A simplified in vitro model for investigation of the antimicrobial efficacy of various antiseptic agents to prevent peri-implantitis.

The biofilm formation by oral bacteria on the implant surface is one of the most remarkable factors of peri-implant infections, which may eventually lead to bone resorption and loss of the dental implant. Therefore, the elimination of biofilm is an essential step for the successful therapy of implant-related infections. In this work we created a basic in vitro model to evaluate the antibacterial effect of three widely used antiseptics.Commercially pure (CP4) titanium sample discs with sand blasted, acid etched, and polished surface were used. The discs were incubated with mono-cultures of Streptococcus mitis and Streptococcus salivarius. The adhered bacterial biofilms were treated with different antiseptics: chlorhexidine-digluconate (CHX), povidone-iodine (PI), and chlorine dioxide (CD) for 5 min and the control discs with ultrapure water. The antibacterial effect of the antiseptics was tested by colorimetric assay.According to the results, the PI and the CD were statistically the most effective in the elimination of the two test bacteria on both titanium surfaces after 5 min treatment time. The CD showed significant effect only against S. salivarius.Based on our results we conclude that PI and CD may be promising antibacterial agents to disinfecting the peri-implant site in the dental practice.

Mutations in cdsA and pgsA Correlate with Daptomycin Resistance in Streptococcus mitis and S. oralis.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

Daptomycin Dose-Ranging Evaluation with Single-Dose versus Multidose Ceftriaxone Combinations against Streptococcus mitis/oralis in an Ex Vivo Simulated Endocarditis Vegetation Model.

The viridans group streptococci (VGS) are a heterogeneous group of organisms which are important components of the normal human oral flora. Among the VGS, the Streptococcus mitis/oralis subgroup is one of the most common causes of infective endocarditis (IE). Daptomycin (DAP) is a potential alternative therapeutic option for invasive S. mitis infections, given high rates of ß-lactam resistance and vancomycin tolerance in such strains. However, the ability of these strains to rapidly evolve high-level and durable DAP resistance (DAP-R) is problematic. Recent data suggest that combination DAP-ß-lactam therapy circumvents this issue. Human-simulated dose-escalating DAP-alone dose regimens (6, 8, 10, or 12 mg/kg/day times 4 days) versus DAP (6 mg/kg/day) plus ceftriaxone (CRO) (2 g once daily times 4 days or 0.5 g, single dose) were assessed against two prototypical DAP-susceptible (DAP-S) S. mitis/oralis strains (SF100 and 351), as measured by a pharmacokinetic/pharmacodynamic (PK/PD) model of simulated endocardial vegetations (SEVs). No DAP-alone regimen was effective, with regrowth of high-level DAP-R isolates observed for both strains over 96-h exposures. Combinations of DAP-CRO with either single- or multidose regimens yielded significant reductions in log10 CFU/g amounts within SEVs for both strains (∼6 log10 CFU/g) within 24 h. In addition, no DAP-R strains were detected in either DAP-CRO combination regimens over the 96-h exposure. In contrast to prior in vitro studies, no perturbations in two key cardiolipin biosynthetic genes (cdsA and pgsA) were identified in DAP-R SEV isolates emerging from strain 351, despite defective phospholipid production. The combination of DAP-CRO warrants further investigation for treatment of IE due to S. mitis/oralis.

Prevention of High-Level Daptomycin-Resistance Emergence In Vitro in Streptococcus mitis-oralis by Using Combination Antimicrobial Strategies.

Among the viridans group streptococci, S. mitis-oralis strains are frequently resistant to multiple ß-lactams and tolerant to vancomycin (VAN). This scenario has led to the proposed clinical use of newer agents, like daptomycin (DAP) for such S. mitis-oralis strains. However, recent recognition of the rapid and durable emergence of high-level DAP-resistance (DAP-R; DAP MICs > 256 µg/ml) induced by DAP exposures in vitro and in vivo has dampened enthusiasm for such approaches. In this study, we evaluated a broad range of DAP combination regimens in vitro for their capacity to prevent emergence of high-level DAP-R in a prototype S. mitis-oralis strain (351) during serial passage experiments, including DAP + either gentamicin (GEN), rifampin (RIF), trimethoprim-sulfamethoxazole (TMP-SMX), imipenem (IMP), ceftaroline (CPT), tedizolid (TDZ), or linezolid (LDZ). In addition, we assessed selected DAP combination regimens for their ability to exert either an early bactericidal impact and/or synergistically kill the S. mitis-oralis study strain. During serial passage, three of the eight antibiotic combinations (DAP + GEN, CPT, or TMP- SMX) exhibited significantly reduced DAP MICs (≈ by 8-40 fold) vs serial exposure in DAP alone (DAP MICs > 256 µg/ml). In addition, combinations of DAP + GEN and DAP + CPT were both bactericidal and synergistic in early time-kill curve interactions.

A Case Report of Penicillin-Tolerant Streptococcus mitis Endocarditis Chang-Hua Chen, MD, MSc, PhD.

There is no clear relationship between the serum inhibition test and clinical outcome for Streptococcus mitis (S. mitis) endocarditis. We report an 84-year-old male with endocarditis caused by penicillin-tolerant S. mitis. The results for the serum inhibitory test (SIT) and serum bactericidal test (SBT) showed a trough level of SIT = 1:256 and SBT = 1:4 and a peak level of SIT ≥ 1:1024 and SBT = 1:16. In addition, the SIT/SBT ratio was 64 at peak level and more than 64 at trough level, which is compatible with penicillin-tolerant S. mitis. Following a 42-day high-dose penicillin treatment (24 M IU/day, via a continuous drip), the patient made a good recovery. In vitro inhibitory and bactericidal test results were not a valid predictor of medical treatment failure. Physicians need to continue to evaluate the surgical indications when treating patients with S. mitis endocarditis.

Antimicrobial Activity of a Colloidal AgNP Suspension Demonstrated In Vitro against Monoculture Biofilms: Toward a Novel Tooth Disinfectant for Treating Dental Caries.

A novel silver nanoparticle (AgNP) formulation was developed as a targeted application for the disinfection of carious dentine. Silver nitrate (AgNO3) was chemically reduced using sodium borohydrate (NaBH4) in the presence of sodium dodecyl sulfate (SDS) to form micelle aggregate structures containing monodisperse 6.7- to 9.2-nm stabilized AgNPs. AgNPs were characterized by measurement of electrical conductivity and dynamic light scattering, scanning electron microscopy, transmission electron microscopy, and inductively coupled plasma mass spectrometry. Antimicrobial activity of AgNPs was tested against planktonic cultures of representative gram-positive and gram-negative oral bacteria using well diffusion assays on tryptic soy broth media and monoculture biofilms grown with brain heart infusion ± sucrose anaerobically at 37°C on microtiter plates. Biofilm mass was measured by crystal violet assay. Effects were compared to silver diamine fluoride and chlorhexidine (negative controls) and 70% isopropanol (positive control) exposed cultures. In the presence of AgNPs, triplicate testing against Streptococcus gordonii DL1, C219, G102, and ATCC10558 strains; Streptococcus mutans UA159; Streptococcus mitis I18; and Enterococcus faecalis JH22 for planktonic bacteria, the minimum inhibitory concentrations were as low as 7.6 µg mL-1 and the minimum bacteriocidal concentrations as low as 19.2 µg mL-1 silver concentration. Microplate readings detecting crystal violet light absorption at 590 nm showed statistically significant differences between AgNP-exposed biofilms and where no antimicrobial agents were used. The presence of sucrose did not influence the sensitivity of any of the bacteria. By preventing in vitro biofilm formation for several Streptococcus spp. and E. faecalis, this AgNP formulation demonstrates potential for clinical application inhibiting biofilms.

Impact of High-Level Daptomycin Resistance in the Streptococcus mitis Group on Virulence and Survivability during Daptomycin Treatment in Experimental Infective Endocarditis.

Among the viridans group streptococci, the Streptococcus mitis group is the most common cause of infective endocarditis. These bacteria have a propensity to be ß-lactam resistant, as well as to rapidly develop high-level and durable resistance to daptomycin (DAP). We compared a parental, daptomycin-susceptible (DAPs) S. mitis/S. oralis strain and its daptomycin-resistant (DAPr) variant in a model of experimental endocarditis in terms of (i) their relative fitness in multiple target organs in this model (vegetations, kidneys, spleen) when animals were challenged individually and in a coinfection strategy and (ii) their survivability during therapy with daptomycin-gentamicin (an in vitro combination synergistic against the parental strain). The DAPr variant was initially isolated from the cardiac vegetations of animals with experimental endocarditis caused by the parental DAPs strain following treatment with daptomycin. The parental strain and the DAPr variant were comparably virulent when animals were individually challenged. In contrast, in the coinfection model without daptomycin therapy, at both the 106- and 107-CFU/ml challenge inocula, the parental strain outcompeted the DAPr variant in all target organs, especially the kidneys and spleen. When the animals in the coinfection model of endocarditis were treated with DAP-gentamicin, the DAPs strain was completely eliminated, while the DAPr variant persisted in all target tissues. These data underscore that the acquisition of DAPr in S. mitis/S. oralis does come at an intrinsic fitness cost, although this resistance phenotype is completely protective against therapy with a potentially synergistic DAP regimen.

Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria.

Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria Streptococcus mitis and S. oralis These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 µg/ml) daptomycin resistance rapidly emerges in S. mitis and S. oralis after a single drug exposure. In this study, we found that inactivating mutations in cdsA are associated with high-level daptomycin resistance in S. mitis and S. oralis IE isolates. This is surprising given that cdsA is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of cdsA not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.

Diversity of Mosaic pbp2x Families in Penicillin-Resistant Streptococcus pneumoniae from Iran and Romania.

Penicillin-resistant Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for ß-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.

Evaluation of daptomycin combinations with cephalosporins or gentamicin against Streptococcus mitis group strains in an in vitro model of simulated endocardial vegetations (SEVs).

Objectives: Among viridans group streptococcal infective endocarditis (IE), the Streptococcus mitis group is the most common aetiological organism. Treatment of IE caused by the S. mitis group is challenging due to the high frequency of ß-lactam resistance, drug allergy and intolerability of mainstay antimicrobial agents such as vancomycin or gentamicin. Daptomycin has been suggested as an alternative therapeutic option in these scenarios based on its excellent susceptibility profile against S. mitis group strains . However, the propensity of many S. mitis group strains to rapidly evolve stable, high-level daptomycin resistance potentially limits this approach. Methods: We evaluated the activity of 6 mg/kg/day daptomycin alone or in combination with gentamicin, ceftriaxone or ceftaroline against two daptomycin-susceptible S. mitis group strains over 96 h in a pharmacokinetic/pharmacodynamic model of simulated endocardial vegetations. Results: Daptomycin alone was not bactericidal and high-level daptomycin resistance evolved at 96 h in both organisms. Combinations of daptomycin + ceftriaxone and daptomycin + ceftaroline demonstrated enhanced killing activity compared with each antibiotic alone and prevented emergence of daptomycin resistance at 96 h. Use of gentamicin as an adjunctive agent neither improved the efficacy of daptomycin nor prevented the development of daptomycin resistance. Conclusions: Addition of ceftriaxone or ceftaroline to daptomycin improves the bactericidal activity against S. mitis group strains and prevents daptomycin resistance emergence. Further investigation with combinations of daptomycin and ß-lactams in a large number of strains is warranted to fully elucidate the clinical implications of such combinations for treatment of S. mitis group IE.

Composition and Antibacterial Activity of the Essential Oils of Orthosiphon stamineus Benth and Ficus deltoidea Jack against Pathogenic Oral Bacteria.

In this study, the essential oils of Orthosiphon stamineus Benth and Ficus deltoidea Jack were evaluated for their antibacterial activity against invasive oral pathogens, namely Enterococcus faecalis, Streptococcus mutans, Streptococcus mitis, Streptococcus salivarius, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum. Chemical composition of the oils was analyzed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The antibacterial activity of the oils and their major constituents were investigated using the broth microdilution method (minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)). Susceptibility test, anti-adhesion, anti-biofilm, checkerboard and time-kill assays were also carried out. Physiological changes of the bacterial cells after exposure to the oils were observed under the field emission scanning electron microscope (FESEM). O. stamineus and F. deltoidea oils mainly consisted of sesquiterpenoids (44.6% and 60.9%, respectively), and ß-caryophyllene was the most abundant compound in both oils (26.3% and 36.3%, respectively). Other compounds present in O. stamineus were α-humulene (5.1%) and eugenol (8.1%), while α-humulene (5.5%) and germacrene D (7.7%) were dominant in F. deltoidea. The oils of both plants showed moderate to strong inhibition against all tested bacteria with MIC and MBC values ranging 0.63-2.5 mg/mL. However, none showed any inhibition on monospecies biofilms. The time-kill assay showed that combination of both oils with amoxicillin at concentrations of 1× and 2× MIC values demonstrated additive antibacterial effect. The FESEM study showed that both oils produced significant alterations on the cells of Gram-negative bacteria as they became pleomorphic and lysed. In conclusion, the study indicated that the oils of O. stamineus and F. deltoidea possessed moderate to strong antibacterial properties against the seven strains pathogenic oral bacteria and may have caused disturbances of membrane structure or cell wall of the bacteria.

Zinc oxide and titanium dioxide nanoparticles induce oxidative stress, inhibit growth, and attenuate biofilm formation activity of Streptococcus mitis.

Streptococcus mitis from the oral cavity causes endocarditis and other systemic infections. Rising resistance against traditional antibiotics amongst oral bacteria further aggravates the problem. Therefore, antimicrobial and antibiofilm activities of zinc oxide and titanium dioxide nanoparticles (NPs) synthesized and characterized during this study against S. mitis ATCC 6249 and Ora-20 were evaluated in search of alternative antimicrobial agents. ZnO and TiO2-NPs exhibited an average size of 35 and 13 nm, respectively. The IC50 values of ZnO and TiO2-NPs against S. mitis ATCC 6249 were 37 and 77 µg ml(-1), respectively, while the IC50 values against S. mitis Ora-20 isolate were 31 and 53 µg ml(-1), respectively. Live and dead staining, biofilm formation on the surface of polystyrene plates, and extracellular polysaccharide production show the same pattern. Exposure to these nanoparticles also shows an increase (26-83 %) in super oxide dismutase (SOD) activity. Three genes, namely bapA1, sodA, and gtfB like genes from these bacteria were identified and sequenced for quantitative real-time PCR analysis. An increase in sodA gene (1.4- to 2.4-folds) levels and a decrease in gtfB gene (0.5- to 0.9-folds) levels in both bacteria following exposure to ZnO and TiO2-NPs were observed. Results presented in this study verify that ZnO-NPs and TiO2-NPs can control the growth and biofilm formation activities of these strains at very low concentration and hence can be used as alternative antimicrobial agents for oral hygiene.

Cell-protection mechanism through autophagy in HGFs/S. mitis co-culture treated with Chitlac-nAg.

Silver-based products have been proven to be effective in retarding and preventing bacterial growth since ancient times. In the field of restorative dentistry, the use of silver ions/nanoparticles has been explored to counteract bacterial infections, as silver can destroy bacterial cell walls by reacting with membrane proteins. However, it is also cytotoxic towards eukaryotic cells, which are capable of internalizing nanoparticles. In this work, we investigated the biological effects of Chitlac-nAg, a colloidal system based on a modified chitosan (Chitlac), administered for 24-48 h to a co-culture of primary human gingival fibroblasts and Streptococcus mitis in the presence of saliva, developed to mimic the microenvironment of the oral cavity. We sought to determine its efficiency to combat oral hygiene-related diseases without affecting eukaryotic cells. Cytotoxicity, reactive oxygen species production, apoptosis induction, nanoparticles uptake, and lysosome and autophagosome metabolism were evaluated. In vitro results show that Chitlac-nAg does not exert cytotoxic effects on human gingival fibroblasts, which seem to survive through a homoeostasis mechanism involving autophagy. That suggests that the novel biomaterial Chitlac-nAg could be a promising tool in the field of dentistry.

Bioassay-guided isolation and evaluation of antimicrobial compounds from Ixora megalophylla against some oral pathogens.

Context Ixora megalophylla Chamch. (Rubiaceae) is a new plant species recently found in southern Thailand. Ethyl acetate extracts of its leaves and stems showed antimicrobial activities. Objectives To isolate and identify the antimicrobial compounds from I. megalophylla leaves and stems. Materials and methods The dried leaves (1.7 kg) and stems (3.5 kg) were consecutively extracted with petroleum ether (5 L × 4), ethyl acetate (5 L × 3) and ethanol (5 L × 4) under reflux conditions. The ethyl acetate extract was subjected to an antimicrobial assay guided isolation with Candida albicans and Streptococcus mutans. Compounds 1-10 were identified by (1)H NMR, (13)C NMR and EI-MS. Minimal lethal concentration (MLC) against C. albicans and Streptococcus spp. was determined using a broth microdilution method for 48 and 24 h, respectively. Results and discussion On the basis of the antimicrobial assay guided isolation, 10 known compounds, including vanillic acid (1), syringic acid (2), 4-hydroxy benzaldehyde (3), scopoletin (4), loliolide (5), syringaldehyde (6), sinapaldehyde (7), coniferaldehyde (8), syringaresinol (9) and 2,2'-dithiodipyridine (10), were identified. Compounds 1-5 were purified from the ethyl acetate extract of the leaves, while 6-9 and 10 were from the ethyl acetate and ethanol extracts of the stems, respectively. Among these isolates, 10 showed the strongest antibacterial activities against S. mutans and Streptococcus mitis, with minimum inhibitory concentrations (MICs) of 2-4 µg/mL, and MLC of 4 µg/mL, as well as having a weak antifungal activity against C. albicans (MIC of 125 µg/mL). This is the first report of the antimicrobial activities of 10.

A dual role for ß1 integrin in an in vitro Streptococcus mitis/human gingival fibroblasts co-culture model in response to TEGDMA.

AIM: To evaluate the effect of TEGDMA on human gingival fibroblasts (HGFs) in vitro co-cultured with Streptococcus mitis, focusing on the signalling pathways underlying cell tissue remodelling and inflammatory response processes. METHODOLOGY: ß1 integrin expression was evaluated by means of imaging flow cytometry. The Western blot technique was used to investigate the expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), matrix metalloproteinase 9 (MMP9) and 3 (MMP3). RT-PCR was performed to quantify nuclear factor-kb subunits (Nf-kb1, ReLa), IkB kinase ß (IkBkB), cyclooxygenase II (COX-2) and tumour necrosis factor-α (TNF-α) mRNA levels. Statistical analysis was performed using the analysis of variance (anova). RESULTS: When HGFs are co-cultured with S. mitis, ß1 integrin intensity, phosphorylated PKC (p-PKC), activated ERK (p-ERK), IkBkB mRNA level and MMP9 expression increased (for all molecules P < 0.05 HGFs versus HGFs co-cultured with S. mitis). A higher level of MMP3 in HGFs treated with TEGDMA was recorded (P < 0.05 HGFs versus HGFs exposed to TEGDMA). COX-2 inflammatory factor mRNA level appeared higher in HGFs exposed to 1 mmol L(-1) TEGDMA (P < 0.01 HGFs versus HGFs exposed to TEGDMA), whereas TNF-α gene expression was higher in HGFs co-cultured with S. mitis (P < 0.05 HGFs versus HGFs co-cultured with S. mitis). CONCLUSIONS: ß1 integrin triggered the signalling pathway, transduced by p-PKCα and involving ERK 1 and 2 and MMPs. This pathway resulted in an unbalanced equilibrium in tissue remodelling process, along with inflammatory response when HGFs are exposed to bacteria or biomaterial alone. On the contrary, the TEGDMA/S. mitis combination restored the balance between extracellular matrix deposition and degradation and prevented an inflammatory response.

Systematic screening of plant extracts from the Brazilian Pantanal with antimicrobial activity against bacteria with cariogenic relevance.

This study proposes a bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. Sixty extracts were obtained from ten plants--(1) Jatropha weddelliana, (2) Attalea phalerata, (3) Buchenavia tomentosa, (4) Croton doctoris, (5) Mouriri elliptica, (6) Mascagnia benthamiana, (7) Senna aculeata, (8) Unonopsis guatterioides, (9) Allagoptera leucocalyx and (10) Bactris glaucescens--using different extraction methods - (A) 70° ethanol 72 h/25°C, (B) water 5 min/100°C, (C) water 1 h/55°C, (D) water 72 h/25°C, (E) hexane 72 h/25°C and (F) 90° ethanol 72 h/25°C. The plants were screened for antibacterial activity at 50 mg/ml using the agar well diffusion test against Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. The active extracts were tested to determine their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), cytotoxicity and chemical characterization. Forty-seven extracts (78%) were active against at least one microorganism. Extract 4A demonstrated the lowest MIC and MBC for all microorganisms except S. gordonii and the extract at MIC concentration was non-cytotoxic. The concentrated extracts were slightly cytotoxic. Electrospray ionization with tandem mass spectrometry analyses demonstrated that the extract constituents coincided with the mass of the terpenoids and phenolics. Overall, the best results were obtained for extraction methods A, B and C. The present work proved the antimicrobial activity of several plants. Particularly, extracts from C. doctoris were the most active against bacteria involved in dental caries disease.

Composition and activity against oral pathogens of the essential oil of Melampodium divaricatum (Rich.) DC.

The chemical composition of the essential oil isolated from the aerial parts of Melampodium divaricatum (Rich.) DC. (Asteraceae) was characterized by GC-FID and GC/MS analyses. (E)-Caryophyllene (56.0%), germacrene D (12.7%), and bicyclogermacrene (9.2%) were identified as the major oil components. The antimicrobial activity of the oil against seven standard strains of oral pathogens from the American Type Culture Collection (ATCC) was evaluated by determining minimum inhibitory concentrations (MICs) using the microdilution method. MIC Values below 100 µg/ml were obtained against Streptococcus sobrinus (90 µg/ml), Lactobacillus casei (30 µg/ml), S. mutans (20 µg/ml), and S. mitis (18 µg/ml). In contrast, the MIC values of the major oil compound (E)-caryophyllene were higher than 400 µg/ml against all pathogens, suggesting that the activity of the oil might depend on minor oil components and/or on synergistic effects. The M. divaricatum essential oil is a promising agent to include in anticariogenic oral rinse formulations for the control of oral pathogens.

Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules.

BACKGROUND: Inflammation within the oral cavity occurs due to dysregulation between microbial biofilms and the host response. Understanding how different oral hygiene products influence inflammatory properties is important for the development of new products. Therefore, creation of a robust host-pathogen biofilm platform capable of evaluating novel oral healthcare compounds is an attractive option. We therefore devised a multi-species biofilm co-culture model to evaluate the naturally derived polyphenol resveratrol (RSV) and gold standard chlorhexidine (CHX) with respect to anti-biofilm and anti-inflammatory properties. METHODS: An in vitro multi-species biofilm containing S. mitis, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was created to represent a disease-associated biofilm and the oral epithelial cell in OKF6-TERT2. Cytotoxicity studies were performed using RSV and CHX. Multi-species biofilms were either treated with either molecule, or alternatively epithelial cells were treated with these prior to biofilm co-culture. Biofilm composition was evaluated and inflammatory responses quantified at a transcriptional and protein level. RESULTS: CHX was toxic to epithelial cells and multi-species biofilms at concentrations ranging from 0.01-0.2%. RSV did not effect multi-species biofilm composition, but was toxic to epithelial cells at concentrations greater than 0.01%. In co-culture, CHX-treated biofilms resulted in down regulation of the inflammatory chemokine IL-8 at both mRNA and protein level. RSV-treated epithelial cells in co-culture were down-regulated in the release of IL-8 protein, but not mRNA. CONCLUSIONS: CHX possesses potent bactericidal properties, which may impact downstream inflammatory mediators. RSV does not appear to have bactericidal properties against multi-species biofilms, however it did appear to supress epithelial cells from releasing inflammatory mediators. This study demonstrates the potential to understand the mechanisms by which different oral hygiene products may influence gingival inflammation, thereby validating the use of a biofilm co-culture model.

Streptococcus mitis enhances metal-induced apoptosis in reconstructed human gingiva but not skin.

BACKGROUND: Commensal bacteria colonizing oral mucosa and skin play an essential role in maintaining host-microbiome homeostasis. It is unknown whether cytotoxicity resulting from metal ions leaching from medical devices may be influenced by commensal microbes. OBJECTIVE: Determine whether the extent of apoptosis triggered by nickel or titanium ions is influenced by Streptococcus mitis and whether apoptosis occurs via the intrinsic or extrinsic apoptosis pathway. METHODS: Reconstructed Human Gingiva (RHG) and Skin (RHS) were topically exposed to titanium or nickel salts in the presence or absence of S. mitis. Cytotoxicity and apoptosis were assessed by histology, immunohistochemistry, TUNEL assay, and Western Blot. RESULTS: S. mitis alone resulted in negligible cytotoxicity. After metal exposure, localized apoptosis was observed in the epithelium and fibroblasts within the lamina propria hydrogel of both RHG and RHS. S. mitis enhanced metal-mediated apoptosis in gingiva but not in skin. Apoptosis was mediated via the extrinsic pathway caspase 8. Activation of the execution phase of apoptosis occurred via caspases 3 and 7, and PARP-1. CONCLUSION: Our study supports the finding that metals have irritant, cytotoxic properties resulting in apoptosis when leaching into skin or gingiva. Particularly for gingiva, commensal microbes exaggerate this detrimental effect.

Early in vitro and in vivo development of high-level daptomycin resistance is common in mitis group Streptococci after exposure to daptomycin.

The development of high-level daptomycin resistance (HLDR; MIC of ≥ 256 mg/liter) after exposure to daptomycin has recently been reported in viridans group streptococcus (VGS) isolates. Our study objectives were as follows: to know whether in vitro development of HLDR after exposure to daptomycin was common among clinical isolates of VGS and Streptococcus bovis; to determine whether HLDR also developed during the administration of daptomycin to treat experimental endocarditis caused by the daptomycin-susceptible, penicillin-resistant Streptococcus mitis strain S. mitis 351; and to establish whether combination with gentamicin prevented the development of HLDR in vitro and in vivo. In vitro studies were performed with 114 VGS strains (mitis group, 92; anginosus group, 10; mutans group, 8; and salivarius group, 4) and 54 Streptococcus bovis strains isolated from 168 consecutive patients with infective endocarditis diagnosed between 1995 and 2010. HLDR was only observed after 24 h of exposure to daptomycin in 27% of the mitis group, including 27% of S. mitis isolates, 47% of S. oralis isolates, and 13% of S. sanguis isolates. In our experimental model, HLDR was detected in 7/11 (63%) and 8/12 (67%) isolates recovered from vegetations after 48 h of daptomycin administered at 6 mg/kg of body weight/24 h and 10 mg/kg/24 h, respectively. In vitro, time-kill experiments showed that daptomycin plus gentamicin was bactericidal against S. mitis 351 at tested concentrations of 0.5 and 1 times the MIC and prevented the development of HLDR. In vivo, the addition of gentamicin at 1 mg/kg/8 h to both daptomycin arms prevented HLDR in 21 out of 23 (91%) rabbits. Daptomycin plus gentamicin was at least as effective as vancomycin plus gentamicin. In conclusion, HLDR develops rapidly and frequently in vitro and in vivo among mitis group streptococci. Combining daptomycin with gentamicin enhanced its activity and prevented the development of HLDR in most cases.