Natural products in caries research: current (limited) knowledge, challenges and future perspective.

Dental caries is the most prevalent and costly oral infectious disease worldwide. Virulent biofilms firmly attached to tooth surfaces are prime biological factors associated with this disease. The formation of an exopolysaccharide-rich biofilm matrix, acidification of the milieu and persistent low pH at the tooth-biofilm interface are major controlling virulence factors that modulate dental caries pathogenesis. Each one offers a selective therapeutic target for prevention. Although fluoride, delivered in various modalities, remains the mainstay for the prevention of caries, additional approaches are required to enhance its effectiveness. Available antiplaque approaches are based on the use of broad-spectrum microbicidal agents, e.g. chlorhexidine. Natural products offer a rich source of structurally diverse substances with a wide range of biological activities, which could be useful for the development of alternative or adjunctive anticaries therapies. However, it is a challenging approach owing to complex chemistry and isolation procedures to derive active compounds from natural products. Furthermore, most of the studies have been focused on the general inhibitory effects on glucan synthesis as well as on bacterial metabolism and growth, often employing methods that do not address the pathophysiological aspects of the disease (e.g. bacteria in biofilms) and the length of exposure/retention in the mouth. Thus, the true value of natural products in caries prevention and/or their exact mechanisms of action remain largely unknown. Nevertheless, natural substances potentially active against virulent properties of cariogenic organisms have been identified. This review focuses on gaps in the current knowledge and presents a model for investigating the use of natural products in anticaries chemotherapy.

Antimicrobial efficacy of lactoferrin, its amidated and pepsin-digested derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens.

AIMS: To evaluate the in vitro bactericidal efficacy of lactoferrin (LF), its amidated (AMILF) and pepsin-digested (PDLF) derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens. METHODS AND RESULTS: PDLF exhibited the most potent bactericidal efficacy on E. coli O157:H7 (>2·5 log(10) CFU ml(-1) reduction at concentrations ≥ 1 mg ml(-1)), and AMILF on Ser. liquefaciens (1 log(10) CFU ml(-1) reduction at 0·25-0·50 mg ml(-1)). Some combinations of LF with PDLF or AMILF showed a slight synergy on E. coli O157:H7 and Ser. liquefaciens. However, all combinations of AMILF with PDLF were less active than the sum of the individual effects of the two antimicrobials. Production of capsular polysaccharide by bacteria might be involved in antimicrobial resistance. CONCLUSIONS: Escherichia coli O157:H7 and Ser. liquefaciens showed marked differences in the sensitivity to LF and its derivatives. E. coli O157:H7 was strongly inhibited by PDLF, whereas the effect of LF and its derivatives on Ser. liquefaciens was weak to negligible. SIGNIFICANCE AND IMPACT OF THE STUDY: PDLF was the most promising of the tested antimicrobials on E. coli O157:H7. However, the resistance of Ser. liquefaciens to LF and its derivatives hinders their use in the food industry.

Subgingival delivery of oral debriding agents: a proof of concept.

OBJECTIVE: This study is a proof of concept to determine the efficacy of a custom-fabricated tray in placing antimicrobial and debriding agents in the periodontal pockets of persons with active gingival infections. Localized subgingival delivery of antimicrobial and antibiotic agents is routinely employed as adjunctive therapy for the treatment and management ofperiopathogens associated with periodontal disease. Because these delivery techniques often face time constraints and impose temporary restrictions on patient brushing and flossing, a custom-formed prescription dental tray can be used to deliver and maintain medications in periodontal pockets between office visits and without brushing or flossing restrictions. The ability of this tray to maintain sufficient concentrations of medication in the periodontal pockets to have a therapeutic effect is evaluated here with theoretical modeling and practical application. METHODS: Hydrogen peroxide is an oral debriding agent and oral wound cleanser with antimicrobial properties. The debriding effect of 1.7% hydrogen peroxide gel was tested in vitro on Streptococcus mutans biofilm using glass carriers for collection. Diffusion modeling tested the potential of the customized tray to place hydrogen peroxide gel into the sulcus in the presence of crevicular fluid flow. Changes in periodontal microflora with scanning electron microscopy analysis of in vivo paper point site sampling were analyzed before and after a thin ribbon of 1.7% hydrogen peroxide gel (approximately 0.7 gm) and a subtherapeutic dose (three drops) of Vibramycin (50 mg/5 ml) were placed via Perio Trays into periodontal pockets, ranging from 4-8 mm at daily prescribed intervals for two to five weeks. RESULTS: In vitro results indicate that 1.7% hydrogen peroxide gel breaks down the exopolysaccharide slime and cell walls ofS. mutans, and begins to debride the cells from glass carriers within 10 minutes. Diffusion modeling indicates that hydrogen peroxide can penetrate into the deeper pockets (9 mm), but also its concentration in these deep pockets will increase over wearing time in the absence of degradation by peroxidases and catalase. Site sampling data confirm diffusion modeling results, with evidence that medication delivered with the prescription tray reduced subgingival bacterial loads and enhanced healing of corresponding oral tissues. CONCLUSION: The prescription Perio Tray effectively placed medication in the gingival sulcus. Mathematical modeling indicated Perio Tray placement of hydrogen peroxide gel in periodontal pockets with depths up to 9 mm over 15 minutes treatment time was theoretically possible. Pathology reports reveal reductions in subgingival bacterial loads and improvements in pretreatment pocket depths of up to 8 mm after 1.7% hydrogen peroxide and Vibramycin Syrup were prescribed for use with the Perio Tray. The in vitro analysis indicating that hydrogen peroxide is the active and effective oral debriding agent needs to be confirmed with additional studies.

The uronic acids assay: a method for the determination of chemical activity on biofilm EPS.

In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity against the production of microbial biofilm exopolysaccharide (EPS). The assay was first applied to biofilms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known to inhibit microbial growth and biofilm formation. The performance of the assay was then characterized through statistical assessment of threshold concentrations for disinfection efficiency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biofilm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biofilm activity and simple disinfection. Activity was characterized as (i) no effect, (ii) a true positive effect (ie increased biofilm EPS), (iii) anti-bacterial activity (ie decreased biofilm EPS and analogous decrease in planktonic growth), and (iv) anti-biofilm EPS activity (ie decreased biofilm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biofilm characterization methods by providing a quantitative measure of biofilm EPS.

Influence of cranberry proanthocyanidins on formation of biofilms by Streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo.

Cranberry crude extracts, in various vehicles, have shown inhibitory effects on the formation of oral biofilms in vitro. The presence of proanthocyanidins (PAC) in cranberry extracts has been linked to biological activities against specific virulence attributes of Streptococcus mutans, e.g. the inhibition of glucosyltransferase (Gtf) activity. The aim of the present study was to determine the influence of a highly purified and chemically defined cranberry PAC fraction on S. mutans biofilm formation on saliva-coated hydroxyapatite surface, and on dental caries development in Sprague-Dawley rats. In addition, we examined the ability of specific PAC (ranging from low-molecular-weight monomers and dimers to high-molecular-weight oligomers/polymers) to inhibit GtfB activity and glycolytic pH drop by S. mutans cells, in an attempt to identify specific bioactive compounds. Topical applications (60-second exposure, twice daily) with PAC (1.5 mg/ml) during biofilm formation resulted in less biomass and fewer insoluble polysaccharides than the biofilms treated with vehicle control had (10% ethanol, v/v; p < 0.05). The incidence of smooth-surface caries in rats was significantly reduced by PAC treatment (twice daily), and resulted in less severe carious lesions compared to the vehicle control group (p < 0.05); the animals treated with PAC also showed significantly less caries severity on sulcal surfaces (p < 0.05). Furthermore, specific A-type PAC oligomers (dimers to dodecamers; 0.1 mg/ml) effectively diminished the synthesis of insoluble glucans by GtfB adsorbed on a saliva-coated hydroxyapatite surface, and also affected bacterial glycolysis. Our data show that cranberry PAC reduced the formation of biofilms by S. mutans in vitro and dental caries development in vivo, which may be attributed to the presence of specific bioactive A-type dimers and oligomers.

Spectroscopic characterization of extracellular polymeric substances from Escherichia coli and Serratia marcescens: suppression using sub-inhibitory concentrations of bismuth thiols.

Free and bound (or capsular) EPS produced by suspended cultures of Escherichia coli and Serratia marcescens were characterized in detail using colorimetric analysis of total proteins and polysaccharides, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and Auger electron spectroscopy (AES) in the presence and absence of bismuth-based antifouling agents. Subtle differences in the chemical composition of free and bound EPS were observed for both bacteria in the absence of bismuth. Total polysaccharides and proteins in free and bound EPS decreased upon treatment with subinhibitory concentrations of lipophilic bismuth thiols (bismuth dimercaptopropanol, BisBAL; bismuth ethanedithiol, BisEDT; and bismuth pyrithione, BisPYR), with BisBAL being most effective. Bismuth thiols also influenced acetylation and carboxylation of polysaccharides in EPS from S. marcescens. Extensive homology between EPS samples in the presence and absence of bismuth was observed with proteins, polysaccharides, and nucleic acids varying predominantly only in the total amount produced. Second derivative analysis of the amide I region of FTIR spectra revealed decreases in protein secondary structures in the presence of bismuth thiols. Hence, antifouling properties of bismuth thiols appear to originate in their ability to suppress O-acetylation and protein secondary structure formation in addition to free and bound EPS secretion.

Effect of antiplaque compounds and mouthrinses on the activity of glucosyltransferases from Streptococcus sobrinus and insoluble glucan production.

INTRODUCTION: The development of therapeutic agents inhibiting the activity of glucosyltransferases (GTF) and their production of glucans is a potential strategy to reduce dental decay. The aim of this study was first to characterize a GTF preparation from Streptococcus sobrinus ATCC 33478 and then to evaluate the effects of select compounds and mouthrinses on insoluble glucan (ISG) formation by combined GTFs. METHODS: The purity of the crude GTF mixture was assessed by electrophoresis. The effects of pH, temperature, sucrose, and dextran T10 concentrations on GTF activity were analyzed and the chemical structure of the products was investigated. Finally, the inhibition of GTF by commercial mouthrinses used in oral hygiene and their active components (chlorhexidine, polyphenolic compounds, fluoride derivatives, polyols, cetylpyridinium chloride, and povidone iodine) was analyzed through the reductions in the overall reaction rate and the quantity of ISG synthesized. RESULTS: The S. sobrinus ATCC 33478 crude GTF preparation obtained contains a mixture of four different GTFs known for this species. For optimal adherent ISG formation, the reaction parameters were 37 degrees C, pH 6.5, sucrose 50 g/l, and dextran T10 2 g/l. Under these conditions, the most effective agents were chlorhexidine, cetylpyridinium chloride, and tannic acid. Eludril, Elmex, and Betadine were the most effective inhibitors of all the mouthrinses tested. CONCLUSION: As the formulation of commercial products considerably influences the efficiency of active components, the fast representative ISG inhibition test developed in this study should be of great interest.

The composition of cell wall skeleton and outermost lipids of Mycobacterium vaccae is modified by ethambutol treatment.

Ethambutol (EMB) is a first line drug in tuberculosis treatment inhibiting the biosynthesis of arabinogalactan, which is a component of the mycobacterial cell wall. The growth of Mycobacterium vaccae cells in the presence of EMB increases cell wall permeability, which was monitored by beta-sitosterol biotransformation. GC/MS and GLC/MS (gas chromatography/mass spectrometry) analysis revealed dramatic changes in the content of covalently bound mycolic acids and in molar ratio galactose (Gal) to arabinose (Ara) in the cell envelopes of EMB-treated cells. The detected variations in the compositions of fatty acids indicate that both the cell wall skeleton and outer layer (free lipids) are decomposed due to EMB treatment.

Effects of Nidus Vespae extract and chemical fractions on glucosyltransferases, adherence and biofilm formation of Streptococcus mutans.

Nidus Vespae (the honeycomb of Polistes olivaceous, P. japonicus Saussure and Parapolybiavaria fabricius) have been extensively used in traditional Chinese medicine, given their multiple pharmacological activities, including antimicrobial, anti-inflammatory, anti-virus, anti-tumor and anesthetic properties. The present study evaluated the anti-glucosyltransferases (GTFs) activity, anti-adherence and anti-biofilm properties of 95% ethanol/water extract, cyclohexane/ethyl acetate, petroleum ether/ethyl acetate and chloroform/methanol fractions of Nidus Vespae. Chloroform/methanol fraction showed a remarkable capacity for inhibiting the adherence of Streptococcus mutans ATCC 25175 to saliva-coated hydroxyapatite disc (S-HA) at sub-MC concentrations. In addition, the Nidus Vespae extract and chemical fractions significantly inhibited the activity of cell-associated and extracellular GTFs at sub-MIC concentrations, and the chloroform/methanol fraction was the most effective one. For the anti-biofilm activity assays, minimum biofilm inhibition concentrations (MBIC50) and minimum biofilm reduction concentrations (MBRC50) were determined using the microdilution method. The chloroform/methanol fraction showed the highest anti-biofilm activities with a MBIC50 of 8mg/ml and a MBRC(50) of 16mg/ml against Streptococcus mutans ATCC 25175. The significant inhibition of GTFs activity and biofilm formation demonstrated by Nidus Vespae shows it to be a promising natural product for the prevention of dental caries.

Relative neutralization of the biological actions of sulfaminoheparosans (K5 derivatives) and heparins by protamine sulfate.

Biosynthetic, semisynthetic, and synthetic analogues of heparins are currently developed as substitute antithrombotic agents for heparin. Sulfaminoheparosan (SAH) represents a bacterial polysaccharide (K5)-derived antithrombotic polymer from which pharmacologically active products with varying molecular weights (5-25 kDa) can be derived. These agents have been shown to exhibit pharmacologic effects comparable to heparins. The objective of this investigation is to determine the relative neutralization profile of various SAH derivatives, also called as bioheparins, by protamine sulfate. Four SAH fractions with varying molecular weights (20, 9, 7, and 6 kDa), a low molecular weight heparin (LMWH), tinzaparin, and unfractionated heparin (UFH) were supplemented to normal human pool plasma over a concentration range of 6.2 to 100 microg/mL. A fixed amount of protamine sulfate at 25 microg/mL (final concentration) was supplemented to determine the neutralization profile by performing tests such as prothrombin time (PT), activated partial thromboplastin time (APTT), Heptest, prothrombin-induced clotting time (PiCT), and amidolytic anti-Xa and anti-IIa assays. Protamine sulfate produced varying degrees of neutralization of all bioheparin fractions in the clotting assays. In the amidolytic anti-IIa assay relatively stronger inhibition was noted for all agents than inhibition of FXa. On a cumulative basis the neutralization profile of SAHs was comparable with heparins. These results suggest that the anticoagulant activities of SAH derivatives can be antagonized by protamine sulfate. These studies warrant further in vivo investigation to validate the relative neutralization profile of sulfaminoheparosans.

Blockade of CTLA-4 (CD152) enhances the murine antibody response to pneumococcal capsular polysaccharides.

The capsular polysaccharides (caps-PS) of Streptococcus pneumoniae are classified as thymus-independent antigens. Nevertheless, T lymphocytes can modulate the antibody response to caps-PS. In this study, we show that anticytotoxic T lymphocyte-associated antigen 4 (CTLA-4) treatment, along with administration of caps-PS to BALB/c mice, resulted in a dose-dependent generation of a strong caps-PS-specific antibody response. Anti-CTLA-4 treatment had no effect on the immunoglobulin G (IgG) antibody production in athymic nu/nu mice. Anti-CTLA-4 treatment stimulated the IgG antibody production in severe combined immunodeficiency (SCID)/SCID mice reconstituted with CTLA-4(-/-) B lymphocytes and wild-type T lymphocytes. This excluded the possibility that anti-CTLA-4 enhanced antibody production by direct interaction with B lymphocytes. Anti-CTLA-4 treatment enhanced the antibody production in SCID/SCID mice reconstituted with B lymphocytes and CD4(+) and CD8(+) T lymphocytes but not in SCID/SCID mice reconstituted with B lymphocytes in the absence of CD4(+) and/or CD8(+) cells. Administration of anti-CTLA-4 in BALB/c mice but not in nu/nu mice resulted in a markedly increased production of interleukin (IL)-2, IL-4, and interferon-gamma. Taken together, these data strongly suggest a role of T lymphocytes and CTLA-4 in the regulation of the antibody response to caps-PS.

The reinforcement of gellan gel network by the immersion into salt solution.

The effect of immersion into salt solutions on rheological properties of gellan gels was investigated. The storage Young's modulus of gellan gels increased with time during the immersion into salt solutions. The increase of the storage Young's modulus can not be explained solely by change in the concentration of gellan. The ellipticity at 202 nm decreased by the immersion, suggesting the formation and aggregation of gellan helices. It was considered that during immersion cations penetrated into gellan gels to induce the formation and aggregation of gellan helices in gels, resulting in reinforcement of the gel network.

Apigenin and tt-farnesol with fluoride effects on S. mutans biofilms and dental caries.

Apigenin (Api) and tt-farnesol (Far) are two naturally occurring agents that affect the development of cariogenic biofilms. Fluoride (F) interferes physicochemically with caries development and also exhibits antibacterial activity. We examined whether the association of Api and Far enhance the anti-caries properties of F by acting cooperatively on the expression of virulence of Streptococcus mutans. The biological effects of each of the agents were greatly enhanced when used in combination with F. In general, biofilms treated with Api and/or Far in combination with F displayed less biomass and fewer insoluble glucans and iodophilic polysaccharides than did those treated with the test agents alone (P < 0.05). The combination of the test agents with F was highly effective in preventing caries development in rats, especially Api+Far+F, and results were comparable with those observed with chlorhexidine + F (positive control). Results from these studies showed that apigenin and tt-farnesol may enhance the cariostatic effectiveness of fluoride.

The inhibiting effect of aqueous Azadirachta indica (Neem) extract upon bacterial properties influencing in vitro plaque formation.

The purpose of this investigation was to examine the inhibitory effects of aqueous extracts derived from the bark-containing sticks (Neem stick) of Azadirachta indica upon bacterial aggregation, growth, adhesion to hydroxyapatite, and production of insoluble glucan, which may affect in vitro plaque formation. Neem stick extracts were screened for minimal bacterial growth inhibition (MIC) against a panel of streptococci by means of a broth dilution assay. Initial bacterial attachment was quantified by the measurement of the adhesion of 3H-labeled Streptococcus sanguis to saliva-conditioned synthetic hydroxyapatite. The effect of the Neem stick extract upon insoluble glucan synthesis was measured by the uptake of radiolabeled glucose from 14C-sucrose. Aggregating activity of the Neem stick extracts upon a panel of streptococci was also examined. No inhibition of bacterial growth was observed among the streptococcal strains tested in the presence of < or = 320 micrograms/mL of the Neem stick extract. The pre-treatment of S. sanguis with the Neem stick extract or the gallotannin-enriched extract from Melaphis chinensis at 250 micrograms/mL resulted in a significant inhibition of the bacterial adhesion to saliva-conditioned hydroxyapatite. Pre-treatment of saliva-conditioned hydroxyapatite with the Neem stick or gallotannin-rich extract prior to exposure to bacteria yielded significant reductions in bacterial adhesion. The Neem stick extract and the gallotannin-enriched extract from Melaphis chinensis inhibited insoluble glucan synthesis. Incubation of oral streptococci with the Neem stick extract resulted in a microscopically observable bacteria aggregation. These data suggest that Neem stick extract can reduce the ability of some streptococci to colonize tooth surfaces.

Effect of calcium hydroxide on bacterial lipopolysaccharide.

Apical periodontitis and its concomitant periapical osteolysis is caused by pulpal infection, and bacterial lipopolysaccharide (LPS) is known to play a major role in the bone resorption process. Little is known concerning the effect of root canal intervisit dressings on residual LPS in root canals after bacterial cell lysis. The purpose of this study was to evaluate the effects of calcium hydroxide on bacterial LPS. Free hydroxy fatty acids were quantified in samples of LPS treated with calcium hydroxide. Calcium hydroxide treatment of LPS was shown to release elevated quantities of hydroxy fatty acids. It was concluded that calcium hydroxide hydrolyzed the lipid moiety of bacterial LPS, resulting in the release of free hydroxy fatty acids. This result suggests that calcium hydroxide-mediated degradation of LPS may be an important reason for the beneficial effects obtained with calcium hydroxide use in clinical endodontics.

Detoxification of endotoxin by endodontic irrigants and calcium hydroxide.

The effects of endodontic irrigants and calcium hydroxide on lipopolysaccharide (LPS; endotoxin) were analyzed using the highly selective technique of mass spectrometry/gas chromatography with selected ion monitoring. An aqueous solution of LPS was mixed with one of a variety of endodontic irrigants for 30 min. Because it is a commonly used interappointment dressing, calcium hydroxide was also applied to LPS for 1, 2, or 5 days. LPS inactivation was measured by quantitation of free fatty acid release. Water, EDTA, ethanol, 0.12% chlorhexidine, chlorhexidine + sodium hypochlorite, and sodium hypochlorite alone showed little breakdown of LPS. Long-term calcium hydroxide--as well as 30-min exposure to an alkaline mixture of chlorhexidine, ethanol, and sodium hypochlorite--did detoxify LPS molecules by hydrolysis of ester bonds in the fatty acid chains of the lipid A moiety.

Structure of an O-acetylated acidic O-specific polysaccharide of Proteus vulgaris O46.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O46 and studied by chemical methods (O-deacetylation, sugar and methylation analyses, partial solvolysis) and 1H and 13C NMR spectroscopy. Solvolysis of the O-deacetylated polysaccharide with trifluoromethanesulfonic acid resulted in a alpha-D-GlcpNAc-(1 --> 3)-D-GlcA disaccharide that demonstrated the usefulness of this reagent for selective cleavage of heteropolysaccharides. The following structure for the polysaccharide was established: --> 4)-alpha-D-Glcp6Ac(1 --> 3)-beta-D-GlcpA4Ac-(1 --> 3)-alpha-D-GlcpNAc-(1 --> 3)-beta-D-GlcpA4Ac-(1 --> where the degree of O-acetylation is approximately 65% at position 6 of Glc and 80-95% at position 4 of GlcA residues.

In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens.

Arnica and propolis have been used for thousands of years in folk medicine for several purposes. They possess several biological activities such as anti-inflammatory, antifungal, antiviral and tissue regenerative, among others. Although the antibacterial activity of propolis has already been demonstrated, very few studies have been done on bacteria of clinical relevance in dentistry. Also, the antimicrobial activity of Arnica has not been extensively investigated. Therefore the aim here was to evaluate in vitro the antimicrobial activity, inhibition of adherence of mutans streptococci and inhibition of formation of water-insoluble glucan by Arnica and propolis extracts. Arnica montana (10%, w/v) and propolis (10%, w/v) extracts from Minas Gerais State were compared with controls. Fifteen microorganisms were used as follows: Candida albicans--NTCC 3736, F72; Staphylococcus aureus--ATCC 25923; Enterococcus faecalis--ATCC 29212; Streptococcus sobrinus 6715; Strep. sanguis--ATCC 10556; Strep. cricetus--HS-6; Strep. mutans--Ingbritt 1600; Strep. mutans--OMZ 175; Actinomyces naeslundii--ATCC 12104, W 1053; Act. viscosus OMZ 105; Porphyromonas gingivalis; Porph. endodontalis and Prevotella denticola (the last three were clinical isolates). Antimicrobial activity was determined by the agar diffusion method and the zones of growth inhibition were measured. To assess cell adherence to a glass surface, the organisms were grown for 18 h at 37 degrees C in test-tubes at a 30 degree angle. To assay water-insoluble glucan formation, a mixture of crude glucosyltransferase and 0.125 M sucrose was incubated for 18 h at 37 degrees C in test-tubes at a 30 degree angle. Arnica and propolis extracts (20 microl) were added to these tubes to evaluate the % of inhibition of cell adherence and water-insoluble glucan formation. The propolis extract significantly inhibited all the microorganisms tested (p < 0.05), showing the largest inhibitory zone for Actinomyces spp. The Arnica extract did not demonstrate significant antimicrobial activity. Cell adherence and water-insoluble glucan formation were almost completely inhibited by the propolis extract at a final concentration of 400 microg/ml and 500 microg/ml, respectively. The Arnica extract showed slight inhibition of the adherence of the growing cells (19% for Strep. mutans and 15% for Strep. sobrinus) and of water-insoluble glucan formation (29%) at these same concentrations. Thus, the propolis extract showed in vitro antibacterial activity, inhibition of cell adherence and inhibition of water-insoluble glucan formation, while the Arnica extract was only slightly active in those three conditions.

Nitric oxide production by macrophages stimulated with Coccidia sporozoites, lipopolysaccharide, or interferon-gamma, and its dynamic changes in SC and TK strains of chickens infected with Eimeria tenella.

Nitric oxide (NO) is an important mediator of innate and acquired immunities. In the studies reported here, we quantified NO produced in vitro by chicken leukocytes and macrophages and in vivo during the course of experimental infection with Eimeria, the causative agent of avian coccidiosis, and identified macrophages as the primary source of inducible NO. Eimeria tenella-infected chickens produced higher levels of NO compared with noninfected controls. In Eimeria-infected animals, SC chickens produced greater amounts of NO compared with infected TK chickens, particularly in the intestinal cecum, the region of the intestine infected by E. tenella. Macrophages that were isolated from normal spleen were a major source of NO induced by interferon (IFN)-gamma, lipopolysaccharide (LPS), and E. tenella sporozoites. Macrophage cell line MQ-NCSU produced high levels of NO in response to Escherichia coli or Salmonella typhi LPS, whereas the HD-11 macrophage cell line was more responsive to IFN-gamma. These findings are discussed in the context of the genetic differences in SC and TK chickens that may contribute to their divergent disease phenotypes.

Evaluation of chlorines' impact on biofilms on scratched stainless steel surfaces.

Biofilms of a wild type Escherichia coli were grown on 316 stainless steel slides in a nutrient starved medium. The stainless steel surfaces were either polished to a smooth finish or scribed. The scribes consisted of lines and crosses. Biofilm samples were taken after 3, 6, 12, 24 and 48 h of growth. After sampling, the slides were soaked in deionized water or 50 or 200 ppm free chlorine prior to vital staining. Images were captured and the areas of viable and total biofilm were estimated. The individual biofilm patches, circularities, total percentage coverage, and viability percentage coverage were analyzed. The biofilms tended to increase in size between 6 and 24 h. A 3-6 h old biofilm on a polished stainless steel surface detached when 200 ppm sodium hypochlorite was applied. When grown in scribes, the circularity decreased up to 24 h, but thereafter increased. As the film grew older, it detached with or without a sodium hypochlorite treatment from the part of the surface that was polished, but remained in the neighborhood of the scribe. Based on the results, we recommend sanitizing at intervals of less than 12 h for this and similar strains of bacteria and protection of stainless steel surfaces to minimize scratching.