An introduction to oral biofilms

Dental plaque is a classic example of a biofilm in which oral bacteria adhere to the tooth surface and to one another in quite specific patterns;³ and are involved in the pathogenesis of caries,⁴ periodontitis,^5,6 dental implant failures,⁷ denture stomatitis and oral yeast infections such as candidiasis.⁸ Several studies have now also established a link between oral microbes and systemic disease.^9–19

Oral biofilms and periodontal health

Over the years, many researchers have examined the composition of oral biofilms. Socransky et al.²⁰ investigated bacterial distribution of biofilm in differing health states of the periodontium. The authors collected 13,261 plaque samples from healthy and periodontitis patients which were then analyzed using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Five major complexes were consistently observed using any of the analytical methods. The authors then put the microbes identified for each of the five complexes in a different colour group.

The first complex consisted of a tightly related group comprising of Bacteroides forsythus, Porphyromonas gingivalis and Treponema denticola (Red Complex). The second complex consisted of a tightly related core group including members of the Fusobacterium nucleatum/periodonticum subspecies, Prevotella intermedia, Prevotella nigrescens and Peptostreptococcus micros (Orange Complex). Species also associated with this group included Eubacterium nodatum, Campylobacter rectus, Campylobacter showae, Streptococcus constellatus and Campylobacter gracilis. The third complex consisted of Streptococcus sanguis, S. oralis, S. mitis, S. gordonii and S. intermedius (Yellow complex). The fourth complex was comprised of three Capnocytophaga species, Campylobacter concisus, Eikenella corrodens and Actinobacillus actinomycetemcomitans serotype a (Green complex). The fifth complex consisted of Veillonella parvula and Actinomyces odontolyticus (Purple complex). A. Actinomycetemcomitans serotype b, Selenomonas noxia and Actinomyces naeslundii genospecies 2 (A. viscosus) were outliers with little relation to each other and the five major complexes. The first complex (Red) related strikingly to clinical measures of periodontal disease particularly pocket depth and bleeding on probing.²⁰

Another study by Ximenez et al.²¹ proposed that Actinomyces species were the dominant taxa in both supragingival and subgingival plaque from healthy and periodontitis subjects. Four Actinomyces species accounted for 63.2 per cent, of supragingival and 47.2 per cent of subgingival plaque in healthy subjects and 48 per cent and 37.8 per cent in periodontitis subjects respectively. Increased proportions of P. gingivalis, B. forsythus, and species of Prevotella, Fusobacterium, Campylobacter and Treponema were detected subgingivally in the periodontitis subjects. P. gingivalis, B. forsythus and T. denticola were significantly more prevalent in both supragingival and subgingival plaque samples from periodontitis subjects. The main differences between supra and subgingival plaque as well as between health and disease were in the proportions and to some extent levels of Actinomyces, ‘orange’ and ‘red’ complex species.

Haffajee et al.²² described similar results to the previously mentioned publications in that the difference between periodontal health and disease was the absence or presence of T. denticola, P. gingivalis, and B. forsythus respectively. These findings have an impact on systemic health as it has been reported extensively in the literature that the presence of these pathogens has been strongly associated with various systemic diseases.^9–19

While most investigators have studied the composition of mature plaque (biofilms),^20–27 there are few good studies that describe the development of biofilms in the oral cavity. Ritz²⁸ used selective media to enumerate seven ‘genera’ of organisms; Streptococcus, Actinomyces, Corynebacterium, Neisseria, Fusobacterium, Veillonella and Nocardia in two pooled samples from each of six adult dentate subjects at one, three, five, seven and nine days. Streptococci were predominant at day one, comprising an average of 46 per cent of the colonies detected. Neisseria (9.1 per cent) and Nocardia (6.2 per cent) were also high in mean proportions at day one, but decreased in counts and proportions over time (1.8 per cent and 0.1 per cent respectively at nine days). Actinomyces were initially low in proportion (0.18 per cent), but rose to 23 per cent of the microbiota by nine days. He suggested that there was microbial succession in plaque development with aerobic or facultative species reducing the environment for the subsequent growth of anaerobic species.

Shifts in the microbial populations that occurred in supragingival plaque were studied by Socransky et al.²⁹ using predominant cultivable microbiota techniques. Samples were taken and dispersed, diluted and plated on nonselective blood agar plates. The data indicated that few shifts occurred in microbial composition from five minutes to eight hours. There was a marked increase in total counts and counts of specific species that occurred at one day, but this levelled off from two to 16 days. Actinomyces species were high in proportion from five minutes to eight hours, but declined in proportions to one day, increased in proportions from one to two days and levelled out by 16 days. Streptococcus sanguis was detected at all time points, increased in proportion at one day and declined thereafter. This study speciated organisms to the level possible at that time, but was limited in that only one sample site in one dentate subject was studied.

Zee et al.³⁰ used culture techniques to examine one pooled plaque sample from each of five ‘rapid’ plaqueforming and six ‘slow’ plaqueforming dentate subjects at one, three, seven and 14 days. At day one, Streptococcus species comprised an average of 30 and 40 per cent of the isolates in ‘slow’ and ‘rapid’ plaque-formers respectively. By 14 days, mean proportions had declined to 12 and 9 per cent in the respective groups. In contrast, the Actinomyces species rose from a mean of about 10 and 5 per cent in the ‘slow’ and ‘rapid’ groups to about 30 and 15 per cent at 14 days. Gram negative anaerobic rods were low in proportion at day one, but increased at three to 14 days and were significantly higher in the ‘rapid’ plaque formers at 14 days than in the ‘slow’ plaque formers.

Theilade et al.³¹ and Moore et al.³² examined the changes that occurred during biofilm development in experimental gingivitis. They suggested that certain species appeared to be associated with the development of experimental gingivitis. Of these, Actinomyces naeslundii (serotype III and phenotypically similar strains that were unreactive with available antisera), Actinomyces odontolyticus (serotype I and phenotypically similar strains that were unreactive with available antisera), Fusobacterium nucleatum, Lactobacillus species D-2, Streptococcus anginosus, Veillonella parvula, and Treponema species A appeared to be the most likely etiological agents of gingivitis. However, the sequence of changes in plaque development over time was not indicated.

A study by Li et al.³³ examined dental biofilm samples from 15 healthy dentate subjects at zero, two, four and six hours after tooth cleaning using the ‘checkerboard’ DNA-DNA hybridization assay for 40 different bacterial species. The composition of these samples was compared with that of whole saliva collected from the same individuals. They found that the bacterial distribution in biofilm samples was distinct from that in saliva, confirming the selectivity of the adhesion process. In the very early stages, the predominant tooth colonizers were found to be Actinomyces species. The relative proportion of streptococci, in particular Streptococcus mitis and Streptococcus oralis, increased at the expense of Actinomyces species between two and six hours while the absolute level of Actinomyces remained unaltered. Periodontal pathogens such as Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola as well as Actinobacillus actinomycetemcomitans were present in extremely low levels at all the examined time intervals in this healthy group of subjects. These results concluded that the early colonizers of the tooth surface predominantly consisted of hostcompatible microorganisms.³³

Oral biofilms and the edentulous subject

Most studies to date have examined oral biofilms in the dentate subject leaving us with limited knowledge regarding biofilms in the edentulous or complete-denture wearing patient. A recent study by Sachdeo et al.³⁴ provided the first step in defining the organisms that are associated with the edentulous on both, the soft (mucosa) and hard surfaces (denture). For the study, sixty-one edentulous subjects with complete maxillary and mandibular dentures were recruited. ‘Supragingival’ biofilm samples were taken from 28 denture teeth for each subject. Biofilm samples were also taken from the dorsal, lateral, and ventral surfaces of the tongue, floor of the mouth, buccal mucosa, hard palate, labial vestibule, ‘attached gingiva,’ and saliva. Samples were individually analyzed for their content of 41 bacterial species using the checkerboard DNA-DNA hybridization technique. The results from this study showed that periodontal pathogens such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were clearly present in the samples from the edentulous patients. It is important to point out that previous literature stated that Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis disappeared from the oral cavity after extraction of all natural teeth and did not re-appear if hard surfaces, in the form of complete dentures, were provided.^35,36 The finding of these periodontal pathogens in the denture-wearing population by Sachdeo et al.³⁴ is of great concern because if there is an association between these microbes and systemic health,^9–19 then the edentulous population is at equal risk, if not higher, than their dentate counterparts.

At the other end of the age spectrum, studies of the edentulous oral cavity of infants prior to tooth eruption suggest that Prevotella melaninoginica was the most frequently isolated anaerobic species found in 70 per cent of infants.³⁷ Other common anaerobes detected in edentulous infants included Fusobacterium nucleatum, Veillonella species, and nonpigmented Prevotella. The source of the anaerobes appeared to be the mother, because there was a correlation between maternal salivary concentration and the infant’s colonization by these species, particularly P. melaninoginica.³⁸ This data is significant in that one does not see the pathogenic microbiota associated with periodontal disease in the infant population.

The above findings were also observed in a cross-sectional study by Cortelli et al.³⁹ who looked at the colonization of Campylobacter rectus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Tannerella forsythia in the tongue and cheek of newborns and elderly individuals with no teeth. For the study, microbiological samples were taken from the dorsum of the tongue and the cheek mucosa of seventy-four edentulous subjects and analyzed using a bacterial DNA-specific PCR. The authors did not detect Porphyromonas gingivalis and Prevotella intermedia in newborns, but periodontal pathogens were found in the oral mucous membranes of the edentulous adult subjects.

Oral biofilms and dental implants

In an attempt to shed light on dental implant success and failure, several investigators have studied the biofilms on dental implants, implant crowns and implant abutments. Lee et al.⁴⁰ suggested that a history of periodontitis had a greater impact on the peri-implant microbiota than implant loading time. While presence of crowns had only a minor impact on the periimplant microbiota, microbial changes were observed the longer the implants had been in function and in those patients with a history of periodontal or peri-implant infections. Although all implants were successfully osseointegrated, the red complex periodontal pathogens, P. gingivalis and B. forsythus, colonized several implants.

Heuer et al.⁴¹ looked at the crevicular fluid around 14 dental implants/healing abutments over a period of 14 days. Despite massive supragingival biofilm formation, their study did not find any periodontal pathogens from the sulcus fluid around the implants/healing abutments during initial bacterial colonization. They concluded that cellular adherence of peri-implant tissue by means of hemidesmosomal, actin filaments and microvilli, reduced the risk of formation of anaerobic subgingival pockets.

In contrast, a study by George et al.⁴² found that dental implants were colonized by the indigenous periodontal microbiota and were well maintained in patients with a history of periodontitis. No significant association between progressing or non-progressing periodontal or peri-implant sampled sites in terms of loss of attachment and infection with at least one of the searched periodontal pathogens was found, suggesting that the presence of putative periodontal pathogens at peri-implant and periodontal sites may not be associated with future attachment loss or implant failure.

In yet another study of implants in the partially edentulous patient, Quirynen et al.⁴³ determined that initial colonization of peri-implant pockets with bacteria associated with periodontitis occurs within two weeks. Per patient, four subgingival plaque samples were taken from shallow and medium pockets around implants (test sites), and teeth within the same quadrant (undisturbed microbiota as control sites), one, two, four, 13, 26 and 78 weeks after abutment connection, respectively. Checkerboard DNA-DNA hybridization and real-time PCR revealed a complex microbiota (including several pathogenic species) in the peri-implant pockets within two weeks after abutment connection. After seven days, the detection frequency for most species, including the red complex periodontitis microbiota, was already nearly identical in samples from the fresh peri-implant pockets (5 per cent and 20 per cent of the microbiota belonging to red and orange complex, respectively) when compared with samples from the reference teeth. Between weeks 2 and 13, the number of bacteria in peri-implant pockets only slightly increased, with minor changes in the relative proportions of bacteria associated with periodontitis (8 per cent and 33 per cent of the microbiota belonging to red and orange complex, respectively). Although small differences were seen between teeth and implants at week two with cultural techniques, a striking similarity in subgingival microbiota was found from month three on, with nearly identical detection frequencies for bacteria associated with periodontitis for both sites (natural teeth and dental implants). These studies vary in their scope as far as the implications for implant health in the presence of periodontal pathogens, but most of them agree that these pathogens are present in the dental implant patient.

Oral biofilms and systemic health

Connections with pre-term births, low birth weight, diabetes, and risk factors associated with cardiovascular disease and stroke, have previously been made with periodontal disease.^9–19 A study by Seymour et al.⁴⁴ specifically demonstrated that P. gingivalis infection enhanced the development and progression of atherosclerosis in apoE-deficient mice. In a recent publication by Fisher et al.,⁴⁵ periodontal disease was also shown to be a clear risk factor for Chronic Kidney Disease. All these studies prove a strong association between oral infection and systemic disease. This information becomes even more significant with the recent finding of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in the complete-denture-wearing population.³⁴ The presence of these periodontal pathogens in the implant patient has already been established^40,43 but it is now apparent that dentures can also in fact harbour these microbes.³⁴ More investigation of this patient pool is most certainly warranted as the elderly edentulous are a significant portion of the population^46,47 Further, with implants becoming an increasing treatment modality for the dental patient, it is important to conduct more long-term investigations to also study the effect of these periodontal pathogens on implant success and/or failure.

Editorial note: A complete list of references is available from the publisher. This article was originally published in Dental Tribune Asia Pacific Vol. 6, No. 7+8, 2008.

Contact info

Dr Amit Sachdeo
Tufts University – School of Dental Medicine
Department of Prosthodontics
One Kneeland Street
Boston, MA 02111
USA