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These organisms are Gram negative bacteria composed of pleomorphic cells which are bounded by a triple-layered membrane and no cell wall.  The appearance of their colonies demonstrate a "fried egg" appearance with a small central nipple which is common in Mycoplasma colonies. It is also considered to be a facultative anarobe although the growth is better under anarobic conditions. Its preferance of habitat for maximal growth is in the human orpharynx at a temperature of 36 degree celsius and pH 7. [Mycoplasma Buccale]

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Oral Disease             (Mycoplasma Buccale)

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Oral Disease             (Mycoplasma Buccale)

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Mycoplasma usually colonizes the throat, it is the bacterial biofilm, or tartar found in the oral cavity. Also it is one of the common causes of acute respiratory infections. Mycoplasma can be separated from root canal infections, gingivitis, and periodontitis clinical specimens, the distribution and pathogenicity in the oral cavity are still unclear. It is can also initiate and progress oral cancer in humans.

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Mycoplasmas are the smallest prokaryotic organisms that can grow in cell-free culture medium. They are found in man, animals, plants, insects, soil and sewage. The first to be recognized, Mycoplasma mycoides ssp. mycoides, was isolated in 1898 from cattle with pleuropneumonia. As other pathogenic and saprophytic isolates accumulated from veterinary and human sources they became known as pleuropneumonia-like organisms (PPLO), a term long superseded by 'mycoplasmas'. Mycoplasma (Greek: mykes, fungus; plasma, something moulded) refers to the filamentous (fungus-like) nature of the organisms of some species and to the plasticity of the outer membrane resulting in pleomorphism.

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PREPARATION OF INOCULUM

Mycoplasmas are grown in Roux bottles containing Hayflick's modified Edward's medium for about 48 hours at 37°C. Only glass-adherent mycoplasmas should be used for an AIA inoculum. It is therefore recommended that the stock be prepared from cultures which show only a slight indicator change from red to orange if phenol red is used as an indicator. The whole supernatant should be removed and the glass-adherent mycoplasmas should be washed with room temperature-equilibrated PBS solution. The mycoplasmas which still adhere to the glass are harvested with a cell scraper in 100 ml of fresh mycoplasma medium and are stored at −70°C in 1-ml aliquots for further use.

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Laboratory personnel

Mycoplasmas from human sources are the most prevalent group and account for approximately one third to half of all strains isolated. Generally M. orale, which is the most common mycoplasma species in the oral cavity of clinically healthy humans, also represents the single most common isolate. Other non-pathogenic mycoplasma species from the normal human microbial flora of the oropharynx that are seen in cell cultures are M. fermentans and M. hominis. As the largest percentage of mycoplasma found in cell cultures are of human origin, it appears to be apparent that the laboratory personnel is directly involved and represents the major source of contamination.

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INTRODUCTION

Mycoplasma and Ureaplasma belong taxonomically to the order Mycoplasmatales in the class Mollicutes ('soft skin'). Apart from being human pathogens, some organisms of the order Mollicutes are economically important plant and animal pathogens. The initial divisions of mycoplasmas into different species were based on inhibition of growth of the organisms on agar media containing homologous antibodies. In later studies, sequencing of the genome of mycoplasmas has confirmed the relevance of the initial classification method.

Over 120 named Mycoplasma species exist; 13 Mycoplasma species, two Acholeplasma species and one Ureaplasma species have been isolated from humans. However, only four are well-established human pathogens:

Apart from the species mentioned above, several other species of Mycoplasma occur in the human endogenous flora of the mouth and the genital tract ( M. buccale, M. faucium, M. orale and M. salivarum).

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Introduction

Mycoplasmas can cause oxidative damage to host cells. By this pathogenic mechanism, the adhering mycoplasmas produce, or induce the cell to produce, reactive oxygen molecules or free radicals. The proximity between the adhering mycoplasmas and the host cells allows the reactive oxygen, which usually has a short half-life, to reach and affect the host cell. The subject has been reviewed by Tryon and Baseman (1992) so only the basics will be summed up here. Many studies suggest that the initial stages of mycoplasma adherence and pathogenesis include the following steps:

1.

Adherence of the mycoplasmas to the host cell membrane via specific receptors (sialoglycoconjugates, proteins, or sulfatides) or to hydrophobic sites.

2.

Production of superoxide anions by mycoplasmas or induction of enhanced production of superoxide anions by host cells.

3.

Partial inhibition of host cell catalase by elevated levels of superoxide anions.

4.

Partial inhibition of host cell Cu,Zn-superoxide dismutase (SOD) by H ₂O ₂ produced by host cells and by the mycoplasmas.

5.

Increased oxidative stress in cytoplasmic and membrane components by reactive oxygen.

6.

Secondary oxidation and breakdown of fatty acids in membranes and production of the cross-linker malonyl dialdehyde.

7.

Cross-linkage of cytoplasmic and membrane components.

8.

Membrane becomes leaky.

9.

Cytoplasmic components ooze through host cell membrane.

10.

Cytadhering mycoplasmas benefit from the nutrients released from host cells.

The events described in steps 2, 3, and 4 can actually proceed in cycles and, therefore, amplify the outcome of all the steps. Altogether, these result in a prolonged, rather than overwhelming, deleterious effect of the mycoplasmas on the host cell, making this model of mycoplasma pathogenesis more attractive since it may explain the chronic nature of mycoplasma diseases.

Methods measuring parameters of oxidative stress will be discussed in this chapter. These include assays for catalase and superoxide dismutase (SOD) activity.

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5.3 Mycoplasma

Mycoplasma can live independently with no cell wall. They are the smallest prokaryotic microbial cells and can be isolated from normal human and animal respiratory mucosa. In the oral cavity, M. pneumoniae, M. oralis, M. salivalis, and M. nominis can be isolated.

Mycoplasma are the smallest of all prokaryotic microbial cells, Figure 5.10(A) and (B).

Mycoplasma have high nutritional demands and can grow on PPLO agar with beef heart infusion and 10-20% horse serum. The serum provides Mycoplasma with the cholesterol and long-chain fatty acids required for growth. The optimum pH for Mycoplasma culture is pH 7.8-8.0. Cells may die when the pH drops below pH 7.0.

Mycoplasma are aerobic or facultative anaerobic microorganisms, but they usually grow better in an aerobic environment. The best culture environment for initial isolation is atmospheric conditions supplemented with 5% CO ₂ or anaerobic conditions with 5% CO ₂ and 95% N ₂.

Mycoplasma colonies are small and can only be observed under a light microscope at low magnification or a dissecting microscope. Characteristic colonies are shown in Figure 5.10(C) and (D).

Mycoplasma and L type bacteria are similar: (1) they both lack a cell wall and the cell is pleomorphic; (2) they can both pass through an antimicrobial filter. The main differences between the two are: (1) Mycoplasma are independent microbes and L-type bacteria are variants of normal bacterial cells that have a cell wall (most L-type cells will revert to their original form when the induction factor is eliminated); (2) Mycoplasma growth requires cholesterol (10-20% serum in the medium) while the growth of L-type bacteria does not; (3) L-type bacteria fade easily after Diane staining, while Mycoplasma do not fade easily.

Mycoplasma usually colonize the throat, bacterial biofilm, or tartar found in the oral cavity. Mycoplasma pneumoniae is one of the common causes of acute respiratory infections. Although previous studies reported that Mycoplasma can be separated from root canal infections, gingivitis, and periodontitis clinical specimens, the distribution and pathogenicity in the oral cavity are still unclear.

Typical colonies of the Mycoplasma are omelet-like in shape. Colony diameter is about 10-15 μm, the center is round, opaque, and extends into the medium, and the edge of the colony is thin and flat, forming a transparent or semitransparent area. Other characteristic colonies of the Mycoplasma include mulberry-shaped colonies of M. pneumoniae. Mycoplasma from the mouth and saliva form visible comet-like colonies in semisolid medium (mostly in the middle and bottom of the culture medium).

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Pathophysiology

Mycoplasma have a high affinity for respiratory epithelial cells. Respiratory infection progresses from tracheal infection to bronchiolitis to peribronchial inflammation. As Mycoplasma have lost almost all genes responsible for biosynthesis of amino acids, these organisms are dependent on host cells to supply biochemical precursors. Adherence to the cell is the first step for pathogenesis, which is associated with disruption of cilia by an action on K+ ion channels. Mycoplasma produce hydrogen peroxide that causes additional disruption of the host cell membrane. Upon fusion with the host cell, release of potent hydrolytic enzymes, particularly nucleases, leads to significant damage to the host cell. Mycoplasma are capable of stimulating the immune system by activating monocytes and macrophages. Mycoplasma also induce the release of tumor necrosis factor (TNF)-alpha, as well as interleukins (IL)-1 and (IL)-6 Rottem (2003).

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Other Mycoplasma Infections

Mycoplasma spp., including M. fermentans, M. penetrans, and other "nonpathogenic" species, have been isolated from patients, including children, with human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). In most instances, infection has appeared to be asymptomatic, but serious and fatal disease has been reported. There is speculation that Mycoplasma spp. may enhance the pathogenicity of HIV by a variety of mechanisms, but evidence is insufficient to prove that Mycoplasma spp. are more than opportunistic pathogens.

Human infections with Mycoplasma spp. that infect animals have been reported predominantly in immunocompromised patients and in individuals in whom trauma and wound contamination have occurred. Because these infections appear to be uncommon, routine attempts to recover Mycoplasma spp. are not warranted. However, in unusual circumstances, such as an immunocompromised patient with negative results on routine bacterial cultures or a patient with an infected wound after a seal bite, culture for Mycoplasma spp. should be considered. "Seal finger," an infection known to occur in seal hunters and wildlife workers, results from seal bites or from skinning or handling seals. Several different species of marine mycoplasma found in mouths and respiratory tracts of seals are the presumed cause. Tetracyclines are considered first-line therapy for seal finger.

A number of illnesses including chronic fatigue syndrome, Gulf War syndrome, and fibromyalgia have been associated with positive results for one or more Mycoplasma spp. by PCR testing. These associations should be considered speculative, at best. Additional studies in subjects with Gulf War syndrome have shown no such association. Although such reports warrant further well-designed studies, PCR testing for Mycoplasma spp. should not be considered part of the clinical investigation for such patients.

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The Pathogen

Mycoplasmas are short rods (10 × 200 nm) that have no cell wall and are bounded by a sterol-containing membrane; thus, they are unaffected by cell wall-inhibiting antimicrobials such as β-lactams. In tissue culture, mycoplasmas are intracellular, but in vivo, infection is primarily extracellular and affects epithelial cells and their organelles, such as cilia. Attachment to respiratory epithelium is by way of terminal adhesin proteins. Mycoplasmas are active in stimulating several components of the immune system. They can act as polyclonal T-cell and B-cell activators and can cause capping of lymphocytes.

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Some mycoplasmas were shown to produce a fraction (named MIaF) that induces major histocompatibility complex class I and class II expression on a variety of animal and human cell types, increasing the antigen presentation function of these cells. Methods for modulation of the expression of MHC molecules by the mycoplasmas are described in Chapter F8. M. pulmonis was found to augment natural killer cell activity in mice, playing a possible role in resistance to M. pulmonis infections by inducing interferon-γ secretion and activating macrophages that phagocytize and kill the mycoplasmas. Methods for investigating this specific aspect of mycoplasma interaction with immune cells are detailed in Chapter F9.

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Mycoplasmas are the smallest prokaryotic organisms that can grow in cell-free culture medium. They are found in man, animals, plants, insects, soil and sewage. The first to be recognized, Mycoplasma mycoides ssp. mycoides, was isolated in 1898 from cattle with pleuropneumonia.

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A pathogenic bacterium, the most frequently found cell morphology is short bacillus, but long bacilli are sometimes observed. They are classified as black pigmented bacteria due to its formation of shiny and smooth colonies which appear either a grey, light brown, or black color on blood agar plates. The cells are nonsporulating, nonmotile, anaerobes, and stain gram-negative. It is commonly isolated from dental cyst, and are a major component of biofilm formation. P. intermediate use steroid hormones as growth factors. They can be found in high concentration in pregnant women and women with bacterial vaginosis [Prevotella Intermedia]

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Periodontal Treatment

Periodontal disease is an infective process usually associated with pathogens such as Actinobacillus actinomycetemcomitans or Prevotella intermedia. Gram-negative anaerobes have developed a selective ecologic niche that leads to the destruction of periodontal tissues. The use of pharmaceutical agents can dramatically reduce the number of these pathogens and is of great benefit in periodontal therapy. Many hygienists currently use ozonated water instead of regular water in their ultrasonic scalers. This ozonated water not only reduces the number of pathogens, but assists with healing. For periodontal surgery, ozonated water also dramatically reduces the number of pathogens and promotes healing. In managing root caries, ozone has been shown to reverse shallow noncavitated lesions when used as part of the full preventive care regimen. This includes reducing the frequency of consuming fermentable carbohydrates, increasing the use of fluoride-containing products, and improving oral hygiene. Ozone is not effective in managing deep root caries. The outer carious lesion must be removed, leaving a maximum of 1 mm of caries over the pulpal floor before ozone is applied. Common sense must be used in determining how much infected tissue can be penetrated by the ozone, and disinfection or at least a dramatic reduction of the microorganisms is needed before restoration.

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Pathophysiology

High prevalences of putative periodontal pathogens including Fusobacterium nucleatum, Peptostreptococcus micros, Porphyromonas gingivalis, Prevotella intermedia and Bacteroides forsythus were found in periodontal abscesses (Herrera et al 2000a).

Significant differences in the levels of prostaglandin E2 (PGE2) and LTB4 were found between patients with and without periodontitis. The PGE2 and LTB4 levels correlated with clinical parameters and reduced markedly after phase-1 of periodontal treatment (Tsai et al 1998). It is interesting that the levels of PGE2 correlated with the severity of the periodontal status, and the levels of LTB4 correlated with gingival inflammation.

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2.7 Periodontal Disease Transmission

Periodontal pathogens may include Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Peptostreptococcus micros, Pophyromonas gingivalis, Prevotella intermedia , Treponema denticola, Treponema forsythia, and putative periodontal pathogens such as Filifactor alocis and Parvimonas micra-especially organisms of the red complex ( P. gingivalis, T. forsythia and T. denticola)-though at least 17 new additional candidate organisms, including species or phylotypes from the phyla Bacteroidetes, Candidatus Saccharibacteria, Firmicutes, Proteobacteria, Spirochaetes, and Synergistetes, as well as possibly from the Archaea domain are reportedly associated with periodontal disease.

In one study, periodontal pathogens were present in 1-year olds in 83% of preterm infants and in 96% of full-term infants. Putative periodontal pathogens may spread between family members which could explain how colonization by periodontal pathogens in early childhood occurs in a larger proportion than previously reported. A marked correlation in the presence of periodontal pathogens, particularly the red complex species was found between mothers and their children. Vertical transmission of A. actinomycetemcomitans between mothers and children ranges from 30% to 60%. Mothers colonized by P. gingivalis are a risk factor for oral colonization of their children and the risk is greatest when both parents are colonized. Likewise, oral T. forsythia, P. Intermedia, and P. nigrescens are more frequently found in children whose parents harbored these bacteria.

The horizontal transmission of A. actinomycetemcomitans between couples has been estimated to be from 14% to 60%, and that of P. gingivalis from 30% to 75%. Many periodontopathic bacteria are detectable in saliva even after removal of all teeth, suggesting that saliva is a vehicle of person-to-person transmission of these bacteria.

Saliva transmission of periodontopathogens via kissing is highly likely but none of these studies absolutely confirmed kissing as the source of transmission which might also, e.g., be spread via fomites.

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Propolis

The research carried out in reference 120 indicated the effectiveness of a propolis extract in reducing the growth of bacteria that belong to the red complex ( Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus and Tannerella forsythia). The studies in references 121-123 confirmed the antibacterial properties of propolis in relation to pathogens of periodontitis. According to some authors, the antibacterial effects are due to flavonoids, phenol acids, and their esters. The results in reference 124 showed the clinical effectiveness of a toothpaste and gel containing a 3% ethanolic extract of propolis in a group of patients with a greater risk of gingivitis caused by dental plaque. As propolis rinses the mouth, and propolis-based toothpastes stop the growth of pathogens of gingivitis and periodontitis, these materials seem to be promising not only as preventive but also as therapeutic agents. The results obtained by reference 126 showed, however, that propolis extracts in concentrations that effectively reduce pathogenic organisms for periodontal diseases are cytotoxic for the gingival fibroblasts.

The first report of the biological activity of Portuguese propolis demonstrated that propolis displays effective antiproliferative activity against human renal cancer cells. Furthermore, a comparison of the in vitro responses of normal and malignant cells to propolis extracts revealed that cancerous cells were more sensitive in terms of growth inhibition than normal cells. The study reported the main constituents of an aqueous extract of Brazilian green propolis from Southeast Brazil and the effects of the orally administered aqueous extract on distinct phases of wound healing (cellular recruitment and extracellular matrix deposition) in a murine sponge model. Their results showed a decrease in cellular recruitment after treatment with propolis. It has been reported that phenolic compounds exert a wide range of biological actions, such as antioxidant activity by scavenging free radicals. Propolis has been found to exert indirect cytotoxic effects on malignant cancer cell lines by activating macrophages. Its anti-inflammatory role and wound healing promoting properties have also been reported: studies have examined the in vitro effect of a Chilean propolis ethanolic extract on human spermatozoa treated with benzo[a]pyrene and exogenous reactive oxygen species. The work demonstrates that the natural drug under investigation is able to protect genomic DNA from damage induced by benzo[a]pyrene, hydrogen peroxide and hydrogen peroxide in combination with adenosine 5V-diphosphate (ADP) and ferrous sulfate (FeSO ₄), producing a significant reduction in intracellular oxidants. A propolis extract was shown to possess the capacity to protect sperm membranes from oxidative attack, suggesting that it may have a role in protection against male infertility.

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Noma (cancrum oris; gangrenous stomatitis)

Noma can result from ANUG in malnourished, debilitated or immunocompromised patients, especially in children in developing areas. Anaerobes have been implicated, particularly Bacteroides ( Porphyromonas) species, Fusobacterium necrophorum (an animal pathogen), Prevotella intermedia , Actinomyces and alpha-haemolytic streptococci. In cases following ANUG, Streptococcus anginosus and Abiotrophia species are the predominant organisms. In early noma, predominant species include Ochrobactrum anthropi, Stenotrophomonas maltophilia, an uncharacterized species of Dialister and an uncultivated phylotype of Leptotrichia. A range of species or phylotypes is found in advanced noma, including Propionibacterium acnes, Staphylococcus species, Stenotrophomonas maltophilia, Ochrobactrum anthropi, Achromobacter species, Afipia species, Brevundimonas diminuta, Capnocytophaga species, Cardiobacterium species, Eikenella corrodens, Fusobacterium species, Gemella haemolysans and Neisseria species. Phylotypes unique to noma infections include those in the genera Eubacterium, Flavobacterium, Kocuria, Microbacterium and Porphyromonas, and the related Streptococcus salivarius and genera Sphingomonas and Treponema. Spreading necrosis penetrates the buccal mucosa, leading to gangrene, an orocutaneous fistula and scarring.

Diagnosis is clinical; an immune defect should always be excluded. Management includes improving nutrition, systemic antibiotics (clindamycin, penicillin, tetracyclines or metronidazole) and plastic surgery.

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Necrotizing Stomatitis/Noma

Necrotizing stomatitis, or noma, is a rapidly progressive condition affecting tissues of the oral cavity and face that occurs during periods of compromised immune function. In humans the condition is referred to as noma cancrum oris, and pathogens including Fusobacterium necrophorum and Prevotella intermedia are frequently isolated (Falker et al., 1999; Enwonwu et al., 2000). To date, in nonhuman primates noma has not been associated with any one etiological agent but as in humans, noma is linked with immunosuppressive conditions (particularly infection with simian retrovirus D) and poor nutritional status (Adams and Bishops, 1980; Schiodt et al., 1988). This condition has been described in macaques and cotton-top tamarins (Brack, 1982; Lackner et al., 1993; Kimura, 2008). Clinically, noma is characterized by rapidly progressive necrosis of the oral soft tissue, which can expose and lead to destruction of the underlying osseous structures with the potential for sequestrum formation (Figure 12.1). The initial presentation may be a small area of mucosal ulceration most often involving the gingiva, lips, or cheeks. The disease process is rapid and quickly progresses to destruction of surrounding tissue. Histologic examination of the lesion reveals circumscribed areas of necrosis surrounded by margins of inflammatory cells and bacteria (Lackner and Wilson, 1993). Treatment of advanced noma in nonhuman primates has met with little success. However, if the condition is detected early and the underlying cause can be identified and corrected, treatments such as debridement of the area and antibiotic therapy may prove more successful (Buchanan et al., 1981; Brack, 1982; Kimura, 2008).

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Adult periodontitis.

Many studies have examined the presence of antibodies to periodontopathic bacteria in the serum of patients with periodontal disease (McArthur and Clark, 1993). Most of these studies have found raised antibodies to Dzink P. gingivalis in patients with CIPD in comparison with controls. Prevotella intermedia has not been associated immunologically with CIPD but has been implicated in acute necrotizing ulcerative gingivitis (ANUG) ( et al., 1985). Overall, the immunologic findings confirm bacteriologic findings and support the association of P. gingivalis with CIPD.

Although antibodies and opsonic activity have been demonstrated in GCF (Challacombe Booth et al., 1992), recently the first demonstration of in vivo activity of antibodies has been demonstrated in humans. Passive immunization with monoclonal antibodies against P. gingivalis was shown to inhibit recolonization for up to 9 months in sites with the most severe periodontitis in a double-blind trial ( et al., 1996). This study suggests that modification of the oral flora by antibodies is feasible and could be a method of altering the disease process (see Abiko, 2000).

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4.2.7 Prevotella

Prevotella is a genus named after the French microbiologist A. R. Prevol. These bacteria belong to the genus Bacteroides and include bile-sensitive strains and melanin-producing, sugar metabolizing strains.

The main species of Prevotella found in the oral cavity are: Prevotella intermedia , Prevotella melaninogenica, Prevotella loescheii, Prevotella nigrescens, Prevotella dentocola, and Prevotella corporis.

The most frequently found cell morphology is short bacillus, but long bacilli are sometimes observed. The cells are nonsporulating, nonmotile, and stain gram-negative.

Bacteria belonging to this genus are obligate anaerobes, and most cultures require supplementing with hemin and vitamin K. On blood agar, Prevotella spp. produce melanin to form black colonies. In PYG liquid medium, these bacteria can produce acetic acid and succinic acid as the major terminal acid products. Species of this genus ferment glucose and hydrolyze gelatin. They are sensitive to bile salt and can thus be distinguished from other bacteroides that can tolerate bile salts.

Prevotella are dominant bacteria in the human gingival groove and are also the suspected pathogens behind periodontitis.

4.2.7.1 Prevotella intermedia

As an obligate anaerobe, cultures require hemin and vitamin K. Most species grow well in temperatures between 25 °C and 45 °C. For other cultural demands, refer to the genus Prevotella. Characteristic colonies are shown in Figure 4.15(D) and (E). Culture in glucose broth is cloudy, with even precipitation and sticky or slightly sticky deposits at times. The final pH value of glucose broth ranges from pH 4.9 to 5.4.

P. intermedia can produce indole and hydrolyze gelatin, but not esculin. Cells ferment glucose and sucrose, but not arabinose, larch sugar, cellobiose, rhamnose, galactose, and salicin. Cells can produce superoxide dismutase, but do not hydrolyze dextran.

The site of colonization is the gingival sulcus, but cells also can be found in saliva, dental calculus, and other plaque specimens. Previous research has shown that this species is the main pathogenic bacteria in pregnancy-related gingivitis. In addition, the bacteria can also be isolated from pericoronitis, the focus of infection after tooth extraction, infected root canals, infections of the head and neck, and pleural infection. P. intermedia is occasionally isolated from blood, abdominal, or pelvic specimens.

The genomic GC content is 41-44%. The type strain is ATCC25611.

4.2.7.2 Prevotella nigrescens

Originally, this species was classified as a strain of P. intermedia. However, since it does not produce lipase, it can be thus distinguished from P. intermedia.

For culture requirements, refer to the information on P. intermedia. Characteristic colonies are shown in Figure 4.16(C) and (D)

Most strains ferment glucose, maltose, sucrose, and maltodextrin to produce acid. They also produce indole, hydrolyze starch, and hydrolyze gelatin. In liquid medium containing glucose, these bacteria can produce acetic acid, succinic acid, isobutyric acid, and isovaleric acid.

The genomic GC content is 40-44%. The type strain is ATCC33563 (namely NCTC9336, VPI8944).

4.2.7.3 Prevotella melaninogenica

Most strains require hemin (1 mg/L) and vitamin K (0.1 mg/L) to grow and can grow at pH 8.5 and 25 °C. Characteristic colonies are shown in Figure 4.17(C), (D), and (E).

Usually, glucose broth culture turns turbid and is accompanied by smooth or filamentous precipitation. The final pH value range is from pH 4.6 to 5.0.

Clinical specimens are isolated from the gingival sulcus.

The genomic DNA GC content is 36-40%. The type strain is ATCC25845.

4.2.7.4 Prevotella corporis

Growth of P. corporis requires chlorinated hemoglobin and vitamin K1. A 10% final concentration of serum can promote growth and improve fermentation in some strains. Characteristic colonies are shown in Figure 4.18C and 4.18D. Glucose broth cultures turn cloudy and often have smooth or coarse precipitates adhered to the bottom flask. The final pH value of glucose broth cultures is 4.8-5.1.

P. corporis can be detected in specimens of all types of clinical infection, including the oral cavity.

The genomic GC content is 43-46%. The type strain is ATCC33457.

4.2.7.5 Prevotella loescheii

Cultures of P. loescheii require chlorinated hemoglobin, while the addition of 10% serum enhances fermentation. Colonies observed by stereomicroscope are shown in Figure 4.19(D) and (E).

Glucose broth cultures turn cloudy and are accompanied by a smooth deposit. The final pH value of cultures grown in glucose broth is between pH 4.9 and 5.4. P. loescheii does not produce H ₂S in SIM medium. But the hydrolysis of esculin and the ability to ferment cellobiose help distinguish this species from P. melaninogenica and P. dentocola.

The genomic GC content is 46% (type strain). The type strain is ATCC15930 (NCTC11321).

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16.4.1 Antimicrobial properties

Infection is the foremost reason for clinical failure of regenerative procedures. Hence, it will be advantageous to have a membrane with antimicrobial properties that tackles postsurgical infection and inflammation at the implantation site. The presence of periodontopathogens, such as Porphyromonas gingivalis ( Pg), Prevotella intermedia , and Fusobacterium nucleatum ( Fn), may negatively affect the success of periodontal regeneration. Therefore, it is extremely important to control and/or reduce bacterial contamination of the periodontal defect in order to enhance periodontal regeneration [18,69].

A wide range of antimicrobial drugs, including but not limited to tetracycline hydrochloride (TCH), metronidazole benzoate (MET), and doxycycline (DOX), have been successfully incorporated into distinct polymer membranes to develop a material with therapeutic properties [70-72]. For example, MET is an antimicrobial drug commonly used in the treatment of periodontal infections [73]. It acts against anaerobic Gram-negative (e.g., Porphyromonas gingivalis) and anaerobic spore-forming Gram-positive bacilli [74]. Zamani et al. [72] fabricated poly(ɛ-caprolactone) (PCL) nanofibrous membranes loaded with MET. Of note, MET release was without a burst and was sustained for at least 19 days, which could be key to the inhibition of related bacteria and an aid in the regeneration of a new periodontium [72]. Xue et al. [75] developed MET-loaded electrospun poly(ɛ-caprolactone)-gelatin nanofibrous membranes, which showed good biocompatibility without infection following subcutaneous implantation in rabbits for 8 months.

In a recent study by our group, we evaluated the antimicrobial properties of MET-containing and ciprofloxacin (CIP)-containing polydioxanone electrospun membranes [7]. The antimicrobial properties were evaluated against those of Pg and Enterococcus faecalis ( Ef). Cytotoxicity was assessed in human dental pulp stem cells (hDPSCs). CIP-containing membranes significantly inhibited the biofilm growth of both bacteria. Conversely, MET-containing membranes only inhibited Pg growth (Fig. 16.1). Agar diffusion confirmed the antimicrobial properties against specific bacteria for the antibiotic-containing membranes. Only the 25 wt% CIP-containing membranes were cytotoxic.

TCH was incorporated into poly(l-lactic acid) (PLLA) fibers by either blending or coaxial electrospinning [71]. Two relevant conclusions were drawn based on the in vitro drug release evaluation. First, it was demonstrated that threads processed from the core-shell fibers had a lower early burst and a more continuous release. Second, threads processed from the blend fibers resulted in a greater early burst release, which can be of great value in avoiding bacterial infection [71]. In another study, Mohammadi et al. [76] fabricated blend and core-shell electrospun membranes using poly-lactic-glycolic acid (PLGA), gum tragacanth (GT), and TCH. Drug release studies showed that both the fraction of GT within blend nanofibers and the core-shell structure can effectively control the TCH release rate. By incorporating TCH into core-shell nanofibers, drug release was sustained for 75 days with only 19% of the burst release within the first 2 h. The prolonged drug release, together with proven biocompatibility, antibacterial, and the mechanical properties of drug-loaded core-shell nanofibers, makes them a promising candidate for use as a drug delivery system to fight periodontal infection.

Incorporation of amoxicillin (AMX) in PCL-based electrospun nanofibers could lead to barrier membranes that can reduce or eliminate the bacterial contamination of periodontal defects. In a new study, Furtos et al. [77] reported on the synthesis of nanocomposite PCL-based membranes modified with both AMX and nanohydroxyapatite (nHAp) to endow antimicrobial and osteoconductive properties, respectively. Meanwhile, recent studies have also reported on the fabrication of electrospun membranes loaded with anti-inflammatory drugs. Farooq et al. [78] incorporated piroxicam, a nonsteroidal anti-inflammatory drug, into novel cytocompatible and biodegradable chitosan (CS)/poly(vinyl alcohol) (PVA)/hydroxyapatite (HA) electrospun composite nanofibrous membranes. In another investigation, Carter et al. [79] blended Aloe vera, proven for its anti-inflammatory properties, with PCL, to prepare GTR nanofibrous membranes aiming to enhance periodontal healing.

It is well recognized that antibiotics may produce some important side effects, mainly those related to bacterial strain resistance, which is a current global concern, since bacteria are becoming resistant to several antibiotic therapies. In light of this, alternative antibacterial agents, rather than antibiotics, have been developed to translate safer regenerative technologies into clinical practice for GTR/GBR applications. Inorganic ions and metallic oxides have extensively demonstrated antibacterial properties. Silver is the most effective antibacterial metal at low concentrations and against both Gram-positive (G +) and Gram-negative (G −) bacteria. Meanwhile, several other metal oxides (e.g., copper, magnesium, titanium, and zinc) have also displayed important antibacterial activity against a wide variety of microorganisms. In this way, a recent study by our group demonstrated the successful synthesis of PCL-based electrospun membranes using ZnO as the antibacterial agent (Fig. 16.2) [80]. The antibacterial activity of the processed fibrous membranes was evaluated against known periodontopathogens, that is, Porphyromonas gingivalis ( Pg) and Fusobacterium nucleatum ( Fn), using agar diffusion assays. All the membranes containing different concentrations of ZnO (5, 15, and 30 wt%) presented antibacterial activity against the targeted bacteria. The membranes, when applied against Pg, showed similar antibacterial activity regardless of the concentration of ZnO; however, when applied against Fn, the most concentrated group (30 wt%) displayed higher antibacterial activity (Table 16.1).

Bacteria

CHX (control)

MIC (μg/mL)

2500

5000

10,000

Pg
A11.7 (± 0.6) a

A8.3 (± 1.2) b

A9.0 (± 1.0) b

A8.0 (± 0.6) b

Fn
A10.3 (± 0.6) a

B0.0 c

B5.3 (± 0.6) b

A9.3 (± 1.2) a

ZnO content (wt%)
Pg Fn

PCL PCL/GEL PCL PCL/GEL

Distinct uppercase letters in a same column and lowercase letters in a same row indicate statistically significant differences among groups tested ( P &lt; .05).

CHX, chlorhexidine; PCL, poly(ɛ-caprolactone); PCL/GEL, mixture of PCL and gelatin (50:50 ratio, w/w).

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2.2.4 Oral Bacteria

Oral bacteria colonization on the surfaces of the teeth can generate dental biofilm, subsequently induce inflammatory response and cause gingivitis. Continuous inflammation leads to the loosening of the teeth, occasional pain, impaired mastication, and eventual tooth loss. This pathological condition is termed periodontal disease and is associated with Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, Prevotella intermedia , Campylobacter rectus, Eubacterium nodatum, Peptostreptococcus micros, Streptococcus intermedius, and Treponema sp. infections. Notably, periodontal disease considerably has a predictive value to AIDS and treatment failure, while some forms of the disease are among the seven cardinal oral lesions indicative of HIV infection, including linear gingivitis erythema (LGE), necrotizing ulcerative gingivitis (NUG), and necrotizing ulcerative periodontitis (NUP). LGE is characterized by a fiery linear erythema along the margin of the gingiva and occasionally with bleeding or petechial-like patches. NUG presents as acute and painful gingivitis with limited destruction of the soft tissues, whereas NUP involves the destruction of the periodontal ligament and the alveolar bone.

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# data[].attributes.attachment_link.description
Periodontal disease is an infective process usually associated with pathogens such as Actinobacillus actinomycetemcomitans or Prevotella intermedia. Gram-negative anaerobes have developed a selective ecologic niche that leads to the destruction of periodontal tissues. The use of pharmaceutical agents can dramatically reduce the number of these pathogens and is of great benefit in periodontal therapy.

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In the picture below (phylogenetic tree), one can see that the most recent common ancestors are Prevotella Bivia and Prevotela Buccalis. The most recent addition to the phylogenetic tree is Prevotella Buccae. Also, the oldest ancestor is Leptotricia Buccalis.

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Domain: | Bacteria
Phylum: | Firmicutes
Class: | Mollicutes
Order: | Mycoplasmatales
Family: | Mycoplasmataceae
Genus: | Mycoplasma
Species: | M.  Buccale

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Initiation and Progression of Oral Cancer      (Mycoplasma Buccale)

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Initiation and Progression of Oral Cancer      (Mycoplasma Buccale)

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Many successful studies have demonstrated the malignant potential of Mycoplasma in prostate cancer, myeloid cell lines, and mouse embryo cells, but there was no substantial evidence linking Mycoplasma to oral cancer. At that time (2005), there was a lack of in-vivo studies illustrating the pathogenesis of Mycoplasma in oral potentially malignant disorders. In vivo studies using animal models like the Syrian hamster cheek pouch may aid us in understanding the molecular pathway of Mycoplasma induced oral carcinogenesis, which in turn will provide us with vital diagnostic markers and potential therapeutic targets.

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Periodontal Disease                   (Prevotella Intermedia)

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Periodontal Disease                   (Prevotella Intermedia)

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Prevotella intermedia has serious implications in the context of medical pathology for humans, It is one of the leading causes of periodontal disease. It is transmissible by saliva and kissing. it can cause subgingival plaque, tissue attachment loss, and probing depth beyond standard ranges. One of the main reasons why P. intermedia has a virulent impact on the connective tissue of the mouth, its because it can form biofilm which then turn into dental plaque; If the plaque is persistant it could leave the mouth vulnerable to infections.
[Prevotella Intermedia]

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 Genome analysis of Prevotella intermedia strain to reveal features related to pathogenicity and adaptation

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 Genome analysis of Prevotella intermedia strain to reveal features related to pathogenicity and adaptation

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Prevotella intermedia ZT belongs to a genus marked with highly dynamic genomes. The specific genes of Prevotella intermedia indicate that adhesion, competing with surrounding microbes and horizontal gene transfer are the main drive of the evolution of Prevotella intermedia.

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Background

Prevotella, previously classified in the genus Bacteroides, was a genus of an obligate anaerobic gram-negative rod-shape bacterium. They are classified as "black pigmented bacteria" as they form shiny and smooth colonies with grey, light brown or black color on blood agar plate [ 1]. Although they generally have a limited ability to ferment amino acid and require hemin and menadione to grow, Prevotella is a versatile genus which has been observed in various niches, such as oral cavity, upper respiratory tract, urogenital tract [ 2], rumen and human feces [ 3]. Many Prevotella taxa from oral cavity are potential/opportunistic pathogens. Gomes et al have shown that Prevotella spp is associated with pain and swelling and 'wet' canal of oral diseases [ 4- 6]. Besides, Prevotella intermdedia, Prevotella dentalis, Prevotella denticola, Prevotella meleninogenica are known to be pathogenic under a wide range of situations and invade into the host tissues [ 7- 9].

Prevotella intermediahas long been known to be associated with periodontal diseases [ 10- 12], periapical periodontitis [ 4, 5, 13] and noma (an acute gangrenous disease) [ 14, 15]. Prevotella intermedia also exists at the diseased site of periapical periodontitis [ 13, 16, 17] and shows a significantly higher detection ratio in symptomatic sites than in asymptomatic sites [ 13]. Besides involved in oral diseases, Prevotella intermedia was also reported to colonize in the respiratory tract and be associated with cystic fibrosis and chronic bronchitis [ 6, 18]. The association of Prevotella intermedia with various diseases raises a question what drives the evolution of Prevotella intermedia in different niches. The question also helps us predict how much gene mutation will take for a strain adapt to another niche.

There have been evidences to show that the intra-species difference exists among Prevotella intermedia strains in different niches, e.g. the degradative enzyme activity of Prevotella intermedia in diseased sites is significantly higher than in healthy sites [ 19]. However, the difference of the phenotype directly related with pathogenicity could be either genetic or non-genetic such as the regulation of gene expression under different niches and interaction with surrounding microbe. Previous study proved that the intra-species differences of some genes caused different bacterial adaptation to the environment [ 20, 21]. The intra-species variation could be an essential factor to these differences [ 22]. Therefore, our approach to solve this issue is to assume the drive is genetic factors and explore the intra-species variation among different strains of Prevotella intermedia.

The first published genome sequence of Prevotella intermedia is that of Prevotella intermedia 17. This is a clinical strain isolated from the periodontal pocket and is one of the most studied of the Prevotella intermedia strains. The genome has facilitated gene discovery in a number of studies [ 23- 25]. DOE Joint Genome Institute also submitted the draft genome sequence of the type strain, Prevotella intermedia ATCC 25611, a strain isolated from empyema. We isolated the clinical strain Prevotella intermedia ZT from the infected root canal of a Chinese patient with chronic apical abscess and obtained its draft genome. To further understand the general character of the genus Prevotella we also compared this strain with other taxa of the genus Prevotella.

Results and discussion

The general feature of the Prevotella intermedia ZT genome

89 contigs of Prevotella intermedia ZT genome were assembled (Figure ) while 91 singletons longer than 200 bp remained. 2165 ORF were predicted. The genome and annotations (BioProject ID: PRJNA208776; accession: ATMK00000000; BioSample SAMN02212661) have been submitted to NCBI.

Genomic comparison of virulence factors in Prevotella intermedia strains

General features of genome comparison

We aligned the assembled contigs of Prevotella intermedia ZT with the published P. intermedia 17 genome (Accession: PRJNA163151; GenBank: CP003502.1 and CP003503.1) and Prevotella intermedia ATCC 25611 DSM 20706 (Accession: PRJNA185645; RefSeq: NZ_JAEZ00000000.1) using MUMmer3.23. One average, 88% of the genome is matched with the weighed identity of 96.2% between any two genomes.

We clustered the genes of Prevotella intermedia ZT, 17 and ATCC 25611 and analyzed the COG classification. The numbers of core genes of the analyzed Prevotella intermedia strains are 1602, 1629 and 1602 (some clusters contains more than one genes from a genome) respectively, which is about 72% of all the genes encoded by each strain (Figure ).

Genes involved in adaptation and pathogenicity

The pathogenicity of anaerobic oral microbes usually initiates with the attachment and invasion of the host tissue. Pathogen will then break down certain cells or proteins of the host and later interfere with the immune system, stimulating inflammatory reaction [ 26]. Most of the genes involved in pathogenicity are shared by the three strains, but there are some strain-specific genes that contribute to adaption to niche and pathogenicity.

Adhesion and attachment

Adhesion to the surface of the tooth or mucosa is the first step and an essential requirement for pathogen survival and pathogenicity in oral cavity or empyema. The structure of cell envelope is the foundation of adhesion to host tissue and surrounding microbes. 85% of the homologous gene clusters predicted to be involved in cell envelope construction are conserved among the three strains while there are strain specific genes: notably, Prevotella intermedia 17 has six strain-specific glycosyltransferase clustered together.

Besides that, some gene was reported to specifically contribute to adhesion. AdpB, an ompA family gene, has been reported to be related to adhesion to host tissue. AdpB, a 29 kDa protein, is the first broad-spectrum ECM (extracellular matrix)-binding protein localized on the cell surface that was identified and characterized in Prevotella intermedia 17 [ 24]. It shared by all the strains 6 orthologous clusters of adhesin are found in Prevotella intermedia strains but only one of them is shared by all the strains. Notably, Prevotella intermedia ZT has a tandem of 4 cleaved adhesin domain protein and 3 of them are strain-specific (Table ).

Proteolysis and Bacteriocin

Protease or peptidase is one of the major virulence factors of Prevotella intermedia. Besides its role in degrading the host tissue, proteolysis is also an important part of the signaling pathway involved in various pathologies including inflammatory diseases [ 27, 28]. Previous studies have shown that Prevotella intermedia degrades proteins such as immunoglobulin and LPS-binding protein (LBP) to impede the defense of the host [ 29- 31].

A large proportion of the proteases locate in cytoplasm and participate in the basic metabolism such as amino acid metabolism and posttranslational modification (Figure ). Interestingly, Among the 24 strain specific proteases, 6 of them are bacteriocin or bacteriocin/lantibiotic exporters. The bacteriocins and lantibiotics also show great strain-specificity: except the two homologous cluster of lantibiotic ABC transporter and one cluster of bacteriocin, the rest of them are all strain specific. Bacteriocin and lantibiotic inhibit the grows of similar or closely bacterial strains. This indicate that the competition with the surrounding strains in the same niche, instead of breaking down proteins in the host tissues as we previously expected, may drive the evolution of Prevotella intermedia (Table ).

Table 2

a. The specific proteases of the Prevotella intermedia strains. Note the genes involved in bacteriocin

Iron uptake

Lysis of erythrocytes and degradation of hemoglobin are the major iron source of Prevotella intermedia [ 32]. This process begins with the accumulation of erythrocytes. According to a previous study, fimbriae of Prevotella intermedia induce hemagglutination [ 33], and the gene encoding hemagglutinin has been isolated [ 34, 35]. Hemagglutinin, or mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase, induces the aggregation of erythrocyte

Prevotella intermedia also has hemolytic activity [ 34, 36- 39]. When Prevotella intermedia is incubated in medium with rabit/sheep/humam red blood cells, beta-hemolytic zones are formed. The hemolytic activity reaches its maximum in weak acid condition [ 36]. The gene encoding hemolysin, the protein responsible for hemolytic activity, has been isolated and characterized [ 38]. Hemolysin gene is also identified in all Prevotella intermedia strains. Its homologous genes with the same/similar function are found in a wide range of Prevotella taxa

As to degredation of hemoglobin, Prevotella intermedia 17 encodes a cysteine protease named Interpain A (InpA), which breaks down oxyhaemoglobin by oxidating it to aquomethaemoglobin (in which the heam iron is oxidated to the Fe(III) state and has H ₂O as sixth co-ordinate ligand) at pH6.0. Aquemethaemoglobin is further completely broken down and hame is released to generate black-colored Fe(III) protoporphyrin IX pigment [ 40, 41]. Interpain A also disturbs immune reaction of the host by degrading complement factor C3 [ 42]. The counterpart gene is identified in other Prevotella intermedia strains and its homologous proteins are found in Prevotella nigrescens and Prevotella pallens.

Prevotella intermedia strains share 2 copies of hemin-binding protein genes and a hemin receptor gene. Although PSORTb [ 43] fails to give any explicit prediction of the location of the two hemin-binding protein, their homologous genes in other Prevotella spp. are annotated to locate on the membrane. Hemin-binding protein identified in oral pathogen Porphyromonas gingivalis [ 44, 45] and Treponema denticola [ 46] is a membrane protein that facilitates the uptake of hemin and the deletion of it will retard the growth of the bacteria in the iron-restricted media.

The whole pathway is intact in all the three strains. Although some genes with on the pathway are annotated to be hypothetic protein, they have more than 95% whole length identity with the genes annotated to be in the iron uptake pathway (Table ).

Drug resistance

Prevotella intermedia strains are predicted to encode different copies of beta-lactamases and multidrug/efflux transporters, which provide resistance to antibiotics. Genes of beta-lactamases and multidrug/efflux transporters are shared by almost all the three strains although in two of the homologous clusters, some of the genes are not annotated to be beta-lactamases despite of the more than 95% whole length identity with beta-lactamase gene (Table ).

Other feature related to virulence

Dipeptidyl peptidase IV(DPP4) are also found in the all the three Prevotella intermedia strains. DPP4 is a protease located on the surfaces of different cells across species including the genus Prevotella and also encoded by Prevotella intermedia 17 [ 47] and other 2 strains. Having a wide range of substrates [ 48], DPP4 is related to various physiological process including immune regulation, signal transduction and carcinogenesis [ 49- 52]. A previous study showed that DPP4 from Prevotella gingivalis in gingival crevicular is associated with its pathogenicity and that Prevotella spp also has strong DPP activity [ 53]. This evidence points to an important role for DPP4 in pathogenicity.

Hypothetical virulence factor BrkB (103055), a 448-amino acid protein, was also found shared by the three strains. Virulence factor BrkB was first identified in Bordetella pertussis and was found to be responsible for serum resistance [ 54]. It is also shared by the three strains.

Virulence-associated protein E and other toxin proteins were identified in the three strains. The strains both share several copies of homologous genes and each possesses strain-specific genes (Table ).

Recombination in Prevotella intermedia

There are more about 130 genes predicted to have functions of replication, recombination and repair in each Prevotella intermedia genome. Recombinase, integrase and type I restriction enzymes and clusters of conjugal transfer protein genes are found in the genomes (data not shown). We also found cluster of strain-specific genes (Additional file 1) in all three strains, which are possibly the relic of previous transpositions.

Some of these clustered strain-specific genes are potentially related with adaptation and pathogenicity: Prevotella intermedia ZT has a cluster containing 1,3-beta-glucan synthase regulator genes; Prevotella intermedia 17 has a cluster of ABC transporter genes and a cluster of glycosyltransferase genes; Prevotella intermedia ATCC 25611 has a cluster containing glycosyltransferases and Na + -driven multidrug efflux pump and a cluster containing BC-type bacteriocin.

Genomic comparison of gene content within the genus Prevotella

In addition to the intro-species genome alignment between Prevotella intermedia ZT and Prevotella intermedia 17, we aligned the Prevotella intermedia ZT genome against the genomes of Prevotella dantalis DSM 3688 (NCBI ID: 184818), Prevotella melaninogenica ATCC 25845 (NCBI ID: 51377), Prevotella denticola F0289 (NCBI ID: 65091), Prevotella ruminicola 23 (NCBI ID: 47507) (Additional file 2). The alignments show that the genome sequences are barely matched between two different species. The matched region count only ~3% of the whole genome in the nucleotide sequence. However a ~49Kb region that is conserved in terms of gene content and partly DNA sequence is shared by all the considered genomes (Table ). This region located around from 56 Kb to 95 Kb of contig00007 in the draft Prevotella intermedia ZT genome sequence. It encodes DNA-directed RNA polymer, 30S and 50S ribosomal proteins and translation elongation factor and five tRNA. And the corresponding regions in other genomes have similar gene contents and arrangement.

Table 6

COG analysis

In addition to the Cluster of Orthologous Group (COG) [ 55] distribution of Prevotella intermedia ZT, the Pan-genome of Prevotella intermedia ZT, Prevotella intermedia 17 (NCBI ID: 163151), Prevotella dantalis DSM 3688 (NCBI ID: 184818), Prevotella melaninogenica ATCC 25845 (NCBI ID: 51377), Prevotella denticola F0289 (NCBI ID: 65091), Prevotella ruminicola 23 (NCBI ID:47507) was also annotated.

Unlike the COG analysis of a single genome, COG analysis of pan-genome gives the number of gene clusters classified with certain annotation instead of the number of genes. The COG result is stratified based on the gene cluster level given by PGAP1.1. The gene cluster level is the number of the genome that distribute to a certain cluster. If a cluster has the cluster level of n, it has the genes from n genome(s). Core clusters consist of genes from all the analyzed genomes (in our study, they have the cluster level of 6). Strain-specific clusters consist of genes from only one genome and have the cluster level of 1. The rest are dispensable clusters. Whole-cluster presents the gene function distribution of all the genes in the pan-genome, namely, the sum of core, dispensable and strain-specific clusters.

As expected, Prevotella intermedia ZT has a similar COG distribution with the whole-cluster of pan-genome (Figure ). The function class with relatively high number of gene clusters in the pan-genome also has relatively high number of genes in Prevotella intermedia ZT genome and verse visa. As taxa of the same species, they reasonably share some common features.

The more striking feature of the analyzed taxa is the specific genes. For every COG class, the major of the clusters are strain-specific (Figure ) which accords with the results drown from the mathematics model that the genus Prevotella has a considerable number of strain-specific genes.

The genes classified into the "intracellular trafficking, secretion and vesicular transport" is absent in the core genome but enriched in strain-specific cluster. The high species specificity of secretion function in the genus Prevotella indicates the high specificity of the interaction between Prevotella taxa and its niche.

There is no gene in "cell motility" class in the core genome either. However, considering the fact that "cell motility" has the least genes in every single genome and that KEGG pathway analysis shows that the genus Prevotella generally has no or only one gene in flagella synthesis pathway, the absence of "cell motility" gene in the core genome should be due to the low motility and probably the absence of flagella of the genus Prevotella. We assume that the genus Prevotella make use of other structures or the help of the surrounding microbe populations to attach on the surface of the oral cavity and invade into the abscess tissue.

Pan-genome analysis and gene distribution mathematics model

Pan-genome is originally used to describe the core genome of all taxa of a species typically the bacteria and archaea species [ 56]. Considering the similarity between different species of the genus Prevotella, the term pan-genome is used to describe the essential genes of the genus Prevotella. For the sake of data validity we use only the completely sequenced Prevotella taxa and Prevotella intermedia ZT to perform the pan-genome analysis. The completely sequenced genomes are Prevotella intermedia 17 (NCBI ID: 163151), Prevotella dantalis DSM 3688 (NCBI ID: 184818), Prevotella melaninogenica ATCC 25845 (NCBI ID: 51377), Prevotella denticola F0289 (NCBI ID: 65091), Prevotella ruminicola 23 (NCBI ID: 47507).

The genes from the six genomes were clustered with PGAP1.1 and only bidirectional best hits were further analyzed. The genus Prevotella has about 165-170 core genes, that is, the genes shared by all the analyzed taxa. This number slightly varies between different genomes because of gene duplications or homology. Each genome has strain-specific genes ranging from 1418 to 2763 (Table ).

Table 7

Venn diagram is usually used to show the distribution of conserved and strain-specific genes. However it will be too fuzzy when more than three genomes are to be considered. Therefore, we take advantage of the mathematics model provided by the PGAP to explicit the gene distribution of the genus Prevotella.

The mathematics model exhausts the pan-genome and core genome size generated from every possible combination of genomes and fit the data with a power model for pan-genome and an exponent model for core-genome:

where y denotes the number of genes in the pan-genome; z denotes the number of genes in the core genome; x denotes the genome number and a, b, c, m, n, k are the fitting parameter.

In the pan-genome model, parameter b is less than 1 because the contribution of each extra single genome added to the pan-genome analysis tends decrease due to the homologous genes between the each genome. From the model we can calculate some important parameter: y(1) is the average gene number of a single genome; y(2)- y(1) is the average difference in gene number of the pan-genome constructed from any two genome and a single genome, that is, the average strain-specific genes in a genome when any two genomes are compared.

In the core genome model, the index of e is minus because the size of the core genome decreases when more genomes are included in the pan-genome analysis. Similarly, z(1) is also the average gene number of a single genome and z(1)- z(2) is the average strain-specific genes in a genome when any two genomes are compared.

The mathematics model based on the six previous mentioned genomes is:

In this model, y(2)- y(1) is 1838.0, which is more than half of the genes in a Prevotella genome. This mathematics model shows that on average more than half of the genes are strain-specific between any two species.

We expand the sample size and build the mathematics from 29 species of the genus Prevotella (either completely sequenced genomes or draft genomes. For each species we only chose one genome in order to prevent that the intra-species differences will not interfere the model) and draw a similar conclusion. In the new model, the average genome size is slightly larger than the previous one and the number of strain-specific genes in a single genome is notably lowered. Even so, the number of strain-specific genes between any two genomes is a considerable 1232, which is nearly half of the genes encoded by a genome.

Phylogenetics of the genus Prevotella

The phylogenetic tree is a common methodology to infer the evolutionary relationship between different species. We selected among the genes that the 36 Prevotella taxa have in common (Additional file 3) and got 80 sets of homologous genes (Additional file 4). These genes are involved in carbohydrate utilization, cell component construction, genetic information process and other basic biochemistry reactions. The 80 sets of genes were used to generate a set of concatenated 34581-nt sequences to build the phylogenetic tree. Maximum likelihood and neighbor joining method were used and as expected generated almost identical results which shared the very same topology and strains of the same species were closest to each other (Figure ).

In order to reveal the influences of niche on the evolution of core genes of the genus Prevotella, we referred to the NCBI websites and previous literature for the isolation sites of each taxon and combined the isolation sites with the phylogenetic tree. Despite the possibility that the bacteria may survive and thrive in the niches other than the isolation sites, the isolation sites at least indicate the niches which the bacteria most successfully adapt to. The majority of the analyzed taxa were isolated from oral cavity but a few taxa were isolated from vagina ( Prevotella bivia, Prevotella disiens), human feces ( Prevotella stercorea, Prevotella copri), rumen and ( Prevotella ruminicola, Prevotella bryantii) and abscesses ( Prevotella bergensis, Prevotella timonensis). These exceptional species are scattered over different branches on the phylogenetic tree, which suggests that adapting to different niches did not play an essential role in the early evolutionary history of the genus Prevotella but accelerated the speciation in the late history.

The phylogenetic tree shows that the Prevotella intermedia has a close evolutionary relationship with Prevotella pallens, Prevotella nigrescens and Prevotella disiens. The former two species are usually isolated from oral cavity while the latter, Prevotella disiens is a pathogen that mainly exists in vagina [ 57, 58].

Amino acid metabolic pathway analysis

As a pathogen which has already adapted to protein-rich niches, Prevotella intermedia ZT lacks many biosynthesis pathways, especially those which synthesize small-molecule nutrients such as amino acid.

The pathways of amino acid metabolism and biosynthesis are very limited. According to the KEGG pathway analysis [ 59], the strain Prevotella intermedia ZT does not have biosynthesis pathway for most of amino acids with only two exceptions: the synthesis of glutamine and glutamate, which plays an important role in lipopolysaccharide biosynthesis, and the biosyntheses of serine from hydroxylpyruvate. Besides that, the strain Prevotella intermedia ZT possesses some enzymes that catalyze the synthesis of amino acid from other amino acid and immediate metabolite: 1) the glycine hydroxymethyltransferase that transform glycine to serine and also the reverse reaction, 2) saccharopine dehydrogenase that degrades saccharopine into lysine, 3) L-asparaginase that transforms asparagines to aspartic acid and 4) branched-chain amino acid aminotransferase that catalyzes the reversible transformation of valine, leucine and isoleucine to their immediate metabolites 2-oxoisovalerate, 4-methyl-2-oxopentanoate and (S)-2,3-methyl-2-oxopentanoate. The previous sequenced strain Prevotella intermedia 17 shares all the amino acid metabolism pathway features with Prevotella intermedia ZT with some minor differences.

The analysis of other Prevotella taxa, that is, Prevotella dantalis DSM 3688 (NCBI ID: 184818), Prevotella melaninogenica ATCC 25845 (NCBI ID: 51377), Prevotella denticola F0289 (NCBI ID: 65091), Prevotella ruminicola 23 (NCBI ID: 47507) shows that the genus Prevotella vary greatly in terms of amino acid metabolism. Prevotella ruminicola has the ability to generate every amino acid from other metabolites, which probably results from the carbonhydrate-rich but peptide-deficient ruminal environment. Other taxa more or less have lost the genes involved in amino acid biosynthesis and the loss of genes is most severe in Prevotella intermedia.

In terms of histidine metabolism, Prevotella intermedia has lost every gene in the histidine biosynthesis pathway but possesses a pathway to degrade histidine. Prevotella denticola and Prevotella melaninogenica have the same histidine degradation pathway as Prevotella intermedia does and they also lost most of the gene in the hisitidine biosynthesis pathway except histidinol-phosphate aminotransferase which generate L-histidinol phosphate with the translocation of an amino group from Imidazole-acetol phosphate to L-glutamate. In sharp contrast with Prevotella intermedia, Prevotella ruminicola has an intact histidine biosynthesis pathway but does not encode any enzymes to degrade hisitdine. Interestingly, Prevotella dentalis is similar with Prevotella ruminicola although it is isolated in oral cavity. It has no enzymes to metabolize histidine but a nearly full biosynthesis pathway with only two genes lost.

As to valine, leucine and isoleucine biosynthesis pathway, Prevotella intermedia, Prevotella denticola and Prevotella melaninogenica have lost all the genes except the branched-chain amino acid aminotransferase which also locates in valine, leucine and isoleucine degradation pathway. On the contrary, Prevotella ruminicola and Prevotella dentalis have a full biosynthesis pathway.

As to the synthesis of phenylalanine and tyrosine, all the analyzed taxa have some genes lost and the pathway in every genome is incomplete. The pathway can be divided into two parts: the biosynthesis of intermediate metabolite prephenate and the synthesis of phenylalanine and tyrosine from prephenate. All the taxa encode some of the enzymes involved in the biosynthesis of chorismate, the immediate metabolite that converts to prephenate reversibly. But they do not encode chorismate mutase, the enzyme that catalyzes this conversion. Both of the Prevotella intermedia strains have lost all the genes for synthesizing phenylalanine and tyrosine from prephenate while other taxa maintain two ( Prevotella melaninogenica) or four genes ( Prevotella dentalis, Prevotella denticola, Prevotella ruminicola).

The summary of the ability to convert other metabolite to certain amino acid is given in the Table . Expectedly, some conclusion about the amino acid metabolism of Prevotella intermedia drawn from the genome analysis accords with a previous bench experiment [ 60].

Table 8

The considerable differences in amino acid biosynthesis indicate the readiness of evolution under proper environment. The ability to synthesize amino acids, which are quite available from the host tissue, is in the process of degradation.

Carbohydrate metabolic pathway and energy production

As an anaerobic microbe, the genus Prevotella only possesses a limited repertoire of enzymes for carbohydrate metabolism and energy production. The enzyme content varies from species to species. Prevotella intermedia ZT and Prevotella intermedia 17 have relatively incomplete pathways but the enzyme repertoire in Prevotella dentalis is the most reduced. Compared with the oral-isolated taxa, the rumen isolated Prevotella ruminicola has the most comprehensive repertoire of carbohydrate usage enzyme (Table ).

Table 9

Glycolysis is the first step to metabolize glucose and energy production for every species. In our study, almost all the taxa that we analyze have a functional glycolysis pathway except Prevotella dentalis, which lacks glucose-6-phosphate isomerase and 6-phosphofructokinase 1. These two enzymes catalyze the second and third reaction (transform α-D-glucose-6P to β-D-Frucose-1,6P ₂) of the glycolysis. Prevotella dentalis also lacks glyceraldehyde 3-phosphate dehydrogenase, the enzyme that catalyze the fifth reaction that transform D-glyceraldehyde 3-phosphate to 3-phospho-D-glyceroyl phosphate and several enzymes in the flanking branches of the glycolysis pathway.

In addition to glycolysis, we analyze other enzymes involved in carbohydrate metabolism. According to the KEGG pathway mapping, the genus Prevotella do not produce lactate or ethanol in cellular respiration due to the absence of the relative enzymes in the glycolysis pathway. Instead, in the pyruvate metabolism pathway, the pyruvate generated during glycolysis are the raw material for the synthesis of formate, oxaloacetate, which accords with the previous research [ 61]. These metabolites, especially noxious ones such as formate may contribute to the pathogenicity of the genus Prevotella.

Synthesis of cell wall components

Considering the analyzed taxa, the genus Prevotella has relatively complete, functionally conserved pathways for the synthesis of complex molecules that are used for cell material such as lipopolysaccharide and peptidoglycan.

Components such as lipopolysaccharides (LPS) [ 62- 70] induce immunological reaction of the host. In the Prevoeolla intermedia ZT genome, we identified genes related to lipopolysaccharides synthesis. The enzymes encoded by these genes compose a nearly full pathway from primal raw material UDP-N-acetyl-D-glucosamine to KDO ₂-lipid IV(A) (KEGG ID: C06025) except that the 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase (KDO 8-P phosphatase) is missing. This enzyme catalyzes the transformation from 3-deoxy-D-manno-octulosonae-8P to 3-deoxy-D-manno-octulosonate. Considering the economy of microbe metabolism, we conjectured that it was not likely that the whole biosynthesis of LPS should be interrupted by one single missing gene and therefore there might be other mechanism that hydrolyzed the phosphate group of 3-deoxy-D-manno-octulosonae-8P. Besides that, no enzyme on other branches of LPS synthesis has been found in this study. From the facts mentioned above, it is natural to draw the conclusion that the main component of LPS of Prevotella intermedia ZT is KDO ₂-lipid IV(A).

Other taxa have a similar LPS biosynthesis pathway. Generally, the Prevotella has a functional pathway to synthesize KDO ₂-lipid IV(A) but a few of enzymes in the branch pathway to modify the final product vary with taxa (Figure ).

The synthesis of another important bacteria cell wall component peptidoglycan is also well equipped with a fully functional pathway (Figure ). Compared with Prevotella intermedia 17, Prevotella intermedia ZT lacks only one gene, undecaprenyl-diphosphatase which hydrolyzes a phosphate from ditrans,octacis-undecaprenyl diphosphate. Although there are some disconnected branches in the peptidoglycan pathway, the main string of the pathway is unblocked. Besides, the blocked branches on the biosynthesis pathway indicate a corresponding biased peptidoglycan composition: The most likely major protein components of the peptidoglycan of genus Prevotella is undecaprenyl-diphospho-N-acetylmuramoyl-(N-acetylglucosamine)-L-alanyl-D-glutamyl-meso-2,6-diaminopimeloyl-D-alanyl-D-alanine (KEGG entry: C05898) and undecaprenyl-diphospho-N-acetylmuramoyl-(N-acetylglucosamine)-L-alanyl-gamma-D-glutamyl-L-lysyl-D-alanyl-D-alanine(KEGG entry: C05893).

Conclusions

As an important pathogen, Prevotella intermedia has long interested stomatologists and microbiologists. It is of clinical significance to study what is the potential drive of its adaptation and pathogenicity in different niches. In previous study, Prevotella intermedia 17, a clinically isolated strain from a human periodontal pocket and Prevotella intermedia ATCC 25611, the type strain isolated from empyema were sequenced. We isolated the strain Prevotella intermedia ZT from the infected root canal of a Chinese patient with chronic apical abscess and compared the genome content of this strain with other Prevotella taxa.

Our study confirmed that Prevotella intermedia ZT possessed a range of virulence factors involved in adhesion, proteolysis, iron uptake etc. For every aspect of pathogenicity, the involved genes have a relatively conserved part and a strain-specific part. The level of conservation varies with the aspect of pathogenicity, which indicates that the different strains of Prevotella intermedia inherited a basic package of genes enabling it to adapt to different niches and cause diseases, and later the package diverged under different conditions and the genes of different functions evolved at different rates. As far as this study shows, strain specificity is greater within genes involves in adhesion and the product and secretion of bacteriocin. It shows that, instead of exploiting the resources from the host, the most important variables of adaptation and pathogenicity are adhesion to the host tissue and/or surrounding microbes and competing with surrounding microbes. Besides, the activity of recombination shows that Prevotella intermedia may gain pathogenic genes through horizontal gene transfer.

Considering the similarity of the living environment and basis gene contents of the genus Prevotella, we analyze other Prevotella taxa to get a better view of what aspect of adaptation and pathogenicity matters most for the survival of Prevotella.

Our study shows that Prevotella taxa have very dynamic genomes with a part of relative high conserved contents. While comparing different taxa across species, we also find the existence of a conserved part. The genus Prevotella has a set of core genes involved in almost every COG class except "intracellular trafficking, secretion and vesicular transport" and "cell motility". The genomes also share an ~49 kb conserved region involved in ribosome biosynthesis and translation. The pathways involved in carbohydrate metabolism and cell wall component synthesis are relatively conserved among the genus Prevotella. The anaerobic respiration product of genus Prevotella is neither lactate nor ethanol but potentially noxious chemicals such as formate. On the other hand, Prevotella taxa have very dynamic genomes. From the mathematics model, we draw the conclusion that the genus Prevotella have highly dynamic genomes that vary greatly in sequences and gene contents from species to species: On average any two genomes of different species share only half of the genes. The strain-specific genes make a large part of each COG cluster in the pan genome, which allows the taxa of the genus Prevotella to adapt to various niches and to show various pathogenicities. The genus Prevotella display great difference in amino acid biosynthesis. Rumen-isolated species Prevotella ruminicola maintains the ability to synthesize all the amino acid while other oral taxa more or less lost some genes to convert other metabolites to amino acids. The gene loss in Prevotella intermedia is most severe.

This result shows that Prevotella taxa which parasitize in oral cavity have enough nitrogen sources so that they gradually lost gene of amino acid synthesis. However, the function of cell wall synthesis remains intact under a highly dynamic context, which accords with our previous hypothesis that adhesion is one of the main drives of the evolution of Prevotella intermedia.

With a highly dynamic genome which enables fast adaptation, metabolic pathway that product potentially noxious products and a wide range of virulence factors, Prevotella intermedia are well equipped for survival in different niches and pathogenicity. Our study shows that instead of exploiting the resources from the host, adhesion and the competition and horizontal gene transfer with the surrounding microbes may be the main drives of the evolution of Prevotella intermedia.

However, since the core content of the Prevotella intermedia has already provide the basic function for each process of pathogenicity, the variation in pathogenic genes is not very significant. Actually it leaves a question whether the variation causes or is caused by adaptation to different niches. Limited by the data resources, we cannot fully answer and validate this question, but we assumed that the almost full function of core content can explain why Prevotella intermedia is able to survive in various niches.

We use a new method to evaluate the variation of genome content. Venn diagram is a common method to visualize the differences in the gene contents. It is explicit and easy to understand but troublesome when more than 5 genomes are analyzed due to the limited expression of flat graph. Here we make use of a mathematics model provided by PGAP to summarize and quantify the tendency of the gene content variation between different genomes. The mathematics model can be served as a supplementary for Venn diagram.

Method

The collection of clinical samples

Selection of patient

The sample was collected from a primary periapical periodontitis patient (male, 34 years old) who had sought treatment for endodontic treatment of his mandibular right canine (tooth 43) diagnosed with apical periodontitis. Biting pain, percussion pain and tenderness to palpation were presented. There was a sinus tract over the buccal mucosa of tooth 44. Gutta-percha tracing of the sinus tract indicated the lesion was originated from the apical region of tooth 43.

The patients had no systematic diseases and had received no previous pulp and periodontal therapy and no antibiotics usage within 3 months before sample collection. The tooth harboring pulpal necrosis was sampled and the tooth was intact enough to allow adequate isolation from saliva.

We obtained the written informed consent from the patient, and the study was approved by the Ethic Committee of Shanghai 9th People's Hospital affiliated to Shanghai Jiao Tong University, School of Medicine, China. The age, gender, general conditions, prior oral treatment and preoperative radiograph were recorded.

Sample collection

The microbiological samples were collected from the infected root canals as described in previous studies [ 16, 71]. After isolated from the oral cavity by a disinfected rubber dam, the tooth was disinfected with 3% hydrogen peroxide followed by 2.5% sodium hypochlorite, which was inactivated with 5% sodium thiosulfate to avoid interference with bacteriological sampling. The enamel was removed with sterile high-speed fissure bur and the operation area was disinfected again. The initial entry to pulp space was made by sterile burs under manual irrigation with sterile 0.9% saline solution. The patency of the root canal was established by a sterile 15 K-type file without any usage of chemical solvents. Consecutively autoclave-sterilized dry paper points were inserted in the full length of the canal using the preoperative radiograph so that the fluid inside the root canal was absorbed. If the canal was dry, a little sterile 0.9% saline would be syringed into it first. The paper points per root canal were pooled into a 1.5 ml centrifuge tube containing 1 ml sterile pre-reduced PBS (Phosphate Buffered Saline) and transported to Shanghai Key Laboratory of Stomatology, China for culture within 20 min.

Culture isolation

The clinical transport fluid mentioned above was diluted 100X and 50 μl dilution was inoculated into anaerobic blood agar CDC-based selective medium for black pigmented bacteria [ 72- 74]. The content of the medium was 48 g CDC powder (Hangzhou Tianhe Microorganism Reagent Co., Ltd), 50 ml sheep blood, 1 ml Vitamin K1, 0.1 g kanamycin (Sangon Biotech (Shanghai) Co., Ltd) and 7.5 mg vancomycin (Japan, Eli Lily Japan KK) dissolved in 1 L distilled water. The plates were cultured in an anaerobic incubator (UK, Ruskinn) in an atmosphere of 85% N ₂, 10% CO ₂, 5% H ₂ at 37°C for 7 days. 95 black colonies appeared in 3 of the 13 samples and were all isolated for further purification by subculture. The pure colonies were picked into eppendorf tubes and stored at -20°C.

PCR testing

Genome DNA of the 95 purified clones and standard strain Prevotella intermedia ATCC25611 supplied by Shanghai Key Laboratory of Stomatology, China were extracted with TIANamp Bacteria DNA Kit (Tiangen Biotech Co., LTD, Beijing, China) according to the manual. The DNA concentration of the samples is measured with spectrophotometer (Thermo Fisher Scientific Co., Ltd, Massachusetts, USA). The samples were diluted to 5 ng/μl and amplified with Prevotella intermedia specific PCR primers with the expected product length of 576 bp: the forward primer: 5′-TTTGTTGGGGAGTAAAGCGGG-3′; the reverse primer: 5′-TCAACATCTCTGTATCCTGCGT-3′ (Sangon Biotech Co., Ltd, Shanghai, China). 1 μl DNA template was added to the PCR mixture containing 12.5 μl Premix Ex Taq vention (Biotechnology Co., LTD, Dalian, China) and 0.5 μl of each primer and 10.5 μl sterilized ultrapure water. The PCR was carried out in a thermal cycle (Applied Biosystems, California, USA) under the program as follows: 94°C for 10 min, 35 cycles at 94°C for 1 min, 60°C for 30 s,72°C for 30 s, and a final 72°C for 7 min. PCR products were analyzed with 1% agarose gel electrophorisis. The agarose gel was stained by ethidium bromide, and the image of the gel was captured under UV light by CCD camera. The sample that yielded the predicted amplicon of 576 bp were predicted to belong to Prevotella intermedia species. They were selected and sent to Shanghai genomePilot Technology, Inc for whole-length 16 s rDNA sequencing. The DNA sample were applified with general 16 s DNA primer Forward 5′-AGAGTTTGATCCTGGCTCAG-3′, Reverse 5′-ACGGCTACCTTGTTACGACTT-3′. The Product is sequenced and compared against NCBI database with nBLAST tool. The result confirmed that the strains belonged to Prevotella intermedia species

Genome sequencing, assembly and annotation

The genomic DNA was sequenced by Roche 454 GS FLX system according to the standard protocol provided by the manufacturer and using the default parameter. 309,368 reads totaling 123,745,995 nucleotides with an average length of 400 bp per read were generated, providing approximately 65X of genome coverage. The raw data was trimmed using SeqClean and Lucy (1.20p version) to eliminate joint sequences generated during library building and sequencing. 871 reads shorter than 50 bp were also left out. 167 contaminated reads from other species were detected by blasting against the NCBI NT Database and omitted from further analysis. 308,007 reads totaling 123,017,649 bp remained, 99.79% of which were assembled by Newbler 2.5.3 into 89 contigs amounting to 2,758,856 bp and 320 singletons amounting to 53,951 bp.

ORF prediction was performed using GLIMMER 3.0. The ORF sequences were in silico translated and search against Non-redundant protein sequences database with blastp with the threshold of e < 1e-3 and identity > 40%. Only the best hits were used for gene function prediction. tRNA, tmRNA, rRNA were predicted by tRNAscan-SE ( http://lowelab.ucsc.edu/tRNAscan-SE/), ARAGORN and RNAmmer1.2 respectively.

Other genomes of the genus Prevotella were downloaded from NCBI ftp ( ftp://ftp.ncbi.nih.gov/genomes/).

The predicted protein sequences were classified with on-line COG engine WebMGA [ 75], which aligned the query sequences to the COG database updated on Feb 2nd 2011 with rpsblast 2.2.15 [ 76].

The protein sequences were mapped to KEGG [ 59] pathway using the online server KAAS [ 77] ( http://www.genome.jp/tools/kaas/).

Genomic sequence comparison

The genome comparison was carried out using MUMmer 3.23. The analysis was performed according to the online manual ( http://mummer.sourceforge.net/manual/) and the result was visualized by mapview.

Gene comparison was carried out using Blastn, with the threshold of e-value = 10 −10, coverage = 80%, identity = 60%.

Pan-genome analysis with PGAP

Pan-Genomes Analysis Pipeline (PGAP) is a perl-script pipeline that consists of orthologous gene clustering, pan-genome analysis, SNP in CDS calling, evolutionary analysis and COG distribution.

The orthologous gene clustering was performed with The GeneFamily Method and ran as the following: The genes were first aligned using BLASTP with the following threshold: e-value of 1e-10 and the score of 40. Only gene pairs of Bidirectional Best Hits results (the identity greater than 50 and coverage greater than 0.5 from both sides) were clustered together. The other analysis was performed using the default parameter provided by the pipeline.

KEGG pathway analysis

The predicted ORFs were mapped to the KEGG pathway by KEGG Automatic Annotation Server (KAAS) [ 77]. The Genes data set was the default "for prokaryotes" and the assignment method was bi-directional best hit.

Prediction of protease

We predicted genes for proteases by two methods: we 1) searched among the annotation of ORFs and 2) searched all the predicted ORFs against the MEROPS release 8.9 Protease Database. The 2 sets of results were then merged. The genes predicted to be protease by MEROPS but annotated to be enzymes other than protease with a high score and identity by BLAST were manually removed.

We further aligned all the predicted protease genes against the PSORTb [ 43] for the cellular location.

Phylogenetics of the genus Prevotella

In order to infer the phylogenetic relationships of the Prevotella intermedia ZT strain and the other 35 Prevotella taxa (Additional file 3), we first downloaded the genome data from NCBI ftp ( ftp://ftp.ncbi.nih.gov/). Then the putative orthologous gene sets were generated through the following process: we first compared the all the 2067 predicted translated ORF sequences of Prevotella intermedia ZT strain against other taxa of the genus Prevotella by BLASTP method with the cutoff e-value of 0.1. 717 genes queries that have at least one hit in every species were selected for further analysis. Every query and its best representations of each species were viewed as a set of putative homologous genes. From these gene sets we manually screened for those that were 1) annotated to have the same biological function and 2) the housekeeping genes which are not likely to be influenced by horizontal gene transfer. 80 gene sets were picked and aligned and by ClustalW2.1 [ 78] with default parameter. The alignment files were clipped and concatenated into a set of 34581-nt multi-gene sequence with the help of BioEdit7.0.

The phylogenetic trees were built with Maximum Likelihood and Neighbor joining method in the Molecular Evolutionary Genetics Analysis (MEGA5.10) [ 79]. For the ML method, Jones-Taylor-Thornton (JTT) model was used and the result was tested by bootstrap method with the bootstrap replications of 1,000. The rates among sites were set as uniform and all the gap and missing data were deleted. The NJ tree was generated using Poisson method and also tested by 1000-replication bootstrap.

Contributor Information

# data[].attributes.attachment_link.title
Comparative genome analysis of Prevotella intermedia strain isolated from infected root canal reveals features related to pathogenicity and adaptation

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Prevotella, previously classified in the genus Bacteroides, was a genus of an obligate anaerobic gram-negative rod-shape bacterium. They are classified as "black pigmented bacteria" as they form shiny and smooth colonies with grey, light brown or black color on blood agar plate [ 1].

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There are different ways to determine a species of bacteria from the genome that it carries without knowing its identity because some genes are specific to the species. One can use a tool that gives access to multiple sequences of RNA/DNA or even protein that can match the sequence with a percentage of similarity to different species. BLAST is this tool and it allows us to  use 16s ribosome to reconstruct phylogeny because it has a slow rate of evolution. Thus it allow us to get the specific species from its nucleotide sequence and eliminate all the species related to it. Also using phylogenetic trees allow me to understand and put into simple terms the ancestral relationship of different organisms in a simplified form and more easy to analyze. My two unknowns were not hard to find information about them once I knew their identity; I was able to find that they were both part of oral disease, they are gram negative bacteria and they do not provide any benefits to the body but dangerous disease. There are a lot research towards them and new findings keep appearing with recent research that will help humans tolerate their dangerous life style in our body. Finally, the TED talk by Dr. Jonathan Elsen was interesting to watch because it showed me that microbes are not all harmful to us humans but they provide benefits that our body alone cannot do. There are more cells from microorganisms than our own cells and the best understanding we can get from their job in our body is by looking at its genome. This will allows us to identify the species as a friend or foe.
Amazing Findings:
Kayla Smith
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