In vitro binding interactions of oral bacteria with immobilized fructosyltransferase

AIMS: The objective of the present study was to explore the role of immobilized fructosyltransferase (FTF) in adhesion process. METHODS AND RESULTS: We investigated real-time biospecific interactions between several types of oral bacteria and recombinant FTF immobilized on a biosensor chip, using surface plasmon resonance technology. Streptococcus mutans, Streptococcus sobrinus and Actinomyces viscosus demonstrated significant binding to FTF. Actinomyces viscosus had a greater binding to FTF, with 373 Resonance Units (RU), than the other tested bacteria. The binding level to FTF of Strep. sobrinus was 320 RU, whereas Strep. mutans and Streptococcus salivarious show binding of 296 and 245 RU, respectively. The binding sensograms displayed different profiles for the tested bacteria at various cell density, suggesting a different affinity to immobilized FTF. CONCLUSIONS: The results from this study suggest that FTF may influence bacterial adherence and colonization of the dental biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: The biomolecular interaction analysis enables real-time monitoring of the interaction between adhesions of intact bacteria and their ligands, which might be crucial in the initial phase of biofilm development in vivo.     

In vitro anti- and pro-oxidative effects of natural polyphenols

The anti- and pro-oxidative effects of phenolic compounds and antioxidants were studied in two different in vitro model systems utilizing ethyl linoleate and 2′-deoxyguanosine (2′-dG) as oxidative substrates, and a Fenton reaction (H₂O₂, Fe²⁺) to initiate oxidation. Oxidation of the biomolecules in both model systems exhibited dose dependency. In the 2′-dG assay, oxidation was closely related to H₂O₂ generation, which occurred during autoxidation of the phenolics. Hydroxylating activity was greatly enhanced by Mn²⁺ and Cu²⁺, but not by Zn²⁺ or Co²⁺. Ethyl linoleate peroxidation was inhibited by low concentrations of catechol, quercitin, and instant coffee. However, peroxidation was promoted by high concentrations of the same compounds, probably by recycling of chelated inactive Fe³⁺ to the active Fe²⁺ state.     

In vitro inhibition of Streptococci binding to enamel acquired pellicle by plant lectins

AIM: Initial colonization of the tooth surface by streptococci involves the attachment of these bacteria to adsorbed salivary components of the acquired pellicle. In dental biofilm this adhesion may also involve lectin-like components, present on the surface of the organisms, which bind to complementary carbohydrates on the surface of the tooth. Therefore, this work aimed to evaluate the potential of six lectins, extracted from seeds of Leguminosae family members, to inhibit the adherence of five streptococci species to acquired pellicle in vitro. METHODS AND RESULTS: The lectins used in this work were extracted from Canavalia ensiformis, Canavalia brasiliensis, Dioclea violacea, Dioclea grandiflora, Cratylia floribunda and Vatairea macrocarpa. Fluorescence micrography was employed to visualize the ability of FITC-labeled lectins to attach to acquire pellicle. Adherence inhibition was performed on saliva-coated microtiter plates at which lectins solutions were previously incubated followed by incubation with the oral streptococci. Glucose-mannose specific lectins attached to acquired pellicle with high intensity, while galactose specific lectins, from V. macrocarpa, exhibits low intensity attachment. CONCLUSIONS: All lectins were able to inhibit the adherence of the microorganisms tested (p < 0.01). SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that lectins may be useful in anti adhesion therapeutics.     

In vitro antibiofilm and anti‐adhesion effects of magnesium oxide nanoparticles against antibiotic resistant bacteria

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic‐resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X‐ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 μg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram‐negative bacteria. Cell adherence assay indicated 25.3–49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time‐dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non‐toxic to HeLa cells at concentrations of 15–120 μg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug‐resistant bacteria.

     

Human mesenchymal stromal cells can uptake and release ciprofloxacin,acquiring in vitro anti-bacterial activity

Background aimsTraditional antibiotic therapy is based on the oral or systemic injection of antibiotics that are often unable to stop a deep infection (eg, osteomyelitis). We studied whether or not bone marrow stromal cells (BM-MSCs) are able to uptake and release ciprofloxacin (CPX), a fluoroquinolone considered the drug of choice for the treatment of chronic osteomyelitis because of its favorable penetration into poorly vascularized sites of infection.MethodsHuman bone marrow stromal cells (BM-MSCs) were primed with CPX (BM-MSCsCPX) according to a methodology previously standardized in our laboratory for paclitaxel (PTX). The anti-microbial activity of CPX released from BM-MSCs cells (BM-MSCsCPX-CM) or supernatant from cell lysate (BM-MSCsCPX-LYS) was evaluated by agar dilution and microdilution methods on three bacterial strains (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). To investigate whether or not primed cells (BM-MSCsCPX) were able to directly act on the bacterial growth, co-colture was performed by mixing E. coli suspension to an increasing number of BM-MSCsCPX. The anti-bacterial activity was determined as number of BM-MSCsCPX that completely inhibited bacterial growth.ResultsThe results demonstrated that BM-MSCsCPX are able to uptake and then release CPX in the conditioned medium. The loaded antibiotic maintains its active form throughout the process as tested on bacteria.ConclusionsOur findings suggest that CPX-loaded MSCs may represent an important device for carrying and delivering CPX (and perhaps other antibiotics) into infected deep microenvironments; they could be used for local application and by systemic infusion when their homing capacity into the bone is cleared.     

In vitro evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells

We investigated the anticancer effects of green and black tea polyphenols alone and in combination with bovine milk lactoferrin (bLF) on human tongue squamous carcinoma (CAL-27) and normal human gingival fibroblast (HGF) cells. Both green (Polyphenon-E;P-E) and black tea polyphenols (Polyphenon-B;P-B) preferentially inhibit the growth of CAL-27 cells in a dose-dependent manner. Based on the IC(50) values, P-E was found to be more effective than P-B and the combination of P-E and bLF (1:2 ratio) exhibited synergistic inhibition of CAL-27 cells. Analysis of the mechanism revealed nuclear fragmentation and condensation with appearance of the A(o) peak indicative of apoptosis. Furthermore, tea polyphenols transduced the apoptosis signal via generation of reactive oxygen species and decrease in the Bcl-2/Bax ratio thereby inducing mitochondrial permeability transition with consequent activation of caspase-3. Overall, the potency of cytotoxic and apoptosis inducing effects of dietary agents on CAL-27 cells was in the order P-E and bLF combination (1:2 ratio)>P-E>P-B. These results suggest that a "designer" approach may be useful for oral cancer prevention strategies.     

Analysis of polyphenolic compounds and radical scavenging activity of four American Actaea species

A reversed-phase high-performance liquid chromatography (RP-HPLC) method with diode array detection has been developed for analysis of the major polyphenols in the roots and rhizomes of black cohosh (Actaea racemosa), an important botanical dietary supplement for women's health, and three closely related American Actaea species, A. rubra, A. pachypoda and A. podocarpa. The method was validated with respect to sensitivity, linearity, precision, accuracy and recovery. The total content of eight major polyphenols in the dried root and rhizome of the four species was determined to be from 0.36 to 2.92% (w/w). The antioxidant activities of Actaea extracts and polyphenolic compounds isolated from A. racemosa were evaluated on 1.1-diphenyl-2-picrylhydrazyl (DPPH) free radicals scavenging assay. The radical scavenging activity of the Actaea extracts correlates to their polyphenolic composition. This validated HPLC method can be used to distinguish A. racemosa from the other major American Actaea species based on this study.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

In vitro growth inhibition of human intestinal bacteria by sarcotoxin IA, an insect bactericidal peptide

Sacotoxin IA, an anti-microbial peptide of insects, inhibited the growth of harmful human intestinal bacteria such as Escherichia coli, Clostridium ramosum and Clostridium paraputrificum, in vitro. Interestingly, E. coli O157 which is well-known to cause food poisoning, was most sensitive to the peptide among the bacteria tested. The peptide, however, did not affect the growth of Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Enterococcus faecalis, Bacteroides thetaiotamicron, Bacteroides uniformis and Bacteroides ovatuss which are abundant in the intestines of healthy people. The evidence suggests that sarcotoxin IA could change the composition of the intestinal flora to improve human health.     

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.     

Identification of a Moraxella catarrhalis gene that confers adherence to various human epithelial cell lines in vitro

Moraxella catarrhalis is a pathogen of the human airways. We found that expression of the M. catarrhalis gene mcmA by Escherichia coli increases adherence to epithelial cells 100-fold. Furthermore, we discovered that disrupting mcmA decreases M. catarrhalis adherence to laryngeal and lung cells, which are relevant to pathogenesis by the bacterium.     

Effects of phenol and natural phenolic compounds on biofilm formation by Pseudomonas aeruginosa

A biofilm is formed as a result of adhesion of microorganisms to various surfaces with the production of extracellular polymers (polysaccharides and proteins). Biofilms cause serious problems in the chemical, medical and pharmaceutical industries. Recent findings indicate that some natural phenolic compounds found in plants have an anti-biofouling effect on biofilm formation by Gram-negative bacteria. The anti-biofouling activities of 14 selected phenol and natural phenolic compounds were tested against Pseudomonas aeruginosa, using a microtiter-plate. A modified microtiter-plate assay was used because it enabled indirect measurement of bacterial cells attached to the surface of the wells. This assay involved fixing the bacterial film with methanol, staining with crystal violet dye and then releasing the bound dye with 33% glacial acetic acid. The optical density (OD) of the solution was measured at 570 nm by using an automated ICN Flow Titertek Multiscan Plus reader. Phenol and natural phenolic compounds except ethyl linoleate and tocopherol showed a significant reduction in biofilm formation by P. aeruginosa.     

In vitro efficacy of bismuth thiols against biofilms formed by bacteria isolated from human chronic wounds

Aims: The purpose of this study was to evaluate the antimicrobial efficacy of thirteen bismuth thiol preparations for bactericidal activity against established biofilms formed by two bacteria isolated from human chronic wounds. Methods: Single species biofilms of a Pseudomonas aeruginosa or a methicillin‐resistant Staphylococcus aureus were grown in either colony biofilm or drip‐flow reactors systems. Biofilms were challenged with bismuth thiols, antibiotics or silver sulfadiazine, and log reductions were determined by plating for colony formation. Conclusions: Antibiotics were ineffective or inconsistent against biofilms of both bacterial species tested. None of the antibiotics tested were able to achieve >2 log reductions in both biofilm models. The 13 different bismuth thiols tested in this investigation achieved widely varying degrees of killing, even against the same micro‐organism in the same biofilm model. For each micro‐organism, the best bismuth thiol easily outperformed the best conventional antibiotic. Against P. aeruginosa biofilms, bismuth‐2,3‐dimercaptopropanol (BisBAL) at 40–80 μg ml^?1 achieved >7·7 mean log reduction for the two biofilm models. Against MRSA biofilms, bismuth‐1,3‐propanedithiol/bismuth‐2‐mercaptopyridine N‐oxide (BisBDT/PYR) achieved a 4·9 log reduction. Significance and Impact of the Study: Bismuth thiols are effective antimicrobial agents against biofilms formed by wound bacteria and merit further development as topical antiseptics for the suppression of biofilms in chronic wounds.     

Anti-biofilm and anti-adherence properties of novel cyclic dipeptides against oral pathogens

Microorganisms embedded in a biofilm are significantly more resistant to antimicrobial agents and the defences of the human immune system, than their planktonic counterpart. Consequently, compounds that can inhibit biofilm formation are of great interest for novel therapeutics. In this study, a screening approach was used to identify novel cyclic dipeptides that have anti-biofilm activity against oral pathogens. Five new active compounds were identified that prevent biofilm formation by the cariogenic bacterium Streptococcus mutans and the pathogenic fungus Candida albicans. These compounds also inhibit the adherence of microorganisms to a hydroxylapatite surface. Further investigations were conducted on these compounds to establish the structure–activity relationship, and it was deduced that the common cleft pattern is required for these molecules to act effectively against biofilms.     

Media- and method-dependent variations in minimal inhibitory concentrations of antiplaque agents on oral bacteria

AIMS: To determine minimal inhibitory concentrations (MICs) and the percentage of nonsusceptible bacteria-- those still cultivable above a threshold concentration--in human supragingival dental plaque and saliva for antiplaque/antimicrobial agents including triclosan (TCS) and trichlorocarbanilide (TCC), and a new potential antimicrobial, 2-t-butyl-5-(4-t-butylphenyl)-phenol (DTBBP). METHODS AND RESULTS: Broth and agar dilution-based MIC tests were performed using 28 oral and nonoral bacterial strains representing 17 species. MICs for TCS were lowest and more than 100-fold lower than DTBBP (P < 0.0005) by both methods. MICs for TCS were lower in broth-based tests compared with TCC. The additions of defibrinated blood to agar and horse serum to broth increased MICs--in the case of TCS, 10- to 15-fold. Significantly higher proportions of nonsusceptible plaque and salivary bacteria were recovered from agar media containing DTBBP or TCC compared with TCS (P < 0.05). CONCLUSIONS: TCS is a more effective antimicrobial agent than either TCC or DTBBP as determined by in vitro testing. SIGNIFICANCE AND IMPACT OF THE STUDY: The utility of in vitro testing for antiplaque agents as a predictor of in vivo efficacy is affected by the methods used.     

In vitro effects of some anabolic compounds on erythrocyte carbonic anhydrase I and II

The in vitro effects of the anabolic compounds, zeranol, 17 β-estradiol, diethylstilbestrol (DES), and trenbolone, on the activity of purified human carbonic anhydrase I and II were evaluated. In vitro CA enzyme activity was determined colorimetrically using the CO₂ hydration method of Maren. IC₅₀ values of the compounds that caused inhibition were determined by means of activity percentage diagrams. The IC₅₀ concentrations of zeranol, 17 β-estradiol, DES and trenbolone on hCA I were 94, 55, 10, 898 µM and for hCA II 89, 159, 439 and 101 μM, respectively.     

In vitro effects of some anabolic compounds on erythrocyte carbonic anhydrase I and II

The in vitro effects of the anabolic compounds, zeranol, 17 β-estradiol, diethylstilbestrol (DES), and trenbolone, on the activity of purified human carbonic anhydrase I and II were evaluated. In vitro CA enzyme activity was determined colorimetrically using the CO? hydration method of Maren. IC?? values of the compounds that caused inhibition were determined by means of activity percentage diagrams. The IC?? concentrations of zeranol, 17 β-estradiol, DES and trenbolone on hCA I were 94, 55, 10, 898 μM and for hCA II 89, 159, 439 and 101 μM, respectively.