In vitro evaluation of yoghurt starter lactobacilli and Lactobacillus rhamnosus GG adhesion to saliva-coated surfaces.
Oral Microbiol Immunol. 2009 Jun;24(3):218-23
Authors: Stamatova I, Kari K, Vladimirov S, Meurman JH
AIM: The aim of the study was to evaluate the adhesion of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus rhamnosus strain GG to saliva-coated surfaces in vitro. METHODS: Fifteen radiolabeled dairy L. delbrueckii subsp. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated hydroxyapatite beads and polystyrene microtiter plates and the radioactivity was measured by liquid scintillation counter. The effects of lysozyme on the adhesion of lactobacilli and of pretreatment with lactobacilli on the adhesion of Streptococcus sanguinis were also assessed. RESULTS: All strains tested adhered to saliva-coated surfaces but with significantly different binding frequencies. The adhesion of the L. delbrueckii subsp. bulgaricus strains remained lower in comparison to L. rhamnosus strain GG. One L. delbrueckii subsp. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment of the samples significantly increased lactobacillus adhesion to saliva-coated surfaces. CONCLUSION: The present results showed significant variations in the adhesion capacity of the Lactobacillus strains studied. Adhesion to oral surfaces is of primary importance for bacterial colonization in the mouth. Only one of the L. delbrueckii subsp. bulgaricus dairy starter culture strains investigated had a high adhesion percentage. This strain might then be considered for further investigations in the oral environment.
Molecular and biological characterization of gtf regulation-associated genes in Streptococcus mutans.
Oral Microbiol Immunol. 2009 Jun;24(3):211-7
Authors: Terao Y, Isoda R, Murakami J, Hamada S, Kawabata S
INTRODUCTION: Surface protein antigen (PAc) and glucosyltransferases (GTF) are major adhesive molecules of Streptococcus mutans, though the mechanism of their regulation has not been fully elucidated. METHODS: To investigate the regulation mechanism, we determined a nucleotide sequence in the upstream region of the pac locus in S. mutans and identified two open reading frames (ORF), designated as orf1 and orf2. Each ORF was inactivated and functional analyses were performed. RESULTS: Western blot analyses revealed that the expression level of PAc was unaffected, while that of cell-associated GTF was diminished in both mutant strains. Furthermore, they showed higher hydrophobicity levels and an impaired sucrose-dependent adherence to smooth surfaces. RNA dot blot analysis demonstrated that transcriptions of the gtfB and the gtfC genes, which encode GTF-I and GTF-SI, respectively, were downregulated, while that of pac was comparable to the wild-type strain. In addition, the GTF activities of the mutant strains were significantly lower than those of the wild-type, though a greater amount of total glucan produced by the mutants was noted in culture supernatants. CONCLUSION: These findings suggest that orf1 and orf2 are associated with positive regulation of the gtfB and gtfC genes.
Genes responsible for dextran-dependent aggregation of Streptococcus sobrinus strain 6715.
Oral Microbiol Immunol. 2009 Jun;24(3):224-30
Authors: Sato Y, Okamoto-Shibayama K, Takada K, Igarashi T, Hirasawa M
INTRODUCTION: Streptococcus sobrinus exhibits more significant dextran-dependent aggregation mediated by glucan-binding proteins than Streptococcus mutans. We have identified four glucan-binding protein C gene (gbpC) homologues designated as gbpC1, gbpC2, dblA and dblB in S. sobrinus in contrast to the single gene gbpC in S. mutans. We attempted to determine which gene is most responsible for the dextran-dependent aggregation of S. sobrinus. METHODS: We introduced mutation with a chemical mutagen, 1-methyl-3-nitro-1-nitrosoguanidine, into S. sobrinus strain 6715 and analysed the four gbpC homologous gene sequences in the parental strain 6715 and an obtained aggregation-negative mutant NUM-Ssg99. We also examined the localization of proteins encoded by these genes in the mutant NUM-Ssg99. RESULTS: The nucleotide sequences of the gbpC1, gbpC2 and dblA genes in NUM-Ssg99 were 100% identical to the homologous genes in parental strain 6715. In contrast, a truncated mutation was detected in the dblB gene and the mutant protein devoid of the LPXTG motif was confirmed by Western blot analysis to be released into the extracellular milieu. CONCLUSION: We conclude that the dblB gene among the four GbpC homologous protein genes is most responsible for aggregation in strain 6715.
Role of Aggregatibacter actinomycetemcomitans in glutathione catabolism.
Oral Microbiol Immunol. 2009 Jun;24(3):236-42
Authors: Chu L, Xu X, Su J, Song L, Lai Y, Dong Z, Cappelli D
INTRODUCTION: Our previous studies demonstrated that three enzymes, gamma-glutamyltransferase (GGT), cysteinylglycinase (CGase) and cystalysin, are required for the catabolism of glutathione to produce hydrogen sulfide (H(2)S) in Treponema denticola. In this study, we examined glutathione catabolism in Aggregatibacter actinomycetemcomitans. METHODS: The GGT and CGase of A. actinomycetemcomitans were determined by biological methods and GGT was characterized using a molecular biological approach. RESULTS: A. actinomycetemcomitans showed GGT and CGase activity, but could not produce H(2)S from glutathione. The addition of recombinant T. denticola cystalysin, an l-cysteine desulfhydrase, to whole cells of A. actinomycetemcomitans resulted in the production of H(2)S from glutathione. Subsequently, we cloned A. actinomycetemcomitans GGT gene (ggt) and overexpressed the 63 kDa GGT protein. The recombinant A. actinomycetemcomitans GGT was purified and identified. The K(cat)/K(m) of the recombinant GGT from N-gamma-l-glutamyl-4-nitroaniline as substrate was 31/microm/min. The activity of GGT was optimum at pH 6.9-7.1 and enhanced by thiol-containing compounds. CONCLUSION: The results demonstrated that A. actinomycetemcomitans had GGT and CGase activities and that the GGT was characterized. The possible role of A. actinomycetemcomitans in glutathione metabolism and H(2)S production from oral bacteria was discussed.
Efficiency of oral fluid collection devices in extracting antibodies.
Oral Microbiol Immunol. 2009 Jun;24(3):231-5
Authors: Chang CK, Cohen ME, Bienek DR
INTRODUCTION: To facilitate diagnoses, this study determined the efficacy of commercial oral fluid collection devices for their ability to recover three human immunoglobulin isotypes; immunoglobulin A (IgA), IgG, and IgM. METHODS: The sandwich enzyme-linked immunosorbent assay was used to determine antibody recovery from the following devices: (i) OraSure oral specimen collection device, (ii) saliva*sampler, (iii) ORALscreen collector, (iv) Dri-Angle, (v) no. 2 cotton roll, (vi) all-gauze sponges device, and (vii) DentaSwabs. For each isotype tested, the recovered eluate was compared with the concentration applied to the device. The performance of each device was determined at various antibody concentrations. RESULTS: Recovery of IgA from the saliva*sampler, ORALscreen collector, Dri-Angle and cotton roll was comparable to that seeded onto the device. When compared with the seeded IgG concentration, the mean concentration of antibody recovered by each product differed by approximately +/- 9 ng/ml. The average amount of IgM recovered by the cotton roll and all-gauze sponges device was approximately 29 and 39 ng/ml, respectively, less (P < 0.0001) than that seeded on the device. For all isotypes tested, the amount of antibody recovered from the device was dependent on the initial seeding concentration. CONCLUSION: Collectively, these data suggest that the product used for specimen collection can affect retrieval of antibodies and potentially confound patient diagnosis.
INTRODUCTION: Helicobacter pylori infection is very prevalent in Brazil, infecting almost 65% of the population. The aim of this study was to evaluate the presence of this bacterium in the oral cavity of patients with functional dyspepsia (epigastric pain syndrome), establish the main sites of infection in the mouth, and assess the frequency of cagA and vacA genotypes of oral H. pylori. METHODS: All 43 outpatients with epigastric pain syndrome, who entered the study, were submitted to upper gastrointestinal endoscopy to rule out organic diseases. Helicobacter pylori infection in the stomach was confirmed by a rapid urease test and urea breath tests. Samples of saliva, the tongue dorsum and supragingival dental plaque were collected from the oral cavity of each subject and subgingival dental plaque samples were collected from the patients with periodontitis; H. pylori infection was verified by polymerase chain reaction using primers that amplify the DNA sequence of a species-specific antigen present in all H. pylori strains; primers that amplify a region of urease gene, and primers for cagA and vacA (m1, m2, s1a, s1b, s2) genotyping. RESULTS: Thirty patients harbored H. pylori in the stomach, but it was not possible to detect H. pylori in any oral samples using P1/P2 and Urease A/B. The genotype cagA was also negative in all samples and vacA genotype could not be characterized (s-m-). CONCLUSION: The oral cavity may not be a reservoir for H. pylori in patients with epigastric pain syndrome, the bacterium being detected exclusively in the stomach.
Oral Candida infection and colonization in solid organ transplant recipients.
Oral Microbiol Immunol. 2009 Jun;24(3):249-54
Authors: Dongari-Bagtzoglou A, Dwivedi P, Ioannidou E, Shaqman M, Hull D, Burleson J
INTRODUCTION: Oral Candida carriage and infection have been reported to be associated with a greater risk for systemic infection in transplant recipients; however, a systematic analysis of the oral Candida titers and species has not been previously conducted. The objectives of this study were to determine the prevalence of oropharyngeal candidiasis, the oral carrier status, Candida titers and species in this population. METHODS: Ninety kidney and heart transplant subjects and 72 age-matched healthy controls were included. Swabs from the oral mucosa and a standardized amount of unstimulated saliva were plated on Chromagar Candida, and colony-forming units per millilitre were calculated. Initial speciation was based on colony color and was confirmed by standard germ tube, biotyping, or polymerase chain reaction assays. RESULTS: Infection with C. albicans was detected in seven transplant subjects and none of the controls. The transplant group had significantly higher oral Candida titers than the control group. There were no statistically significant relationships between the dose or type of immunosuppressants and oral Candida titers or infection. A significantly higher percentage of transplant subjects were colonized by more than one species, compared with control subjects. The most frequent species combination in transplant subjects was C. albicans and C. glabrata. C. glabrata was isolated from 13.5% of transplant carriers and none of the controls. CONCLUSIONS: Increased oral Candida infection and carriage titers were found in the transplant population. Although the majority of transplant patients were colonized by C. albicans, C. glabrata appears to emerge as the second most prevalent species.
Human cytomegalovirus and Epstein-Barr virus inhibit oral bacteria-induced macrophage activation and phagocytosis.
Oral Microbiol Immunol. 2009 Jun;24(3):243-8
Authors: Lin YL, Li M
INTRODUCTION: Periodontal disease is an inflammatory condition caused by periodontal microorganisms. Viruses such as human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) are associated with certain types of periodontal disease, but their roles in promoting the disease are unclear. Because both viruses infect human macrophages, cells which play key roles in the clearance of pathogenic bacteria, it is likely that the viruses alter the functional capacity of macrophages by inhibiting their defense mechanisms against invading pathogens. METHODS: Macrophages preinfected with HCMV or EBV were evaluated following stimulation by selected oral bacteria. Bacteria-induced macrophage activation was assayed by measuring the levels of tumor necrosis factor-alpha (TNF-alpha) produced in the media, and phagocytic activity was analysed by a phagocytosis assay with fluorescein isothiocyanate-labeled bacteria. The virus-infected macrophages were also subjected to semi-quantitative polymerase chain reaction to measure the expression of toll-like receptor 9, which is involved in the activation of phagocytosis-related pathways. RESULTS: Both HCMV and EBV significantly diminished the TNF-alpha production typically induced by oral bacteria, inhibited the phagocytic activity of macrophages, and downregulated the expression of toll-like receptor 9. CONCLUSION: Infection by HCMV or EBV inhibits the functional ability of macrophages to respond to bacterial challenge, thereby suggesting their pathogenic role in the development of periodontal disease.
Fluoride, triclosan and organic weak acids as modulators of the arginine deiminase system in biofilms and suspension cells of oral streptococci.
Oral Microbiol Immunol. 2009 Aug;24(4):265-71
Authors: Barboza-Silva E, Castro AC, Marquis RE
INTRODUCTION: The arginine deiminase system (ADS) of oral bacteria is a major generator of alkali (ammonia) in dental plaque and is considered to have anticaries effects. However, many of the antimicrobial agents used in oral care products may reduce alkali production by the ADS. The objective of our work was to assess the sensitivity of the ADS of oral streptococci to commonly used antimicrobials, fluoride, triclosan and organic weak acids. METHODS: Streptococcus sanguinis NCTC 10904 and Streptococcus ratti FA-1 were grown in suspension cultures and mono-organism biofilms. ADS activity at pH values of 4, 5 and 6 was assessed, and the actions of the agents was determined in terms of reduced production of alkali from arginine, inhibition of ADS enzymes and changes in uptake of arginine. RESULTS: ADS activity was not greatly affected by pH changes between 4 and 6 and was greater per unit of biomass for cell suspensions than for biofilms. NaF was a poor inhibitor, while triclosan was highly effective with a 50% inhibitory dose for the two organisms between 0.03 and 0.05 and between 0.10 and 0.15 mm-h for suspension cells and biofilms, respectively. The weak acid indomethacin was nearly as potent at pH 4.0 as triclosan, while capric and lauric acids were less potent, especially for biofilms. The methyl ester of lauric acid was slightly stimulatory. The major targets for the inhibitors appeared to be transport systems for arginine uptake, although carbamate kinase was a secondary target. CONCLUSION: Triclosan, indomethacin, caprate and laurate can reduce ADS activity in dental plaque.
Homotypic biofilm structure of Porphyromonas gingivalis is affected by FimA type variations.
Oral Microbiol Immunol. 2009 Jun;24(3):260-3
Authors: Kuboniwa M, Amano A, Inaba H, Hashino E, Shizukuishi S
INTRODUCTION: Porphyromonas gingivalis is a periodontal pathogen whose long fimbriae (FimA) are classified into six genotypes (types I-V and Ib) based on the diversity of the fimA genes. FimA variations were previously shown to be related to the onset and development of adult periodontitis in a general population, while FimA were recently found to be critical mediators of initial biofilm formation. However, it is unclear if FimA variations have effects on biofilm features. Here, we compare the characteristic structures of homotypic biofilms developed by P. gingivalis strains with different FimA types. METHODS: Biofilms were formed on saliva-coated glass bottom wells in phosphate-buffered saline and their structures were analysed using confocal laser scanning microscopy. Furthermore, the biovolumes of the biofilms were quantified with a three-dimensional fluorophotometric method. RESULTS: Biofilm structures formed by the six representative FimA-type strains apparently differed. Type I and Ib P. gingivalis formed biofilms with a dense basal monolayer and dispersed microcolonies, whereas those formed by types II, III and IV strains had markedly luxuriant biofilms filled with widely clumped and tall colonies, and their biovolumes were significantly greater than those of types I and Ib. These characteristic features were confirmed to be closely related to FimA type in assays that utilized fimA-substituted mutants from type I to II and those from type II to I. CONCLUSION: Our results suggest that FimA variations have effects on the structures of biofilms formed by P. gingivalis, which may be an important factor in the pathogenesis of periodontitis.
PMID: 19416458 [PubMed - indexed for MEDLINE]
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