Antiseptic effect of slightly acidic electrolyzed water on dental unit water systems.

Biofilm formation in dental unit water systems (DUWSs) can contaminate water from three-in-one syringes, air rotors, and low-speed handpieces. This may serve as a potential source of infection for dentists, dental staff, and patients, so these systems must be sterilized. Because slightly acidic electrolyzed water (SAEW) is often used as a disinfectant for food, the aim of this study was to investigate the possibility of using SAEW as a DUWS disinfectant. Slightly acidic electrolyzed water was injected into a dental unit and its effects evaluated. Chemical properties such as chlorine ion and potential hydrogen in the SAEW were measured. Detection of both ordinary and heterotrophic bacteria from the DUWS was performed by culture, and biofilm formation of the bacteria in the DUWS evaluated. Polymerase chain reaction (PCR) was used to detected contamination by nosocomial pathogens. Almost all the chlorine ions in the SAEW were exhausted during the two-day trials, and the pH value of the SAEW fell from 5 to 4. No viable cells were detected in the SAEW collected. Biofilm formation in the water from the DUWS with SAEW was almost at a baseline level, whereas that without SAEW was 4 times higher. The PCR analysis showed that no nosocomial infecting pathogens were detected in the SAEW. The present study demonstrated the antiseptic effect of SAEW in DUWS.

Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts.

OBJECTIVE: Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL)-6, IL-8, and the chemical mediator prostaglandin E2 (PGE2) are known to play important roles in inflammatory responses and tissue degradation. Recently, we reported that the protein kinase A (PKA) inhibitor H-89 suppresses lipopolysaccharide (LPS)-induced IL-8 production by human gingival fibroblasts (HGFs). In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8) and PGE2 by HGFs were examined. METHODS: HGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP), aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE2 levels were evaluated by ELISA. RESULTS: H-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE2 production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE2 production. Up to 10 µg/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at 100 µg/ml. Similarly, 0.01 µg/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE2 production, but they were significantly increased at concentrations of 0.1 and 1 µg/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE2 production. CONCLUSION: These results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE2 production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 µg/ml in serum). These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.

The short-term effects of various oral care methods in dependent elderly: comparison between toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine.

OBJECTIVES: To explore the short-term effects from toothbrushing, tongue cleaning with sponge brush and wiping on oral mucous membrane by chlorhexidine. BACKGROUND: Numerous reports have been seen in recent years proving the effectiveness of mouth cleaning with a toothbrush for the prevention of respiratory infections among the dependent elderly. However, the short-term effects from each oral care method have not yet been clarified. Hence, an investigation was conducted by having each subject independently perform various oral care methods for five consecutive days. MATERIALS AND METHODS: The subjects consisted of 12 assistance-dependent elderly who have difficulties with tooth brushing by themselves, have 10 or more residual teeth and are not yet using plate dentures. After the pre-intervention examination, each of the following oral care methods were performed on the same subject on an approximately three week basis: 1) Tooth brushing 2) Tongue cleaning with sponge brush 3) Wiping on oral mucous with sponge brush by chlorhexidine. Each method was performed independently, once a day for 5 consecutive days and the subjects were reexamined on the sixth day for comparative verification. RESULTS: Consequently, toothbrushing decreased the plaque index and gingival index significantly and an improvement of oral malodour was also acknowledged (p < 0.01). Tongue cleaning with a sponge brush decreased the tongue coat score significantly (p < 0.05) and oral malodour was also improved (p < 0.01). Wiping on oral mucous with a sponge brush soaked in chlorhexidine significantly decreased opportunistic infections in the pharynx region (p < 0.05). CONCLUSIONS: It was suggested that the use of not only a toothbrush but also chlorhexidine gluconate may be indicated for dependent elderly people in whom pathogens of opportunistic infection are detected.

Effects of deuterium oxide on Streptococcus mutans and Pseudomonas aeruginosa.

A complex aggregation of microorganisms growing on a solid substrate is termed a biofilm and is considered to be an etiological agents. Pseudomonas aeruginosa and Streptococcus mutans are representative bacteria in such biofilms. It is well known that deuterium oxide (D2O) causes toxic effects on a number of biological systems. We investigated the effects of D2O on growth and biofilm formation of P. aeruginosa and S. mutans. These bacteria were incubated in medium containing D2O (100%, 75% or 0%) at 37°C for 24hr, 48 hr or 72 hr. Growth of P. aeruginosa was inhibited by D2O within the first 48 hr. However, after 72 hr, growth rate was seen to increase in the D2O-containing medium compared with in medium without D2O. In contrast, the growth of S. mutans in the D2O medium was inhibited within 72 hr. The biofilm formation of P. aeruginosa was increased in the D2O medium. Biofilm formation of S. mutans in the D2O medium increased compared with in the medium without D2O, but this increase was only temporary in the case of P. aeruginosa. Compared to biofilm formation in 0% D2O medium marked as 100%, the biofilm formation rate of S. mutans in 75% D2O medium was 143% at 24 hr, 146% at 48 hr and 130% at 72 hr. In other D2O concentration media biofilm formation was lower. In 100% D2O medium, biofilm formation rate decreased from 114% at 24 hr to 56% at 72 hr. The biofilm formation rate of P. aeruginosa in 100% D2O medium was 172% at 24 hr, but decreased to 88% at 72 hr. Biofilm formation of P. aeruginosa in 75% and 0% D2O media showed no significant difference. We consider that these results were due to stress or alteration in bacterial metabolisms.

Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease.

BACKGROUND AND OBJECTIVE: A major factor in the pathogenesis of periodontal disease, which is one of the biofilm infectious diseases, is thought to be lipopolysaccharide (LPS), owing to its ability to cause inflammation and promote tissue destruction. Moreover, the elimination of pathogens and their component LPSs is essential for the successful treatment of periodontal disease. Lipopolysaccharide tolerance is a mechanism that prevents excessive and prolonged responses of monocytes and macrophages to LPS. Since persistence of inflammation is necessary for inflammatory cytokine production, cells other than monocytes and macrophages are thought to maintain the production of cytokines in the presence of LPS. In this study, we investigated whether human gingival fibroblasts (HGFs), the most abundant structural cell in periodontal tissue, might be able to maintain inflammatory cytokine production in the presence of LPS bynot displaying LPS tolerance. MATERIAL AND METHODS: Human gingival fibroblasts were pretreated with LPS (from Porphyromonas gingivalis and Escherichia coli) and then treated with LPS, and the amounts of interleukin (IL)-6 and IL-8 in the cell culture supernatants were measured. The expression of negative regulators of LPS signalling (suppressor of cytokine signalling-1, interleukin-1 receptor-associated-kinase M and SH2 domain-containing inositol-5-phosphatase-1) was also examined in LPS-treated HGFs. RESULTS: Human gingival fibroblasts did not display LPS tolerance but maintained production of IL-6 and IL-8 when pretreated with LPS, followed by secondary LPS treatment. Lipopolysaccharide-treated HGFs did not express negative regulators. CONCLUSION: These results demonstrate that HGFs do not show LPS tolerance and suggest that this characteristic of HGFs sustains the inflammatory response in the presence of virulence factors.

Sixteen homologs of the mex-type multidrug resistance efflux pump in Bacteroides fragilis.

Sixteen homologs of multidrug resistance efflux pump operons of the resistance-nodulation-cell division (RND) family were found in the Bacteroides fragilis genome sequence by homology searches. Disruption mutants were made to the mexB homologs of the four genes most similar to Pseudomonas aeruginosa mexB. Reverse transcription-PCR was conducted and indicated that the genes were transcribed in a polycistronic fashion and that the promoter was upstream of bmeA (the mexA homolog). One of these disruption mutants (in bmeB, the mexB homolog) was more susceptible than the parental strain to certain cephems, polypeptide antibiotics, fusidic acid, novobiocin, and puromycin. The gene for this homolog and the adjacent upstream gene, bmeA, were cloned in a hypersensitive Escherichia coli host. The resultant transformants carrying B. fragilis bmeAB were more resistant to certain agents; these agents also had lower MICs for the B. fragilis bmeB disruption mutants than for the parental strain. The putative efflux pump operon is composed of bmeA, bmeB, and bmeC (a putative outer membrane channel protein homologous with OprM). Addition of the efflux pump inhibitors, carbonyl cyanide m-chlorophenylhydrazone (a proton conductor that eliminates the energy source) and Phe-Arg beta-naphthylamide (MC-207,110) (the first specific inhibitor described for RND pumps in P. aeruginosa), resulted in lowered MICs in the parental strain but not in the bmeB disruption mutant, indicating that the bmeB pump is affected by these inhibitors. This is the first description of RND type pumps in the genus Bacteroides.

Comparison of ability of apoptosis induction by lipopolysaccharide of Porphyromonas gingivalis with Escherichia coli.

We compared effects of Porphyromonas gingivalis LPS with Escherichia coli LPS to the murine peritoneal macrophage. E. coli LPS possessed a threshold dose between 100 micro g and 10 micro g, the higher dose induced apoptosis at the murine peritoneal macrophage while the lower dose did not. The ability of apoptosis induction at the murine peritoneal macrophage of P. gingivalis LPS was weaker than E. coli LPS. P. gingivalis LPS did not induce significant apoptosis in all tested dose. However, the morphology of the peritoneal macrophage treated by P. gingivalis LPS was obviously different from that of the unstimulated cells.

Isolation and properties of a tripeptidyl peptidase from a periodontal pathogen Prevotella nigrescens.

Prolyltripeptidyl amino peptidase activity was found in a crude extract of Prevotella nigrescens and this enzyme was purified by procedures including concentration with ammonium sulfate, ion exchange chromatography, gel filtration, and isoelectric focusing. This peptidase hydrolyzed Ala-Ala-Pro-p-nitroanilide as well as Ala-Phe-Pro-p-nitroanilide. Furthermore, several p-nitroanilide derivatives of dipeptides with a proline residue in the second position from the amino-terminal end (Xaa-Pro) were also cleaved detectably. The molecular mass of this tripeptidase was calculated as 56 kDa and its isoelectric point was 5.8. The enzyme was inactivated completely by heating at 60 degrees C for 5 min and inhibited significantly by specific serine enzyme inhibitors.

Dipeptidyl peptidase with strict substrate specificity of an anaerobic periodontopathogen Porphyromonas gingivalis.

A dipeptidyl peptidase which hydrolyzed Xaa-Ala-p-nitroanilide was purified to homogeneity by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration and isoelectric focusing from the cell extract of Porphyromonas gingivalis. The purified enzyme hydrolyzed p-nitroanilide derivatives of Lys-Ala, Ala-Ala, and Val-Ala, but not Xaa-Pro. Enzyme activity was maximum at neutral pHs. Its molecular mass was 64 kDa with an isoelectric point of 5.7. The enzyme belonged to the family of serine peptidases.