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ABSTRACT: Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors, and aimed to elucidate the mechanism involved in occurrence of STSS from the aspect of the complement system. First, we revealed that culture supernatant of given S. pyogenes strain and recombinant SpeB degraded the C1-INH. And then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity in normal as compared to heat-inactivated serum, while it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of the C9, one of the component of membrane attack complex, in greater amounts rate on the surface of the speB mutant, while lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.
Journal of Biological Chemistry 04/2013; · 4.77 Impact Factor
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ABSTRACT: MicroRNAs are single-stranded RNAs that regulate gene expression by forming imperfect base pairs, which have also been speculated to play regulatory roles in gene expression of Streptococcus pyogenes itself. We hypothesized that bacterial microRNAs cause molecular interference in host, when there is high homology to human microRNAs. Total RNA from cultured S. pyogenes strain SSI-1 was isolated and the cDNA fragments were then inserted into vector plasmid and transformed to competent cells, after which genomic sequence analyses were performed. Cell transfection, evaluation of mRNA transcription, measurement of inflammatory mediators, and assessment of surviving bacteria with murine splenocytes were also performed. Three microRNAs were selected from about 600 candidates according to their homology with human genome DNA. In the quantitative method, transcription of nasopharyngeal cells with microRNA was significantly lower in 2 of 11 targets, and greater in 10 of 11 targets. The ELISA findings revealed that transcription of MIP-2 was significantly greater with miR-SSI1-221 and miR-SSI1-281. Furthermore, strain SSI-1 had significantly higher survival in the supernatant of the control as compared to the miR-SSI1-221 and miR-SSI1-281 transfected cells. In conclusion, microRNA fragments derived from S. pyogenes have a high homology to the human genome and contribute to enhancement of the host immune system. (203 words).
Microbes and Infection 12/2012; · 3.10 Impact Factor
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ABSTRACT: Bacterial infection associated with the use of medical or dental devices is a serious concern. Although devices made of ethylene vinyl acetate (EVA) are often used in the oral cavity, there are no established standards for their storage. We investigated bacterial survival on EVA sheets under various storage conditions to establish a standard for hygienic storage of such dental devices. Bacterial counts were evaluated, which showed a significant decrease after washing with sterilized water, mechanical brushing and rinsing, and using Mouthguard Cleaner as compared to untreated samples. In addition, no bacteria were detected on samples stored 2 days or longer in a ventilated environment, whereas they were detected for up to 14 days on samples without any cleaning stored in a closed environment. Bacterial counts for the untreated samples gradually declined, while surviving bacteria on samples treated with sterilized water and mechanical brushing showed a rapid decrease. Additionally, bacterial identification using polymerase chain reaction (PCR) revealed that Streptococcus oralis was dominantly detected on salivary samples after 14 days of storage among both two subjects. For effective hygienic storage of dental devices made of EVA, washing with sterilized water is important to remove absorbed salivary compounds along with storage in a ventilated environment.
International Journal of Oral Science 03/2012; 4(1):14-8. · 1.41 Impact Factor
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ABSTRACT: The aim of this study was to investigate the cross-sectional association of dental restorations with salivary cariogenic pathogens among the elderly to establish effective parameters of caries risk for this population.
Stimulated whole saliva was collected from 289 community-dwelling older adults (66.2 ± 3.9 years old) who had 20 or more teeth. Salivary levels of three cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus and lactobacilli) were estimated using quantitative polymerase chain reaction (real-time PCR) method.
The mean number of residual teeth was 26.4, and restored teeth with crowns, inlays and composite resin were 7.35, 3.88 and 0.68, respectively. The number of crowns correlated positively with salivary S. mutans, S. sobrinus and lactobacilli. Multiple linear regression analyses showed that the number of restored teeth with crowns was independently associated with salivary S. mutans, S. sobrinus and lactobacilli after controlling for age, gender, number of residual teeth and salivary flow rate. Salivary flow rate was independently associated with salivary S. mutans and lactobacilli.
The number of crowns had an association with salivary levels of cariogenic bacteria, suggesting that this parameter may be a caries risk indicator for the elderly population.
Gerodontology 10/2011; 29(2):e845-50. · 1.03 Impact Factor
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ABSTRACT: To determine how patients and dentists in Japan value the outcomes of different treatment options for missing molars. Materials and
Elderly removable-denture wearers and dentate persons (senior group), preclinical dental students (young group), and prosthodontists were presented with five possible treatment options for missing lower bilateral first and second molars. The participants indicated on a visual analogue scale how they would value the treatment (utility value: UV), if they had received each of the treatments. Two-way repeated measures anova was used for statistical analyses.
The UV for the shortened dental arch (SDA) without replacement was the lowest in every group. The young group rated the value of cantilever fixed partial dentures (FPD) and implants significantly higher than removable partial dentures (RPD), while the senior group rated the value of FPD and RPD significantly higher than implants. Those having experience with removable dentures were likely to place a higher value on the acrylic RPD. The prosthodontists rated the value of implants significantly higher than any other options.
The participants in every group placed the lowest value on the outcome from the SDA in Japan. The denture wearers preferred the RPD, while prosthodontists preferred implants.
Gerodontology 09/2011; 28(3):192-6. · 1.03 Impact Factor
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ABSTRACT: Streptococcus pyogenes causes a broad spectrum of acute infections and is the bacterium most frequently isolated from patients with pharyngitis. A number of antibiotics including penicillin have been shown to be effective, although antibiotic treatment failure in cases of streptococcal pharyngitis have been reported. Herein, we aimed to elucidate the features of recurrent strains using clinical isolates. Ninety-three S. pyogenes organisms were obtained from Japanese patients with recurrent pharyngitis. Following genetic characterization, M-type isolates from patients with recurrent pharyngitis differed from those obtained at initial onset in 11 of 49 episodes, and pulsed field gel electrophoresis analysis showed different patterns in those cases. Additionally, spe genotyping revealed that the Spe type of the strains obtained at secondary onset corresponded with those from the initial onset in 22 cases. Furthermore, antibiotic susceptibility testing revealed that more than half of the strains were resistant to macrolides and lincosamides, which was a much greater ratio as compared with the strains obtained from initial onsets in previous studies. Our results suggest that recurrence and reinfection are often confused during the diagnosis of repetitive and persistent streptococcal pharyngitis. Moreover, the present S. pyogenes organisms were less susceptible to antibiotics, which raises caution about their appropriate use in clinical practice.
FEMS Microbiology Letters 03/2011; 318(2):143-51. · 2.04 Impact Factor
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ABSTRACT: Streptococcus pyogenes is the bacterium most frequently isolated from patients with pharyngitis. Although various antibiotics including penicillin are effective, antibiotic treatment failure in cases of streptococcal pharyngitis have been reported. Herein, we investigated mechanisms associated with recurrent streptococcal pharyngitis. Clinically isolated S. pyogenes strains showed serotype-specific features, with emm12 strains most frequently detected and emm6 strains more likely to produce biofilm. The architectures of formed biofilms were observed using a fluorescence microscope with Live/Dead staining. Furthermore, various cationic antimicrobial peptides were tested to evaluate their inhibitory activities toward biofilms formed by S. pyogenes. After treatments with high concentrations of antibiotics, S. pyogenes survived in biofilm even when dead bacterial cells covered the surface. Other findings demonstrated that some antimicrobial peptides have inhibitory effects on forming and formed biofilm. Moreover, emm4, emm6, and emm75 strains showed significantly higher levels of invasion capacity into Detroit 562 cells than strains with other genotypes. Additionally, more than half of the strains temporarily escaped killing by penicillin alone by internalization into epithelial cells, even when the antibiotic concentration used was greater than the 10-fold minimum inhibitory concentration (MIC) for planktonic S. pyogenes. Also, combined administrations of multiple antibiotics were more effective to eradicate strains more likely to be internalized. Finally, flow cytometric analyses demonstrated that emm12 strains with higher invasive capabilities expressed PrtF1 protein on the bacterial surface. These findings suggest that S. pyogenes isolated from patients with recurrent streptococcal pharyngitis have emm type-specific features that allow escape from eradication by antibiotics.
Microbial Pathogenesis 03/2011; 51(1-2):58-68. · 1.94 Impact Factor
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ABSTRACT: Pneumonia is reported to be associated with high morbidity in elderly and compromised individuals, with poor oral health demonstrated to be a significant risk factor for pneumonia. Several opportunistic pathogens, such as Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus, have been detected in patients with pneumonia. We investigated the prevalence of opportunistic pathogens in the oral cavity of healthy independent living Japanese elderly subjects and analysed factors related to harbouring those pathogens.
We studied 265 subjects, each of whom received a dental examination, during which specimens were collected with a tongue swab and examined for the presence of 10 oral opportunistic pathogens using single or multiple selective media. Furthermore, the presence of occult blood in saliva was examined using test paper strips.
Oral opportunistic pathogens were detected in 13.6% of the subjects. Those positive for occult blood in saliva had a significantly higher rate of harbouring the pathogens (p < 0.05). In addition, age was a significant factor for the presence of pathogenic microbes in the oral cavity (p = 0.033).
Positive findings of occult blood in saliva and older age are suggested to be significant factors for harbouring opportunistic pathogens in the oral cavity.
Gerodontology 11/2010; 29(2):e229-33. · 1.03 Impact Factor
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ABSTRACT: Streptococcus pneumoniae is a major pathogen of community-acquired pneumonia and one of its major virulence factors is pneumolysin, which functions as a cholesterol-dependent cytolytic pore-forming toxin. In this study, we identified the ply-like gene spd0729 in a BLAST search. Unexpectedly, hemolytic and cytotoxic assays showed no significant differences between a Deltaspd0729 mutant strain and the wild-type strain, whereas the mutant strain exhibited weaker anti-phagocytic activity in human peripheral blood. In addition, real-time RT-PCR analysis revealed that four capsular biosynthesis genes in the mutant strain had expressions 7- to 432-fold greater than those of the wild type, while an enzyme-linked immunoassay showed a mean 3-fold greater amount of total capsular polysaccharide in the mutant strain. These results suggest that Spd0729 is not a cytolysin, though it plays crucial roles in anti-phagocytosis and regulation of capsule expression. Thus, we named Spd0729 as a negative regulator of capsular polysaccharide synthesis (Nrc).
Biochemical and Biophysical Research Communications 10/2009; 390(1):155-60. · 2.48 Impact Factor
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ABSTRACT: We hypothesized that improvement of occlusal force would cause an increase in salivary flow rate (SFR) even in elderly complete denture wearers. The purpose of this study was to examine the change of SFR after the replacement of complete dentures.
Subjects were 22 edentulous outpatients of the Osaka University Dental Clinic. Unstimulated whole saliva was collected for 5 minutes. Stimulated whole saliva was collected for 2 minutes. The maximal occlusal force was measured with pressure-sensitive sheets. All measurements were done before and after replacement of the dentures.
The replacement of complete dentures improved maximal occlusal force and increased their stimulated and unstimulated SFR. Stimulated SFR was positively correlated with unstimulated SFR both before and after the replacement. Maximal occlusal force, stimulated SFR, and unstimulated SFR after the replacement were significantly correlated to the measurements before the replacement.
These results suggest that proper prosthetic treatment can increase not only maximal occlusal force but also salivary flow rate.
Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 09/2009; 108(2):211-5. · 1.50 Impact Factor
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ABSTRACT: To examine the relationship between bite force and stimulated salivary flow rate in older adults.
The study sample consisted of 934 independent-living, cognitively healthy Japanese older adults 60 to 84 years of age. The maximal bite force was measured with pressure-sensitive sheets, and stimulated whole saliva was collected using the mastication method.
The mean stimulated salivary flow rate was 1.36 +/- 0.97 mL/min. In bivariate analysis, the stimulated salivary flow rate was significantly lower in females and subjects with daily multiple medications (P < .01). Multiple linear regression analysis showed that the stimulated salivary flow rate was significantly associated with sex (standardized partial regression coefficient: partial beta = .22; P < .001), bite force (partial beta = .18; P < .001), and multiple daily medications (partial beta = -.12; P < .001).
It is suggested that reduced bite force is associated with a decline in stimulated whole salivary flow rate independent of sex and medication in older adults.
Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 10/2007; 104(4):510-5. · 1.50 Impact Factor
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ABSTRACT: Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.
Microbes and Infection 11/2006; 8(12-13):2791-6. · 3.10 Impact Factor
