Clearly salivary flow and content as well as mucin composition can modulate microbial community dynamics (61). Both immune associated and immune-independent salivary factors are involved (61). Microbiome regulatory immune related salivary components such as; lactoferrin, lysozyme, lactoperoxidase, and IgA modulate community life (57). Further, mucins derived from non-immune related cells can also play a distinctive role in adherence/clearance ratio of supragingival bacterial communities (62). Moreover, as will be described below, both host and microbial factors can have a major impact on another paramount dental disease, periodontitis (63).

Of particular relevance to this review are studies by Dige et al., 2020 using Fluorescent In Situ Hybridization (FISH) showing Streptococcus spp., Actinomyces spp., Lactobacillus spp., and Bifidobacterium spp., in pits and fissures of demineralized occlusal carious lesions (44, 64). In this case the anatomy of the pit or fissure provides a home for bacteria that can actively survive in a protected site without having to possess adhesive factors. Therefore, this torturous anatomy obviates the need for the acid producing bacteria to supply its own adhesins (51, 52). Strategies to modify these torturous landscapes over the years have included; (1) application of silver nitrate to infiltrate the deep fissures to kill the microbes and prevent caries (48) including site directed application to deep pits and fissures for annihilation of these microbes (65), (2) prophylactic odontotomy to modify the deep fissures into smooth accessible, cleansable areas (66), (3) use of amalgam to fill or eliminate the pits and fissures after surgical enlargement and (67) and most recently (4) use of plastic sealants to fill the fissure and smooth them out (Table 1) (51). These measures have one thing in common they make-an-effort to change the pit and fissure anatomy to make it less torturous (68), less fissure-like and less likely that microbes can use the original habitat to find fitness in a niche that permit the microbes to multiply, produce acid and cause decay (50).

Smooth surface caries occurs on the buccal, lingual, and proximal surfaces of the tooth. For these surfaces to be colonized microorganisms need to form a biological bond with material on the tooth surface (60). This boundary between the microbe and the salivary coated tooth surface occurs via electrostatic, physicochemical, and biological interactions typically requiring adhesin/receptor contact for prolonged associations (52, 57, 69). While lactobacilli are routinely found in occlusal pits and fissures, they lack surface adhesive properties, and require a retentive area (62). Lactobacilli spp. also thrive at a low pH and require access to carbohydrates (45, 52, 70). Lactobacilli are not found as pioneer colonizers and are considered by many as opportunistic microbes. In contrast, S. mutans has many attributes (adhesins, extra-cellular polysaccharides, glycosyl binding proteins) that enable it to adhere to smooth surfaces (71). Moreover, sucrose metabolism by S. mutans results in a thick sticky polymeric extracellular polysaccharide, glucan, that tends to bind the cells together to form a protective shield (72). S. mutans is not a pioneer colonizer of enamel but in the presence of sucrose with the production of a S. mutans derived gelatinous glucan coupled with fimbrial extensions, other adhesins such as Antigen I/II, Glucosyl transferase, the combination of these factors makes S. mutans a formidable early tooth colonizer (45). S. mutans is both acidogenic and aciduric and thus, once it has colonized, and the pH of the environment changes, S. mutans has an ecological advantage as compared to other acid vulnerable microorganisms. As mentioned, Streptococci spp. for the most part do not require retentive surfaces. In many ways, S. mutans is more adaptable than Lactobacilli spp. and thus can be considered more of a generalist. Damage to the smooth surface of enamel and dentin can change the local environment to one that is favorable to non-adherent bacteria and thus permit lactobacilli spp., a minimally adaptable specialist type microbe to gain a foot-hold in retentive spaces (52). Recent studies indicate that S. mutans produces an extracellular scaffold that forms a rotund structure with an inner S. mutans core. This core “patch” district is surrounded by non-mutans microbes and the patch can be located directly above a demineralized enamel region Figure 2 (60).

While pit and fissure environments can be altered by sealants, smooth surfaces can also be altered. In this case, S. mutans can colonize in the interproximal area below the contact points of two adjacent tooth surfaces making it difficult to remove the biofilm by physical means. Flossing, a physical method of biofilm removal is difficult while antimicrobial rinsing has proven to be somewhat effective and trials have been conducted using chlorhexidine or other antimicrobial rinses and varnishes to interfere with microbial attachment capacity (72, 73). This method plus frequent professional cleanings have been used quite successfully in public health efforts in Sweden in the past (74, 75). Other smooth surface altering methods include, (1) enzymatic effects on salivary proteins (76), (2) antiseptic treatment of tooth surfaces to prevent or reduce attaching bacteria (74, 75), (3) passive application of antibodies to tooth surfaces to block adhesin receptor interactions (77), and (4) as mentioned physical removal of plaque by brushing and flossing. Local application of plant developed antibody to S. mutans Antigen I/II has been successful in showing a reduction in primate recolonization of S. mutans as a passive immunization strategy (78) (Table 1). These studies while showing modest success in changing the local environment have not been applied to practice more than likely because of cost-benefit ratios. Nevertheless, these studies provide additional evidence that alteration of the environment can alter disease. Unlike the carious process where there is a direct effect of biofilm acid production on tooth enamel resulting in demineralization, periodontal disease is more closely aligned with indirect effects more typically aligned with inflammatory soft and hard tissue responses to complex bacterial communities on their surrounding environment (79). Thus, periodontitis is provoked by bacterial biofilms that result in niche activity resulting in tissue inflammation. This inflammatory insult has many wide-reaching and complex effects as seen below (80, 81).

Inflammation, the hallmark of periodontitis, can activate a wide range of tissue damage to epithelial cells, cementum, alveolar bone, and collagen in a time dependent manner (96, 97). Damage to these tissues can cause the release of nutrients, components of collagen, iron, and other proteins and small chain fatty acids (98). These host derived elements can provide new environments compatible with further growth of pathobionts in the anerobic periodontal pocket habitat (99).

The subgingival habitat is a very different than the supragingival habitat. Each habitat is composed of distinct patch or patches that house very different niche types. Serum, the predominant subgingival fluid supplies nutrients such as proteins and fatty acids (100). The inflammatory response to the accumulating subgingival microbiome is intense and clearly directed to prevent invasion of the overwhelming microbial load infringing on the underlying tissue. The dynamic nature of these subgingival microbial interactions is not well elucidated because the habitat is not as readily accessible as the supragingival habitat. However, there are methodologies emerging that can provide a window into this intriguing subgingival habitat.

Methodologies have been introduced that permit study of the subgingival domain. Initially, Transmission Electron Microscopic (TEM) methods were used to study teeth designated for extraction (101). However, these methods suffered from the disadvantage that examination of subgingival plaque biogeography could only be done in a cross-sectional manner, and only gross microscopic identification could be determined (101, 102). Mylar strips were placed subgingivally to assess biofilm development in the subgingival habitat (103) but the mylar surfaces had no relationship to cementum (103). Next cemental strips were acquired from premolar teeth slated for extraction for orthodontic purposes (104). These teeth were cleaned of connective tissue, gamma irradiated for sterilization purposes, cut into 4 strips from one pre-molar, and then placed into pockets that were cleaned. Strips with their microbial contents were removed 7, 14 and 21 days after cleaning. These studies were designed to compare colonization of sites from patients with chronic adult periodontitis (CAP) to those with juvenile localized aggressive periodontitis (LAP) and to those who were “healthy” and had shallow pockets. Microscopic differences were seen as follows, (1) strips from CAP had dense subgingival plaque that often penetrated a demineralized root surface; (2) strips from LAP had a globular deposit on the root surface early on, had significantly less plaque until 14 days after cleaning and had no root penetration, and (3) strips from relatively healthy sites had almost no plaque, had PMNs migrating on either root surfaces or through intercellular spaces on reformed junctional epithelial cells (Figure 3). It was impossible to determine specific microbes using TEM methods. Since then, several methods have shown that microbial identification can be done; first using antibodies directed to the specific usual suspects, then using DNA technologies and most recently and most illuminating using CLASI-FISH which provides a greater potential for defining biogeography (5, 42, 43).

These methods should provide data that will illuminate a better understanding of; (1) who's there, (2) where they are, and (3) how they interact with both the microbial community, and (4) the host response to that challenge. These advanced technologies provide an exciting future for oral microbiologists who are interested in landscape ecology which can lead to a better definition of subgingival niche activities.

Treatment of periodontal disease has had an illustrious history dating back to 1918 when two oral care providers introduced a novel way of using local delivery to retard disease progression. Kritchevsky and Seguin at the Pasteur Institute in Paris introduced the use of neosalvarsan in abscessed pockets that contained fusobacteria and spirochetes (105). Their treatment consisted of local infiltration of a combination of glycerin and 10% neosalvarsan followed by intravenous injections of three low doses of neosalvarsan in addition to a thorough root scaling and polishing (1921). This treatment reduced the presence of the spirochetes as well as the inflammation in the local tissues but at times had adverse side-affects. At some time later Harold Box a prominent scientist and dentist from Toronto introduced a method called oxygen insufflation which was designed to change the subgingival environmental atmosphere from one that was anaerobic to one that was aerobic. While partially successful the substantiveness of this methodology was absent and as such the results were not long lasting (106).

In the late 50's Waerhaug made a point of demonstrating that overhanging restorations posed a serious threat to sustained periodontal health and showed that iatrogenic or poorly performed dentistry can act as a provocative irritant resulting in microbial collections and inflammation in the underlying gingiva (107). The resulting inflammation was due to a challenge to the vasculature directly below the junctional epithelium that caused an exudation of fluid emanating from the underlying vessels and emptying into the gingival crevice and then into the saliva (108). The amount and contents of GCF was dependent on the inflammatory response and could act as a mirror of inflammation in the gingival tissue (109). Work by Page and Schroeder (82) demonstrated that the tissue cellular response could be correlated with inflammation in response to microbial irritants. These classical experiments paved the way toward a better understanding of the challenges of tooth related biofilms to both the supragingival and subgingival space.

One dramatic illustration of this dysfunctional relationship between subgingival biofilm gathering combined with irregular/irritating restorative dental margins was elegantly demonstrated by Lang and associates (110). A restoration was made in the form of a gold inlay that extended across the occlusal surface to both the mesial (front facing side) and distal (back facing side) of a molar tooth. In this experimental model the overhanging restorative margin on the mesial side of the inlay restoration was placed and then the rough irregular surface was allowed to percolate and to collect subgingival plaque for 19 weeks during which time inflammation occurred (110). In conjunction with the inflammation, the subgingival plaque showed an increased growth of Bacteroides app now Porphyromonas gingivalis which gained its iron and menadione from the exacerbated bleeding caused by the irritating dental restoration that plunged into the tissue below (110). This irritant caused a tissue response that resulted in inflammation and bleeding and encouraged the overgrowth of Bacteroides type microbes (specialists). The opposite inlay side on the same tooth (the distal surface) had a well-placed smooth margin that resulted in minimal to no inflammation and no Bacteroides spp. To demonstrate the process was marginal overhang/irritant related, the restoration was redesigned, and the experiment was repeated now placing the overhanging margin on the opposite side (distal) of the inlay (110). This new restoration was kept in place for a similar time-period. Once again, the irritant provoked inflammation, bleeding, and a change of the growth pattern in the subgingival microbiota favored overgrowth of P. gingivalis, while the opposite side remained healthy. This well-designed experiment demonstrated once again how the environment dictates who lives and thrives (Table 1).

Several less invasive but equally dramatic experiments demonstrate that meticulous and consistent removal of supragingival plaque biofilm influences the subgingival microbiota (111, 112). In the first case consistent and precise removal of supragingival plaque with a toothpick by a researcher from volunteers with 4–6 mm pockets, showed a change in the subgingival pattern of microbes in pockets that were 4 mm in depth but had little effect on pockets that were 6 mm or deeper. We can assume from this modest study that removal of the nutrient continuum produced by members of the supragingival habitat which extends from above the gum-line to below has a range of influence that is limited in terms of its impact on the adjacent subgingival microbiome members. In the second case frequent supragingival scaling described a dramatic effect on reducing “periodontopathic” microorganisms in the subgingival domain in shallow pockets (83). Thus, pocket extension of greater than 3 mm is too great a span for consistent passage of metabolic agents required to support growth of subgingival microorganisms. For this health response related affect to be sustainable, the supragingival cleaning method needs to be repeated over time in a consistent manner (83, 111). These results illustrate that both space and time are effective influencers with regard to patch dynamics in these experiments where subjects already had periodontal pockets (113). Bacteria can alter the environment by induction of inflammation and encouraging overgrowth by inflammophilic pathobionts conducive to fitness for the growth of species that favor continuance of disease. Treatment strategies that are designed to remove these modulating bacterial/inflammophilic forces to restore an environment that favors growth of commensals is a desirable goal.

The sequential development of dental plaque from a clean tooth surface provides a clear demonstration that early tooth colonizers referred to as pioneers have great adaptability that then pave the way for secondary residents that follow in a sequential order (111, 114). Most of these pioneers are commensal/generalist while in their designated habitat. However, these commensal microbes can change in circumstances where tissue ulceration occurs (an over-zealous tooth cleaning in a vulnerable patient). Thus a commensal such as S. sanguis has been shown to exacerbate infective endocarditis when heart valves are infected by S. sanguis derived from plaque and deposited on damaged heart valves (115).

Further, recent evidence has shown that Fusobacterium nulceatum, a common oral microbe, can move from the oral cavity and lodge onto colo-rectal cancerous lesions and cause an exacerbated reaction at the cancer site. Elimination of these bacteria have been shown to reduce the lesion size (116). In another example, Aa, a pathobiont in some cases, can colonize the buccal mucosa but the microbe only becomes relevant when it moves to the tooth surface and then moves into a habitat below the gum-line. These movements from one habitat to another are often related to signals created by the up-regulation of genes provoked by hazardous insults (117). Once in the subgingival domain Aa can play a role in periodontitis but it can also move into the blood stream, colonize damaged heart valves in a specific EmA adhesin-receptor interaction and provoke an uptick of infectious endocarditis (118). These examples show how generalist microbes due to their fitness adaptability can be dangerous when they escape their ‘natural” habitat.

Many years ago, a “clear zone” was discovered in the area directly above the gingival margin that was observed three days after study participants abstained from all oral hygiene procedures in the experimental gingivitis protocol Figure 4 (119). This zone occurred in 13 of 16 participants, however, was seen to vanish after one day of abstention in three subjects who were considered to be “heavy plaque formers”. In the context of habitat designation, it was unclear whether this zone was due to saliva that accumulated at the gingival margin or whether it was derived from the inflammatory exudate emanating from the vasculature underlying the gingival epithelium. As time went on plaque overcame this “clear zone” and thereafter the plaque biofilm migrated to the area below the gumline (119). In context of landscape ecology in the early stages of biofilm development on a clean tooth surface this “clear zone” could be an important landmark that separates one habitat (supra-gingival) from a second subgingival habitat. Studying the way in which bacteria move from the zone above the gum-line to the area below the gum-line could reveal a transition zone that could alter disease. Questions posed by this mysterious zone can answer whether there are hazardous signals that bacteria in the supragingival zone receive that up-regulate dispersal genes that permit them to move away from their original habitat? Alternatively, is the supragingival habitat too competitive for less hardy bacteria to compete for available metabolic resources?