Curriculum Vitae Hirschel Wohl Degree and Date to Be Conferred: M.S

Metagenome and Metatranscriptome Analysis of the Subgingival Bacteria in Periodontal Disease. A Systematic Narrative Review

Item Type dissertation

Authors Wohl, Hirschel

Publication Date 2021

Abstract The main purpose of this systematic narrative review is to determine the difference in abundance of bacteria and bacterial genes of subgingival microflora of human periodontal pockets compared to healthy sites, via Metagenomic and Metatranscriptomic ...

Keywords metatranscriptome; Metagenome; Periodontal Pocket-- microbiology; Periodontitis

Download date 02/10/2021 01:38:25

Link to Item http://hdl.handle.net/10713/15785 Curriculum Vitae Hirschel Wohl Degree and Date to be conferred: M.S. May 2021 Collegiate Institutions Attended: 2017-2020 University of Maryland School of Dentistry Candidate for Master of Science in Biomedical Science Periodontics Resident

2013-2017 Howard University College of Dentistry Doctor of Dental Surgery

Professional Positions Held: Postgraduate Resident University of Maryland Baltimore, School of Dentistry

Publication: Issa, K., Wohl, H., Naziri, Q., McDermott, J. D., Cherian, J. J., & Mont, M. A. (2013). Early results of total hip arthroplasty in the super-obese patients. Journal of long-term effects of medical implants, 23(4).

Current Committee Memberships: Dr. Mary Elizabeth Aichelmann-Reidy Dr. Robert Ernst Dr. Harlan Shiau Dr. Christine Hsu

Abstract

Title: Metagenome and Metatranscriptome Analysis of the Subgingival Bacteria in Periodontal Disease. A Systematic Narrative Review Hirschel Wohl, Master of Science, 2021 Thesis Directed by: Dr. Mary Elizabeth Aichelmann-Reidy

The main purpose of this systematic narrative review is to determine the difference in abundance of bacteria and bacterial genes of subgingival microflora of human periodontal pockets compared to healthy sites, via Metagenomic and Metatranscriptomic analyses. Databases EMBASE and MEDLINE were searched for articles, with earliest records from 1978. Main outcome measures included: 1) Bacterial genera and/or species significantly increased 2) Most prevalent or significantly upregulation of genes. Ten studies met selection criteria and were included in the study. Nine studies were cross- sectional, and one was longitudinal. Main results showed trends of specific bacteria and genes found in periodontal pockets. However, within the limitations of this narrative review, trends of abundant bacteria and genes does not imply these specific species or genes are actively participating in disease progression. Nine of ten included studies were cross-sectional in design with eight studies being metagenome based and not able to measure gene expression.

Metagenome and Metatranscriptome Analysis of the Subgingival Bacteria in Periodontal Disease. A Systematic Narrative Review

by Hirschel Wohl

Thesis submitted to the Faculty of Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Master of Science 2021

Table of Contents

Introduction……………………………………………………………………………..1 Background of periodontitis…………………………………………………….1 Prevalence……………………………………………………………………….2 Etiology of Periodontitis………………………………………………………...3 Putative Periodontal pathogens…………………………………………………4 Studies of Association ………………………………………………….5 Studies of Host Response ………………………………………………7 Studies of Elimination…………………………………………………..9 Virulence Factors………………………………………………………..10 Studies of Animal Pathogenicity………………………………………..11 Keystone Pathogens and Dysbiosis …………………………………………….12 Pathogenesis and Host Response ……………………………………………….13 T and B Cells……………………………………………………………15 Osteoimmunology……………………………………………………….16 Methodology of Studying Bacteria……………………………………………...17 16s rRNA and Culture Independent Methods…………………………..17 Metagenomics and Metatranscriptomics………………………………..17 Purpose…………………………………………………………………...... 19

Materials and Methods………………………………………………………………….19 Search Protocol…………………………………………………………………19 Selection Criteria………………………………………………………………..20 Inclusion Criteria……………………………………………………….20 Exclusion Criteria………………………………………………………20 Data Collection and Analysis…………………………………………………..20 Data Extraction and Outcome Measures……………………………………….21

Results…………………………………………………………………………………..22

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Study Characteristics……………………………………………………………22 Bacteria of Most Abundance…………………………………………...... 26 Healthy Sites…………………………………………………………….26 Periodontitis Sites……………………………………………………….31

Most Prevalent or Upregulated Genes in Periodontitis Sites…………………...38 Upregulated Genes at Progressing sites………………………………………...39 Species Expressing Increased Virulence factors at Periodontitis

Sites……………………………………………………………………………..40

Species Expressing Increased Virulence Factors at Progressing sites………….40

Upregulated Genes and Virulence Factors in the Red complex Bacteria………41

Viruses and Archaea Proportion………………………………………………..44

Discussion………………………………………………………………………………45 Limitations……………………………………………………………………...49

Conclusion………………………………………………………………………………49 References………………………………………………………………………………51

List of Tables

Table 1: Excluded Studies from Systematic Review……………………………………22 Table 2: Included Studies for Systematic Review………………………………………24 Table 3: Most Abundant Bacterial Genera at Healthy Sites…………………………….27 Table 4: Most Abundant Bacterial Species at Healthy Sites……………………………28 Table 5: Most Abundant Bacterial Genera at Periodontitis Sites……………………….31 Table 6: Most Abundant Bacterial Species at Periodontitis Sites………………………34 Table 7: Most Prevalent or Upregulated Genes in Periodontitis Sites………………….38 Table 8: Most Upregulated Genes in Periodontitis Sites (Longitudinal Study)………..40 Table 9: Upregulated Genes and Virulence Factors in the Red Complex Bacterial Species in Periodontitis Sites……………………………………………………………………42 Table 10: Virus and Archaea Proportion in Periodontitis Sites………………………...44

List of Figures

Figure 1: Summary of Screening Process……………………………………………..21 Figure 2: Most Abundant Bacterial Genera at Healthy Sites by the Number of Reported Studies…………………………………………………………………………………29 Figure 3: Most Abundant Bacterial Species at Healthy Sites by the Number of Reported Studies…………………………………………………………………………………30 Figure 4: Most Abundant Bacterial Genera at Periodontitis Sites by the Number of Reported Studies………………………………………………………………………36 Figure 5: Most Abundant Bacterial Species at Periodontitis Sites by the Number of Reported Studies………………………………………………………………………37

List of Abbreviations Abbreviation Meaning CP Chronic Periodontitis

AgP Aggressive Periodontitis

AAP American Academy of Periodontology

EFP European Federation of Periodontology

NHANES National Health and Nutrition Examination Survey

JP Juvenile Periodontitis

AP Aggressive Periodontitis

DNA Deoxyribonucleic acid

IgG Immunoglobulin G

IgA Immunoglobulin A

IgM Immunoglobulin M

CDT Cytolethal distending toxin

C5a Complement factor 5a

C5aR Complement factor 5a receptor

PSD Polymicrobial synergy and dysbiosis

RANKL Receptor activator for nuclear factor kappa beta ligand

NK Cell Natural killer cell

Th Cell (Th1,Th2,Th17) T helper cell

IL (IL-1,IL-4,IL-17) Interleukin

TNF-alpha Tumor necrosis factor

M-CSF Macrophage colony stimulating factor

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OPG Osteoprotegerin rRNA Recombinant RNA rDNA Recombinant DNA

HOMIM Human oral microbe identification microarray

PCR Polymerase chain reaction

HOMD Human oral microbiome database

NGS Next generation sequencing

WGS Whole genome sequencing

HMP Human Microbiome Project mRNA Messenger RNA

PPD Pocket probing depth

GO Gene ontology

KEGG Kyoto encyclopedia of gene and genome

CAL Clinical attachment loss

LPS Lipopolysaccharide

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Introduction Background and Terminology

Periodontitis is “microbially-associated, host mediated inflammation that results in loss of periodontal attachment” (Tonetti 2017). Various forms of periodontitis have long been recognized, and previously categorized based on the age of the individual. The diagnoses, adult periodontitis and early onset periodontitis, were used to describe periodontal disease with juvenile periodontitis, pre-pubertal and rapidly progressive periodontitis being under the umbrella term “early onset periodontitis” (AAP 1989). The descriptive terms for these diagnoses implied age was the distinguishing difference between the forms of periodontitis. However, in 1999, the International Workshop for Periodontal Diseases and

Conditions replaced the terms adult and early onset with chronic and aggressive periodontitis. This was because age alone could not be considered the distinguishing characteristic between the various forms of the disease. The term chronic periodontitis

(CP) was used because it is non-specific, and characterizes a disease entity that is many times slowly progressive (Armitage 1999). Aggressive periodontitis (AgP) was used on the other hand since it has certain features that distinguish it from chronic periodontitis.

These features are characterized and divided into primary and secondary. The primary features include rapid attachment loss, an otherwise systemically healthy patient, and familial aggregation. Secondary features are many and can include elevated proportions of Aggregatibacter actinomycetemcomitans, as well as Porphyromonas gingivalis in some populations (Geurs 2015).

More recently, in the 2017 AAP and EFP world workshop, the classification system for periodontitis changed to a staging and grading modality. Where staging classifies the

1 current severity and grading estimates the current future risk of disease progression. The diagnosis aggressive periodontitis was “retired” from the diagnostic system due to the lack of evidence of a specific pathophysiologic process to differentiate aggressive from chronic periodontitis. Within the current classification system there remains an acknowledgement of a rapid rate of periodontal progression which is representative of a grade C diagnosis. However, the pathophysiologic process appears to be the same regardless of the grade (Tonetti 2017).

Prevalence

The prevalence of periodontal disease in the United States ranges from 35%-47% according to epidemiological studies (Albander 1999, Eke 2012, Eke 2015). Albander et al. found 35% of adults to have periodontitis with 21% being mild and 12% being moderate to severe (Albander,1999). Using NHANES data, Eke et al. found 47% of adults to have periodontitis with 8% being mild, 30% moderate, and 8% severe (Eke

2012). In an update in 2015, Eke found the prevalence to be 46% with the highest prevalence found among Hispanics (63%) and non-Hispanic Blacks (59%) (Eke 2015).

However, the prevalence of the former disease classification known as aggressive periodontitis (AgP) is reduced compared to chronic periodontitis (CP). Melvin et al determined the prevalence and sex ratio of juvenile periodontitis from a military recruit population aged 17-26. They found an overall prevalence of 0.76%, with Blacks having a higher prevalence than Caucasians, 3.8% compared to 0.09%. Also, Black males had a higher prevalence than females 3.8% compared to 1.9% (Melvin 1991). Cogen et al in a

2 retrospective cross-sectional study determined the prevalence among Whites to be 0.3%, with a female/male ratio 4:1 and Blacks the prevalence was 1.5%, with a female/male ratio 1:1 (Cogen 1992). More recent epidemiologic studies have shown overall prevalence levels ranging from 0.6% to as high as 6.7%, with higher prevalence levels found with African and Middle eastern individuals, as compared to Caucasians (Eres

2009, Levin 2006, Susin 2011, Albander 2002)

Etiology

The bacterial plaque biofilm has been well established as a primary etiologic factor in the development of gingivitis and periodontitis. Loe et al studied 12 dental students (2 females, 10 males) with a mean age of 23, who went 10-21 days without oral hygiene.

They showed that removal of tooth brushing leads to marginal gingivitis between 10-21 days depending on the subject. Moreover, it was found that bacterial flora changed from

Gram-positive cocci to Gram-negative cocci and spirochetes with removal of oral hygiene. However, upon reinstitution of oral hygiene the gingiva returned to health in less than 7 days and the bacterial flora returned to baseline (Loe 1965). Waerhaug in a study on extracted teeth, found a high degree of congruence between the plaque front and the attachment loss. Attachment loss occurred when the plaque was 0.2- 1.5mm or less from the attachment fibers (Waerhaug 1977). Based on these studies, the bacterial plaque biofilm seems to be associated with the process of gingivitis and the loss of clinical attachment in periodontal disease. Also, elimination of the etiology and re-establishing health, gives further evidence that plaque is an etiologic factor.

Longitudinal studies further substantiate the claim of bacteria being the etiologic agent for periodontal disease (Lindhe 1982, 1984, Ramfjord 1982, Axelson 1981). Lindhe et al reported 5 year results of patients comparing scaling and root planing to flap surgery. It was found that patients who maintained good oral hygiene had superior clinical results compared to patients who failed to perform adequate oral hygiene (Lindhe 1984).

Axelson et al evaluated the efficacy of maintenance for subjects with advanced periodontal disease. Subjects were followed for 6 years and placed in maintenance program which consisted of a maintenance interval every 2 weeks followed by a 2 to 3 month intervals. They found that subjects in the maintenance program maintained their clinical attachment levels, however subjects not in the maintenance program showed signs of recurrent disease (Axelson 1981).

Putative periodontal pathogens

In 1992, Socransky et al outlined criteria that help define bacterial species as periodontal pathogens. These criteria include an association, elimination, host response, virulence factors, and animal pathogenicity ( Socransky 1992). An association requires elevated levels or increased frequency of a pathogen in periodontal lesions. Elimination of the bacteria requires a successful therapeutic response clinically following elimination of the bacteria. Host response shows elevated levels of antibodies to specific bacteria in subjects with periodontal disease. Established virulence factors of bacteria help support its purported role as a pathogenic bacterium. Finally, animal studies show that the bacteria can induce disease in an animal model. These types of studies will be highlighted for the different bacteria that have been shown to be periodontal pathogens.

Studies of Association

Several studies have shown an association between specific periodontal pathogens and periodontal disease (Listgarten 1978, Slots 1980, Zambon 1983, Loesche 1985,

Socransky 1998). Using darkfield microscopy, Listgarten et al analyzed the bacterial flora in healthy and diseased sites in subjects with chronic periodontitis. They found that diseased sites had a higher ratio of spirochetes compared to healthy sites. This established that a different microbial flora can exist in periodontal disease sites (Listgarten 1978).

Similarly, Loesche et al analyzed the bacterial profiles of adult periodontitis subjects, as well as localized juvenile periodontitis. They obtained plaque samples from subjects and used anaerobic chambers to analyze the anaerobic bacteria in the sample. They found higher proportion of spirochetes in subjects with adult periodontitis and to a lesser extent the Bacteroides phylum B. gingivalis and B. intermedius. They also noted that subjects with localized juvenile periodontitis had higher proportions of B. gingivalis and B. intermedius but did not have detectable amounts of A. actinomycetemcomitans. It should be noted that other studies at this time period found a higher prevalence of A. actinomycetemcomitans in these subjects (Loesche 1985). Perhaps this was due to the small sample size (N=4) of the localized juvenile periodontitis subjects.

In comparing adult periodontitis (AP) to juvenile periodontitis (JP), Slots et al found that

9 out of 10 subjects with JP were more extensively infected with A. actinomycetemcomitans compared to AP patients with 6 out of 12 subjects infected with

A. actinomycetemcomitans (Slots 1980). Zambon et al further showed a higher

5 prevalence of A. actinomycetemcomitans in subjects with JP compared to AP. They found

A. actinomycetemcomitans was present in 28 out of 29 subjects (96.5%) with JP compared to 28 out of 134 (18%) AP subjects (Zambon 1983).

Further studies have analyzed periodontal pathogens involved in periodontitis using molecular-based approaches. In 1998, Socransky et al described the red complex organisms as those bacteria associated with periodontal disease. Using genomic DNA probes and checkerboard DNA-DNA hybridization, the presence of 40 subgingival bacteria were analyzed. Using cluster analysis, bacteria were grouped into 5 different clusters/complexes based on the association between bacterial species. These clusters included the red, orange, yellow, purple, and green complexes. Firstly, they showed that there appears to be a certain synergy between bacterial species based on cluster analysis.

Secondly, they detailed the bacterial species in the red complex which are associated with periodontal disease. The organisms described in the red complex consists of P. gingivalis,

T. denticola, and B. forsythus. With community ordination analysis, they found that sites harboring all three bacteria were higher than expected. Sites lacking red complex bacteria showed the shallowest probing depth while sites with bacteria from the red complex showed deeper probing depths (Socransky 1998).

Using DNA hybridization, Faveri et al identified the microbial etiology of localized aggressive periodontitis. A. actinomycetemcomitans was found more in shallow and intermediate pockets than deep pockets and it was shown to have a negative correlation with age, and proportion of red complex bacteria. It was shown that A. actinomycetemcomitans plays a role in early stage of the disease and Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Campylobacter gracilis,

Eubacterium nodatum, and Prevotella intermedia have roles in disease progression

(Faveri 2009).

Studies of Host Response

The ability of the bacteria to elicit a host response to specific periodontal pathogens has been shown in many studies and has been suggested to be protective (Offenbacher 1996).

In general, antibodies can attach to bacteria blocking their ability to attach to the host, as well as trigger complement mediated damage and opsonize bacteria for phagocytosis

(Male 2013). However, there appears to be host related factors as well as environmental factors like smoking which may impact the level of antibody titers (Lu 1994,

Graswinckel 2004). Mandell et al longitudinally followed eight patients with juvenile periodontitis and found that A. actinomycetemcomitans was related to a marked increase in attachment loss. Moreover, seven out of the eight subjects were evaluated for elevated serum IgG antibody levels. The results showed that all the patients screened had elevated serum IgG levels to the same A. actinomycetemcomitans serotype B. Other elevated serum antibody levels were noted for F. nucleatum, B intermedius, B. gracilus, B. gingivalis and E. corrodens. (Mandel 1987).

Ebersole et al evaluated the antibody response to Bacteroides species including B. gingivalis and B. intermedius in subjects with periodontal disease. They found a significant increase in serum IgG levels to B. gingivalis in adult and advanced destructive periodontitis subjects. Also, B. intermedius was significantly increased in the advanced destructive periodontitis subjects (Ebersole 1986). Lopatin et al further evaluated

7 antibodies reactive with P. gingivalis in subjects with adult periodontitis. They found mean IgG levels were increased to P. gingivalis in periodontitis subjects compared to controls. Furthermore, they found higher avidity levels of antibody levels in periodontitis patients compared to controls. However, they noted that the absolute level of avidity was relatively low compared to a rabbit model. They hypothesized the low avidity in humans may be due to chronic exposure to the oral flora (Lopatin 1991).

Lu et al evaluated the levels of IgG2 to A. actinomycetemcomitans in localized juvenile periodontitis subjects, adult periodontitis, early onset periodontitis and control subjects.

IgG2 has been reported to be the predominant IgG isotype produced in response to lipopolysaccharide found on bacteria (Scott 1988). They found that serum IgG2 levels were elevated about 30-40% in localized juvenile periodontitis subjects compared to matched controls. They concluded that some of the elevated levels of IgG2 is probably attributable to the high response to A. actinomycetemcomitans, but more likely that localized juvenile periodontitis subjects have higher levels of IgG2 subclass which may make them more susceptible (Lu 1994).

Graswinckel et al evaluated the total levels of IgG, IgA, and IgM as well as the isotypes for IgG in periodontitis subjects and further compared smoking subjects to non-smokers.

IgG has 4 isotypes (IgG1, IgG2, IgG3, and IgG4) which are structurally and functionally different and therefore have different binding affinities to antigens (Male 2013). Firstly, they found higher levels of IgG1 and IgG2 were observed compared with healthy control subjects. However, smoking was associated with no or minimal rise in serum levels of

IgG1 and IgG2 even though they had increased levels of periodontal pathogens. This lack of increase was irrespective of severity of periodontitis and or having a positive culture of

A. actinomycetemcomitans and P. gingivalis (Graswinckel 2004). Thus, it appears that smoking may modify the antibody response which may be a factor in smoking being a risk factor for periodontitis as seen in other studies (Stoltenburg 1993, Haber 1993,

Tomar 2000).

Studies of Elimination

In a longitudinal study, Slots et al treated six subjects with localized juvenile periodontitis via a three stage approach. Scaling and root planing with subsequent 10% betadine solution administration followed by a 14 day administration of systemic tetracycline (1g/day). Microbiological sampling was performed for all 6 subjects. It was found that tetracycline lowered subgingival counts of A. actinomycetemcomitans and

Capnocytophaga and spirochetes to below detectable levels in most periodontal pockets for 8 months, whereas scaling alone did not. They also found that deep pockets with continued periodontal destruction harbored A. actinomycetemcomitans whereas those with attachment gain did not harbor A. actinomycetemcomitans (Slots 1983).

Mandell et al evaluated the clinical and microbiological effects of surgery plus doxycycline in 1988. In this study, eight localized juvenile periodontitis patients were followed for one year. Seven of the eight patients showed mean attachment gain, however there were two sites in two separate subject that had continued loss of attachment and recurrence of disease. The continued presence of A. actinomycetemcomitans was hypothesized to be responsible for the further destruction in

9 these patients since both patients harbored increased biomass of A. actinomycetemcomitans. (Mandell 1988).

Mombelli et al evaluated the effects of scaling and modified Widman flap on seventeen periodontitis subjects. Clinical parameters and microbial sampling were assessed prior to and after therapy in fourteen of the seventeen patients. They found a significant relationship between the tendency for a site to bleed upon sampling and the number of P. intermedia/nigrescens sites. It was also found that persisting pockets deeper than 4mm after therapy had a higher presence of P. gingivalis. For every millimeter of residual probing depth, the odds of detecting P. gingivalis increased by a factor of 2.47 (Mombelli

2000).

Virulence Factors

The virulence of several periodontal pathogens namely P. gingivalis , T. denticola, T. forsythia, and A. actinomycetemcomitans have been studied extensively. The virulence of these bacteria allows them to evade and invade the host, as well as colonize and multiply within the host. Fives-Taylor et al described and detailed many of the virulence factors for A. actinomycetemcomitans (Fives-Taylor 1999). A. actinomycetemcomitans contains leukotoxin and is therefore capable of killing leukocytes allowing it to evade the host (

Lally 1989). Also, A. actinomycetemcomitans releases Cdt (cytolethal distending toxin) which can inhibit growth of human fibroblast cells (Sugai 1998). Other examples of virulence factors of A. actinomycetemcomitans include the release of bacteriocin, lipopolysaccharide (endotoxin), and antibiotic resistance markers (Fives-Taylor 1999).

Both P. gingivalis and A. actinomycetemcomitans are capable of invading the hosts epithelial cells (Lamont 2000, Meyer 1996). P. gingivalis has also been shown to release gingipains which can lead to hemoglobin breakdown as well dysregulation of the immune response (Lewis 1999, Curtis 2001). T. denticola contains a trypsin-like protease gene designated opdB which allow it to hydrolyze peptide substrates (Fenno 2001). It also has the ability to induce and degrade cytokines, as well as inhibit fibroblast and neutrophil migration (Ishihara 2010). T. forsythia has been shown to have surface proteins which can mediate attachment to epithelial cells (Lee 2006). Collectively, these virulence factors further validate these bacteria as periodontal pathogens.

Animal Pathogenicity Studies

Holt et al compared the affects of ligatures inoculated with B. gingivalis to a control without B. gingivalis in cynomolgus monkeys. They found that monkeys with implanted

B. gingivalis had increased systemic antibody level to B. gingivalis, as well as significant bone loss (Holt 1988). In a murine model, Kimura et al induced experimental periodontitis via silk ligatures pre-incubated with P. gingivalis and compared to a negative control (no ligature) and sham control (ligature without P. gingivalis). They found an increase in bone loss after 9 weeks of infection in mice infected with P. gingivalis and in the sham control. After 13-15 weeks of infection, the mice infected with

P. gingivalis showed a significantly higher amount of bone loss compared with the sham- infected mice. This suggests that P. gingivalis enhances alveolar bone loss in a murine model (Kimura 2000).

Kesavalu et al showed a possible polymicrobial synergy between P. gingivalis, T. denticola, and T. forsythia in a rat model. Rats were infected either with P. gingivalis, T. denticola, or T. forsythia or all 3 bacteria species along with and without F. nucleatum.

The results showed that rats infected with all 3 bacteria species irrespective of F. nucleatum had increased bone resorption as measured by radiographs compared to rats infected with just one bacteria species (Kesavalu 2007). These results seem to show a synergy which may result in periodontitis progression.

Keystone Pathogens and Dysbiosis

In a review paper, Hajishengallis et al described the term keystone pathogen and its relationship to the microbial community (Hajishengallis 2012). A keystone pathogen supports and stabilizes the microbiota associated with disease. This can occur through indirect or direct effects or by both mechanisms. An example of this can be seen with the keystone pathogen P. gingivalis. The indirect mechanism occurs through modulation of the host response leading to dysbiosis of the microbiota. This can occur when P. gingivalis releases gingipains which cleave the complement construct C5a. C5a induces the activation of C5aR which can lead to inflammation and impaired leukocyte killing

(Liang 2011). Impaired leukocyte killing may then allow uncontrolled growth of other species in the biofilm leading to dysbiosis. The direct mechanism occurs through direct effects on the microbiota. Frias-Lopez et al showed via metatranscriptome analysis that introducing P. gingivalis into a healthy biofilm can alter the community gene expression

(Frias-Lopez 2012). Thus, the keystone pathogen hypothesis proposes that these

12 pathogens can exert their effects on the symbiotic microbiota leading to a dysbiosis and further diseased state.

Hajishengallis et al further described the polymicrobial synergy and dysbiosis (PSD) model of periodontal disease pathogenesis which contrasts with the specific plaque hypothesis theory. Evidence against the specific plaque hypothesis include the finding of periodontal pathogens like P. gingivalis in healthy sites. Furthermore, the periodontal microbiota is very diverse containing over 700 species with many novel species correlating with disease even more so than the red complex bacteria (Kumar 2005). In the proposed model by Hajishengallis, specific interaction and communication amongst species of bacteria result in a dysbiotic synergistic microbiota. Keystone pathogens such as P. gingivalis interact with oral streptococci and can increase virulence of the entire microbial community leading to a dysbiosis within the community (Hajishengallis 2012).

Pathogenesis and Host response

Based on the studies cited above, the bacteria plaque biofilm can be considered the primary etiology and specific periodontal pathogens or “keystone species” may lead to the development of periodontitis via dysbiosis of the microbiota. Although bacteria may have the ability to directly cause degradation of the periodontium (Uitto 1983), most studies find that the host immune response is responsible for the breakdown of the periodontium (Offenbaucher 1996).

Page and Schroeder described based on animal and human studies, a histopathologic process leading to gingivitis and periodontitis. They defined 4 types of lesions beginning

13 with initial, early, and established lesions which are representative of gingivitis. The final lesion is an advanced lesion representing periodontitis. They described a sequence of cellular and structural events. The hallmarks of initial and early lesion are an increase in neutrophils and subsequent increase in macrophages and T cells. The established and advanced lesions have an increase in plasma cells. Structurally, there is an apical migration of the junctional epithelium, cytopathological changes in fibroblasts along with loss of collagen, and finally alveolar bone loss. Importantly, they noted that not every established gingivitis lesion develops into an advanced lesion. Rather the established lesion can be stable indefinitely without becoming periodontitis (Page 1976). It is thought therefore that the host immune response is the factor involved in the transition from gingivitis to periodontitis.

Various immune response mechanisms have been proposed that may cause breakdown of the periodontium. Cells such as neutrophils, macrophages, T cells or B cells, along with proinflammatory cytokines have all been implicated in connective tissue breakdown and bone resorption (Gemmel 2007, Hajishengallis 2017, Silva 2015). At the innate immunity level, the polymorphonuclear neutrophil has a protective function and is one of the first responders toward the site of periodontal inflammation controlling the bacteria

(Miyasaki 1991). It is hypothesized that periodontal disease can result from failure of specific aspects of the neutrophil in its interaction with the pathogen. This failure is either because of a phenotypic variation within the host or bacterial evasive strategies as both P. gingivalis and A. actinomycetemcomitans have been shown to affect neutrophil migration

(Gemmel 2007). Furthermore, it is now recognized that neutrophils can express RANKL

(a ligand involved in bone hemostasis) along with releasing matrix metalloproteinases,

14 both of which are implicated in the breakdown of the periodontium. Another potential mechanism involves neutrophils downregulating other leukocyte cell types such as macrophages, NK cell, dendritic cells, and T cells (Hajishengallis 2015).

T and B cells

T cell mediated adaptive immune response involves the differentiation of a Th precursor cell into Th1, Th2, Th17, or Treg. Each of these cells can produce various cytokines and further influence the immune response. Th1 cells are associated with macrophage activation while Th2 are associated with activation of B cells and antibody production along with mast cells and eosinophils. Th17 is associated with neutrophil recruitment via

IL-17, while Treg is associated with protective features which help regulate the immune response (Male 2013, Silva 2015).

In a in situ study, Reinhardt et al found that B cells were found in high amounts in infiltrate from active disease sites (Reinhardt 1988). This confirmed Page and Schroders findings (Page 1976), and it was thought that this increase in B cells was due to T helper cells. Seymor et al proposed that there is a shift in the lymphocyte population from T cells in gingivitis to B cells in periodontitis. Th1 cells release interferon-gamma and have been shown to have protective effects in periodontal lesions, while Th2 are associated with B cells and humoral immunity (Gaffen 2008). Thus, the Th2 cell response may be involved in the progression of periodontitis.

Recent studies have looked at the Th17 cell subset and its role in periodontal disease

(Gaffen 2008). Cytokines that are secreted from Th17 cells are seen in periodontally

15 diseased tissue. It is proposed that Th17 cells secrete Il-17 which can contribute periodontal disease. Zhao et al in a human study looked at cytokines of T cell subsets

(Th1,Th2, and Th17) after non-surgical therapy. They found that there was a significant decrease in cytokines for Th17 cells compared to Th1 cells. They also found the Th2 cytokine IL-4 increased after therapy and therefore may provide a protective effect in periodontitis progression (Zhao 2011). Thus, the Th17 cell may play a more substantial role in periodontal lesions compared to other T helper cells as previously thought.

Osteoimmunology

The biological process of bone resorption involves osteoblasts secreting RANKL and macrophage colony stimulating factor (M-CSF) which bind to macrophages via receptors. Both secreted products help differentiate a macrophage into an osteoclast which leads to osteoclastogenesis. This process is highly regulated as osteoblasts also secretes OPG which acts as decoy and compete with RANKL for the RANK receptor

(Teitelbaum, 2000). Proinflammatory cytokines including TNF-alpha, IL-17, IL-1 beta can induce RANKL expression thereby implicating their involvement with periodontal disease. However, in periodontal lesions it has been shown that immune cells including T and B cell are the major source of RANKL thereby further substantiating their involvement in bone resorption and periodontal disease progression (Hajishengallis

2017).

Methodology of studying bacteria

The methodology for elucidating bacteria in a periodontal pocket has changed drastically over the years. In the early 20th century studies, used culturing techniques along with microscopy to describe the microflora (Lindhe 2015). By the 1970s and 1980s, researchers cultivated subgingival bacteria in anerobic jars and chambers (Socransky

1970, Moore 1982, 1983). The studies by Moore et al looked at chronic and aggressive periodontitis and found 190 taxa, although half of the taxa could not be identified. These methods elucidated the bacteria in great detail, however at the same time were very time consuming and labor intensive (Wade 2011). Thereafter, the use of molecular techniques began with using DNA probes to target specific bacteria in a checkerboard format. The

DNA-DNA-checkerboard hybridization method identified around 40 bacterial species in a relatively efficient manner (Socransky 1998).

16s rRNA and culture independent methods

Further technology has taken advantage of 16s rRNA and PCR methodology. The 16s rRNA is a component of the 30s small subunit of bacterial ribosomes. The genes coding for 16s rRNA are on 16s rDNA and contain hypervariable regions that provide species specific sequences. The HOMIM (Human oral microbe identification microarray) has allowed investigators to detect up to 300 bacterial species (Lindhe 2015). This is achieved via isolating 16s rRNA from clinical samples and PCR amplification of the sequences. These sequences are then fluorescently labeled and hybridized to 16s rRNA oligonucleotide probes on glass slides. The bacterial profiles are elucidated via bands in

17 which band intensity signifies the relative abundance of an organism. In 2010, the

HOMD (Human Oral Microbiome Database) was launched. This is a database of over

700 bacterial species based on 16s rRNA technology and it allows a bacterial strain to be referenced using this library. Some of the disadvantages of using 16s rRNA include that it may miss sequences not on the 16s rRNA genes and some species have similarities and can be difficult to separate using this technology (Wade 2011).

Metagenomics and Metatranscriptomics

More novel methods of describing the subgingival microbiome include whole genome shotgun (WGS) sequencing also known as next generation sequencing (NGS). This can be achieved via different NGS technology such as Illumina and 454 pyrosequencing (Xu

2014). The basic technology involves DNA or RNA samples which are isolated from a sample and broken into fragments. These fragments are then sequenced using a specific platform where sequence adaptors are added and are then PCR amplified (Illumina-An

Introduction to NGS). Sequences can then be aligned to genome databases such as

HOMD or HMP (Human Microbiome Project) and differential expression analysis or gene set / pathway analysis can be achieved (Solbiati 2018). Metatrancriptome analysis is similar to metagenome but instead isolates RNA from the plaque sample and is sequenced using next generation sequencing technology. The advantage of this technology is that it can analyze gene expression of the entire microbiota in the sample based on the mRNA transcripts.

To date, there are no systematic narrative reviews of metagenome and metatranscriptome studies of subgingival periodontal pockets compared to healthy control sites.

Purpose

The main purpose of this systematic narrative review is to determine the difference in abundance of bacteria and gene expression of subgingival microflora of human periodontal pockets compared to healthy sites, via Metagenomic and Metatranscriptomic analyses reported in the literature. A secondary aim is to determine these differences

(bacteria abundance and gene expression) in progressing pockets.

Materials and Methods

Search Protocol

The databases EMBASE and MEDLINE were searched for articles by one investigator

Mary-Ann Williams (MAW). Searches were not limited by year and earliest records were from 1978. The basic search strategy was the following:

1) exp Periodontitis/ (30354)

2) exp *Periodontitis/ (21411)

3) *Metagenomics/ or metagenom$3.ti,ab. or *Metagenome/ or *Transcriptome/ or

Transcriptome$1.ti,ab. (76363)

4) exp *Microbiota/ or microbiota.ti,ab. or microbiome.ti,ab. (62281)

5) 1 and 3 (87)

6) 2 and 4 (596)

7) 5 or 6 (656)

8) limit 7 to (English language and humans) (569)

Searches were supplemented by reviewing The Journal of Dental Research as well as screening review articles.

Selection Criteria

Inclusion criteria: Searches were limited to human subjects with active periodontal disease, subgingival plaque sampling of disease and healthy control (site or subject), metagenome or metatranscriptome analysis. Metagenome studies included 16s rRNA, pyrosequencing, and whole genome shotgun sequencing.

Exclusion criteria: Subjects that had non-surgical therapy or antibiotic use within three months of sampling, subjects with significant systemic disease impacting the periodontium, smokers, subjects under 12 years of age, in vitro studies, case reports and review articles.

Data Collection and Analysis

Six hundred seventy-six abstracts were reviewed independently by two investigators

Mary Elizabeth Aichelmann-Reidy and Hirschel Wohl (MEA-R, HW). Six hundred nine articles were excluded after initial review for failing to meet selection criteria. Full text publications of 67 articles were reviewed for inclusion based on the criteria for this review. Fifty-seven articles were excluded due to not meeting the selection criteria set forth above. After exclusion of the articles, ten articles were included in the systematic review. Study selection summary is summarized in Fig 1.

Database search resulted in 676 articles

609 articles excluded after abstract review

67 full text publications reviewed

57 articles excluded after not meeting inclusion criteria

10 articles included for

analysis

Fig 1: Summary of screening process

Data Extraction and Outcome Measures

One investigator (HW) extracted data related to demographics (age, sex, ethnicity, population location), study design, sample size, methodology, and periodontal status. The following outcome measures were extracted from each article if available depending on study design.

1. Bacterial genera and/or species significantly abundant (P< 0.05) when comparing

periodontitis sites and healthy sites. Top ten most abundant bacterial genera

and/or species reported.

2. Most prevalent genes or upregulated genes in disease sites compared to healthy

sites (P< 0.05).

3. Bacterial species with increased expression of virulence factors in disease sites

compared to healthy sites.

4. Upregulated genes expressed in the red complex bacterial species (P. gingivalis,

T. denticola, and T. forsythia).

5. Abundance of viruses or archaea in disease sites compared.

Data was compiled and tabulated qualitatively using the software Tableau.

Results

Study characteristics

Fifty-seven full text articles were reviewed and excluded from the study for not meeting inclusion criteria (Table 1).

Table 1: Excluded Studies from Systematic Review

Reference Rationale for Exclusion Li et al. 2015, Camillo-Castillo et al. 2015, Han et al. 2017, Lacking healthy

Junemann et al. 2012, Kageyma et al. 2017, Shi et al. 2015, control site

Albander et al. 2012, Beyer et al. 2018, Bizzaro et al. 2013,

Longo et al. 2018, Shchipkova et al. 2010, Hong et al. 2015,

Moon et al. 2015

Table 1 continued Baek et al. 2018, Sousa et al. 2017, Zhang et al. 2015, Previous treatment or unclear if

Yu et al. 2019, Payungporn et al. 2017, Tsai et al. previous treatment was

2018 excluded

Duran-Pinedo et al. 2011,2014, Sakamoto et al. 2004, Not applicable due to study

You et al. 2013, Babev et al. 2017, Marchesan et al. design or technology used

2015, Colombo et al. 2013, Joaquim et al. 2017, Yost et al. 2017, Tian et al 2016, Gaetti-Jardim et al. 2015,

Guglielmetti et al. 2014, Deng et al. 2018, Ram-

Mohan et al. 2020, Fine et al. 2013, Shaddox et al.

2012, Velsko et al. 2018, Ai et al. 2017

Casarin et al. 2013, Cao et al. 2018, Scher et al. 2012, Systemic disease impacting the

Ge et al. 2013, Schulz et al. 2019, Jorth et al. 2014, periodontium or smokers

Chen et al. 2018, Szafranski et al. 2015, Abusleme et included or lack of information al. 2013, Griffen et al. 2012, Liu et al. 2012, Li et al. if these were excluded

2014

Rylev et al. 2011 Reporting bacteria on phylum

level

Downes et al. 2009, Belda-Ferre et al. 2012, Coretti Sampling of supragingival et al. 2017, Chen et al. 2015 plaque

Correa et al. 2017, Horz et al. 2011, No periodontal disease or

unclear periodontal status

Ten studies met the selection criteria and were included in the study (Table 2). Nine of the studies were case control/cross-sectional and one was a longitudinal/cohort study following progression of periodontal disease. All studies compared a periodontal disease site to a healthy site. Primarily the comparison was between periodontal disease subjects and healthy control subjects. Two studies further used a split mouth analysis to compare shallow healthy sites <3mm to diseased sites in periodontal subjects. Most studies looked at subjects with chronic periodontitis, however two studies further included subjects with aggressive periodontitis. All ten studies used metagenome analysis via either 16s rRNA, pyrosequencing or whole genome shotgun analysis with two studies also incorporating metatranscriptome analysis. Five of the ten studies had a combined (health and periodontitis) sample size of 30 or more while the other five studies had a combined sample size of 16 or less.

Table 2: Included Studies from Systematic Review

Reference Study Type Sample Population Technology used Period

Size (N) Age ontitis:

CP or

AGP

Dabdoub Case 50 (25 50-59 Metagenomics via CP et al. 2016 control/Cross periodontitis shotgun analysis

-sectional 25 healthy)

Table 2 continued Duran- Case 13 (7 Not Metatranscriptomics CP

Pinedo et control/Cross periodontitis reported and Metagenomics

al. 2014 -sectional , 6 healthy)

Farina et Case 12 (9 50-59 Metagenomics via CP

al. 2019 control/Cross periodontitis shotgun analysis

-sectional , 3 healthy)

Kirst et al. Case 50 (25 Not Pyrosequencing 454 CP

2015 control/Cross periodontitis reported

-sectional , 25 healthy)

Park et al. Case 32 (20 50-59 Pyrosequencing 454 CP and

2015 control/Cross periodontitis Gingivi

-sectional , 12 healthy) tis

Perez- Case 16 (9 40-49 Metagenomics via CP

Chaparro control/Cross periodontitis 16s rRNA

et al. 2018 -sectional , 7 healthy)

Shi et al. Case 9 (6 20-29 Metagenomics via CP and

2018 control/Cross periodontitis 16s rRNA AgP

-sectional , 3 healthy)

Shi et al. Case 31 (23 40-49 Metagenomics via CP

2020 control/Cross periodontitis shotgun analysis

-sectional , 8 healthy)

Table 2 continued Wei et al. Case 32 (23 30-39 Metagenomics via CP and

2019 control/Cross periodontitis 16s rRNA AgP

-sectional , 9 healthy)

Yost et al. Cohort/Longi 15 (9 50-59 Metatranscriptomics CP

2015 tudinal periodontitis and Metagenomics

, 6 healthy)

Bacteria of Most Abundance

Healthy sites

Four studies were included that reported on significant increases in bacteria genera in

healthy sites compared to disease sites. All studies reported healthy sites as ≤3mm PPD.

Five studies reported on significant increases in bacteria genera and/or species at healthy

sites (Tables 3 and 4). Of these studies, Perez-Chaparro et al compared periodontitis sites

to healthy sites, as well as healthy subjects. This allowed for an added set of data for both

genera and species. Corynebacterium, Streptococcus, and Rothia were one of the most

abundant genera in most of the studies (Fig 2). At the species level, C. matruchotii was

reported in three of the studies. R. dentocariosa, R. aeria, S. anginosus and C.durum were

reported in two of the five studies. (Fig 3).

Table 3: Most Abundant Bacterial Genera at Healthy Sites

Reference Healthy subject or Results

site

Farina et Healthy subject Microcella al. 2019

Perez- Healthy subject Corynebacterium, Streptococcus, Bergeyella

Chaparro et al. 2018

Perez- Healthy site (split Cardiobacterium, Streptococcus,

Chaparro et mouth) Corynebacterium, Rothia al. 2018

Shi et al. Healthy subject Rothia, Corynebacterium, Mobiluncus

2018

Wei et al. Healthy subject Streptococcus, Neisseria, Haemophilus, Rothia,

2019 Corynebacterium, Lautropia, Granulicatella,

Gemella

Table 4: Most Abundant Bacterial Species at Healthy Sites

Reference Healthy subject or Results

healthy site

Dabdoub et Healthy subject A. oris, S. sanguinis, R. dentocariosa, C. al.2016 hominis, R. aeria, L. goodfellowii, L. mirabilis,

L. fusiformis, A. johnsonii, C. matruchotii

Duran-Pinedo Healthy subject E. saburreum, C. matruchotii, L. hofstadii, P. et al. 2014 indolicus, Lachnospiraceae oral taxon 107,

Lachnospiraceae oral taxon 082,

Peptoniphilus sp oral taxon 386, Oribacterium

sp oral taxon 108, R. aeria, S. anginosus

Farina et al. Healthy subject M. alkaliphila

2019

Kirst et al. Healthy subject S. mitis, G. adiacens, S. sanguinis,

2015 Actinomyces sp oral taxon 170, G.

haemolysans

Perez- Healthy site (split R. dentocariosa, C. matruchotii, C. durum

Chaparro et al. mouth)

2018

Table 4 continued

Perez- Healthy subject C. matruchotii, H. parainfluenzae, C. durum

Chaparro et al.

2018

Bergeylla Cardiobacterium Corynebaacterium Gemella Granulicatella Haemophilus Lautropia Microcella Mobiluncus

Neisseria Abundant Abundant Bacterial Genera Rothia Streptococcus

0 1 2 3 4 5 Number of Studies

Fig 2: Most Abundant Bacterial Genera at Healthy Sites by the Number of

Reported Studies

Actinomyces johnsonii Actinomyces oris Actinomyces sp oral taxon 170 Cardiobacterium hominis Corynebacterium durum Corynebacterium matruchotii Eubacterium saburreum Gemella haemolysans Granulicatella adiacens Haemophilus parainfluenzae Lachnospiraceae oral taxon 082 Lachnospiraceae oral taxon 107 Lautropia mirabilis Leptotrichia goodfellowii Leptotrichia hofstadii Lysinibacillus fusiformis Microcella alkaliphila

Abundant Abundant Bacterial Species Oribacterium sp oral taxon 108 Peptoniphilus indolicus Peptoniphilus sp oral taxon 386 Rothia aeria Rothia dentocariosa Streptococcus anguinosus Streptococcus mitis Streptococcus sanguinis 0 1 2 3 4 5 Number of studies

Fig 3: Most Abundant Bacterial Species in Healthy Sites by the Number of Reported

Studies

Periodontitis sites

Six studies were included that reported on significant increases in bacteria genera at diseased sites compared to healthy sites (Table 5). Two of the six studies included

30 aggressive, as well as chronic periodontitis and stratified data according to each group with all diseased sites having a PPD ≥4mm. The bacterial genera Porphyromonas and

Filifactor were found in the majority of these studies with the Treponema genus represented in half of the studies (Table 5).

Six studies were included that reported on significant increases of bacterial species at diseased sites (Table 6). Only chronic periodontitis subjects were studied, and all diseased sites had a PPD ≥4mm. P. gingivalis was reported to be one of the most abundant species in all studies. T. forsythia was one of the most abundant in five out of the six studies. T. denticola , F. nucleatum, and F. alocis were increased in abundance in half of the included studies.

Table 5: Most Abundant Bacterial Genera at Periodontitis Sites

Reference Mean PPD at Type of Results

Diseased Site Periodontitis

Dabdoub 6.1mm CP Fusobacterium, Tannerella, et al. 2016 Porphyromonas, Freitibacterium,

Filifactor, Parvimonas, Selenomonas,

Kingella

Farina et 6.6mm CP Porphyromonas, Campylobacter, al. 2019 Anaerolineaceae

Table 5 continued

Park et al. ≥4mm CP Porphyromonas, Freitibacterium, Rothia,

2015 Filifactor

Perez- ≥4mm CP Fusobacterium, Treponema,

Chaparro Porphyromonas, Tannerella,

et al. 2018 Freitibacterium, Peptostreptococcaceae

[XI][G-6], Desulfobulbus,

Peptostreptococcaceae [XI][G-1],

Bacteroidetes [G-3], Bacteroidaceae [G-

1],

Shi et al. ≥4mm CP Peptostreptococcus, Filifactor,

2018 Eubacterium, Butyrivibrio, Klebsiella,

Bulleidia, Lactococcus, Wolinella,

Acholeplasma

Shi et al. ≥4mm AgP Treponema, Porphyromonas,

2018 Filifactor,Eubacterium, Desulfobulbus,

Prevotella, Butyrivibrio,Mycoplasma,

Lactococcus

Table 5 continued

Wei et al. ≥4mm CP Veillonella, Treponema, Filifactor,

2019 Freitibacterium, Tannerella,

Peptostreptococcaceae [XI][G-6],

Peptostreptococcaceae [XI][G-5],

Bacteroidales [G-2], Bacteroidetes [G-5],

Johnsonella

Wei et al. ≥4mm AgP Treponema, Freitibacterium, Filifactor,

2019 Tannerella, Peptostreptococcaceae

[XI][G-6], Peptostreptococcaceae [XI][G-

5], Peptococcus, Bacteroidetes [G-5],

Bacteroidales [G-2], Anaeroglobus

Table 6: Most Abundant Bacterial Species at Periodontitis Sites

Mean Results

Reference PPD at

Diseased

Site

Dabdoub et 6.1mm F. nucleatum, F. alocis, T. forsythia, P. gingivalis, F. al. 2016 naviforme, V. parvula, E. brachy, Desulfobulbus HOT.041,

Fretibacterium HOT.452, S. sputigena

Duran- ≥6mm P. gingivalis, S. wiggsiae, S. mutans S. parasanguinis L.

Pinedo et lactis, R. pickettii, T. forsythia, B. cepacia, Actinomyces sp al. 2014 oral taxon 448, P. fluorescens

Farina et al. ≥5mm P. gingivalis, C. rectus, Anaerolineaceae bacterium oral

2019 taxon 439

Kirst et al. ≥6mm P. gingivalis, F. nucleatum subsp vincentii, T. forsythia, T.

2015 denticola, P. endodontalis, Synergistetes G 3 sp

oraltaxon360, Synergistetes G 3 sp oraltaxon363, F. alocis,

T. socranskii

Table 6 continued

Perez- ≥4mm Fusobacterium unclassified, Treponema unclassified, P.

Chaparro gingivalis, T. forsythia, F. alocis, T. denticola, et al. 2018 Freitibacterium unclassified, P. endodontalis

Bacteroidetes_[G-5] unclassified Eubacterium [XI][G-6]

unclassified

Shi et al. ≥5mm P. endodontalis, P. gingivalis, F. nucleatum, P. nigrescens,

2020 T. forsythia, T. medium, P. intermedia, T. denticola, P.

tannerae, C. rectus

Acholeplasma Anaeroglobus Anaerolineaceae Bacteroidaceae [G-1] Bacteroidales [G-2] Bacteroidetes [G-3] Bacteroidetes [G-5] Bulleidia Butyrivibro Campylobacter Desulfobulbus Eubacterium Filifactor Freitibacterium Fusobacterium Johnsonella Kingella Klebsiella Lactococcus Mycoplasma Parvimonas

Abundant Abundant Bacterial Genera Peptococcus Peptostreptococcaceae [XI][G-1] Peptostreptococcaceae [XI][G-5] Peptostreptococcaceae [XI][G-6] Peptostreptococcus Porphyromonas Prevotella Rothia Selenomonas Tannerella Treponema Veillonella Wolinella 0 1 2 3 4 5 6 7 Number of Studies

Fig 4: Most Abundant Bacterial Genera at Periodontitis Sites by the Number of

Reported Studies. (G-1, G-2 etc. represent unnamed novel genera)

Actinomyces sp oral taxon 448 Anaerolineaceae bacterium oral… Burkoholderia cepacia Camylobacter rectus Desulfobulbus HOT. 041 Eubacterium brachy Filifactor alocis Fretibacterium fastidiosum Fretibacterium HOT. 452 Fretibacterium sp HMT 360 Fusobacterium naviforme Fusobacterium nucleatum Lactococcus lactis Porphyromonas endodontalis Porphyromonas gingivalis Prevotella intermedia Prevotella nigrescens Prevotella tannerae

Pseudomonas fluorescens Abundant Abundant Bacterial Species Ralstonia picketti Scardovia wiggsiae Selenomonas sputigena Streptococcus mutans Streptococcus parasanguinis Tannerella forsythia Treponema denticola Treponema medium Treponema socranskii Veillonella Parvula 0 1 2 3 4 5 6 7 Number of Studies

Fig 5: Most Abundant Bacterial Species at Periodontitis Sites by the Number of

Reported Studies

Most Prevalent or Upregulated Genes in Periodontitis Sites

In total, four of the cross-sectional studies reported on significantly abundant genes or significantly expressed genes. The three metagenome studies (Dabdoub et al 2016, Kirst et al 2015, Shi et al 2020) reported on the significantly abundant genes in periodontitis sites compared to healthy sites, while the metatranscriptome study (Duran-Pinedo et al

2014) reported on the genes significantly expressed at periodontitis sites compared to healthy sites. The included studies compared the genes from the bacterial samples to gene databases such as GO (Gene ontology) and KEGG (Kyoto encyclopedia of gene and genome). All the studies found increases in genes related to bacterial motility or chemotaxis (Table 7). Genes related to lipopolysaccharide biosynthesis were found to be increased in most studies. Genes related to antibiotic resistance and beta lactam degradation as well as iron acquisition genes were increased in two of the four studies.

Table 7: Most Prevalent or Upregulated Genes in Periodontitis Sites (Cross- sectional studies)

Reference Genes

Dabdoub et Bacterial chemotaxis Flagellar assembly al. 2016 Antibiotic resistance Gram negative and gram-positive cell wall components Iron acquisition Lipopolysaccharide metabolism Oxidation of primary alcohols Genes associated with short chain fatty acids Anaerobic reductases

Table 7 continued

Duran- Bacterial chemotaxis Bacterial motility proteins Pinedo et Translation Peptides transport al. 2014 Iron acquisition Beta lactam degradation Lipopolysaccharide metabolism Energy metabolism Carbohydrate metabolism Lipid metabolism Metabolism of cofactor and vitamins Amino acid metabolism

Kirst et al. Bacterial motility proteins Lipopolysaccharide biosynthesis 2015 proteins Flagellar assembly Bacterial chemotaxis Peptidases Lipopolysaccharide biosynthesis Energy metabolism Lipid metabolism Methane metabolism Metabolism of cofactor and vitamins

Shi et al. Bacterial motility proteins Flagellar assembly 2020 Bacterial chemotaxis Signal transduction Carbohydrate metabolism Lipid metabolism

Upregulated Genes at Progressing sites

Only one longitudinal study (Yost et al 2015) was included that reported on sites of disease progression (Table 8). Progression of disease was defined as loss of clinical attachment loss (CAL) equal or greater than 2mm. This study compared baseline of

39 progressing sites to non-progressing sites. Upregulated genes included those related to cell motility, lipopolysaccharide biosynthesis, peptidoglycan biosynthesis, transport, protein kinase C-activating G-protein signaling pathway, and synthesis of aromatic compounds (Table 8).

Table 8: Most Upregulated Genes in Periodontitis Sites (Longitudinal Study)

Reference Genes

Yost et al. Cell motility Transport of iron, potassium, chloride, citrate and amino acids 2015 Lipopolysaccharide biosynthesis Peptidoglycan biosynthesis PKC activating G-protein coupled receptor signaling pathway Synthesis of aromatic compounds

Species Expressing Increased Virulence Factors at Periodontitis sites

Duran-Pinedo et al reported 64 species that expressed ≥40 virulence factors. All 3 red complex bacterial species along with A. actinomycetemcomitans and members from the orange complex (C. gracilis, C. rectus, C. showae, P intermedia) expressed high levels of virulence factors. Unexpectedly species such as Neisseria flavescens, Corynebacterium matruchotii, Neiseria sicca, Streptococcus oralis and Rothia dentocariosa all expressed high levels of virulence factors.

Species Expressing Increased Virulence Factors at Progressing Sites

Yost et al. evaluated subjects clinically at a baseline initial visit and monitored them every two months up to one year. They evaluated changes in subgingival plaque during

40 disease progression as well as comparing baselines from initial visits from subjects with disease progression, compared to those without disease progression. Progression of disease was defined as loss of CAL ≥2mm. In the comparison of baseline to after disease progression, it was found that 207 species had upregulated virulence factors in the progressing sites. Forty-seven of those species showed upregulation of ≥50 virulence factors. Both P. gingivalis, as well as T. forsythia showed high levels of virulence factors in progressing sites. Members of the orange complex with upregulation included C. gracilis, P. intermedia, F. nucleatum, S constellatus, P. nigrescens, and P. micra.

Interestingly, the species with the most upregulated virulence factors included several

Streptococcus spp. (S. mutans, S. oralis, S. parasanguinis, S. intermedius and S. mitis) along with P. fluorescens, V. parvula, and C. matruchotii which upregulated > 200 virulence factors. In the comparison of baseline from subjects with disease progression compared to those without disease progression, most members of the red and orange complex bacteria were not especially active. P. gingivalis upregulated 34 virulence factors, while T. denticola had three and T. forsythia had one, respectively. Three

Streptococcus spp. (S. oralis, S. mitis, and S. salivarius) were upregulating some of the highest amount of virulence factors ( >200) along with Lautropia mirabilis, Actinomyces sp. oral taxon 170, and P. fluorescens.

Upregulated genes and virulence factors in the red complex bacterial species

Duran-Pinedo et al reported that metalloproteases, peptidases, and iron metabolism proteins and genes involved with cell invasion were upregulated in all three members of the red complex bacteria (Table 9). Yost et al in comparing subgingival plaque at baseline visit to after disease progression, reported genes related to proteolysis, protein

41 kinase C-activating G-protein coupled receptor, response to antibiotic, and transport of iron, cation, lactate, citrate, sodium, and phosphate to be upregulated in members of the red complex bacteria. Additionally, T. denticola upregulated genes related to flagellar biosynthesis and P. gingivalis and T. forsythia upregulated TonB-dependent receptors, aerotolerance genes, hemolysins, and CRISPR-associated genes.

Table 9: Upregulated Genes and Virulence Factors in the Red Complex Bacterial

Species in Periodontitis Sites

Bacteria Reference Genes

P. gingivalis Duran-Pinedo et al. Metalloproteases and peptidases, iron

2014 metabolism proteins, hemolysin, vitamin

B12 ABC transporter, genes involved in

cell invasion (ClpB,GroEL, and DnaK)

Yost et al 2015 * Peptidases, protein kinase C-activating G-

protein coupled receptor, response to

antibiotic (beta lactamase), c, TonB-

dependent receptors, aerotolerance genes,

hemolysins, CRISPR-associated genes,

biotin synthesis, capsular polysaccharide

biosynthesis proteins, conjugative

transposons

Table 9 continued

T. denticola Duran-Pinedo et Metalloproteases and peptidases, iron

al 2014 metabolism proteins, vitamin B12 ABC

transporter, oligopeptide transport, flagellar

assembly, genes involved in cell invasion

(internalin homologs)

Yost et al. 2015 Protein kinase C-activating G-protein

* coupled receptor, transport (iron, cation

,lactate ,citrate ,sodium, and phosphate),

flagella biosynthesis

T. forsythia Duran- Pinedo et Metalloproteases and peptidases, iron

al. 2014 metabolism proteins, genes involved in cell

invasion (homologs of NanH and internalins)

Yost et al. 2015 Peptidases, sulfur compound metabolism,

* response to antibiotic (beta lactamase),

transport (iron, cation ,lactate ,citrate

,sodium, and phosphate), TonB-dependent

receptors, aerotolerance genes, iron transport

genes, hemolysins ,CRISPR-associated

genes, transposases, polysaccharide binding

*Comparisons were made from baseline to endpoint of study for subjects with 2mm CAL and did not compare to a healthy control group.

Virus and Archaea proportion

Only three of the studies reported on viruses and/or archaea in the subgingival sample

(Table 10). Overall, it appears from these studies that viruses are in relatively small proportion with Yost et al reporting that herpes virus had increased activity in progressing sites. Similarly, archaea appear to make up a small fraction of the subgingival flora.

Table 10: Virus and Archaea Proportion in Periodontitis Sites

Reference Virus Archaea

Dabdoub et al 5% total including viruses, 5% total including viruses,

fungi, and archaea fungi, and archaea

Duran-Pinedo et al None Small fraction (number not

reported)

Yost et al 0.04-.7% (increased gene Number not reported

expression of phages and (Methanobrevibacter sp.

herpes virus at progressing more active in progressing

sites) sites)

Discussion

Putative periodontal pathogens P. gingivalis , T. denticola, T. forsythia, and A. actinomycetemcomitans have been implicated as key bacterial species in the development of periodontitis (Socransky 1998, Slots 1983, Mandell 1988). This study found that P. gingivalis and T. forsythia were found in statistically significant amounts in periodontal pockets in almost all the studies, and T. denticola was found in significant amounts in three out of the six studies (Fig.5). A. actinomycetemcomitans on the other hand was not found in statistically higher amounts in periodontal pockets compared to healthy sites.

However, all the studies reporting on bacterial species evaluated chronic periodontitis subjects, whereas A. actinomycetemcomitans has been shown to be associated with aggressive periodontitis subjects (Slots 1983, Mandell 1988). Although this study found trends of specific bacterial species in periodontal pockets, this does not implicate these species as actively participating in disease progression. Most of the included studies are cross sectional and do not show longitudinal evidence that these bacteria are in higher amounts. Therefore, it is difficult to establish these bacteria as the cause and not a result of the periodontal pocket. The increased abundance may be due to the environment of the pocket changing allowing for specific bacteria to be favored (Socransky 1992).

Therefore, to show evidence of causation, prospective longitudinal studies are still necessary.

From the perspective of gene expression, a bacterial species of high abundance in the metagenome does not automatically mean that it is a highly active member expressing genes. This was shown by Duran-Pinedo et al, where of the top ten significantly abundant

45 bacterial species in disease, only half had an accompanying significantly increased gene expression (Duran-Pinedo 2014). Interestingly, in their study both P. gingivalis and T. forsythia were increased in the metagenome as well as the metatranscriptome which would seem to imply that these bacteria are highly active in periodontal pockets. In fact, all three red complex bacteria along with A. actinomycetemcomitans, as well as many orange complex bacteria released increased virulence factors in the diseased state.

However, since this study was cross-sectional the increased gene expression may have occurred after the formation of the periodontal pocket.

Yost et al in the only longitudinal study included in this review, reported that at baseline, progressing pockets had very few red and orange complex bacteria releasing up-regulated virulence factors compared to non-progressing sites. P. gingivalis released 34 virulence factors, T. denticola released three and T. forsythia released one. In fact, many of the

Streptococcus spp. and Actinomyces spp. released the most virulence factors in sites that eventually lost ≥2mm of attachment level. This would seem to imply that perhaps many of the red and orange complex bacteria are not as active in the development of periodontitis. Interestingly, the study by Yost et al. would seem to agree with the polymicrobial synergy and dysbiosis (PSD) model of periodontal disease as presented by

Hajishengallis et al As noted previously, keystone pathogens such as P. gingivalis may interact with other bacterial species such as oral streptococci and can increase virulence of the entire microbial community (Hajishengallis 2012). This co-operation has been shown in animal models and may lead to increased alveolar bone loss (Daep 2011).

Therefore, the increased virulence factors of various bacteria like Streptococcus species and Actinomyces species may be due to this synergy.

In general, the studies reporting significantly abundant genes or upregulated gene expression, found genes related to bacterial motility, lipopolysaccharide biosynthesis, antibiotic resistance, and iron acquisition among others increased in periodontitis sites.

This narrative review also found that virulence factors such as metalloproteases, peptidases, and genes involved with cell invasion were upregulated in all three members of the red complex (Table 9). Many of these are established virulence factors which help the bacteria evade and multiply within the host (Saglie 1990, Fives-Taylor 1999). For instance, lipopolysaccharide from A. actinomycetemcomitans has been shown to activate macrophages leading to production of Il-1 and TNF-α (Saglie 1990). Similarly, P. gingivalis has been shown to release high amounts of LPS which can activate toll-like receptors which have been implicated in a pro-inflammatory response (Darveau 2004,

Silva 2015). Iron acquisition is necessary for nutrition as it facilitates electron transport and other essential cellular functions (Dabdoub 2016). Indeed, iron has been shown to be necessary for growth of P.gingivalis (Bramanti 1991). Antibiotic resistance is an established virulence factor allowing the bacteria to multiply even after administration of antibiotics (Fives-Taylor 1999). Rams et al in a in vitro study found that 74.2% of subjects with chronic periodontitis had periodontal pathogens resistant to antibiotics.

From the bacterial species they tested in vitro, they found that 55% of the subjects were resistant to doxycycline, 43.3% resistant to amoxicillin, 30.3% resistant to metronidazole and 26.5% resistant to clindamycin (Rams 2014). Therefore, the findings in this narrative review further validates that upregulated virulence factors may play an important role in periodontitis sites.

Primarily, in most studies the comparisons made were between subjects with periodontal disease compared to a healthy control subject. However, two studies used a split mouth design comparing disease sites to healthy sites within subjects (Perez-Chaparro 2018,

Dabdoub 2016). In general, a split mouth design can remove the variability between subjects such as systemic and environmental differences. Therefore, a split mouth design can be more advantageous than comparisons between different subjects. Interestingly,

Dabdoub et al showed that healthy sites had similarities in gene functionality to disease sites in subjects with periodontal disease, although the microbiome was different between the healthy and diseased sites. This may imply that even healthy sites in periodontitis subjects may be more susceptible to disease. However, this would have to be confirmed with long-term longitudinal studies.

In the reported available studies, viruses and archaea were found in low proportion in periodontitis and progressing sites. It is interesting that Yost et al found increased activity of herpes virus in progressing sites. Although its role in periodontal disease is controversial, previous studies have found high levels and high frequency of herpes virus in periodontally involved sites (Teles 2013). Regarding archaea, although it is in low abundance it may play an important role as it has been shown that it may provide favorable conditions for growth of anaerobic bacteria (Curtis 2020).

Limitations

Several limitations are evident within this systematic narrative review. Firstly, the heterogeneity amongst the studies was very apparent with respect to technology used. Of the metagenome studies, two used pyrosequencing, three used shotgun analysis and three used 16s rRNA. Also, of the two metatranscriptome studies, the study designs differed, one was cross sectional and the other longitudinal making it difficult to synchronize the data. Of the cross-sectional studies, three of the four used metagenome technology and therefore may not give an accurate picture of the actual gene expression. This is because the metagenome data is analyzing the DNA, while the metranscriptome studies analyze the RNA which represents actual expression of genes. Furthermore, many studies only reported on the bacteria genus level while others reported on the species level which limited the ability to compare and determine trends. Another major limitation was that many studies pooled the data from different periodontal pockets or did not relate the data to the clinical parameters of probing depths and clinical attachment levels. Without clinical measurements one cannot relate metagenome and metatranscriptome data to disease severity.

Conclusion

This study found trends of specific bacteria found in periodontal pockets. Of the six studies reporting on bacterial species, P. gingivalis and T. forsythia were found in statistically significant amounts in periodontal pockets in almost all the studies. T. denticola, F. nucleatum, P. endodontalis and F. alocis were significantly increased in

49 abundance in half of the included studies. Regarding studies looking at abundance of genes or upregulation of genes, all the studies found increases in genes related to bacterial motility or chemotaxis. However, within the limitations of this narrative review, trends of abundant bacteria and upregulation of genes in periodontal pockets does not imply these specific species or genes are actively participating in disease progression as nine at out the ten included studies were cross-sectional in design. Also, eight of the ten included studies were metagenome-based studies and therefore not able to measure true gene expression. In fact, the one metatranscriptome longitudinal study found that

Streptococcus spp. and Actinomyces spp. released the most virulence factors in sites that eventually lost ≥2mm of attachment level. Whereas bacteria from the red and orange complex expressed lower levels of virulence factor. Therefore, well-designed metatranscriptome longitudinal studies are needed to further elucidate specific bacteria and genes in the process of disease progression.

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