Aims: To characterize the plantaricin IIA-1A5 crude extract that biosynthesized by Lactobacillus plantarum IIA-1A5 using corn steep liquor (CSL) based medium. Methodology and results: Lactobacillus plantarum IIA-1A5 was grown in several media containing different components including corn steep liquor (CSL), molasses and MRS (de Man Rogosa Sharpe) as control medium for 24 h at 37 °C. Antibacterial activities of the cell-free supernatant were expressed as diameter of inhibition zones observed by paper disc method. The results showed that CSL medium produced cell-free supernatant of L. plantarum IIA-1A5 with significantly higher antibacterial activity againts Staphylococcus aureus ATCC 25923 (9.81 mm), Lactobacillus monocytogenes ATCC 7644 (9.61 mm), Bacillus cereus (8.97 mm) and Escherichia coli ATCC 25922 (9.23 mm) were not significantly different compared to control MRS broth media (9.59 mm). CSL medium added only with 3% yeast extract and Tween 80 produced supernatant which showed similar antibacterial activity either to 10% molasses or control medium (Medium K and B). The CSL medium was considered more efficient and low cost, therefore this medium was selected for production and characterization of plantaricin IIA-1A5 crude extract. Further characterization performed by SDS PAGE analysis showed that crude plantaricin had molecular weight of approximately 9.9 kDa, higher than that produced in control medium (8.0 kDa). However, both plantaricins were categorized under the same class for small bacteriocin (class II). This study also revealed the plantaricin IIA-1A5 produced in CSL medium was stable to heat and pH and not significantly different compared to control MRS broth media. The antibacterial activity of plantaricin IIA-1A5 crude extract against S. aureus ATCC 25923 (10.09 mm) was not significantly different with 1000 ppm sodium benzoate (9.70 mm) and 300 ppm sodium nitrite (9.82 mm). Conclusion, significance and impact of study The CSL medium produced cell-free supernatant of L. plantarum IIA 1A5 had significant antibacterial activity characterization againts S. aureus ATCC 25923, L. monocytogenes ATCC 7644, B. cereus and E. coli ATCC 25922. Comparison of the inhibition activity of plantaricin IIA-1A5 crude extract against pathogen with synthetic preservatives indicated that plantaricin IIA-1A5 crude extract have the potency to replace synthetic preservatives. CSL based medium is potential to be used for low-cost plantaricin IIA-1A5 production.
Anti-Bacterial Agents--metabolism ; Bacteriocins--metabolism ; Lactobacillus plantarum ; Microbial Viability--drug effects ; Staphylococcus aureus

Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great potential for the development of novel antifungal strategies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the antifungal mechanism of Neosartorya fischeri antifungal protein (NFAP), a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disrupting-effect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP activates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any influence on the cell wall integrity pathway, but an unknown cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken together, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related proteins from Aspergillus giganteus and Penicillium chrysogenum.
Actins ; metabolism ; Antifungal Agents ; pharmacology ; Apoptosis ; drug effects ; Aspergillus nidulans ; cytology ; drug effects ; growth & development ; Cell Membrane ; drug effects ; metabolism ; Cell Wall ; drug effects ; metabolism ; Chitin ; metabolism ; Endocytosis ; drug effects ; Fungal Proteins ; pharmacology ; GTP-Binding Proteins ; metabolism ; Hyphae ; cytology ; drug effects ; Microbial Viability ; drug effects ; Neosartorya ; chemistry ; Signal Transduction ; drug effects

OBJECTIVETo investigate the resistant effect of houttuyfonate sodium (SH) combined with imipenem (IMP) against Pseudomonas aeruginosa (Pa) biofilms.

METHODThe two-fold dilution method was used to examine the minimum inhibitory concentration (MIC) of the tested drug. The crystal violet staining was applied to detect the effect of the combination of 1/2MIC, 1MIC, 2MIC of SH, single IMP, 1/2MIC of SH and IMP of various concentrations on the clearance rate of adherent bacteria, growth of biofilms and alginate production. Fluorescein diacetate (FDA)-propidium iodide (PI) doubling staining assay was employed to observe the bacterial viability and morphological changes after membrane dispersion of each drug group.

RESULTSodium houttuyfonate could enhance the effect of IMP against pseudomonas aeruginosa biofilms. Particularly, the combination group with the concentration of 2MIC showed the highest effect, with P < 0.001 compared with the negative control group. The above results were proved by the bacterial viability and biofilm morphology under fluorescence microscope.

CONCLUSIONAfter being combined with imipenem, sodium houttuyfonate shows a higher effect against biofilms. It is expected that the combination of the two drugs could improve the clinical efficacy of associated infections.

Alkanes ; pharmacology ; Biofilms ; drug effects ; growth & development ; Drug Synergism ; Imipenem ; pharmacology ; Microbial Sensitivity Tests ; Microbial Viability ; drug effects ; Pseudomonas aeruginosa ; drug effects ; physiology ; Sulfites ; pharmacology

The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media: Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy (CLSM)-based analyses of the microbial vitality, respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride, CTC) and production of extracellular polysaccharides (EPS) were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters: culture growth, vitality, CTC activity and EPS production. However, xylitol exposure caused a difference in gene expression compared to the control. GtfC was upregulated only in the presence of xylitol. Under xylitol exposure, gtfB was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three. Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms.
Bacterial Load ; drug effects ; Bacteriological Techniques ; Biofilms ; drug effects ; Cariogenic Agents ; metabolism ; pharmacology ; Culture Media ; Dental Enamel ; microbiology ; Fluorescent Dyes ; Gene Expression Regulation, Bacterial ; drug effects ; Gene Expression Regulation, Enzymologic ; drug effects ; Genotype ; Glucose ; metabolism ; Glucosyltransferases ; metabolism ; Humans ; Microbial Viability ; drug effects ; Microscopy, Confocal ; Polysaccharides, Bacterial ; biosynthesis ; Streptococcus mutans ; drug effects ; enzymology ; metabolism ; Sucrose ; metabolism ; pharmacology ; Sweetening Agents ; metabolism ; pharmacology ; Tetrazolium Salts ; Time Factors ; Up-Regulation ; Xylitol ; metabolism ; pharmacology

Candida species have been associated with the emergence of strains resistant to selected antifungal agents. Plant products have been used traditionally as alternative medicine to ease mucosal fungal infections. This study aimed to investigate the effects of Piper betle extract on the growth profile and the ultrastructure of commonly isolated oral candidal cells. The major component of P. betle was identified using liquid chromatography-mass spectrophotometry (LC-MS/MS). Seven ATCC control strains of Candida species were cultured in yeast peptone dextrose broth under four different growth environments: (i) in the absence of P. betle extract; and in the presence of P. betle extract at respective concentrations of (ii) 1 mg⋅mL(-1); (iii) 3 mg⋅mL(-1); and (iv) 6 mg⋅mL(-1). The growth inhibitory responses of the candidal cells were determined based on changes in the specific growth rates (µ). Scanning electron microscopy (SEM) was used to observe any ultrastructural alterations in the candida colonies. LC-MS/MS was performed to validate the presence of bioactive compounds in the extract. Following treatment, it was observed that the µ-values of the treated cells were significantly different than those of the untreated cells (P<0.05), indicating the fungistatic properties of the P. betle extract. The candidal population was also reduced from an average of 13.44×10(6) to 1.78×10(6) viable cell counts (CFU)⋅mL(-1). SEM examination exhibited physical damage and considerable morphological alterations of the treated cells. The compound profile from LC-MS/MS indicated the presence of hydroxybenzoic acid, chavibetol and hydroxychavicol in P. betle extract. The effects of P. betle on candida cells could potentiate its antifungal activity.
Antifungal Agents ; pharmacology ; Candida ; drug effects ; growth & development ; ultrastructure ; Candida albicans ; drug effects ; growth & development ; ultrastructure ; Candida glabrata ; drug effects ; growth & development ; ultrastructure ; Candida tropicalis ; drug effects ; growth & development ; ultrastructure ; Chromatography, Liquid ; methods ; Colony Count, Microbial ; Culture Media ; Eugenol ; analogs & derivatives ; analysis ; Humans ; Hydroxybenzoates ; analysis ; Microbial Viability ; drug effects ; Microscopy, Electron, Scanning ; Mouth ; microbiology ; Phytotherapy ; Piper betle ; chemistry ; Plant Extracts ; analysis ; pharmacology ; Spectrophotometry ; methods ; Tandem Mass Spectrometry ; methods ; Time Factors

This study investigated the efficacy of calcium hydroxide and chlorhexidine gel for the elimination of intratubular Candida albicans (C. albicans). Human single-rooted teeth contaminated with C. albicans were treated with calcium hydroxide, 2% chlorhexidine gel, calcium hydroxide plus 2% chlorhexidine gel, or saline (0.9% sodium chloride) as a positive control. The samples obtained at depths of 0-100 and 100-200 µm from the root canal system were analyzed for C. albicans load by counting the number of colony forming units and for the percentage of viable C. albicans using fluorescence microscopy. First, the antimicrobial activity of calcium hydroxide and the 2% chlorhexidine gel was evaluated by counting the number of colony forming units. After 14 days of intracanal medication, there was a significant decrease in the number of C. albicans colony forming units at a depth of 0-100 µm with chlorhexidine treatment either with or without calcium hydroxide compared with the calcium hydroxide only treatment. However, there were no differences in the number of colony forming units at the 100-200 µm depth for any of the medications investigated. C. albicans viability was also evaluated by vital staining techniques and fluorescence microscopy analysis. Antifungal activity against C. albicans significantly increased at both depths in the chlorhexidine groups with and without calcium hydroxide compared with the groups treated with calcium hydroxide only. Treatments with only chlorhexidine or chlorhexidine in combination with calcium hydroxide were effective for elimination of C. albicans.
Antifungal Agents ; administration & dosage ; pharmacology ; Calcium Hydroxide ; administration & dosage ; pharmacology ; Candida albicans ; drug effects ; Chlorhexidine ; administration & dosage ; pharmacology ; Colony Count, Microbial ; Coloring Agents ; Dental Pulp Cavity ; microbiology ; Drug Combinations ; Humans ; Microbial Viability ; drug effects ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Root Canal Irrigants ; administration & dosage ; pharmacology ; Root Canal Preparation ; methods ; Smear Layer ; Time Factors

OBJECTIVETo evaluate antioxidant, antimicrobial and cytotoxic activity of different parts (root, flower, leaf and stem) of Leucas aspera (L. aspera) (Labiatae).

METHODSDifferent parts of L. aspera were extracted with 80% (v/v) methanol. The methanol extracts were subjected to antioxidant, antimicrobial and brine shrimp lethality assay.

RESULTSAll the extracts showed moderate to potent antioxidant activity, among which the root extract demonstrated the strongest antioxidant activity with the IC50 value of 6.552 µg/mL. Methanol extract of root possessed antioxidant activity near the range of vitamin E and thus could be a potential rich source of natural antioxidant. In case of antimicrobial screening, crude extracts of root, flower, leaf and stem showed notable antibacterial activity against tested microorganisms. The root extract showed the highest mean zone of inhibition ranging from 9.0-11.0 mm against tested microorganisms, at a concentration of 100 mg/mL. In the brine shrimp lethality bioassay, it was evident that the methanol root extract did not show significant toxicity. The LC50 value for 12 h and 24 h observation was 2.890 mg/mL and 1.417 mg/mL, respectively.

CONCLUSIONSThe present finding suggests that the methanol root extract of L. aspera could be developed as pharmaceutical products.

Animals ; Anti-Bacterial Agents ; chemistry ; pharmacology ; toxicity ; Antioxidants ; chemistry ; pharmacology ; toxicity ; Artemia ; drug effects ; Bacteria ; drug effects ; Biphenyl Compounds ; metabolism ; Lamiaceae ; chemistry ; Methanol ; Microbial Viability ; drug effects ; Picrates ; metabolism ; Plant Components, Aerial ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; toxicity ; Plant Roots ; chemistry

The purpose of our study was to evaluate the effect of photodynamic therapy (PDT), using erythrosine as a photosensitizing agent and a dental halogen curing unit as a light source, on Streptococcus mutans in a biofilm phase. The S. mutans biofilms were formed in a 24-well cell culture cluster. Test groups consisted of biofilms divided into four groups: group 1: no photosensitizer or light irradiation treatment (control group); group 2: photosensitizer treatment alone; group 3: light irradiation alone; group 4: photosensitizer treatment and light irradiation. After treatments, the numbers of colony-forming unit (CFU) were counted and samples were examined by confocal laser scanning fluorescence microscopy (CLSM). Only group 4 (combined treatment) resulted in significant increases in cell death, with rates of 75% and 55% after 8 h of incubation, and 74% and 42% at 12 h, for biofilms formed in brain-heart infusion (BHI) broth supplemented with 0% or 0.1% sucrose, respectively. Therefore, PDT of S. mutans biofilms using a combination of erythrosine and a dental halogen curing unit, both widely used in dental clinics, resulted in a significant increase in cell death. The PDT effects are decreased in biofilms that form in the presence of sucrose.
Bacterial Load ; drug effects ; Bacteriological Techniques ; Biofilms ; drug effects ; Curing Lights, Dental ; classification ; Erythrosine ; therapeutic use ; Humans ; Microbial Viability ; drug effects ; Microscopy, Confocal ; Photochemotherapy ; methods ; Photosensitizing Agents ; therapeutic use ; Sonication ; Streptococcus mutans ; drug effects ; Sucrose ; pharmacology ; Time Factors

Trans-trans farnesol (tt-farnesol) is a bioactive sesquiterpene alcohol commonly found in propolis (a beehive product) and citrus fruits, which disrupts the ability of Streptococcus mutans (S. mutans) to form virulent biofilms. In this study, we investigated whether tt-farnesol affects cell-membrane function, acid production and/or acid tolerance by planktonic cells and biofilms of S. mutans UA159. Furthermore, the influence of the agent on S. mutans gene expression and ability to form biofilms in the presence of other oral bacteria (Streptococcus oralis (S. oralis) 35037 and Actinomyces naeslundii (A. naeslundii) 12104) was also examined. In general, tt-farnesol (1 mmol x L(-1)) significantly increased the membrane proton permeability and reduced glycolytic activity of S. mutans in the planktonic state and in biofilms (P < 0.05). Moreover, topical applications of 1 mmol x L(-1) tt-farnesol twice daily (1 min exposure/treatment) reduced biomass accumulation and prevented ecological shifts towards S. mutans dominance within mixed-species biofilms after introduction of 1% sucrose. S. oralis (a non-cariogenic organism) became the major species after treatments with tt-farnesol, whereas vehicle-treated biofilms contained mostly S. mutans (>90% of total bacterial population). However, the agent did not affect significantly the expression of S. mutans genes involved in acidogenicity, acid tolerance or polysaccharide synthesis in the treated biofilms. Our data indicate that tt-farnesol may affect the competitiveness of S. mutans in a mixed-species environment by primarily disrupting the membrane function and physiology of this bacterium. This naturally occurring terpenoid could be a potentially useful adjunctive agent to the current anti-biofilm/anti-caries chemotherapeutic strategies.
Actinomyces ; physiology ; Biofilms ; drug effects ; Cell Membrane Permeability ; drug effects ; Colony Count, Microbial ; Durapatite ; Farnesol ; pharmacology ; Gene Expression Regulation, Bacterial ; drug effects ; Glycolysis ; Humans ; Hydrogen-Ion Concentration ; Microbial Viability ; drug effects ; Plankton ; drug effects ; Saliva ; microbiology ; Streptococcus mutans ; drug effects ; genetics ; physiology ; Streptococcus oralis ; physiology

OBJECTIVETo study the effect of phage lysin on the growth of lysogens.

METHODSSputum specimens processed by modified Petroff's method were respectively treated with phagebiotics in combination with lysin and lysin alone. The specimens were incubated at 37 °C for 4 days. At the end of day 1, 2, 3 and day 4, the specimens were streaked on blood agar plates and incubated at 37 °C for 18-24 hours. The growth of normal flora observed after day 1 was considered as lysogens.

RESULTSSputum specimens treated with phagebiotics-lysin showed the growth of lysogens. When specimens treated with lysin alone, lysogen formation was avoided and normal flora was controlled.

CONCLUSIONSLysin may have no effect on the growth of lysogens.

Bacteria ; drug effects ; growth & development ; Bacteriophages ; enzymology ; Lysogeny ; Microbial Viability ; drug effects ; Mucoproteins ; metabolism ; Sputum ; microbiology ; Temperature ; Time Factors