Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways.

Porphyromonas gingivalis (Pg) is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. Pg-OMVs are involved in the pathogenesis of periodontitis. Pg-OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of Pg-OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. Pg-OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. Pg-OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that Pg-OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that Pg-OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.

Inactivation of human norovirus by chlorous acid water, a novel chlorine-based disinfectant.

INTRODUCTION: Human norovirus (HuNoV) is a leading cause of infectious gastroenteritis. Since HuNoV shows resistance to alcohol, chlorine-based sanitizers are applied to decontaminate the virus on environmental surfaces. Chlorous acid water (CA) has been recently approved as a novel chlorine-based disinfectant categorized as a Type 2 OTC medicine in Japan. In this study, we aimed to evaluate the capability of CA to inactivate HuNoV. METHODS: HuNoV (genogroups GII.2 and GII.4) was exposed to the test disinfectants including CA and sodium hypochlorite (NaClO), and the residual RNA copy was measured by reverse transcription quantitative PCR (RT-qPCR) after pretreatment with RNase. In addition, the log10 reduction of HuNoV RNA copy number by CA and NaClO was compared in the presence of bovine serum albumin (BSA), sheep red blood cells (SRBC), polypeptone, meat extract or amino acids to evaluate the stability of these disinfectants under organic-matter-rich conditions. RESULTS: In the absence of organic substances, CA with 200 ppm free available chlorine provided >3.0 log10 reduction in the HuNoV RNA copy number within 5 min. Even under high organic matter load (0.3% each of BSA and SRBC or 0.5% polypeptone), 200 ppm CA achieved >3.0 log10 reduction in HuNoV RNA copy number while less than 1.0 log10 reduction was observed with 1,000 ppm sodium hypochlorite (NaClO) in the presence of 0.5% polypeptone. CA reacted with only cysteine, histidine and glutathione while NaClO reacted with all of the amino acids tested. CONCLUSIONS: CA is an effective disinfectant to inactivate HuNoV under organic-matter-rich conditions.

The Human Gut Microbe Bacteroides thetaiotaomicron Suppresses Toxin Release from Clostridium difficile by Inhibiting Autolysis.

Disruption of the human gut microbiota by antibiotics can lead to Clostridium difficile (CD)-associated diarrhea. CD overgrowth and elevated CD toxins result in gut inflammation. Herein, we report that a gut symbiont, Bacteroides thetaiotaomicron (BT), suppressed CD toxin production. The suppressive components are present in BT culture supernatant and are both heat- and proteinase K-resistant. Transposon-based mutagenesis indicated that the polysaccharide metabolism of BT is involved in the inhibitory effect. Among the genes identified, we focus on the methylerythritol 4-phosphate pathway gene gcpE, which supplies the isoprenoid backbone to produce the undecaprenyl phosphate lipid carrier that transports oligosaccharides across the membrane. Polysaccharide fractions prepared from the BT culture suppressed CD toxin production in vitro; the inhibitory effect of polysaccharide fractions was reduced in the gcpE mutant (ΔgcpE). The inhibitory effect of BT-derived polysaccharide fraction was abrogated by lysozyme treatment, indicating that cellwall-associated glycans are attributable to the inhibitory effect. BT-derived polysaccharide fraction did not affect CD toxin gene expression or intracellular toxin levels. An autolysis assay showed that CD cell autolysis was suppressed by BT-derived polysaccharide fraction, but the effect was reduced with that of ΔgcpE. These results indicate that cell wall-associated glycans of BT suppress CD toxin release by inhibiting cell autolysis.

Microbiome analysis of contact lens care solutions and tear fluids of contact lens wearers: Possible involvement of streptococcal antigens in allergic symptoms related to contact lens wear.

The present study elucidated the pathogenesis of allergic symptoms (AS) related to contact lens (CL) wear by assaying CL care solutions in lens storage cases and tears from subjects with AS using molecular biology techniques. A total of 15 CL storage cases were collected from subjects with AS (n=9) and healthy, asymptomatic control CL wearers (n=6). Bacterial populations in CL care solutions and tears were assayed by culture and 16S rDNA sequencing. Histamine levels in tears were measured by high­performance liquid chromatography. Western blot analysis was performed to identify the bacteria recognized by tear IgE from subjects with AS. No significant differences were found in the culture results between the subjects with AS and asymptomatic subjects. Histamine was detected in 2 subjects with AS. Meta­16S rDNA sequencing identified a cluster of 4 subjects with AS that were distinguished from others by principal coordinate analysis. Detailed population analysis revealed that the abundance of Gram­positive bacteria in the microbiomes of CL care solutions used by the subjects with AS were higher than those of asymptomatic subjects (42.24±9.47 vs. 16.85±22.76% abundance). Among these, Streptococcus was the dominant genus (12.1­18.3% abundance). Tear microbiome analysis revealed that the abundance of Streptococcus in the subjects with AS was significantly higher than that in other subjects (19.02±5.50 vs. 3.08±3.35%, P<0.01). Western blot analysis demonstrated that the tear IgE in all subjects with AS reacted with Streptococcus (100%), but not with Staphylococcus. On the whole, these results provide novel insight into the pathogenesis of AS and identify Streptococcus as an important factor in AS associated with CL wear.

D-Tagatose Effectively Reduces the Number of Streptococcus mutans and Oral Bacteria in Healthy Adult Subjects: A Chewing Gum Pilot Study and Randomized Clinical Trial.

We examined the effect of D-Tagatose on the growth of oral bacteria including Streptococcus mutans (S. mutans). Saliva collected from 10 healthy volunteers was plated on BHI medium (to culture total oral bacteria) and MBS medium (to culture S. mutans, specifically). Agar plates of BHI or MBS containing xylitol or D-Tagatose were cultured under aerobic or anaerobic conditions. We then counted the number of colonies. In BHI plates containing D-Tagatose, a complete and significant reduction of bacteria occurred under both aerobic and anaerobic conditions. In MSB medium, significant reduction of S. mutans was also observed. We then performed a doubleblind parallel randomized trial with 19 healthy volunteers. They chewed gum containing xylitol, D-Tagatose, or both for 4 weeks, and their saliva was collected weekly and plated on BHI and MSB media. These plates were cultured under anaerobic conditions. Total bacteria and S. mutans were not effectively reduced in either the D-Tagatose or xylitol gum group. However, S. mutans was significantly reduced in volunteers chewing gum containing both D-Tagatose and xylitol. Thus, D-Tagatose inhibited the growth of S. mutans and many types of oral bacteria, indicating that D-Tagatose intake may help prevent dental caries, periodontitis, and many oral diseases.

Effect of thymoquinone on Fusobacterium nucleatum­associated biofilm and inflammation.

Periodontitis affects oral tissues and induces systemic inflammation, which increases the risk of cardiovascular disease and metabolic syndrome. Subgingival plaque accumulation is a trigger of periodontitis. Fusobacterium nucleatum (FN) contributes to subgingival biofilm complexity by intercalating with early and late bacterial colonizers on tooth surfaces. In addition, inflammatory responses to FN are associated with the progression of periodontitis. Nigella sativa Lin. seed, which is known as black cumin (BC), has been used as a herbal medicine to treat ailments such as asthma and infectious diseases. The current study examined the inhibitory effect of BC oil and its active constituents, thymol (TM) and thymoquinone (TQ), on FN­associated biofilm and inflammation. FN­containing biofilms were prepared by co­cultivation with an early dental colonizer, Actinomyces naeslundii (AN). The stability and biomass of FN/AN dual species biofilms were significantly higher compared with FN alone. This effect was retained even with prefixed cells, indicating that FN/AN co­aggregation is mediated by physicochemical interactions with cell surface molecules. FN/AN biofilm formation was significantly inhibited by 0.1% TM or TQ. Confocal laser scanning microscopy indicated that treatment of preformed FN/AN biofilm with 0.01% of BC, TM or TQ significantly reduced biofilm thickness, and TQ demonstrated a cleansing effect equivalent to that of isopropyl methylphenol. TQ dose­dependently suppressed TNF­α production from a human monocytic cell line, THP­1 exposed to FN, yet showed no toxicity to THP­1 cells. These results indicated that oral hygiene care using TQ could reduce FN­associated biofilm and inflammation in gingival tissue.

Ethanolamine utilization supports Clostridium perfringens growth in infected tissues.

Clostridium perfringens possesses the ethanolamine (EA) utilization (eut) system encoded within the eut operon, which utilizes the EA as a carbon, nitrogen and energy source. To determine the role of the eut system in C. perfringens growth, an in-frame deletion of the eutABC genes was made in strain HN13 to generate the eutABC-deleted mutant strain HY1701. Comparison of HN13 and HY1701 growth in media supplemented with 1.0% glucose and/or 1.0% EA showed that glucose enhanced the growth of both strains, whereas EA enhanced HN13 growth, but not that of HY1701, indicating that the eut system is necessary for C. perfringens to utilize EA. The two-component regulatory system EutVW is needed to induce eut gene expression in response to EA whereas the global virulence regulator VirRS differentially controlled eut gene expression depending on glucose and EA availability. To assess the role of the eut system in vivo, an equal number of HN13 and HY1701 cells were injected into the right thigh muscles of mice. Mice infected with HY1701 showed fewer symptoms than those injected with HN13. The mortality rate of mice infected with HY1701 tended to be lower than for mice infected with HN13. In addition, in infected tissues from mice injected with a mixture of HN13 and HY1701, HN13 outnumbered HY1701. PCR screening demonstrated that C. perfringens isolated from gas gangrene and sporadic diarrhea cases carried both eut genes and the perfringolysin O gene (pfoA) as well as the phospholipase C gene (plc). However, pfoA was not detected in isolates from food poisoning patients and healthy volunteers. Culture supernatants prepared from HN13 grown in media containing 7.5% sheep red blood cells induced significantly higher eutB expression levels compared to those from plc- and/or pfoA-deletion mutants. Together, these results indicate that the eut system plays a nutritional role for C. perfringens during histolytic infection.

Characterization of a recombinant Bacteroides fragilis sialidase expressed in Escherichia coli.

The human gut commensal Bacteroides fragilis produces sialidases that remove a terminal sialic acid from host-derived polysaccharides. Sialidase is considered to be involved in B. fragilis infection pathology. A native B. fragilis sialidase has been purified and characterized, and was shown to be post-translationally modified by glycosylation. However, the biochemical properties of recombinant B. fragilis sialidase expressed in a heterologous host remain uncharacterized. In this study, we examined the enzymatic properties of the 60-kDa sialidase NanH1 of B. fragilis YCH46, which was prepared as a recombinant protein (rNanH1) in Escherichia coli. In E. coli rNanH1 was expressed as inclusion bodies, which were separated from soluble proteins to allow solubilization of insoluble rNanH1 in a buffer containing 8 M urea and renaturation in refolding buffer containing 100 mM CaCl2 and 50 mM L-arginine. The specific activity of renatured rNanH1 measured using 4-methylumberiferyl-α-D-N-acetyl neuraminic acid as a substrate was 6.16 µmol/min/mg. The optimal pH of rNanH1 ranged from 5.0 to 5.5. The specific activity of rNanH1 was enhanced in the presence of calcium ions. rNanH1 preferentially hydrolyzed the sialyl α2,8 linkage and cleaved sialic acids from mucin and serum proteins (e.g., fetuin and transferrin) but not from α1-acid glycoprotein, which is similar to the previously observed biochemical properties for a native sialidase purified from B. fragilis SBT3182. The results and methods described in this study will be useful for preparing and characterizing recombinant proteins for other B. fragilis sialidase isoenzymes.

L­histidine augments the oxidative damage against Gram­negative bacteria by hydrogen peroxide.

Excessive damage to DNA and lipid membranes by reactive oxygen species reduces the viability of bacteria. In the present study, the proliferation of recA­deficient Escherichia coli strains was revealed to be inhibited by 1% L­histidine under aerobic conditions. This inhibition of proliferation was not observed under anaerobic conditions, indicating that L­histidine enhances oxidative DNA damage to E. coli cells. Reverse transcription­quantitative polymerase chain reaction analysis demonstrated that the expression of recA in E. coli MG1655 increased ~7­fold following treatment with 10 mM hydrogen peroxide (H2O2) plus 1% L­histidine, compared with that following exposure to H2O2 alone. L­histidine increased the genomic fragmentation of E. coli MG1655 following exposure to H2O2. In addition, L­histidine increased the generation of intracellular hydroxyl radicals in the presence of H2O2 in E. coli cells. Next, our group investigated the disinfection properties of the H2O2 and L­histidine combination. The combination of 100 mM H2O2 and 1.0% L­histidine significantly reduced the number of viable cells of extended­spectrum­ß­lactamase­producing E. coli and multidrug­resistant Pseudomonas aeruginosa, and this treatment was more effective than 100 mM H2O2 alone, but this effect was not observed in methicillin­resistant Staphylococcus aureus or vancomycin­resistant Enterococcus faecium. The combination of L­histidine and H2O2 may be a useful strategy to selectively increase the microbicidal activity of oxidative agents against Gram­negative bacteria.

D­Tagatose inhibits the growth and biofilm formation of Streptococcus mutans.

Dental caries is an important global health concern and Streptococcus mutans has been established as a major cariogenic bacterial species. Reports indicate that a rare sugar, D­tagatose, is not easily catabolized by pathogenic bacteria. In the present study, the inhibitory effects of D­tagatose on the growth and biofilm formation of S. mutans GS­5 were examined. Monitoring S. mutans growth over a 24 h period revealed that D­tagatose prolonged the lag phase without interfering with the final cell yield. This growth retardation was also observed in the presence of 1% sucrose, although it was abolished by the addition of D­fructose. S. mutans biofilm formation was significantly inhibited by growth in sucrose media supplemented with 1 and 4% D­tagatose compared with that in a culture containing sucrose alone, while S. mutans formed granular biofilms in the presence of this rare sugar. The inhibitory effect of D­tagatose on S. mutans biofilm formation was significantly more evident than that of xylitol. Growth in sucrose media supplemented with D­tagatose significantly decreased the expression of glucosyltransferase, exo­ß­fructosidase and D­fructose­specific phosphotransferase genes but not the expression of fructosyltransferase compared with the culture containing sucrose only. The activity of cell­associated glucosyltransferase in S. mutans was inhibited by 4% D­tagatose. These results indicate that D­tagatose reduces water­insoluble glucan production from sucrose by inhibiting glucosyltransferase activities, which limits access to the free D­fructose released during this process and retards the growth of S. mutans. Therefore, foods and oral care products containing D­tagatose are anticipated to reduce the risk of caries by inhibiting S. mutans biofilm formation.

Microbicidal effects of weakly acidified chlorous acid water against feline calicivirus and Clostridium difficile spores under protein-rich conditions.

Sanitation of environmental surfaces with chlorine based-disinfectants is a principal measure to control outbreaks of norovirus or Clostridium difficile. The microbicidal activity of chlorine-based disinfectants depends on the free available chlorine (FAC), but their oxidative potential is rapidly eliminated by organic matter. In this study, the microbicidal activities of weakly acidified chlorous acid water (WACAW) and sodium hypochlorite solution (NaClO) against feline calcivirus (FCV) and C. difficile spores were compared in protein-rich conditions. WACAW inactivated FCV and C. difficile spores better than NaClO under all experimental conditions used in this study. WACAW above 100 ppm FAC decreased FCV >4 log10 within 30 sec in the presence of 0.5% each of bovine serum albumin (BSA), polypeptone or meat extract. Even in the presence of 5% BSA, WACAW at 600 ppm FAC reduced FCV >4 log10 within 30 sec. Polypeptone inhibited the virucidal activity of WACAW against FCV more so than BSA or meat extract. WACAW at 200 ppm FAC decreased C. difficile spores >3 log10 within 1 min in the presence of 0.5% polypeptone. The microbicidal activity of NaClO was extensively diminished in the presence of organic matter. WACAW recovered its FAC to the initial level after partial neutralization by sodium thiosulfate, while no restoration of the FAC was observed in NaClO. These results indicate that WACAW is relatively stable under organic matter-rich conditions and therefore may be useful for treating environmental surfaces contaminated by human excretions.

Counterselection employing mutated pheS for markerless genetic deletion in Bacteroides species.

Markerless gene deletion is necessary for multiple gene disruptions due to the limited number of antibiotic resistant markers for some bacteria. However, even in transformable strains, obtaining the expected mutation without a marker requires laborious screening of a large number of colonies. Previous studies had success in various bacteria with a counter-selection system where a conditional lethal gene was incorporated into the vector. We examined the efficacy of the mutated pheS gene (pheS*) as a counter-selective marker for gene deletion in Bacteroides. This mutation produces an amino acid substitution (A303G) in the alpha subunit of Bacteroides phenylalanyl tRNA synthetase, which in E. coli alters the specificity of the tRNA synthetase resulting in a conditional lethal mutation due to the incorporation of p-chloro-phenylalanine (p-Cl-Phe) into protein. B. fragilis YCH46 and B. thetaiotaomicron VPI-5482 transformed with a pheS*-harboring shuttle vector were clearly growth-inhibited in the presence of >5 mM p-Cl-Phe in liquid defined minimal media (DMM) and on DMM agar plates. A targeting plasmid was constructed to delete the genetic region for capsular polysaccharide PS2 in B. fragilis or PS1 in B. thetaiotaomicron. After counterselection, p-Cl-Phe-resistant colonies were generated at a frequency of 8.1 × 10-3 for B. fragilis and 1.7 × 10-3 for B. thetaiotaomicron. Of the p-Cl-Phe-resistant colonies, 4.2% and 72% harbored the correct genetic deletion for B. fragilis and B. thetaiotaomicron, respectively. These results indicate that mutated pheS is a useful counter-selective gene to construct markerless genetic deletions in Bacteroides.

Cleansing effect of acidic L-arginine on human oral biofilm.

BACKGROUND: Dental plaque formed on tooth surfaces is a complex ecosystem composed of diverse oral bacteria and salivary components. Accumulation of dental plaque is a risk factor for dental caries and periodontal diseases. L-arginine has been reported to decrease the risk for dental caries by elevating plaque pH through the activity of arginine deiminase in oral bacteria. Here we evaluated the potential of L-arginine to remove established oral biofilms. METHODS: Biofilms were formed using human saliva mixed with Brain Heart Infusion broth supplemented with 1 % sucrose in multi-well plates or on plastic discs. After washing the biofilms with saline, citrate (10 mM, pH3.5), or L-arginine (0.5 M, pH3.5), the retained biofilms were analyzed by crystal violet staining, scanning electron microscopy, and Illumina-based 16S rDNA sequencing. RESULTS: Washing with acidic L-arginine detached oral biofilms more efficiently than saline and significantly reduced biofilm mass retained in multi-well plates or on plastic discs. Illumina-based microbiota analysis showed that citrate (pH3.5) preferentially washed out Streptococcus from mature oral biofilm, whereas acidic L-arginine prepared with 10 mM citrate buffer (pH3.5) non-specifically removed microbial components of the oral biofilm. CONCLUSIONS: Acidic L-arginine prepared with citrate buffer (pH3.5) effectively destabilized and removed mature oral biofilms. The acidic L-arginine solution described here could be used as an additive that enhances the efficacy of mouth rinses used in oral hygiene.