Normal Oral Flora Research Articles

Macrolides and changes in the oral flora

Macrolides have been used in dental practice for many years, and may have a role in treating periodontal disease. Increased numbers of antibiotic-resistant oral streptococci have been reported after administration of both penicillins and macrolides. We confirm these findings for erythromycin, josamycin and azithromycin, and show that small numbers of macrolide-resistant streptococci are part of the normal oral flora at baseline. Resistant organisms fill the vacuum created by the removal of sensitive strains by antibiotic treatment. Following treatment with azithromycin, periodontal bacterial pathogens such as black pigmented anaerobes and spirochaetes decrease, whereas numbers of oral streptococci increase. These changes in the oral flora indicate a return to a healthier oral environment. In our studies, no clinical problems resulted from the transient increase in macrolide-resistant streptococci.

Local chemokine paralysis, a novel pathogenic mechanism for Porphyromonas gingivalis.

Periodontitis, which is widespread in the adult population, is a persistent bacterial infection associated with Porphyromonas gingivalis. Gingival epithelial cells are among the first cells encountered by both P. gingivalis and commensal oral bacteria. The chemokine interleukin 8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by gingival epithelial cells in response to components of the normal oral flora. In contrast, P. gingivalis was found to strongly inhibit IL-8 accumulation from gingival epithelial cells. Inhibition was associated with a decrease in mRNA for IL-8. Antagonism of IL-8 accumulation did not occur in KB cells, an epithelial cell line that does not support high levels of intracellular invasion by P. gingivalis. Furthermore, a noninvasive mutant of P. gingivalis was unable to antagonize IL-8 accumulation. Invasion-dependent destruction of the gingival IL-8 chemokine gradient at sites of P. gingivalis colonization (local chemokine paralysis) will severely impair mucosal defense and represents a novel mechanism for bacterial colonization of host tissue.

Biochemical and genetic characterization of a Prevotella intermedia/nigrescens-like organism.

Thirty-three previously non-typable faintly pigmented Gram-negative anaerobic bacterial isolates, biochemically most closely related to Prevotella intermedia and Prevotella nigrescens, were analysed for enzymic reactions, cellular fatty acid (CFA) composition, electrophoretic mobility of malate and glutamate dehydrogenases, hybridization with P. intermedia and P. nigrescens species-specific oligonucleotide probes and, for genetic heterogeneity, by arbitrarily primed PCR (AP-PCR). P. intermedia ATCC 25611T and P. nigrescens ATCC 33563T were run in parallel for comparison. Twenty-nine isolates originated from the normal oral flora of 18 subjects (including five mother-child pairs), and four isolates from various infections. Except for a negative lipase reaction, enzymic profiles of the test isolates were similar to those of P. intermedia and P. nigrescens. Clustering of CFAs, electrophoretic mobility patterns, hybridization with DNA probes for P. intermedia and P. nigrescens, and AP-PCR band patterns of the test isolates differed from those of the type strains of P. intermedia and P. nigrescens, suggesting the existence, in humans, of a new anaerobic species of pigmented, moderately saccharolytic, indole-positive Gram-negative rods.

PRESENÇA DE LEVEDURAS DO GÊNERO CANDIDA NA SALIVA DE PACIENTES COM DIFERENTES FATORES PREDISPONENTES E DE INDIVÍDUOS CONTROLE

Os microrganismos do gênero Candida são, em geral, comensais da cavidade bucal, mas, em determinados indivíduos e em situações específicas, podem transformar-se na forma parasitária, produzindo candidoses bucais. O objetivo deste trabalho foi analisar a presença das espécies do gênero Candida na saliva de indivíduos controle, comparando com pacientes que apresentavam diferentes fatores predisponentes. Foram avaliados 493 pacientes, divididos em 7 grupos: prótese total, prótese parcial removível, periodontite crônica de adulto, respiração bucal, aparelho ortodôntico fixo, aparelho ortodôntico removível e aparelho extrabucal. O grupo controle consistiu de 570 indivíduos que não apresentavam nenhum desses fatores, divididos em 2 grupos: infantil e adulto. Foram realizados o isolamento e a identificação das espécies do gênero Candida. Os resultados mostraram maior porcentagem de pacientes positivos para Candida nos grupos com fatores predisponentes em relação aos controles. Houve predominância de isolamento de C. albicans em todos os grupos, porém, os pacientes com fatores predisponentes apresentaram diversidade maior de espécies

Widespread geographic distribution of oral Candida dubliniensis strains in human immunodeficiency virus-infected individuals

Candida dubliniensis is a recently identified chlamydospore-positive yeast species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected (HIV+) patients and is closely related to Candida albicans. Several recent reports have described atypical oral Candida isolates with phenotypic and genetic properties similar to those of C. dubliniensis. In this study 10 atypical chlamydospore-positive oral isolates from HIV+ patients in Switzerland, the United Kingdom, and Argentina and 1 isolate from an HIV-negative Irish subject were compared to reference strains of C. albicans and Candida stellatoidea and reference strains of C. dubliniensis recovered from Irish and Australian HIV+ individuals. All 11 isolates were phenotypically and genetically similar to and phylogenetically identical to C. dubliniensis. These findings demonstrate that the geographical distribution of C. dubliniensis is widespread, and it is likely that it is a significant constituent of the normal oral flora with the potential to cause oral candidiasis, particularly in immunocompromised patients.

Effect of use of 0.05% cetylpyridinium chloride mouthwash on normal oral flora

Objectives: The aims of this study were to examine the effect of the use of a mouthwash containing 0.05% cetylpyridinium chloride (CPC) on (1) the composition of the normal oral microbial flora and (2) the establishment of non-oral bacterial species in the mouth. Method: In a double-blind study, 129 subjects used, twice daily for 6 weeks, either a randomly assigned test or placebo mouthwash. At baseline and at the completion of the study, a sample of the oral flora was obtained by asking each subject to “swish” around the mouth for 30 s, 10 ml of distilled water. The numbers of total colony forming units (CFU), facultative bacteria, streptococci, mutans streptococci, lactobacilli, staphylococci, yeasts and enterococci were enumerated after culture on eight non-selective and selective media. Results: After 6 weeks use of either the test or placebo mouthwashes, there were no significant differences (analysis of variance) between the counts (CFU/ml of sample) for any of the microbial taxa. Conclusions: These data indicate that the use of a CPC-containing mouthwash does not alter the composition of the normal oral flora or result in the establishment of non-oral and potentially pathogenic bacteria in the mouth.

Intracellular multiplication and toxic destruction of cultured macrophages by Capnocytophaga canimorsus

Capnocytophaga canimorsus is a gram-negative rod that causes opportunistic infections resulting in bacteremia, septicemia, meningitis, and death in immunocompromised, splenectomized, and alcoholic individuals. Infections caused by a related species, Capnocytophaga cynodegmi, remain localized at the site of the wound where the organism is introduced. Both organisms are part of the normal canine oral flora and are introduced through puncture wounds via dog bites. We found that both C. canimorsus and C. cynodegmi attach, are phagocytized, and multiply intracellularly in J774 mouse macrophage cells. After 48 h of infection by C. canimorsus, large sections of the macrophage cell layer were observed to detach and lyse, while the monolayer infected with C. cynodegmi demonstrated no cytotoxic effects. Tissue culture supernatants from the C. canimorsus-infected J774 cells filtered through a 0.22-micron-pore membrane produced a similar effect on fresh monolayers, while filtrates from C. cynodegmi and uninfected controls produced no effect. No endotoxin release was observed in these supernatants. We conclude that the cytotoxic phenotype of C. canimorsus is the likely result of a toxin produced by this organism.

Bacterial adhesion to dental amalgam and three resin composites

Objectives: The ability of three oral bacteria to adhere to hydrophobic amalgam (water contact angle 60 °) and hydrophobic resin composites (Prisma-AP.H 56 °, Herculite XRV 82 ° and Z100 89 °) was compared using an in vitro assay. Methods and results: Following preincubation of the materials with human saliva, X-ray photoelectron spectroscopy showed the surfaces to adsorb carbon and nitrogen-containing compounds in a conditioning film that appeared to block the detection of Na and others in 2100 resin, Hg and Ag in amalgam, Si and Zn in Prisma AP.H resin and Ag and Na in Herculite resin. The precoating of the substrata by a proteinaceous conditioning film led to decreased binding of viable cells of Streptococcus sanguis CH3, Streptococcus salivarius HB and Actinomyces viscosus WG as compared with the adhesion to bare composites. With and without salivary coating, there was a correlation between increased bacterial hydrophobicity and increased retention on the substrata. However, there was no statistical difference in binding to the amalgam compared with the resin composites. In vitro studies showed that the bacteria autoaggregated in the presence of saliva. Conclusion: The results indicate the potential ability of normal oral flora to colonize resin composite.

上気道細菌感染症における扁桃常在菌叢の意義

Bacterial interference between normal oral flora and pathogens is known to play a significant role in non-specific host defense mechanisms against bacterial infection. In particular, much attension has been focused on a-streptococci with inhibitory activity against group A streptococcus in the oral cavity and throat.We developed a new method named “filter paper stamp method” to investigate the bacterial interference and this method was found to be simpler and more accurate than previous procedures. The detection rate of a-streptococci with inhibitory activity against group A streptococcus was examined in both healthy individuals and patients with tonsillitis. In healthy individuals, the inhibitory strains increased with age and may reflect the fact that group A streptococcal infection decrease with age. In patients with tonsillitis, the detection rate of inhibitory α-streptococcus in patients for whom tonsillectomy was clinically indicated was much lower than in those not indicated for this surgery. In the patients who received tonsillectomy, the detection rate of the inhibitory strains steadily increased after surgery. It is suggested that studies of α-streptococcus with inhibitory activity against group A streptococcus in the oral cavity and throat are of clinical significance in determining the indication for tonsillectomy and in evaluating the effect of surgery.We also found that non-pathogenic oral bacteria such as β-lactamase-producing H. parainfluenzae as well as α-streptococci with inhibitory activity against group A streptococcus were of considerable significance in the antimicrobial therapy for tonsillar infection. As a result, bacteriological follow-up studies of both pathogenic and non-pathogenic strains in the oral cavity and throat are of importance in patients with tonsillitis.

Isolation of alpha-streptococci with inhibitory activity against pathogens, in the oral cavity and the effect of tobacco and gargling on oral flora

We investigated the distribution of oral alpha-streptococci with inhibitory activity against pathogens, which compose an oral defense mechanism. Detection rate of alpha-streptococci with inhibitory activity against S. pyogenes and S. aureus derived from the tonsil was higher than in other portions, the tongue, cheek, gingiva, or saliva. It has suggested that tonsillar bacterial flora are mainly a defense mechanism. The oral flora in healthy smokers was compared with healthy non-smokers to investigate the influence of tobacco on oral bacterial flora. The results showed that the detection rate of S. aureus in smokers was higher while that of alpha-streptococci with inhibitory activity against S. aureus was lower. However, the detection rate of alpha-streptococci with inhibitory activity against S. pyogenes in smokers was as high as in non-smokers. It is suggested that it was easy for S. aureus to adhere to the oral mucosa in smokers, and was considered to influence the strain which produces beta-lactamase for medical treatment. We investigated the influence of gargling on oral bacterial flora by comparing the amount of bacteria before and after gargling with popidine-iodine gargle and saline solution. It was shown that alpha-streptococci, a main component of normal oral flora were decreased after gargling in both smokers and non-smokers. Furthermore, it was shown that group A Streptococcus was not decreased after gargling, and it was concluded that use of gargle medicinal mouth wash in bacillus carriers should be studied further.

The Production of Protease Activities byStreptococcus oralisStrains Isolated from Endocarditis

Nine Streptococcus oralis strains isolated from cases of endocarditis ( n =5) and from the normal oral flora ( n =4) were examined for their ability to produce a number of protease activities measured using the following 7-amido-4-methylcoumarin (-AMC) linked fluorogenic substrates: BOC-leu-ser-thr-arg-AMC, gly-pro-AMC, CBZ-lys-AMC, arg-AMC and BOC-val-pro-arg-AMC, a synthetic substrate for thrombin. The influence of glucose and porcine gastric mucin on their production was determined. The distribution of the protease activities between cell-associated and supernatant was not significantly associated with the origin of the strains. However, BOC-leuser-thr-arg-AMC and BOC-val-pro-arg-AMC hydrolysing activity was greater in the supernatant while the gly-pro-AMC, CBZ-lys-AMC and arg-AMC hydrolysing activity was more cell associated, irrespective of whether the cells were grown in minimal media supplemented with either glucose or PGM. Inclusion of increasing concentrations of glucose in media containing a constant PGM concentration (0.25 per cent w/v) resulted in significant reductions in the supernatant protease activity hydrolysing BOC-leu-ser-thr-arg-AMC and BOC-val-pro-arg-AMC while the cell-associated activity hydrolysing CBZ-lys-AMC and arg-AMC increased and the hydrolysis of gly-pro-AMC was essentially unaltered. The response of S. oralis proteolytic activities to changes in media composition, including the inclusion of a model glycoprotein, PGM, was not predictable but indicated that strains from endocarditis and from the normal oral flora were indistinguishable. The production of proteases in vivo may depend upon the level of fermentable carbohydrates in the circulation but at low concentrations elevated levels of protease activity, including a thrombin-like activity, may be found within fibrin-platelet thrombi associated with endocarditis. Keywords: Streptococcus oralis , protease, mucin, endocarditis

Fine antigen specificity of human gamma delta T cell lines (V gamma 9+) established by repetitive stimulation with a serotype (KTH-1) of a gram-positive bacterium, Streptococcus sanguis.

We have established human gamma delta T cell lines specific for Streptococcus sanguis (S. sanguis) KTH-1 present in normal oral cavity flora. The CD4-CD8-CD3+V gamma 9+V delta 1-CD45RO+ CD25+ T cell lines showed a proliferative response to the streptococcal antigen (Ag) in the presence of autologous antigen-presenting cells without apparent evidence of HLA restriction. The proliferative response of the gamma delta T cell lines was completely blocked by anti-TcR gamma delta monoclonal antibody (mAb) and anti-HLA class I mAb (W6/32), whereas anti-HLA classical class Ia mAb (B-H9; anti-HLA-A,B,C), anti-HLA class II mAb (anti-DR, anti-DQ, and anti-DP) and anti-CD4 mAb did not have any inhibitory effects. Surprisingly, the gamma delta T cell lines showed the proliferative response against the original bacterial Ag KTH-1 exclusively, and exhibited no cross-reactivity with nominal Ag such as purified protein derivative of tuberculin, tetanus toxoid and Mycobacterium tuberculosis, or the same species but different strain of S. sanguis, American Type Culture Collection (ATCC) standard strain (10556), or even with the same strain but different serotype of S. sanguis, KTH-3. Moreover, cytokine production of the gamma delta T cell lines was similar to the Th1 pattern [interferon-gamma, tumor necrosis factor (TNF)-alpha and TNF-beta]. They also produced interleukin-8 that functions as one of chemoattractants for polymorphonuclear cells. Using direct sequencing technique of the polymerase chain reaction products, we found that junctional diversity of the T cell receptor (TcR) used by the parental KTH-1 specific gamma delta T cell line and its subclones is rather limited. It is suggested that gamma delta T cells with canonical TcR could preferentially respond to KTH-1 Ag. Thus, in addition to a broad or cross-reactivity of gamma delta T cells against phylogenetically conserved stress/heat-shock protein, which is well characterized by others, some peripheral blood gamma delta T cells could recognize and kill exogenous agents with fine antigenic specificity to protect the body against them.

Use of the Syrian golden hamster for the induction of intraoral abscesses by sutures contaminated with human subgingival plaque.

Analysis of normal oral flora in 150 cheek pouches of hamsters (Mesocricetus auratus) defined the microbial working environment and demonstrated the absence of human oral black-pigmented bacteria. Silk sutures saturated with Porphyromonas gingivalis, Prevotella intermedia or subgingival plaque were used to close wounds made in hamster's cheek pouches. Abscesses were formed when sutures had solitary P. gingivalis or other bacteria mixed with P. gingivalis or when P. intermedia was mixed with other bacteria besides P. gingivalis. A concentration of black-pigmented bacteria emanating from 3 x 10(5) colony-forming units/inoculum was required for abscess formation. Six abscesses (14.3%) were developed in association with the presence of other odontopathic bacteria, primarily Fusobacterium nucleatum and Actinomyces viscosus. The hamster cheek pouch with iatrogenic wounds closed with plaque-impregnated sutures is a novel and effective model to study the pathology of wound infections and virulence of human subgingival organisms.

Microbiology of acute and chronic sinusitis in children

To determine the bacteriologic cause of acute sinusitis, a sample of sinus secretions must be obtained from one of the paranasal sinuses without contamination by normal respiratory or oral flora that colonize mucosal surfaces. When maxillary sinus aspiration is performed on children who have signs and symptoms of acute sinusitis, bacteria are recovered in high density from 70%. In patients with acute, subacute, or chronic sinusitis who are generally well except for persistent respiratory symptoms, of nasal discharge or cough or both, the usual bacterial isolates are Streptococcus pneumoniae, Haemophilus influenzae, an Moraxella catarrhalis. In contrast, anaerobic organisms and staphylococci should be suspected in patients who have very long-standing symptoms or in those whose symptoms are so severe or complicated that sinus surgery is undertaken.

HIV-Related Infections of the Oral Cavity

Oral infections are among the first manifestations of infection by the human immunodeficiency virus (HIV). They include fungal, viral, and bacterial infections and range from being essentially trivial, through troublesome to life threatening. Although some infections are due to overproliferation of the normal oral flora, others are due to organisms that normally are not found in the mouth. The clinical features of many of these infections have now been characterized, and clinical trials have indicated the optimal management. However, for many infections, the underlying processes are still not clear, and improvements in treatment are necessary.

The predominant microflora of the palatal mucosa in an elderly island population.

The prevalence and microbiology of macrophotographically documented denture stomatitis were studied in denture wearers participating in an interdisciplinary health-monitoring project (Koster Health Project) on the Koster islands, Sweden. Upper dentures were used by 26.6% of the adult population, and 59.2% of the denture wearers had stomatitis. Denture stomatitis type I was identified as sialadenitis. The more severe forms of denture stomatitis (types II and III) demonstrated increased recovery of microorganisms in the palatal mucosa in addition to sialadenitis. Only one proband showed increased growth of fungi. Hemophilus spp. and Bacteroides spp. were the predominating microorganisms in stomatitis types II and III. Shifts in the normal oral flora are suggested to be an important factor for the development of denture stomatitis. It is concluded that bacterial colonization on the palatal mucosa may play an important role in denture stomatitis in this relatively healthy population.

DNA fingerprinting of oral bacteria

This elective study was conducted at the Dental School, University of Wales College of Medicine and at the University of Wales College of Cardiff School of Pure and Applied Biology. The student's interest in oral microbiology and desire to contribute to the work being performed in the field of oral microbiology, led him to concentrate his study on developing a DNA fingerprinting method to evaluate the putative new species of the genus Capnocytophaga. The genus Capnocytophaga, which contains Gram-negative, capnophilic fusiform-shaped bacteria, has been implicated in juvenile periodontitis and prepubertal gingivitis. However, the genus is also part of the normal oral flora. The student believed that there was an implication of the existence of both pathogenic and non-pathogenic strains of Capnocytophaga and developed a method of DNA fingerprinting to distinguish Capnocytophaga strains from clusters identified by Khwaja et al. The student felt that the study allowed insight into the tremendous potential of molecular techniques for furthering the understanding of dental disease and confirmed his ambition to pursue a career in academic oral microbiology. This area of study allows new associations to be made between specific bacteria and diseases, aiding the possibility of rapid and early diagnosis.

Significance of normal oral flora, particularly group oral streptococci as defense mechanism against infection in healthy individuals (normal defense mechanism by oral Streptococcus group)

The alpha-streptococci, consisted of normal oral flora mainly, with inhibitory activity against pathogenic microbes in healthy individuals was investigated by group A Streptococcus (indicator strain 6-22 nonmucoid T-12). Rate of alpha-streptococci with inhibitory activity against group A Streptococcus was increased as aging, and the rate in pre-school children was higher than that in school children. These results suggested that more than 90% of the tested alpha-streptococci with strong inhibitory activities (S. salivarius) against indicator strain had inhibitory activities against group A Streptococcus (mucoid T-6), H. influenzae, S. pneumoniae, group C Streptococcus, and from 40% to 70% of the tested strains had also inhibitory activities against other pathogens. As there were many strains of alpha-streptococci with inhibitory activities against pathogens, that usually detected in the upper respiratory infection, the problem on the strains in the future will explain significance of the defense mechanism against upper respiratory infection and this can be applied clinically.

Detection of capnocytophaga species in simulated blood cultures by bactec-NR

Capnocytophaga is a genus of Gram negative, fusiform, capnophilic bacteria which are part of the normal oral flora of man and animals. They are rare causes of bacteraemia particularly in neutropenic patients. C.canimorsus (formerly DF-2) may cause overwhelming sepsis following animal bites. A study using clinical isolates of C. ochracea and C. canimorsus in simulated blood cultures was performed to determine the sensitivity of detection by BACTEC-NR. 48 hour HBA cultures were harvested and inoculated into BHI broth. Serial ten fold dilutions were performed (100 to 108 CFU/ml) and 6A and 7A BACTEC bottles were inoculated along with 5 ml of sterile human blood. A Growth Index of > 35 was taken as a positive signal. The results suggest that C. ochracea should be detectable by BACTEC using standard procedures as long as cultures were processed beyond 5 days. C. canimorsus however may not signal up to 7 days and requires terminal subculture. Some strains of C. canimorsus are inhibited by SPS in the blood culture media and require blind subculture within 48 hours or possibly the use of SPS free media.

Are certain oral pathogens part of normal oral flora in denture‐wearing edentulous subjects?

A complete loss of teeth may eliminate some pathogenic bacteria for lack of a suitable habitat for colonization. The occurrence of Actinobacillus actinomycetemcomitans, black-pigmented Bacteroides, mutans streptococci, lactobacilli and yeasts was investigated in 51 edentulous subjects with complete denture(s); mean age 74 years (range 44-92 years). None of the subjects had received antibiotics during the preceding 6 months. Four swab samples per subject were taken from the fitting surface of the upper denture, from the palate, buccal mucosae and dorsum of the tongue. At least 0.5 ml of stimulated saliva was collected into a sterile tube. The samples were cultivated aerobically and anaerobically using nonselective and selective media. A. actinomycetemcomitans was not found in any of the samples. Black-pigmented Bacteroides were found in 49 (96%) and yeasts in 25 (49%) of the subjects. Of the 49 saliva samples, 41 (84%) yielded mutans streptococci and 45 (92%) lactobacilli. In contrast to black-pigmented Bacteroides, mutans streptococci and lactobacilli, A. actinomycetemcomitans may not belong to the normal oral flora of edentulous subjects wearing dentures.