Sites of infection associated with Streptococcus anginosus group among children.

Streptococcus anginosus group (SAG) are parts of normal flora of the oral cavity and associated with abscess forming in various sites on the body. Although the clinical features of infections caused by each member of the SAG in adults has been reported, it has not well been known in children. The aim of this study was to clarify the site of infections associated with individual SAG species among children. Medical records from March 2010 to July 2016 were reviewed at Tokyo Metropolitan Children's Medical Center. Any SAG species (S. anginosus, S. constellatus, or S. intermedius) isolated from clinical samples and recorded in the microbiological database were included for analysis. Analysis of 52 infectious episodes found that S. anginosus was most frequently isolated from the genitourinary tract, and 73% of genitourinary tract infection was balanoposthitis. All genitourinary tract infections were associated with S. anginosus. These findings were different from those of a previous study of adults. Of all the patients, 45 patients (87%) had polymicrobial infections. More than 70% of patients infected by S. anginosus and S. constellatus were co-infected by obligate anaerobes, in comparison with only 21% of S. intermedius cases. Among the obligate anaerobes species, Bacteroides spp. was significantly accompanied with S. anginosus. Susceptibility to penicillin, ampicillin, cefotaxime, erythromycin, clindamycin, levofloxacin, and vancomycin was 100%, 100%, 100%, 77%, 89%, 97% and 100%, respectively. S. anginosus was often isolated from balanoposthitis among children.

Positive- and Negative-Control Pathways by Blood Components for Intermedilysin Production in Streptococcus intermedius.

Streptococcus intermedius is an opportunistic bacterial pathogen secreting a human-specific cytolysin called intermedilysin (ILY) as a major pathogenic factor. This bacterium can degrade glycans into monosaccharides using two glycosidases, multisubstrate glycosidase A (MsgA) and neuraminidase (NanA). Here, we detected a stronger hemolytic activity mediated by ILY when S. intermedius PC574 was cultured in fetal bovine serum (FBS) than when it was grown in the standard culture medium. FBS-cultured cells also showed higher MsgA and NanA activity, although overproduction of ILY in FBS was undetectable in mutants nanA-null and msgA-null. Addition of purified MsgA and NanA to the FBS resulted in a release of 2.8 mM galactose and 4.3 mM N-acetylneuraminic acid; these sugar concentrations were sufficient to upregulate the expression of ILY, MsgA, and NanA. Conversely, when strain PC574 was cultured in human plasma, no similar increase in hemolytic activity was observed. Moreover, addition of human plasma to the culture in FBS appeared to inhibit the stimulatory effect of FBS on ILY, MsgA, and NanA, although there were individual differences among the plasma samples. We confirmed that human plasma contains immunoglobulins that can neutralize ILY, MsgA, and NanA activities. In addition, human plasma had a neutralizing effect on cytotoxicity of S. intermedius toward HepG2 cells in FBS, and a higher concentration of human plasma was necessary to reduce the cytotoxicity of an ILY-high-producing strain than an ILY-low-producing strain. Overall, our data show that blood contains factors that stimulate and inhibit ILY expression and activity, which may affect pathogenicity of S. intermedius.

Hydroalcoholic extract of Carum carvi L. in oral mucositis: a clinical trial in male golden hamsters.

OBJECTIVES: Several studies have attempted to prevent or improve oral mucositis (OM) but have not produced a qualified treatment yet. This study evaluates the effects of Carum carvi L. (caraway) hydroalcoholic extract (CHE) as one of the traditional medicinal plants in 5-fluorouracil (5-FU)-induced OM in golden hamsters. MATERIALS AND METHODS: OM was induced in 54 male golden hamsters by 5-FU and cheek pouch scratching. Starting from day 12, 500 and 1000 mg kg(-1) per day topical CHE were administered. Pouch histopathology score, malondialdehyde and reduced glutathione contents, and activity of myeloperoxidase plus microbial cultures of cheek pouch, antimicrobial properties of CHE, and essential oil constituents were evaluated. RESULTS: Lower histopathology score (0, 1, and 2) and malondialdehyde level, higher reduced glutathione level and activities of myeloperoxidase were detected in 1000 and 500 mg kg(-1) per day topical CHE and control groups, respectively (P < 0.001). The CHE was more potent against Staphylococcus epidermidis and Streptococcus intermedius. γ-Terpinene (37.2%) was identified as the main constituent of essential oil. CONCLUSION: The use of CHE in topical form may be associated with reduced intensity of OM. This may be due to appropriate antibacterial activity and terpinene contents.

Effects of extracellular DNA and DNA-binding protein on the development of a Streptococcus intermedius biofilm.

AIMS: The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone-like DNA-binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity. METHODS AND RESULTS: Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7-hydoxyl-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) and anti-HLP antibody without fixation, respectively. DNase I treatment (200 U ml(-1)) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 µg ml(-1)) purified from Strep. intermedius, other Gram-positive bacteria, Gram-negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild-type, HLP-downregulated strain and control strains. In contrast, the addition of eDNA (>1 µg ml(-1)) decreased the biofilm mass of all Strep. intermedius strains. CONCLUSIONS: These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity. SIGNIFICANCE AND IMPACT OF THE STUDY: eDNA- and HLP-targeting strategies may be applicable to novel treatments for bacterial biofilm-related infectious diseases.

Effect of saliva viscosity on the co-aggregation between oral streptococci and Actinomyces naeslundii.

BACKGROUND: The co-aggregation of oral bacteria leads to their clearance from the oral cavity. Poor oral hygiene and high saliva viscosity are common amongst the elderly; thus, they frequently suffer from pneumonia caused by the aspiration of oral microorganisms. OBJECTIVES: To examine the direct effect of saliva viscosity on the co-aggregation of oral streptococci with actinomyces. MATERIALS AND METHODS: Fifteen oral streptococcal and a single actinomyces strain were used. Co-aggregation was assessed by a visual assay in phosphate buffer and a spectrophotometric assay in the same buffer containing 0-60% glycerol or whole saliva. RESULTS: Nine oral streptococci co-aggregated with Actinomyces naeslundii ATCC12104 in the visual assay and were subsequently used for the spectrophotometric analysis. All tested strains displayed a decrease in co-aggregation with increasing amounts of glycerol in the buffer. The co-aggregation of Streptococcus oralis with A. naeslundii recovered to baseline level following the removal of glycerol. The per cent co-aggregation of S. oralis with A. naeslundii was significantly correlated with the viscosity in unstimulated and stimulated whole saliva samples (correlation coefficients: -0.52 and -0.48, respectively). CONCLUSION: This study suggests that saliva viscosity affects the co-aggregation of oral streptococci with actinomyces and that bacterial co-aggregation decreases with increasing saliva viscosity.

Comparison of the antibacterial effect of modified 3-mix paste versus Ultrapex over anaerobic microorganisms from infected root canals of primary teeth: an in vitro study.

OBJECTIVE: The aim of this study was to evaluate in vitro the antimicrobial efficacy of a modified 3-mix paste and to compare it with an iodoform paste (Ultrapex) against anaerobic microorganisms isolated from root canals of infected or necrotic primary teeth. STUDY DESIGN: An in vitro experimental assay was performed over isolated and identified anaerobic microorganisms of 21 samples, in order to compare the antimicrobial ability of both root canal filling materials, using a disc-diffusion method. RESULTS: A total of 21 microbial samples (15 polymicrobial and 6 monomicrobial) were obtained, from which 19 different strains were identified. Modified 3-mix paste showed an excellent antimicrobial effect against most of both kinds of microbial samples, although some of them exhibited resistance; on the other hand, Ultrapex showed only minimal antimicrobial ability (null or low categories). Clostridium ramosum exhibited the most resistance to both materials. CONCLUSION: The bactericidal effect of the modified 3-mix paste was superior to Ultrapex, with a statistically significant difference, against anaerobic microorganisms isolated from infected root canals of primary teeth.

Comparative inhibitory effects of 4-allylpyrocatechol isolated from Piper betle on Streptococcus intermedius, Streptococcus mutans, and Candida albicans.

Streptococcus intermedius, Streptococcus mutans, and Candida albicans are harmful oral pathogens and prone to resist chemical antimicrobial agents. Active ingredients from plants are of increasing interest as an alternative. This study aims to compare antimicrobial effects of 4-allylpyrocatechol (APC) extracted from Piper betle on these oral pathogens. Minimum concentration of APC against the tested pathogens was determined using a broth microdilution assay. Killing kinetic study of APC was carried out within 24 h. Morphology of the pathogenic cells was assessed using scanning electron microscopy (SEM). Anti-biofilm was investigated using crystal violet assay and confocal laser scanning microscopy (CLSM). The results showed that the mechanism of inhibition of APC was bactericidal and fungicidal effects. APC at minimum concentration of 400 µg/mL could completely kill Streptococcus and Candida spp., however, the killing rate on S. intermedius and C. albicans was significantly faster than on S. mutans. APC inhibited biofilm formation of C. albicans more efficiently than that of the bacterial cells. Cell morphology from SEM indicated that APC caused bacterial cell membrane destruction and inhibited fungal budding or tubing formation. CLSM images confirmed the killing potential of APC and suggested that bacterial dead cells could be easier washed out than the fungal dead cells. It is concluded that APC potentially inhibits growth and biofilms of oral Streptococcus and Candida spp. in different mechanism of action and killing rate. APC can be considered as a promising agent for preventing and treating dental disorders caused by S. intermedius, S. mutans, and C. albicans.

Effects of Diluents, Saliva and Other Organics on the Microbicidal Activity of Cetylpyridinium Chloride and Povidone-iodine.

Povidone-iodine (PVP-I) is used for infection control and preoperative sterilization of the oral and pharyngeal regions. Marketed preparations containing cetylpyridinium chloride (CPC) are used to inhibit growth of oral bacteria. We conducted an in vitro study of the sterilizing effects of these microbicides on 10 oral bacterial strains and fungi related to pneumonia and periodontal disease, after dilution with phosphate-buffered saline (PBS), saliva, and components in saliva. The CPC solution was evaluated at 50 mg/100 mL, which is the concentration used in products. CPC sterilized all strains within 1 minute. Prolongation of the sterilization time associated with dilution was more gradual in comparison to PVP-I solution. CPC sterilized 7 of 10 microbial strains within 3 minutes at 3 mg/100 mL. At 500 mg/100 mL, which is near the upper limit of the concentration that is actually used, PVP-I solution sterilized 7 microbial strains within 3 minutes. However, PVP-I had no sterilization effect when diluted to 100 mg/100 mL or lower. With addition of saliva, PVP-I sterilized 2 microbial strains within 3 minutes at 500 mg/100 mL, whereas CPC solution sterilized 9 microbial strains within 1 minute at 50 mg/100 mL. Our results show that in use influenced by dilution with saliva, CPC is likely to maintain a strong sterilization effect, whereas PVP-I may have a reduced effect.

AI-2/LuxS is involved in increased biofilm formation by Streptococcus intermedius in the presence of antibiotics.

Bacteria utilize quorum-sensing communication to organize their behavior by monitoring the concentration of bacterial signals, referred to as autoinducers (AIs). The widespread detection of AI-2 signals and its enzymatic synthase (LuxS) in bacteria suggests that AI-2 is an inter- and intraspecies communication signal. We have previously shown that antibiotic susceptibility is affected by AI-2 signaling in Streptococcus anginosus. Since chronic infections involve persistent biofilms resilient to antibiotic treatment, we explored the role of AI-2/LuxS in Streptococcus intermedius biofilm formation and cell viability when the organism was exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. The S. intermedius wild type (WT) and its isogenic luxS mutant, strain SI006, were exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. Biofilms were formed on polystyrene discs in microtiter plates. To assess planktonic cell viability, the ATP microbial viability assay was performed and the numbers of CFU were determined. For complementation assays, the AI-2 precursor dihydroxy pentanedione (DPD) was used as a supplement for SI006. Relative luxS expression was quantified by real-time PCR. The sub-MICs of all three antibiotics increased biofilm formation in S. intermedius WT. However, biofilm formation by SI006 was either unaffected or reduced (P < or = 0.05). Bacterial viability tests of biofilm and planktonic cell cultures indicated that SI006 was more susceptible to antibiotics than the WT. DPD complemented the luxS mutant phenotype. Real-time PCR revealed modest yet significant changes in luxS expression in the presence of antibiotic concentrations that increased biofilm formation. In conclusion, in S. intermedius, AI-2/LuxS was involved in antibiotic susceptibility and increased biofilm formation at sub-MICs of antibiotic.

Effects of Piper betle fractionated extracts on inhibition of Streptococcus mutans and Streptococcus intermedius.

The overgrowth of certain strains of normal flora in oral cavity can cause many kinds of oral infections or diseases such as carries, periodontitis, and gingivitis. Prevention and treatment of these diseases are usually achieved by chemical antiseptics. However, these chemicals are found as negative impacts of human health hazards and accession of microbial resistance. The present study explores the potential of Piper betle extracts on inhibition of two oral pathogenic bacteria; Streptococcus mutans DMST 41283 and Streptococcus intermedius DMST 42700. P. betle demonstrated significantly higher inhibitory activity against both pathogenic strains than Acacia catechu, Camellia sinensis, Coccinia grandis, Solanum indicum, and Streblus asper. Among fractionated extracts of P. betle from several solvents, the extract from ethyl acetate (Pb-EtOAc) possessed the widest inhibition zone of 11.0 ± 0.1 and 11.3 ± 0.4 mm against both bacterial strains, respectively. Pb-EtOAc showed the same minimum inhibitory concentration of 0.5 mg/mL against both strains, whereas its minimum bactericidal concentrations were 2.0 and 0.5 mg/mL against S. mutans and S. intermedius, respectively. HPLC analysis demonstrated that the major active compound of Pb-EtOAc was 4-allylpyrocatechol. It was found that the killing kinetics of Pb-EtOAc against both test strains were time and dose dependent. Scanning electron microscopy micrographs showed the morphological changes and depletion of the tested pathogens indicating cell destruction after exposure to Pb-EtOAc. It is confirmed that Pb-EtOAc is potentially effective against both oral pathogens and might be used as natural alternative agents in prevention and treatment of oral infections caused by oral pathogenic bacteria.

Antibacterial Efficacy and Discoloration Potential of Endodontic Topical Antibiotics.

INTRODUCTION: The optimal concentration for the use of endodontic topical antibiotics is not known. The aims of this study were to determine the minimum bactericidal concentrations (MBCs) and minimum inhibitory concentrations (MICs) of metronidazole, ciprofloxacin, minocycline, Augmentin (GlaxoSmithKline, Research Triangle Park, NC), and tigecycline against common endodontic pathogens and to evaluate ex vivo the antibacterial efficacy and discoloration effect of triple antibiotic paste (TAP), Augmentin, and tigecycline at different concentrations using a slow-release hydrogel scaffold. METHODS: Using the Epsilometer test method (Etest; bioMérieux USA, St Louis, MO), MICs and MBCs of selected antibiotics were determined against Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus intermedius, and Enterococcus faecalis. Biofilms of these bacterial species were then grown in extracted single-rooted teeth anaerobically for 3 weeks. Root canals were filled with TAP, Augmentin, and tigecycline at concentrations of 1 or 0.1 mg/mL in a degradable hydrogel scaffold or pure TAP at 1 g/mL for 7 days. Coronal discoloration was evaluated spectrophotometrically at 1, 2, and 3 weeks after dressing. RESULTS: MIC/MBC data showed significant efficacy of tigecycline, Augmentin, and minocycline compared with the other antibiotics (P < .05). Significant differences were found when comparing the log10 colony-forming units of all experimental groups (P < .05). TAP at 1 g/mL had no bacterial growth but caused the greatest discoloration. Hydrogel mixtures with TAP, Augmentin, or tigecycline at 1 mg/mL significantly reduced bacterial growth and the number of positive samples compared with those at 0.1 mg/mL (P < .05) with minimal discoloration. CONCLUSIONS: TAP, Augmentin, and tigecycline in a hydrogel at 1 mg/mL reduced bacterial growth significantly with minimal color change.

Effects of Caesalpinia sappan on pathogenic bacteria causing dental caries and gingivitis.

The present study explores antimicrobial activities of Caesalpinia sappan extracts against three strains of oral pathogenic bacteria; Streptococcus mutans DMST9567 (Smu9), Streptococcus mutans DMST41283 (Smu4), and Streptococcus intermedius DMST42700 (Si). Ethanol crude extract of C. sappan (Cs-EtOH) was firstly compared to that of other medicinal plants using disc diffusion method. Cs-EtOH showed significantly higher effective inhibition against all tested strains than other extracts and 0.12% chlorhexidine with the inhibition zone of 17.5 ± 0.5, 18.5 ± 0.0, and 17.0 ± 0.0 mm against Smu9, Smu4, and Si, respectively. Three fractionated extracts of C. sappan using hexane, ethyl acetate, and ethanol, respectively, were further investigated. The fractionated extract from ethanol (F-EtOH) presented the strongest activities with the minimum bactericidal concentration (MBC) of 125-250 µg/mL. Killing kinetics of F-EtOH was depended on the bacterial species and the concentration of F-EtOH. Two-fold MBC of F-EtOH could kill all tested strains within 12 h whereas its 4-fold MBC showed killing effect against Si within 6 h. Separation of F-EtOH by column chromatography using chloroform/methanol mixture as an eluent yielded 11 fractions (F1-F11). The fingerprints of these fractions by high-performance liquid chromatography at 280 nm revealed that F-EtOH consisted of at least 5 compounds. F6 possessed the significantly highest antimicrobial activity among 11 fractions, however less than F-EtOH. It is considered that F-EtOH is the promising extract of C. sappan for inhibiting oral pathogenic bacteria and appropriate as natural antiseptic for further develop of oral hygiene products.

Antibacterial action of photoactivated disinfection {PAD} used on endodontic bacteria in planktonic suspension and in artificial and human root canals.

OBJECTIVES: To measure antibacterial action of photoactivated disinfection (PAD) on endodontic bacteria in planktonic suspension and root canals. METHODS: Four bacteria, Fusobacterium nucleatum,Peptostreptococcus micros, Prevotella intermedia and Streptococcus intermedius, were tested in suspension. After mixing equal volumes of Tolonium chloride and bacterial suspension for 60s, each 200 microL of concentration (>10(6)cfu mL(-1)) was irradiated with light at 633+/-2 nm. Each energy dose/Tolonium chloride concentration combination was tested eight times, with controls. Prepared root canals in Training Blocs and extracted human teeth were inoculated with S. intermedius followed by 10 mg L(-1) Tolonium chloride or saline. Bacteria in canals were sampled before and after light irradiation. Student t-test assessed significance of changes in viable bacteria produced by treatment of either light or Tolonium chloride alone and light/Tolonium chloride combinations. RESULTS: In suspension, reductions in bacteria were highly significant (P<0.01) for light/Tolonium chloride combinations compared to light or Tolonium chloride alone. Maximum mean log reductions of 1.14 (P. intermedia), 2.48 (P. micros), 2.81 (F. nucleatum) and 6.73 (S. intermedius) were at 4.8 J/20 mg L(-1). Antibacterial action was increased by energy dose increase (not always significantly), but not by Tolonium chloride concentration. In control canals mean log reductions of 0.42 (Blocs) and 0.38 (teeth) from initial levels were not significant. PAD mean log reductions of 2.40 (Blocs) and 2.01 (teeth) were highly significant. Changes for PAD/energy dose combinations were not significant. CONCLUSION: PAD killed endodontic bacteria at statistically significant levels compared to controls. Kills varied with bacterial species.

Identification of Anion Channels Responsible for Fluoride Resistance in Oral Streptococci.

Recently, it has been reported that eriC and crcB are involved in bacterial fluoride resistance. However, the fluoride-resistance mechanism in oral streptococci remains unclear. BLAST studies showed that two types of eriCs (eriC1 and eriC2) and two types of crcBs (crcB1 and crcB2) are present across 18 oral streptococci, which were identified in ≥ 10% of 166 orally healthy subjects with ≥ 0.01% of the mean relative abundance. They were divided into three groups based on the distribution of these four genes: group I, only eriC1; group II, eriC1 and eriC2; and group III, eriC2, crcB1, and crcB2. Group I consisted of Streptococcus mutans, in which one of the two eriC1s predominantly affected fluoride resistance. Group II consisted of eight species, and eriC1 was responsible for fluoride resistance, but eriC2 was not, in Streptococcus anginosus as a representative species. Group III consisted of nine species, and both crcB1 and crcB2 were crucial for fluoride resistance, but eriC2 was not, in Streptococcus sanguinis as a representative species. Based on these results, either EriC1 or CrcBs play a role in fluoride resistance in oral streptococci. Complementation between S. mutans EriC1 and S. sanguinis CrcB1/CrcB2 was confirmed in both S. mutans and S. sanguinis. However, neither transfer of S. sanguinis CrcB1/CrcB2 into wild-type S. mutans nor S. mutans EriC1 into wild-type S. sanguinis increased the fluoride resistance of the wild-type strain. Co-existence of different F- channels (EriC and CrcB) did not cause the additive effect on fluoride resistance in oral Streptococcus species.

Intermedilysin release by Streptococcus intermedius: effects of various antibacterial drugs at sub-MIC levels.

Intermedilysin is a cytolytic toxin produced by Streptococcus intermedius, a pathogen of humans. In vitro studies showed that exposure of S. intermedius to sub-minimum inhibitory concentration (MIC) levels (1/2 MIC) of protein-inhibiting antibiotics and nucleic acid-inhibiting antibiotics decreased intermedilysin release by S. intermedius. The most potent antibiotic was clindamycin. On the other hand, exposure to cell wall-inhibiting antibiotics generally showed insignificant changes in intermedilysin release at sub-MIC concentrations. Investigations into possible mechanisms underlying this sub-MIC effect with clindamycin showed that there was selective decrease in biosynthesis and release of toxin after exposure to 1/2 MIC condition. However, no significant differences in the mRNA levels of the intermedilysin gene were observed.

Effect of pH modified EDTA solution to the properties of dentin.

The purpose of this study was to evaluate the influence of smear layer removal with 3% EDTA solution (pH of 9.0) on the dentin in terms of the permeability of root canal disinfectants into the dentin, wetting by endodontic sealer, and adhesive strength of the sealer. Three types of disinfectant (formalin cresol, phenol, and calcium hydroxide) and 4 types of endodontic sealers (included eugenol, non-eugenol, polycarboxylic acid, and resin) were used. The contact angle between endodontic sealer solution and dentin decreased in the 3% EDTA group but increased in the 15% EDTA. The adhesive strength of endodontic sealer to dentin increased in the EDTA groups for all types of sealers. The permeability of root canal disinfectants increased to similar degrees in the 3% and the 15% EDTA groups. In comparing these properties, we propose that the 3% EDTA is more useful for clinical applications.