tannerella forsythia are involved in protein–protein interactions with each other and with Fusobacterium nucleatum [93].

Late colonizers have weak colonizing ability and require partner species primarily constituted of anaerobic Gram-negative bacteria to become attached into the dental biofilms [13]. Among this, Treponema denticola is unable to adhere on oral surfaces as it is only able to colonize to form oral biofilms when Porphyromonas gingivalis is present. Moreover, in sub-gingival dental biofilms, Treponema denticola is typically found associated to Porphyromonas gingivalis [94]. A chymotrypsin-like proteinase (CTLP) found within a high molecular mass complex on the cell surface of Treponema denticola mediates its adherence to other potential periodontal pathogens such as Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Parvimonas micra [95]. Interaction between Veillonella atypica and Streptococcus sp. depends on metabolic cooperation provided by their growth in close proximity.

Metabolite mediated cooperation also favors physiological interactions among the biofilm bacteria. Metabolic communications among oral microbes may occur through the excretion of a metabolite by one bacterium that nourishes another species [96] or through syntrophic biochemical enzymes to cooperatively metabolize a substrate. For instance, the growth of Streptococcus sp. leads to production of lactic acid which acts as a substrate for growth of Veillonella sp. [97]. Also, the short-chain acids like acetate that are produced by the early colonizers via sugar metabolism typically serve as sources of carbon and energy for succeeding colonizers [83]. Interactions occurring among the major cariogens that lead to biofilm formation are shown in (Fig. 4).

Dental caries and periodontitis are the two most important biofilm mediated oral infections. The progression and prevention of dental caries can be arrested by daily habits and clinical therapies for promoting the remineralization. The replacement of lost hydroxyapatite of the tooth tissue by fluorides or other ions and prevention of oral biofilm formation that acts to reduce the pH are mostly employed. Measures of biofilm control like mechanical removal of plaque, systematic and local antibiotics or interventions that artificially increase saliva production are mostly preferred. A prominent reason of primary and secondary endodontic infection can be attributed to the Enterococcus faecalis biofilms. The widespread occurrence of Enterococcus faecalis in primary endodontic infection has been reported to be 40% and in persistent endodontic infection 24 to 77%. Enterococcus faecalis, by virtue of its ability to survive severe environments including detergents, azide, heavy metals, desiccation, and ethanol, colonizes the dentinal tubules and other inaccessible areas within the oral cavity. It also survives high temperature, alkaline pH, salt concentration and bile salts. The biofilm forming ability enables Enterococcus faecalis to survive phagocytosis, antimicrobials and antibodies (Mohamed and Huang, 2007). The inter canal medicaments and dressings even though offer solution, but its failure in inhibiting such microbes and biofilm results in failure of typical root canal therapies. Though canal cannot be reliably rendered free of all bacteria in all of the cases, applying a temporary filling material with an inherent or incorporated microbicidal agent may reduce the overall microbial load.

Microbial Etiology of Secondary Caries

The microorganisms occupying the root canal and various endodontic regions were well established [106]. Bacteria present in dental pulp are associated with periapical diseases. The chemo-mechanical preparation of the tooth during the root canal treatment helps in the removal of the bacterial flora, but the failure in complete removal of the bacterial cells during the treatment or entry of the bacterial cells and nutrients into the root canal via micro-leakage can be a reason for residual caries or secondary caries [107]. Still, microbiology of the secondary caries has not received much attention and also is a disputed matter [108].

Gonzalez-Cabezas elucidated the active roles of Streptococcus mutans, Lactobacilli, and Actinomyces naeslundii in the development of secondary caries [87]. Mo et al. explored the most predominant micro-flora of secondary caries with the major candidates including streptococci mutans, Lactobacilli, Prevotella, Veillonella, Neisseriae followed by Actinomyces, PeptoStreptococcus, Fusobacterium, Porphyromonas gingivalis, and occasionally Capnocytophaga [99]. Actinomyces odontolyticus and Candida spp. were also reported to be key players [109]. The presence of obligate anaerobic species and facultative anaerobes in the secondary caries suggests a bacterial spectrum similar to the micro-flora of subgingival plaque associated with periodontal disease and pulp pathogens. Gonzalea-cabezas also reported the distribution of three cariogenic bacteria: mutans streptococci, Actinomyces naeslundii, and Lactobacillus sp. Among these, 88.9% were mutan streptococci in secondary carious lesions occurring around amalgam restorations [110].

Residual caries persists because of sustenance and viability of primary infection throughout the treatment procedure as well as failure of the endodontic irrigant to eliminate bacteria or the result of an inability of chemo-mechanical instrumentation due to survival of bacteria in various inaccessible locations (isthmuses, accessory canal and apical regions of canals) [111]. Zivko et al. identified undetected residual caries under old fillings that led to the failure of prosthetic dental treatment [112]. Nair et al., reported the difficulties to differentiate between the microorganisms existing from primary infections and the ones that contribute to the secondary infection [111].

It is to be noted that the dentin organic materials contain inherent proteases particularly MMPs and cysteine cathepsins which are activated in the acidic environment. This would further degrade the treated tooth. It is already proven that MMP inhibition controls dentin caries progression and allows natural remineralization of the treated tooth. Hence, during the treatment of coronal caries (mainly in deep and active caries lesions) direct application of MMP solution is performed after the mechanical removal of carious residues before restoration. Chlorhexidine digluconate (CHX) is frequently used MMP inhibitor in dentistry acts by calcium-chelating mechanism but its long term affectivity in a treated tooth is not sure [113, 114]. These inhibitors have been shown to improve the integrity of the hybrid layers obtained by a simplified etch-and-rinse adhesive after dentin caries removal. Another indicative is to incorporate in mouthwashes and toothpastes. In periodontal infections oral administration of MMP inhibitors are also practiced [15]. The acidic activation elevates the level of MMP-2 and MMP-9 which could be due to the inhibition of TIMPs. This could also contribute to further degradation of endodontically treated tooth and aggravate the residual caries [115].