Nonoral Sites Research Articles

Exploring the presence of oral bacteria in non-oral sites of patients with cardiovascular diseases using whole metagenomic data

Cardiovascular diseases (CVDs) encompass various conditions affecting the heart and its blood vessels and are often linked with oral microbes. Our data analysis aimed to identify oral bacteria from other non-oral sites (i.e., gut, arterial plaque and cultured blood) that could be linked with CVDs. Taxonomic profiling identified bacteria to the species level and compared with the Human Oral Microbiome Database (HOMD). The oral bacteria in the gut, cultured blood and arterial plaque samples were catalogued, with their average frequency calculated for each sample. Additionally, data were filtered by comparison with the Human Microbiome Project (HMP) database. We identified 17,243 microbial species, of which 410 were present in the HOMD database and further denominated as “oral”, and were found in at least one gut sample, but only 221 and 169 species were identified in the cultured blood and plaque samples, respectively. Of the 410 species, 153 were present solely in oral-associated environments after comparison with the HMP database, irrespective of their presence in other body sites. Our results suggest a potential connection between the presence of specific species of oral bacterial and occurrence of CVDs. Detecting these oral bacterial species in non-oral sites of patients with CVDs could help uncover the link between oral health and general health, including cardiovascular conditions via bacterial translocation.

Virulence factors of Streptococcus anginosus – a molecular perspective

Streptococcus anginosus together with S. constellatus and S. intermedius constitute the Streptococcus anginosus group (SAG), until recently considered to be benign commensals of the human mucosa isolated predominantly from oral cavity, but also from upper respiratory, intestinal, and urogenital tracts. For years the virulence potential of SAG was underestimated, mainly due to complications in correct species identification and their assignment to the physiological microbiota. Still, SAG representatives have been associated with purulent infections at oral and non-oral sites resulting in abscesses formation and empyema. Also, life threatening blood infections caused by SAG have been reported. However, the understanding of SAG as potential pathogen is only fragmentary, albeit certain aspects of SAG infection seem sufficiently well described to deserve a systematic overview. In this review we summarize the current state of knowledge of the S. anginosus pathogenicity factors and their mechanisms of action.

Microcribriform Adenocarcinoma of Salivary Glands: A Unique Tumor Entity Characterized by an SS18::ZBTB7A Fusion.

The landscape of salivary gland carcinomas is ever-changing, with a growing list of new tumors and newly elucidated variants of well-known tumor entities. The routine use of next-generation sequencing has been instrumental in identifying novel fusions and tumor entities, which has helped bring the classification to a more objective and evidenced-based model. However, morphology remains critical in assessing the validity of these novel molecular findings, and most importantly, in assessing which of these findings will have an impact on the prognosis and treatment decisions for patients. The recognition of microsecretory adenocarcinoma (MSA) as a distinct low-grade malignancy of salivary glands, underpinned by MEF2C::SS18 , and a single possibly related case of SS18::ZBTB7A , recently expanded this growing list of distinctive tumors. It was not until now, however, that the morphology of the latter case was known to be unique and reproducible. The authors have now seen 4 of these distinctive tumors that show a combination of distinctive oncocytic cells forming compact glandular growth as well as amphophilic cells forming tubular growth, and suggest the appellation "microcribriform adenocarcinoma" (MCA). So far, these tumors appear to preferentially occur in nonoral sites (2 parotid, 1 submandibular gland, and 1 bronchial seromucous glands). By immunohistochemistry, they express S100 and SOX-10 with focal outer myoepithelial cells marked by circumferential p63, p40, and smooth muscle actin staining around some of the nests and tubules. The tumors show infiltrative growth within a hyalinized and myxoid stroma. Cytologically, they appear generally low grade, similar to MSA. The morphologic and molecular uniformity of these 4 microcribriform adenocarcinoma cases warrants their recognition, and while related to MSA, they are sufficiently different to be classified as a distinct tumor. So far, in limited follow-up, these tumors appear to be relatively indolent.

Periodontal herpesvirus morbidity and treatment.

Four billion individuals worldwide have a history of periodontitis, with the poorest people in society most affected. Periodontitis can lead to unsightly drifting of teeth and tooth loss that may interfere with the wellbeing of daily living and has also been linked to at least 57 medical diseases and disabilities. The etiology of severe periodontitis includes active herpesviruses, specific bacterial pathogens, and destructive immune responses, but herpesviruses seem to be the major pathogenic determinant. Periodontal herpesviruses that disseminate via the systemic circulation to nonoral sites may represent a major link between periodontitis and systemic diseases. Current treatment of periodontitis focuses almost exclusively on bacterial biofilm and will require revision. Periodontal therapy that targets both herpesviruses and bacterial pathogens can provide long-term clinical improvement and potentially reduces the risk of systemic diseases. Molecular diagnostic tests for periodontal pathogens may enable early microbial identification and preemptive therapy. This review details an efficient and reliable anti-infective treatment of severe periodontitis that can be carried out in minimal time with minimal cost.

A renewed focus on extra-oral manifestations of oral infections

Dental focal infections are extraoral manifestations caused by oral pathogens. Pathological oral conditions, such as periapical inflammation and periodontitis, can cause bacteremia. Dissemination of oral pathogens to nonoral sites can subsequently cause infections in extraoral tissues and organs. Cardiovascular infections and brain abscesses are the most common of these. The course of such infections can be lethal. In order to improve patient care, a closer collaboration between dental and medical caregivers is necessary.

Structural studies of surface adhesins expressed by oral bacteria

Dental plaque is one of the most complex biofilms known and consists of hundreds of bacterial species. Plaque development is initiated by the attachment of salivary proteins to the tooth surface, followed by adherence of early colonizers, such as the Gram-positive Actinomyces oris and oral streptococci. These bacteria form an initial biofilm that is used as attachment surface for late colonizers. The key factors for these microorganisms to network with other bacteria and cells are their surface adhesins and pili. These Gram-positive surface proteins are assembled by very different mechanisms. One form represents the pili, that consists of polymerized proteins linked by covalent bonds with a large adhesin located at the tip. A second form consists of large monomeric proteins with N-terminal adhesive domains presented on stalks formed by repetitive small domains. A third form is represented by the antigen I/II proteins, expressed by oral streptococci. Antigen I/II have a unique fold where a central domain is presented on a stalk formed by intertwining flanking regions bringing both the N- and C-termini close to the cell wall. We have solved structures representing all three aforementioned groups; the pilin protein FimP from A. oris, the surface adhesin sgo0707 from Streptococcus gordonii and antigen I/II proteins from S. gordonii, Streptococcus mutans as well as from Streptococcus pyogenes. The late colonizers are often Gram-negative and one of these bacteria is the periodontal pathogen Porphyromonas gingivalis. This bacteria causes tooth loss and chronic inflammation and increasing evidence points to that P. gingivalis also is involved in the onset of disease at non-oral sites, causing cancer, cardiovascular disease and diabetes. This bacteria expresses two forms of pili with hitherto unexplored structures. Since these pili are important virulence factors we are focusing on structure determination also of these.

Structural and Functional Analysis of the N-terminal Domain of the Streptococcus gordonii Adhesin Sgo0707

The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.

Oral Malodor: A Common Oral Problem

Oral malodor, also called halitosis or bad breath, is universally experienced condition that has a variety of etiologic factors. It is extremely common and majority of adult population have had it at some point in time. Halitosis may be physiological, pathological, and sometimes psychological also. Although numerous non-oral sites and many different causes have been correlated to bad breath, an estimated 80 percent to 90 percent of all bad breath odors originate from the mouth, and bacteria are directly responsible for most of the offensive gases. Specific groups of bacteria have been identified with the production of oral malodor, in particular, gram- negative, anaerobic bacteria. The unpleasant smell of breath mainly originates from volatile sulfide compounds (VSCs), especially hydrogen sulfide (H2S), methylmercaptan (CH3SH), and dimethylsulfide [(CH3)2S] result from the proteolytic degradation of peptides present in saliva, shed epithelium, food debris and gingival crevicular fluid (GCF). This article reviews the etiology, diagnosis, and treatment of oral malodor and gives a brief description of Halitosis Associated Life-quality Test (HALT) questionnaire, a specific 20-item quality-of-life measure for halitosis.

Plasmablastic Lymphoma Involving the Parotid Gland

Plasmablastic lymphoma is a rare form of non-Hodgkin lymphoma. It is strongly associated with HIV infection, although it has been recognized in immunocompetent patients. Plasmablastic lymphoma has a predilection for the oral cavity. Its occurrence in the parotid gland has not been previously described. We report a case of an HIV positive man who developed a rapidly enlarging parotid mass. A core biopsy of the parotid mass was evaluated by routine microscopy, immunohistochemistry, and in situ hybridization. The tumor was comprised of sheets of large cells with abundant cytoplasm, eccentric nuclei and prominent nucleoli. The cells exhibited a plasmacytic immunophenotype including expression for CD38 and CD138. An in situ hybridization assay for Epstein-Barr virus was positive. These findings were diagnostic of plasmablastic lymphoma. Plasmablastic lymphoma is notoriously difficult to diagnose, particularly when it arises in unexpected sites outside of the oral cavity. As an aggressive lymphoma, plasmablastic lymphoma must be considered in the differential diagnosis of a high-grade malignant neoplasm not just in the oral cavity but at non-oral sites including the parotid gland, particularly in an HIV-positive individual.

The Mouthwash Question

The Mouthwash Question

Effect on polymorphonuclear cell function of a human-specific cytotoxin, intermedilysin, expressed by Streptococcus intermedius.

Streptococcus intermedius is a member of the normal flora of the mouth but is also an opportunistic pathogen associated with purulent infections at oral and nonoral sites. Intermedilysin (ILY) has been shown to be a cytolysin capable of generating pores in the cell membrane of erythrocytes demonstrable by electron microscopy. This effect has been shown to be specific for human cells. Since polymorphonuclear cells (PMNs) are the main cell involved in innate immunity we investigated the effect of purified intermedilysin from Streptococcus intermedius on PMN function. Active ILY at a concentration of 40 ng/microl caused a significant decrease in the number of intact PMNs after 60 min. The active cytolysin, when compared with heat-inactivated ILY, did not appear to be chemotactic for the PMNs but did cause an increase in intracellular calcium, with increased cell surface CD11b expression, metabolic burst, and phagocytosis of Staphylococcus aureus. These findings may have implications for the role of ILY in deep-seated abscesses.

Calcium and vitamin D supplements reduce tooth loss in the elderly

Purpose Oral bone and tooth loss are correlated with bone loss at nonoral sites. Calcium and vitamin D supplementation slow the rate of bone loss from various skeletal sites, but it is not known if intake of these nutrients affects oral bone and, in turn, tooth retention. Subjects and methods Tooth loss was examined in 145 healthy subjects aged 65 years and older who completed a 3-year, randomized, placebo-controlled trial of the effect of calcium and vitamin D supplementation on bone loss from the hip, as well as a 2-year follow-up study after discontinuation of study supplements. Teeth were counted at 18 months and 5 years. A comprehensive oral examination at 5 years included assessment of caries, oral hygiene, and periodontal disease. The odds ratio (OR) and 95% confidence interval (CI) of tooth loss were estimated by stepwise multivariate logistic regression. Initial age (mean ± SD) of subjects was 71 ± 5 years, and the number of teeth remaining was 22 ± 7. Results During the randomized trial, 11 of the 82 subjects (13%) taking supplements and 17 of the 63 subjects (27%) taking placebo lost one or more teeth (OR = 0.4; 95% CI: 0.2 to 0.9). During the 2-year follow-up period, 31 of the 77 subjects (40%) with total calcium intake of at least 1000 mg per day lost one or more teeth compared with 40 of the 68 subjects (59%) who consumed less (OR = 0.5; 95% CI: 0.2 to 0.9). Conclusion These findings suggest that intake levels of calcium and vitamin D aimed at preventing osteoporosis have a beneficial effect on tooth retention.

Genetic heterogeneity in Prevotella intermedia, Prevotella nigrescens, Prevotella corporis and related species isolated from oral and nonoral sites.

Prevotella intermedia (43 isolates), Prevotella nigrescens (55) and Prevotella corporis (8) from oral and nonoral sites were distinguished by species-specific DNA fragments, after hybridization of DNA fragments with ribosomal RNA (ribotyping). Eight strains previously identified as P. intermedia did not have these specific fragments. P. nigrescens, P. intermedia and P. corporis formed separate clusters in dendrograms constructed using clustering with an unweighted pair group method with arithmetic averages of similarity values derived from ribotype patterns, with 10 subclusters in P. intermedia isolates and 26 in P. nigrescens. Nine groups of P. intermedia isolates and 6 of P. nigrescens shared identical patterns. Specific ribotypes or species were not associated with particular diseases when all isolates were analyzed. However, results from organisms isolated by one laboratory using consistent clinical reporting indicated that P. intermedia was associated with more severe forms of periodontitis and P. nigrescens with mild to moderate disease.

The role of black-pigmented Bacteroides in human oral infections.

Today, 10 black-pigmented Bacteroides (BPB) species are recognized. The majority of these species can be isolated from the oral cavity. BPB species are involved in anaerobic infections of oral and non-oral sites. In the oral cavity, BPB species are associated with gingivitis, periodontitis, endodontal infections and odontogenic abscesses. Cultural studies suggest a specific role of the various BPB species in the different types of infection. Bacteroides gingivalis is closely correlated with destructive periodontitis in adults as well as in juveniles. Bacteroides intermedius seems to be less specific since it is found in gingivitis, periodontitis, endodontal infections and odontogenic abscesses. The recently described Bacteroides endodontalis is closely associated with endodontal infections and odontogenic abscesses of endodontal origin. There are indications that these periodontopathic BPB species are only present in the oral cavity of subjects suffering from periodontal breakdown, being absent on the mucosal surfaces of subjects without periodontal breakdown. BPB species associated with healthy oral conditions are Bacteroides melaninogenicus, Bacteroides denticola and Bacteroides loescheii. There are indications that these BPB species are part of the normal indigenous oral microflora. Many studies in the past have documented the pathogenic potential and virulence of BPB species. This virulence can be explained by the large numbers of virulence factors demonstrated in this group of micro-organisms. Among others, the proteolytic activity seems to be one of the most important features. Several artificial substrates as well as numerous biological proteins are degraded. These include anti-inflammatory proteins such as alpha-2-macroglobulin, alpha-1-antitrypsin, C3 and C5 complement factors and immunoglobulins. B. gingivalis is by far the most proteolytic species, followed by B. endodontalis. Like other bacteria, the lipopolysaccharide of B. gingivalis has shown to be active in bone resorption in vitro and is capable in stimulating interleukin-1 production in human peripheral monocytes. Based on the well documented association with periodontal disease and the possession of relevant virulence factors, BPB species must be considered as important micro-organisms in the etiology of oral infections. B. gingivalis seems to be the most pathogenic and virulent species.

Long-wave UV light fluorescence for identification of black-pigmented Bacteroides spp.

Black-pigmented Bacteroides strains were grown on blood agar, and the colonies were evaluated for fluorescence from long-wave UV light. Most test strains of Bacteroides melaninogenicus subsp. intermedius exhibited a brilliant red fluorescence. B. melaninogenicus subsp. melaninogenicus fluoresced mostly red-orange. Bacteroides asaccharolyticus showed a yellow or red fluorescence. The intensity of the Bacteroides fluorescence weakened when the black pigment of the colonies developed. In contrast, neither young nor old colonies of the oral species Bacteroides gingivalis displayed fluorescence. Since B. gingivalis can produce severe oral infections and also can seed to nonoral sites, awareness of the inability of this organism to fluoresce is important for microbiologists utilizing UV light fluorescence to screen for black-pigmented Bacteroides spp. The present data also indicate that UV light fluorescence may be a rapid method of distinguishing some black-pigmented Bacteroides spp.

Antigenic studies of oral and nonoral black-pigmented Bacteroides strains.

Antigens of several oral and nonoral strains of Bacteroides asaccharolyticus (proposed classification of oral B. asaccharolyticus, Bacteroides gingivalis), Bacteroides melaninogenicus subsp. intermedius, B. melaninogenicus subsp. melaninogenicus, and B. melaninogenicus subsp. levii were identified in soluble preparations obtained by sonication, autoclaving, and NaOH treatment of whole bacterial cells. The sonicate preparations contained the most complete representation of soluble antigens using antisera to the whole organism in gel precipitation tests. Among strains of B. melaninogenicus subsp. intermedius many common antigens were detected, and no consistent antigenic differences were seen between strains from oral and nonoral sites. None of the antigens of B. melaninogenicus subsp. intermedius reacted with sera raised to several strains of oral or nonoral B. asaccharolyticus, nor did antigens prepared from the latter strains react with antisera to B. melaninogenicus subsp. intermedius. At least one common antigen was shared by strains of B. melaninogenicus subsp. intermedius and strains of B. melaninogenicus subsp. melaninogenicus; however, subspecies-specific antigens were also found. Antigens from and antisera to oral and nonoral strains of B. asaccharolyticus did not react with sera to and antigens from B. melaninogenicus subsp. melaninogenicus. Strains of B. asaccharolyticus isolated from the oral cavity were antigenically distinct from strains of B. asaccharolyticus obtained from nonoral sites and lesions. This lack of cross-reactivity between the oral and nonoral strains of B. asaccharolyticus together with recent findings of marked genetic differences between oral and nonoral strains of B. asaccharolyticus suggest that these groups of organisms may represent different species.

Production of phenylacetic acid by strains of Bacteroides asaccharolyticus and Bacteroides gingivalis (sp. nov.)

Strains of Bacteroides asaccharolyticus and Bacteroides melaninogenicus subspecies isolated from human and animal sources were examined for the production of phenylacetic acid. B. asaccharolyticus strains isolated from sites in humans and monkeys always produced phenylacetic acid. B. asaccharolyticus strains isolated from human nonoral sites consistently failed to produce this product. This metabolic difference correlates with the genetic dichotomy recently found to exist between oral and nonoral B. asaccharolyticus strains.

Genetic Heterogeneity in Bacteroides asaccharolyticus (Holdeman and Moore 1970) Finegold and Barnes 1977 (Approved Lists, 1980) and Proposal of Bacteroides gingivalis sp. nov. and Bacteroides macacae (Slots and Genco) comb. nov.

Strains of Bacteroides asaccharolyticus (Holdeman and Moore 1970) Finegold and Barnes 1977 (Approved Lists, 1980) which had been isolated from human mouths, most often from periodontal pockets, showed remarkably little deoxyribonucleic acid base sequence homology with the deoxyribonucleic acid of strains of the same species which were isolated from nonoral sites. In addition, four strains of catalase-positive Bacteroides melaninogenicus subsp. macacae Slots and Genco 1980 isolated from periodontal pockets in monkeys were distinct from B. asaccharolyticus. With deoxyribonucleic acid immobilized on membrane filters and index deoxyribonucleic acid made radioactive by translation repair with [3H] thymidine triphosphate, we found that the deoxyribonucleic acid homology among the three groups could readily be distinguished by their deoxyribonucleic acid base contents. The nonoral strains had 51 to 52 mol% guanine plus cytosine, the human oral strains had 46.5 to 48.5 mol% guanine plus cytosine, and the monkey catalase-positive strains had 42 to 43 mol% guanine plus cytosine in their deoxyribonucleic acids. Some strains which fit the species description of B. asaccharolyticus were not genetically related to the three groups. A new species, Bacteroides gingivalis sp. nov., which comprises those strains that resemble B. asaccharolyticus but have 46.5 to 48.5 mol% guanine plus cytosine, is described. These strains can also be distinguished serologically and by their production of phenylacetic acid. The type strain is Slots’ strain 2561 (=ATCC 33277). Bacteroides melaninogenicus subsp. macacae, which is comprised of the monkey isolates which resemble B. asaccharolyticus but which have a guanine plus cytosine content of 42 to 43 mol% and give a positive catalase reaction, is elevated to species status. Its name is thus Bacteroides macacae (Slots and Genco) comb. nov. The type strain of this species is Slots’ strain 7728-L6C (=ATCC 33141).

Evaluation of Fluoretec-M for detection of oral strains of Bacteroides asaccharolyticus and Bacteroides melaninogenicus.

Fluoretec-M is a polyvalent conjugate used in direct fluorescent-antibody staining for identification of the Bacteroides asaccharolyticus-Bacteroides melaninogenicus group. The Fluoretec-M reagent detected all oral and nonoral test strains of B. melaninogaenicus subsp. intermedius, all test strains of B. melaninogenicus subsp. melaninogenicus, and the nonoral strains of B. asaccharolyticus. However, the Fluoretec-M polyvalent reagent and the monovalent conjugates which constitute Fluoretec-M did not detect the oral strains B. asaccharolyticus. The use of Fluoretec-M can therefore generate false-negative results in studies of specimens from oral cavity and from nonoral sites in which an infection with B. asacacharolyticus of oral origin may have taken place. It is suggested that antibodies reactive with the oral antigenic type of B. asaccharolyticus be included in the preparative procedure of the Fluoretec-M reagent.

Direct hemagglutination technique for differentiating Bacteroides asaccharolyticus oral strains from nonoral strains

A simple and economical method for differentiating Bacteroides asaccharolyticus of oral sources from nonoral sources is described. The present data indicate that oral strains of B. asaccharolyticus strongly agglutinate sheep erythrocytes, whereas isolates from various nonoral sites typically are devoid of hemagglutination activity. The direct hemagglutination test may aid in determining the source of B. asaccharolyticus present in an infection, and thus the procedure has potential value as a means of biotyping.