The Commensal Microbe Veillonella as a Marker for Response to an FGF19 Analog in NASH.

BACKGROUND AND AIMS: The composition of the human gut microbiota is linked to health and disease, and knowledge of the impact of therapeutics on the microbiota is essential to decipher their biological roles and to gain new mechanistic insights. Here we report the effect of aldafermin, an analog of the gut hormone FGF19, versus placebo on the gut microbiota in a prospective, phase 2 study in patients with NASH. APPROACH AND RESULTS: A total of 176 patients with biopsy-confirmed nonalcoholic steatohepatitis (NASH) (nonalcoholic fatty liver disease activity score ≥ 4), fibrosis (F1-F3 by NASH Clinical Research Network criteria), and elevated liver fat content (≥ 8% by magnetic resonance imaging-proton density fat fraction) received 0.3 mg (n = 23), 1 mg (n = 49), 3 mg (n = 49), and 6 mg (n = 28) aldafermin or placebo (n = 27) for 12 weeks. Stool samples were collected on day 1 and week 12 and profiled using 16S ribosomal RNA gene sequencing; 122 patients had paired stool microbiome profiles at both day 1 and week 12. Overall, the state of the gut microbial community was distinctly stable in patients treated with aldafermin, with all major phyla and genera unaltered during therapy. Patients treated with aldafermin showed a significant, dose-dependent enrichment in the rare genus Veillonella, a commensal microbe known to have lactate-degrading and performance-enhancing properties, which correlated with changes in serum bile acid profile. CONCLUSIONS: Veillonella may be a bile acid-sensitive bacteria whose enrichment is enabled by aldafermin-mediated suppression of bile acid synthesis and, in particular, decreases in toxic bile acids. This study provides an integrated analysis of gut microbiome, serum bile acid metabolome, imaging, and histological measurements in clinical trials testing aldafermin for NASH. Our results provide a better understanding of the intricacies of microbiome-host interactions (clinicaltrials.gov trial No. NCT02443116).

Bacteremia caused by Veillonella dispar in an oncological patient.

Veillonella dispar is a Gram-negative anaerobic coccus involved in only a few human diseases. We report the second case of bacteremia due to this microorganism in an elderly patient. A 72-year-old man with a history of bladder cancer presented with diarrhea, vomiting, and fever for 48 hours. After the diagnosis of septic shock, four sets of blood cultures were taken, and three of them yielded V. dispar. Resistance to metronidazole, penicillin, and piperacillin-tazobactam was documented. Treatment with clindamycin was started, and the patient was discharged after improvement in his general condition.

Variability in nitrate-reducing oral bacteria and nitric oxide metabolites in biological fluids following dietary nitrate administration: An assessment of the critical difference.

There is conflicting evidence on whether dietary nitrate supplementation can improve exercise performance. This may arise from the complex nature of nitric oxide (NO) metabolism which causes substantial inter-individual variability, within-person biological variation (CVB), and analytical imprecision (CVA) in experimental endpoints. However, no study has quantified the CVA and CVB of NO metabolites or the factors that influence their production. These data are important to calculate the critical difference (CD), defined as the smallest difference between sequential measurements required to signify a true change. The main aim of the study was to evaluate the CVB, CVA, and CD for markers of NO availability (nitrate and nitrite) in plasma and saliva before and after the ingestion of nitrate-rich beetroot juice (BR). We also assessed the CVB of nitrate-reducing bacteria from the dorsal surface of the tongue. It was hypothesised that there would be substantial CVB in markers of NO availability and the abundance of nitrate-reducing bacteria. Ten healthy male participants (age 25 ±â€¯5 years) completed three identical trials at least 6 days apart. Blood and saliva were collected before and after (2, 2.5 and 3 h) ingestion of 140 ml of BR (∼12.4 mmol nitrate) and analysed for [nitrate] and [nitrite]. The tongue was scraped and the abundance of nitrate-reducing bacterial species were analysed using 16S rRNA next generation sequencing. There was substantial CVB for baseline concentrations of plasma (nitrate 11.9%, nitrite 9.0%) and salivary (nitrate 15.3%, nitrite 32.5%) NO markers. Following BR ingestion, the CVB for nitrate (plasma 3.8%, saliva 12.0%) and salivary nitrite (24.5%) were lower than baseline, but higher for plasma nitrite (18.6%). The CD thresholds that need to be exceeded to ensure a meaningful change from baseline are 25, 19, 37, and 87% for plasma nitrate, plasma nitrite, salivary nitrate, and salivary nitrite, respectively. The CVB for selected nitrate-reducing bacteria detected were: Prevotella melaninogenica (37%), Veillonella dispar (35%), Haemophilus parainfluenzae (79%), Neisseria subflava (70%), Veillonella parvula (43%), Rothia mucilaginosa (60%), and Rothia dentocariosa (132%). There is profound CVB in the abundance of nitrate-reducing bacteria on the tongue and the concentration of NO markers in human saliva and plasma. Where these parameters are of interest following experimental intervention, the CD values presented in this study will allow researchers to interpret the meaningfulness of the magnitude of the change from baseline.

Identification of an early stage biofilm inhibitor from Veillonella tobetsuensis.

Oral biofilm, the cause of dental caries and periodontal diseases, consists of multiple bacterial species. Streptococcus spp. and Veillonella spp. have been reported as to be initial and early colonizers of oral biofilms. Our previous studies showed that Veillonella tobetsuensis may play an important role on the development of S. gordonii biofilms without coaggregation involving extracellular biomolecules. In this study, the effect of a cyclic dipeptide autoinducer from culture supernatants from V. tobetsuensis at late-exponential growth phase on S. gordonii biofilm was examined. The cyclic dipeptide, identified as cyclo (-L-Leu-L-Pro) by gas chromatography/mass spectrometry, inhibited the development of S. gordonii biofilm. Furthermore, cyclo (-L-Leu-L-Pro) appeared not to cause bactericidal effects on planktonic cells of S. gordonii. This is the first report that oral Veillonella produces cyclo (-L-Leu-L-Pro) in their culture supernatants. Moreover, the results of this study suggest that cyclo (-L-Leu-L-Pro) may have an application to inhibit early stage development of oral biofilms.

Antimicrobial efficiency of mouthrinses versus and in combination with different photodynamic therapies on periodontal pathogens in an experimental study.

BACKGROUND AND OBJECTIVE: In the therapy of destructive periodontal disease, chemical antimicrobial agents and increasingly photodynamic therapy (PDT) play an important adjunctive role to standard mechanical anti-infective treatment procedures. However, both antiseptic methods have their shortcomings in terms of eliminating periodontal pathogens. The aim of the study was to compare the antibacterial efficacy of different antiseptic mouthrinses, of a conventional and a new, modified PDTplus as well as of the different antiseptic mouthrinses combined with either the conventional or the modified PDTplus against periopathogens. MATERIAL AND METHODS: Six representative periodontitis-associated bacterial strains were grown for 24 h under anaerobic conditions. After mixing the individual cell pellets they were exposed to 10 different antiseptic mouthrinse formulations: chlorhexidine (0.2%, 0.06%, CHX); CHX + cetylpyridinium chloride (each 0.05%); sodium hypochlorite (0.05%); polyhexanide (0.04%, PHMB1; 0.1%, PHMB2); octenidine dihydrochloride (0.1%); fluoride (250 ppm); essential oils; povidone iodine (10%); and saline (0.9%, NaCl) as control. Furthermore, the bacteria were treated with conventional PDT based on light-emitting diodes and a new modified photodisinfection combining photosensitizer with hydrogen peroxide to PDTplus also based on light-emitting diodes. In addition to the single treatments, a combined application of antiseptic exposure followed by use of PDT or PDTplus was performed. The microbial viability was characterized by analyzing colony growth and fluorescence-based vitality proportions. RESULTS: Nearly all mouthrinses caused a statistically significant growth inhibition. The most effective antiseptics, CHX (0.2%), CHX/cetylpyridinium chloride and octenidine dihydrochloride, inhibited bacterial growth completely. Conventional PDT resulted in moderate reduction of colony growth. The modified PDTplus achieved maximum antimicrobial effect. The combination of antiseptic exposure and PDT against periopathogens predominantly increased antibacterial efficacy compared to the single applications. The mouthrinse containing essential oil seemed to interfere with PDT. CONCLUSION: A combination therapy of preceding chemotherapeutical exposure and subsequent photodisinfection may be a more effective and promising antibacterial treatment than single applications of the antiseptic methods. The modified PDTplus using oxygen-enriched toluidine showed a superior antibacterial effect on periodontal pathogens to conventional PDT and to the majority of the investigated mouthrinses.

A new treatment of sepsis caused by veillonella parvula: A case report and literature review.

WHAT IS KNOWN AND OBJECTIVES: Veillonella parvula is usually regarded as a contaminant bacteria associated with polymicrobial infection. Here we report a rare case of sepsis caused by V. parvula, which was successfully treated by chloramphenicol. In addition, the therapeutic strategy for V. parvula infection was reviewed. CASE SUMMARY: A 55-year-old woman underwent surgery for lumbar spinal stenosis and developed high fever (39.5°C) after surgery, accompanied by chills and fatigue. V. parvula was isolated in blood culture and shown to be probably sensitive to both meropenem and chloramphenicol. No improvement was seen after 3 days of treatment with meropenem, and then, iv chloramphenicol (1.5 g once daily) was added to the regimen. The symptoms and fever resolved in 2 days. She was continuously treated with chloramphenicol for another 10 days before discharge. WHAT IS NEW AND CONCLUSION: The reported antibiotics in the therapy of infections caused by veillonella species include penicillins, metronidazole, cephalosporins, aminoglycosides, imipenem, clindamycin, doxycycline, erythromycin and chloramphenicol. To our knowledge, this is the first case describing successful treatment of V. parvula-caused sepsis with chloramphenicol.

Effect of smokeless tobacco products on human oral bacteria growth and viability.

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log10 fold, 18 strains showed enhanced growth of 0.3-0.97 log10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log10 fold, 8 strains showed enhanced growth of 0.3-1.0 log10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 µg/ml; while the growth of P. micros was enhanced 0.31-0.54 log10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log10 fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria.

Antimicrobial Susceptibility of Microorganisms Isolated from Periapical Periodontitis Lesions.

Periapical periodontitis usually results from microbial infection, with these microorganisms occasionally migrating to the root canal, which can lead to further, potentially life-threatening, complications. Here, the susceptibility of 27 bacterial strains to various antimicrobial agents was evaluated. These strains comprised 13 species; 16 of the strains were clinical isolates from periapical lesions. Each strain was inoculated onto blood agar plates containing one of the antimicrobial agents. The plates were incubated anaerobically at 37°C for 96 hr and the minimal inhibitory concentrations (MICs) determined. Ten strains required an MIC of 32 µg/ml or greater for amoxicillin, 6 for cefmetazole, and 5 for cefcapene among ß-lactam antibiotics; 8 strains required an MIC of 32 µg/ml or greater for clindamycin, 4 for azithromycin, and 11 for clarithromycin among macrolide antibiotics; 3 strains required an MIC of 32 µg/ml or greater for ciprofloxacin and 2 for ofloxacin among fluoroquinolones. The effect of cefcapene on 5 strains was evaluated after biofilm formation to investigate the relationship between biofilm formation and susceptibility. All strains showed a decrease in susceptibility after biofilm formation. The results revealed that several antimicrobial agents commonly used in a clinical setting, including amoxicillin, cefmetazole, and clindamycin, are potentially effective in the treatment of orofacial odontogenic infections. The development of resistant strains, however, means that this can no longer be guaranteed. In addition, azithromycin, ciprofloxacin, and ofloxacin were more effective than the 3 ß-lactam antibiotics tested. These results suggest that sensitivity testing is needed if odontogenic infections are to be treated safely and effectively.

Effects of azithromycin, metronidazole, amoxicillin, and metronidazole plus amoxicillin on an in vitro polymicrobial subgingival biofilm model.

Chronic periodontitis is one of the most prevalent human diseases and is caused by dysbiosis of the subgingival microbiota. Treatment involves primarily mechanical disruption of subgingival biofilms and, in certain cases, adjunctive use of systemic antibiotic therapy. In vitro biofilm models have been developed to study antimicrobial agents targeting subgingival species. However, these models accommodate a limited number of taxa, lack reproducibility, and have low throughput. We aimed to develop an in vitro multispecies biofilm model that mimics subgingival plaque, to test antimicrobial agents. Biofilms were cultivated using the Calgary Biofilm Device and were exposed to amoxicillin (AMX), metronidazole (MTZ), azithromycin (AZM), and AMX-MTZ at four different concentrations for 12, 24, or 36 h. Chlorhexidine (CHX) (0.12%) was used as the positive control. The compositions of the biofilms were analyzed by checkerboard DNA-DNA hybridization, and the percent reduction in biofilm metabolic activity was determined using 2,3,5-triphenyltetrazolium chloride and spectrophotometry. Thirty-five of the 40 species used in the inoculum were consistently recovered from the resulting in vitro biofilms. After 36 h of exposure at the 1:27 dilution, AMX-MTZ reduced metabolic activity 11% less than CHX (q = 0.0207) but 54% more than AMX (q = 0.0031), 72% more than MTZ (q = 0.0031), and 67% more than AZM (q = 0.0008). Preliminary evidence of a synergistic interaction between AMX and MTZ was also observed. In summary, we developed reproducible biofilms with 35 subgingival bacterial species, and our results suggested that the combination of AMX and MTZ had greater antimicrobial effects on these in vitro multispecies biofilms than expected on the basis of the independent effects of the drugs.

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.

Antibacterial Efficacy of a Propolis Toothpaste and Mouthrinse Against a Supragingival Multispecies Biofilm.

PURPOSE: To determine in vitro the antibacterial properties of propolis toothpaste and mouthrinse against an in vitro multispecies biofilm model. MATERIALS AND METHODS: Six-species biofilms grown anaerobically on pellicle-coated hydroxyapatite disks were fed with glucose/sucrose-supplemented medium 3 times daily for 45 min and incubated in 37°C saliva between feedings for up to 64.5 h. At each interval, biofilms were exposed to six different slurries and solutions, including: 1) toothpaste without propolis, 2) toothpaste with propolis, 3) toothpaste with chlorhexidine, 4) mouthrinse with propolis, 5) mouthrinse with chlorhexidine, 6) saline solution (control). Afterwards, biofilms were harvested and the number of colony forming units were determined (CFU). The results were analysed using ANOVA, followed by the Bonferroni test at a 5% significance level. RESULTS: The strongest CFU reduction was shown after treatment with 0.12% chlorhexidine (p<0.0004). When comparing the different toothpastes, there was no statistically significant difference (p<0.05) in CFU reduction. However, they all showed a significant reduction in CFU of more than one log-step vs the saline control group. Nevertheless, the propolis-containing mouthrinse showed no significant reduction in CFU. CONCLUSION: All toothpastes under investigation displayed some growth inhibition in this supragingival biofilm model, which accounted for an approximately 80%-88% linear reduction. However, the propolis mouthwash had no effect.

Effects of pH and lactate on hydrogen sulfide production by oral Veillonella spp.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H2S), one of the major components of oral malodor. However, there is little information on the physiological properties of H2S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H2S production. Thus, in the present study, the H2S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H2S during growth in the presence of l-cysteine. Resting cells of these bacteria produced H2S from l-cysteine, and the cell extracts showed enzymatic activity to convert l-cysteine to H2S. H2S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H2S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H2S, ammonia was produced in cell extracts of all the strains, indicating that H2S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine ß-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H2S from l-cysteine and that their H2S production can be regulated by oral environmental factors, namely, pH and lactate.

Antibacterial epipolythiodioxopiperazine and unprecedented sesquiterpene from Pseudallescheria boydii, a beetle (coleoptera)-associated fungus.

Pseudallescheria boydii residing in the gut of coleopteran (Holotrichia parallela) larva produces four new epipolythiodioxopiperazine (ETP) boydines A-D (3-6) and two novel sesquiterpene boydenes A (7) and B (10), in addition to bisdethiobis(methylthio)-deacetylaranotin (1), bisdethiodi(methylthio)-deacetylapoaranotin (2), AM6898 A (8) and ovalicin (9). The structure elucidation was accomplished by a combination of spectral methods with quantum chemical calculations of optical rotations and electronic circular dichroism (ECD) spectra. Boydine B (4) was shown to be active against the clinical strains Bifidobacterium sp., Veillonella parvula, Anaerostreptococcus sp., Bacteroides vulgatus and Peptostreptococcus sp. with an MIC range of 0.2-0.8 µM, and the pharmacophore 3-hydroxy-2,4,6-trimethyl-5-oxooct-6-enoyl chain of 4 was shown to have (2R,3S,4S)-configurations. Boydene A (7) possessed an unprecedented carbon skeleton, suggesting an unusual biochemistry that allows an intramolecular Aldol addition in the fungus. Collectively, the finding may inspire the discovery of new antibacterial agents and the understanding on biosyntheses of polythiodioxopiperazine and sesquiterpene metabolites.

Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model.

BACKGROUND AND OBJECTIVES: Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. MATERIAL AND METHODS: PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 µm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. RESULTS: When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10 . P. gingivalis showed a vitality reduction in the biofilm of 3 log10 , while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. CONCLUSION: These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.

No evidence of triclosan-resistant bacteria following long-term use of triclosan-containing toothpaste.

BACKGROUND AND OBJECTIVE: There is a paucity of data in relation to the possible emergence of triclosan (TCS)-resistant bacteria following long-term exposure to TCS toothpaste. Therefore, this study investigated whether long-term continuous exposure to TCS in toothpaste selects for TCS-resistant bacteria within the oral biofilm. MATERIAL AND METHODS: Dental plaque samples were collected from 40 individuals during year 5 of a randomised controlled trial. Participants had been randomly assigned to use TCS (3000 µg/mL TCS) (n = 18) or placebo toothpaste (n = 22). Diluted plaque samples were plated on to Wilkins-Chalgren agar plates containing 5% (v/v) laked sheep red blood cells and TCS (concentrations ranging from 25 to 150 µg/mL) and incubated at 37 °C under microaerophilic and anaerobic conditions for 2-10 d. Selected bacterial isolates were identified by partial 16S rDNA sequencing and TCS minimum inhibitory concentration (MIC) determined for each isolate. RESULTS: At 3000 µg/mL TCS no growth was observed under microaerophilic or anaerobic conditions in either group. The MICs of TCS for all isolates ranged from 125 to 1000 µg/mL in both groups. Species common to both groups had similar MICs. Veillonella parvula and Campylobacter gracilis were the most frequent isolates from both groups, with similar MICs in both groups. CONCLUSION: The use of TCS-containing toothpaste did not appear to lead to an increase in MIC of TCS of oral bacterial isolates.

Antibacterial effects of blackberry extract target periodontopathogens.

BACKGROUND AND OBJECTIVE: Antimicrobial agents provide valuable adjunctive therapy for the prevention and the control of oral diseases. Limitations in their prolonged use have stimulated the search for new, naturally occurring agents with more specific activity and fewer adverse effects. Here we sought to determine the antibacterial properties of blackberry extract (BBE) in vitro against oral bacterial commensals and periodontopathogens. MATERIAL AND METHODS: The effects of whole and fractionated BBE on the metabolism of 10 different oral bacteria were evaluated using the colorimetric water-soluble tetrazolium-1 assay. The bactericidal effects of whole BBE against Fusobacterium nucleatum were determined by quantitating the numbers of colony-forming units (CFUs). Cytotoxicity was determined in oral epithelial (OKF6) cells. RESULTS: BBE at 350-1400 µg/mL reduced the metabolic activity of Porphyromonas gingivalis, F. nucleatum and Streptococcus mutans. The reduced metabolic activity observed for F. nucleatum corresponded to a reduction in the numbers of CFUs following exposure to BBE for as little as 1 h, indicative of its bactericidal properties. An anthocyanin-enriched fraction of BBE reduced the metabolic activity of F. nucleatum, but not of P. gingivalis or S. mutans, suggesting the contribution of species-specific agents in the whole BBE. Oral epithelial cell viability was not reduced following exposure to whole BBE (2.24-1400 µg/mL) for ≤ 6 h. CONCLUSION: BBE alters the metabolic activity of oral periodontopathogens while demonstrating a minimal effect on commensals. The specific antibacterial properties of BBE shown in this study, along with its previously demonstrated anti-inflammatory and antiviral properties, make this natural extract a promising target as an adjunct for prevention and/or complementary therapy of periodontal infections.

Comparative evaluation of bactericidal potential of four root canal filling materials against microflora of infected non-vital primary teeth.

BACKGROUND AND OBJECTIVES: Since complete debridement of the root canals of the primary teeth is not practically possible due to the highly variable root canal anatomy, success of the endodontic therapy depends partly on the use ofantibacterial irrigating agents and root canal filling materials. Recent literature indicates that anaerobes comprise a majority of the bacteria in necrotic root canals ofprimary teeth. The study determined the antibacterial effectiveness of four root canal filling materials namely Calcium hydroxide, Zinc oxide eugenol, Vitapex and Metapex against microbial specimens obtained directly from necrotic root canals of primary teeth. METHOD: Microbial specimens were collected using sterile paper points, from 15 primary maxillary and mandibular posterior teeth of randomly selected children in the age group of 4-10 years with infected non vital primary teeth, requiring pulpectomy procedure. The microbial specimens collected were subjected to microbiological analysis and the antimicrobial potential of root canal filling materials were tested using Agar diffusion technique. RESULTS: were statistically analyzed using one-way ANOVA. Facultative/Aerobic organisms were isolated in all the cases, anaerobic organisms were isolated in 80% of the cases, and Candida albicans was isolated in 1 case. ZOE showed superior inhibitory activity against most of the organisms isolated followed by Vitapex, Calcium hydroxide and Metapex in descending order. CONCLUSION: Our data may be useful as a guide for relative antimicrobial effectiveness or non-effectiveness of the materials employed. In vivo studies are required to state the specific antimicrobial activity and merits and demerits of any of the test filling material.

Liquid-Infused Structured Titanium Surfaces: Antiadhesive Mechanism to Repel Streptococcus oralis Biofilms.

To combat implant-associated infections, there is a need for novel materials which effectively inhibit bacterial biofilm formation. In the present study, the antiadhesive properties of titanium surface functionalization based on the "slippery liquid-infused porous surfaces" (SLIPS) principle were demonstrated and the underlying mechanism was analyzed. The immobilized liquid layer was stable over 13 days of continuous flow in an oral flow chamber system. With increasing flow rates, the surface exhibited a significant reduction in attached biofilm of both the oral initial colonizer  Streptococcus oralis and an oral multispecies biofilm composed of S. oralis, Actinomyces naeslundii, Veillonella dispar, and Porphyromonas gingivalis. Using single cell force spectroscopy, reduced S. oralis adhesion forces on the lubricant layer could be measured. Gene expression patterns in biofilms on SLIPS, on control surfaces, and expression patterns of planktonic cultures were also compared. For this purpose, the genome of S. oralis strain ATCC 9811 was sequenced using PacBio Sequel technology. Even though biofilm cells showed clear changes in gene expression compared to planktonic cells, no differences could be detected between bacteria on SLIPS and on control surfaces. Therefore, it can be concluded that the ability of liquid-infused titanium to repel S. oralis biofilms is mainly due to weakened bacterial adhesion to the underlying liquid interface.

Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm.

INTRODUCTION: Our previous studies showed that Streptococcus mutans and Veillonella parvula dual-species biofilms have a different acid production profile and a higher resistance to chlorhexidine than their single-species counterparts. The aim of the current study was to test whether the susceptibility of S. mutans grown in the presence of V. parvula is also decreased when it is exposed to various other antimicrobials. Furthermore, the aim was to identify other changes in the physiology of S. mutans when V. parvula was present using transcriptomics. METHODS: Susceptibility to antimicrobials was assessed in killing experiments. Transcript levels in S. mutans were measured with the help of S. mutans microarrays. RESULTS: When V. parvula was present, S. mutans showed an increase in survival after exposure to various antimicrobials. Furthermore, this co-existence altered the physiology of S. mutans. The expression of genes coding for proteins involved in amino acid synthesis, the signal recognition particle-translocation pathway, purine metabolism, intracellular polysaccharide synthesis, and protein synthesis all changed. CONCLUSION: Growing in a biofilm together with a non-pathogenic bacterium like V. parvula changes the physiology of S. mutans, and gives this bacterium an advantage in surviving antimicrobial treatment. Thus, the study of pathogens implicated in polymicrobial diseases, such as caries and periodontitis, should be focused more on multispecies biofilms. In addition, the testing of susceptibility to currently used and new antimicrobials should be performed on a multispecies microbial community rather than with single pathogens.

An experimental study for rapid detection and quantification of endodontic microbiota following photo-activated disinfection via new multiplex real-time PCR assay.

BACKGROUND: The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. MATERIALS AND METHODS: Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. RESULTS: Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). CONCLUSION: As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively.