Agar Overlay Assay Research Articles

The sponges Hymeniacidon perlevis and Halichondria panicea are reservoirs of antibiotic-producing bacteria against multi-drug resistant Staphylococcus aureus.

Evaluation of the antibacterial activity of cultivable bacteria associated with the marine sponges Hymeniacidon perlevis and Halichondria panicea against multi-drug-resistant Staphylococcus aureus. One hundred and fourteen bacterial isolates were recovered from H. perlevis and H. panicea. Antibacterial action was demonstrated by 70% of the isolates against reference strain Staphylococcus aureus ATCC 29213 and by 31·6% against Pseudomonas aeruginosa ATCC 27853 in agar overlay assays. Antibacterial potential was further analysed against 36 multi-drug-resistant hospital Staphylococcus aureus strains with diverse resistance profiles. Among the 80 isolates positive against S. aureus ATCC 29213, 76·3% were active against at least one clinical S. aureus pathogen and 73·6% inhibited one or more methicillin-resistant (MRSA) and vancomycin non-susceptible S. aureus strains. In addition, 41·3% inhibited all vancomycin nonsusceptible MRSA strains. Culturable bacteria associated to H. perlevis and H. panicea are promising sources of antibacterial compounds of great pharmaceutical interest. This study was the first to explore the antibacterial potential of culturable bacteria associated with the marine sponges H. perlevis and H. panicea against MDR bacteria. This is the first report of antibacterial activity by Aquimarina, Denitrobaculum, Maribacter and Vagococcus isolates against MDR S. aureus strains, including vancomycin nonsusceptible and methicillin-resistant ones, against which new antibiotics are urgently needed.

Developing a functional lozenge with microencapsulated Lactiplantibacillus pentosus to improve oral and dental health

In this study, a functional lozenge was developed by using microencapsulated Lactiplantibacillus pentosus NRRL-B 227. Antimicrobial activity of L. pentosus was determined to find out the efficiency on cariogenic and pathogen microorganisms that are known to cause dental caries and gum diseases. Streptococcus mutans is a well-known cariogenic strain, which easily converts sugars to acids and exert adverse effects on dental health. Candida albicans is an opportunistic pathogen when body resistance is weak, it becomes dominant in the mouth, causing disruption of oral health. Within this context, by applying several methods (e.g. broth microdilution, disc diffusion, agar overlay and planktonic culture assays), L. pentosus showed antibacterial and antifungal activities against S. mutans ATCC 25175 and C. albicans DSMZ 5817, respectively. Thus, three different lozenge formulation were produced such as; CL - control formulation without cells, CPL - formulation containing microencapsulated cells and FPL - formulation containing free cells. The microbiological, physicochemical and sensorial studies were carried out for all formulations stored at 4 °C and 25 °C. Among these, CPL was found to be more stable than FPL, since microencapsulation with Pullulan/WPC water-in-oil emulsion play a protective role. L. pentosus viability has been lost after 1 month at 25 °C, however the viability was maintained without significant reduction at 4 °C in case of CPL samples. In the light of findings, lozenge formulation incorporated with L. pentosus can be considered as potential antimicrobial to improve oral health and therefore may become a promising candidate to contribute to the range of functional food products.

Isolation, characterization, and application of bacteriophages against several pathogenic and food spoilage bacteria

Background: Food is a fundamental requirement of everyday life, but contamination can cause food spoilage and food-borne diseases. Bacteriophage can be used as natural antimicrobials and biocontrol in the food industry without interfering with the natural microflora, as well as enhancing the safety of food products. This study is aimed to isolate and characterize bacteriophages from food and soil samples and analyze their activity as biocontrol for food safety. Methods and materials: This study used Ready-to-Eat (RTE) food such as dawet and ketoprak, seafood, and soil sample against pathogenic and food spoilage bacteria such as V. cholera, V. parahaemolyticus, E. cloacae, EPEC, EHEC, ETEC, B. cereus, and B. subtilis. Bacteriophage isolation, enrichment, and titer determination were done using agar overlay assay method. Bacteriophage then screened against pathogenic bacteria by the Efficiency of Plating (EOP) method. The inhibition of bacteria were determined using Minimum Inhibitory Multiplicity of Infection (miMOI) method and for the bacteriophage morphology was determined using Transmission Electron Microscopy (TEM). Results: The positive result of bacteriophage isolation was shown by the formation of clear plaque on plate, indicating bacteriophage lyses the bacteria. The titer determination of bacteriophage (>108 PFU/mL) tend to be more effective to reduce bacterial counts in foods which proved by 95.41% of EHEC and 99.24% of ETEC can be reduced by bacteriophage from dawet, while 62.15% of EPEC and 80.11% of ETEC can be reduced by bacteriophage from ketoprak. Bacteriophage that attacked V. parahaemolyticus had an efficiency of 99.03% and a concentration of 2.28 × 1010 PFU/mL. Bacteriophages that attacked V. cholera had an efficiency of 99.87% and a concentration of 1.08 × 108 PFU/mL. Bacteriophages that attack E. cloacae had an efficiency of 95.36% and a concentration of 1.81 × 1010 PFU/mL. Based on the TEM result, all the isolated bacteriophage were suspected from family Myoviridae. Conclusion: This study revealed that all the isolated bacteriophage have great potential to be used as biocontrol for food safety. Further study still needs to be conducted to characterize and analyze the bacteriophage against B. cereus and B. subtilis.

Antifungal effects of Lactobacillus acidophilus and Lactobacillus plantarum against different oral Candida species isolated from HIV/ AIDS patients: an in vitro study

ABSTRACT Oropharyngeal Candidiasis (OPC) is an opportunistic fungal infection occurring in immunocompromised patients such as HIV/AIDS. The purpose of this study was to evaluate the antifungal properties of Lactobacillus acidophilus and Lactobacillus plantarum on different Candida species isolated from oral cavity of HIV/AIDS patients compared to Fluconazole (FLC). In this study, the antifungal effects of both cells and cell-free supernatants (CFSs) of L. acidophilus and L. plantarum were investigated against different oral Candida species by co-aggregation, agar overlay interference and broth microdilution assays, respectively. Our results showed that the highest co-aggregation ratio of the two tested Lactic acid bacteria (LAB) was observed for C. krusei. Both L. acidophilus and L. plantarum at cell concentrations 1010 to 102 cfu/ml were able to inhibit the growth of most of the oral Candida species, except for C. albicans, and to some C. krusei. In this study, MIC and MFC values for CFS of L. acidophilus ranged from 100 to 200 µl/ml and 100 to 200 µl/ml, respectively, and MIC and MFC values for CFS of L. plantarum were 50 to 200 µl/ml and 50 to 200 µl/ml, respectively. The ranges of MIC and MFC for FLC were 256–1024 µg/ml and 512–2048 µg/ml, respectively. C. albicans and C. parapsilosis displayed the highest and least susceptibility to CFSs of two LAB, respectively. Our findings showed that both cells and CFSs of L. acidophilus and L. plantarum had antifungal effects against oral Candida species.

Screening for Antibiotic-Producing Bacteria from Imbak Canyon Conservation Area (ICCA)

Antibiotic resistance is an escalating threat to public health. Therefore, there is an urgent need for new antibiotics. This study aims to screen for antibiotic-producing microorganisms from the forest soil of Batu Timbang. Soil samples were collected, diluted and spread plated onto 1/5 Nutrient Agar (NA) and Actinomycete Isolation Agar (AIA) for the isolation of antibiotic-producing microorganisms. A total of 180 bacterial isolates were screened for their antibiotic-producing ability, and ten were tested positive for inhibitory activity against one or more test pathogens via agar overlay assay (Staphylococcus aureus ATCC BAA-1717, Enterococcus faecalis ATCC 700802, and Acinetobacter baumannii ATCC BAA-1605). Ten bacterial isolates were subjected to 16S rRNA gene amplification and gene sequence analysis. The isolates were identified to be closely related to the genus Variovorax, Streptomyces, Kitasatospora, Chromobacterium, Burkholderia, Pseudomonas and Massilia. Three isolates (Variovorax sp. A5, Variovorax sp. A6 and Kitasatospora sp. H8) are potentially novel as these isolates form a different clade from their respective closely related species via phylogenetic tree analysis using reference sequences obtained from GenBank/EMBL/DDBJ databases. The antibiotics produced by the bacterial isolates might potentially be new, as novel species might possess unique biosynthetic gene clusters to produce new compounds. Nevertheless, further taxonomic identification and antibiotic isolation work is required. This study has revealed the potential of antibiotic discovery from Batu Timbang (Imbak Canyon Conservation Area) and its implications in tackling antibiotic resistance.

ISOLATION OF PHAGES INFECTING LISTERIA MONOCYTOGENES

It was aimed to isolate, purify and determine host ranges of lytic phages that infect Listeria monocytogenes in this study. Out of 68 samples screened, 4 positive isolates were recovered from feces, food processing waste water, fisheries waste water, and fish samples. Recovery status of the L. monocytogenes phage was found to be 5.88%. To determine host ranges of phages, soft agar overlay plaque assay was used. While eleven L. monocytogenes strains showed resistant to all four isolated phages, 24 strains were sensitive. The plaque sizes of the 4 phages against 24 L. monocytogenes strains ranged from 0.4 and 2.6 mm. None of the phages had identical host ranges.

Genome annotation and antimicrobial properties of Bacillus toyonensis VU‐DES13, isolated from the Folsomia candida gut

AbstractAntibiotic resistance necessitates the search for new bioactive compounds with novel mechanisms of action. Natural products derived from bacteria and fungi are widely used in the field of medicine and new environments can be explored as sources of antimicrobials. Bacteria associated with springtails have shown high inhibitory activity against pathogens. Here, we characterized a bacterial strain with high potential for antimicrobial activity, isolated from the gut of the springtail Folsomia candida Willem (Collembola: Isotomidae). The strain was characterized using the ‘analytical profile index’ and the ‘minimal inhibitory concentration’ assay to test for antibiotic resistance. Agar overlay and agar disk diffusion assays were used to test the inhibitory activity of the strain and its extract against a variety of pathogens, and reporter assays were used to investigate the mode of action. High‐performance liquid chromatography was used to analyze and fractionate the extract of bacterial culture, followed by additional assays on the fractions. The genome of the strain was screened for presence of antibiotic resistance genes and secondary metabolite gene clusters. The isolate was identified as Bacillus toyonensis Jiménez et al., but it displayed differences in metabolic profile when compared to the type species. The isolate was highly resistant to penicillin and inhibited the growth of a variety of pathogenic microorganisms. Genome analysis revealed an enrichment of resistance genes for β‐lactam antibiotics compared to the type isolate. Also, secondary metabolite clusters involved in the production of siderophores, bacteriocins, and nonribosomal peptide synthetases were identified. In conclusion, a unique Bacillus strain was isolated from the gut of F. candida, for which we provide evidence of inhibitory activity against an array of pathogens. This, coupled with high resistance to penicillin as substantiated by the presence of resistance genes, points to the potential of B. toyonensis VU‐DES13 to provide a new source of antimicrobial compounds.

Anti-Candida activity of beneficial vaginal lactobacilli in in vitro assays and in a murine experimental model.

Vulvovaginal candidiasis (VVC) is one of the most frequent infections affecting women worldwide. Healthy vaginal microbiota is dominated by lactobacilli, which form a strong defense line against pathogens. In this work, in vitro antimicrobial properties of thirty vaginal Lactobacillus strains were evaluated against eleven Candida vaginal clinical isolates, employing three different methods. Also, the effect of intravaginal (i.va.) administrations (preventive, therapeutic and preventive-therapeutic) of L. reuteri CRL1324 or L. rhamnosus CRL1332 strains against the i.va. challenge with Candida albicans C2 (C.a.) was evaluated in a murine experimental model. From the results of agar overlay and liquid medium assays the selected lactobacilli strains have shown to inhibit the growth of at least one Candida strain. The inhibition was mainly due to the effect of organic acids. Anti-Candida activity was not evidenced in the agar plate diffusion method. In the experimental murine model, only preventive-therapeutic administration of both lactobacilli was able to significantly reduce viable C.a. numbers recovered in vaginal washes and the leukocyte influx induced by the fungi. In conclusion, lactobacilli exhibited in vitro and in vivo antimicrobial effects on Candida, suggesting that they could be promising candidates for protection against VVC.

In vitro biocompatibility of orthodontic miniscrews with human gingival fibroblast and SAOS-2 osteoblast cultures

Miniscrews are an important choice for orthodontic anchorage. Yet reports on failures do exist, and attempts have been made to elucidate the causes. Clinical outcomes may be compromised not only by the mechanical implications of miniscrew design and the location of anchorage but also by poor biocompatibility. Hence, this study deals with the surface roughness and elemental composition of miniscrews and how these properties may affect the in vitro biocompatibility of four commercially available miniscrews. Most of the currently available miniscrews are made of TiAl6V4, an alloy widely considered to be biocompatible. The samples tested in this study included four similarly dimensioned TiAl6V4 products from different manufacturers: tomas® by Dentaurum, OrthoEasy® by Forestadent®, Dual Top™ by Jeil Medical/Promedia, and LOMAS by Mondeal®. The surface properties of these products were characterized by scanning electron microscopy (SEM) and energy-dispersive X‑ray spectroscopy (EDX). Cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and agar overlay assays according to ISO10993-5. The miniscrew products were found to show variations in surface-finish quality pertaining to topography and chemical composition, with the latter departing slightly from the manufacturers' specifications. MTT assays yielded rates of cell culture viability in excess of 90%, and agar overlay assays did not reveal decoloration beyond the specimen outlines in any of the experimental groups tested. The four miniscrew products exhibited some minor, but statistically significant, differences in microtopography, alloy composition, and biological inertness. Cytotoxicity testing revealed that all four products should be considered non-cytotoxic, thus, ruling out poor biocompatibility as acause of miniscrew failure.

Spathaspora passalidarum selected for resistance to AFEX hydrolysate shows decreased cell yield.

This study employed cell recycling, batch adaptation, cell mating and high-throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability. The most promising candidate YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and an ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Quantitative Assessment of Antimicrobial Activity of PLGA Films Loaded with 4-Hexylresorcinol.

Profound screening and evaluation methods for biocide-releasing polymer films are crucial for predicting applicability and therapeutic outcome of these drug delivery systems. For this purpose, we developed an agar overlay assay embedding biopolymer composite films in a seeded microbial lawn. By combining this approach with model-dependent analysis for agar diffusion, antimicrobial potency of the entrapped drug can be calculated in terms of minimum inhibitory concentrations (MICs). Thus, the topical antiseptic 4-hexylresorcinol (4-HR) was incorporated into poly(lactic-co-glycolic acid) (PLGA) films at different loadings up to 3.7 mg/cm2 surface area through a solvent casting technique. The antimicrobial activity of 4-HR released from these composite films was assessed against a panel of Gram-negative and Gram–positive bacteria, yeasts and filamentous fungi by the proposed assay. All the microbial strains tested were susceptible to PLGA-4-HR films with MIC values down to 0.4% (w/w). The presented approach serves as a reliable method in screening and quantifying the antimicrobial activity of polymer composite films. Moreover, 4-HR-loaded PLGA films are a promising biomaterial that may find future application in the biomedical and packaging sector.

Inhibition of Fish Pathogenic Aeromonas hydrophila and Edwardsiella tarda by Centella asiatica In-vitro

The present study assessed the in-vitro inhibition of fish pathogenic bacteria, viz., Aeromonas hydrophila and Edwardsiella tarda by aqueous, methanol and chloroform extracts of Centella asiatica by agar-disc diffusion, agar overlay well-diffusion, and broth dilution assays. The agar-disc diffusion assay with 10 μL of sterile crude C. asiatica extracts failed to inhibit A. hydrophila; while the crude chloroform extract inhibited E. tarda (11.25 ± 0.35 mm). In agar overlay well-diffusion assay, the methanol and chloroform extracts of C. asiatica(50 μL) inhibited E. tarda at varying levels exhibiting zones of 7.50 ± 0.70 mm and 30.50 ± 6.40 mm, respectively. With the increasing concentration of crude chloroform C. asiatica extract (0-10%/mL), an increased growth inhibition of E. tarda was noted in broth dilution assay. These results demonstrated that the chloroform extract of C. asiatica has the highest antibacterial activity against E. tarda in-vitro, which can be applied as an alternative to the commercial antibiotic to control E. tarda infection in aquaculture.

Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production

This study evaluated in situ expression of the nisA gene by an indigenous, nisin A-producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A-mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

Protective effects of indigenous Escherichia coli against a pathogenic E. coli challenge strain in pigs.

To investigate the inhibitory effect of indigenous enterobacteria on pathogenic Escherichia coli, a challenge trial with postweaning pigs was conducted. A pathogenic E. coli strain was administered to all animals and their health was closely monitored thereafter. Faecal samples were taken from three healthy and three diarrhoeic animals. Samples were cultivated on MacConkey agar and isolates were subcultured. A soft agar overlay assay was used to determine the inhibitory activity of the isolates. A total of 1,173 enterobacterial isolates were screened for their ability to inhibit the E. coli challenge strain. Colony forming units of enterobacteria on MacConkey agar were not different between healthy and diarrhoeic animals in the original samples. Furthermore, numbers of isolates per animal were also not significantly different between healthy (482 isolates) and diarrhoeic animals (691 isolates). A total of 43 isolates (3.7%) with inhibitory activity against the pathogenic E. coli challenge strain were detected. All inhibitory isolates were identified as E. coli via MALDI-TOF. The isolates belonged to the phylotypes A, C and E. Many isolates (67.4%) were commensal E. coli without relevant porcine pathogenic factors, but toxin- and fimbrial genes (stx2e, fae, estIb, elt1a, fas, fan) were detected in 14 inhibitory isolates. Healthy animals showed significantly (P=0.003) more inhibitory isolates (36 of 482 isolates; 7.5%) than diseased animals (7 of 691 isolates; 1.0%). There were no significant correlations regarding phylotype or pathogenic factors between healthy and diseased animals. This study has shown that a small proportion of indigenous E. coli is able to inhibit in vitro growth of a pathogenic E. coli strain in pigs. Furthermore, healthy animals possess significantly more inhibitory E. coli strains than diarrhoeic animals. The inhibition of pathogenic E. coli by specific indigenous E. coli strains may be an underlying principle for the containment of pathogenic E. coli in pigs.

Characterisation of Lactococcus lactis isolates from herbs, fruits and vegetables for use as biopreservatives against Listeria monocytogenes in cheese

The control of Listeria monocytogenes in soft cheese manufacture remains a challenge due to its widespread occurrence, ability to grow at refrigerated temperatures and salt resistance. The application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives for improving cheese quality and safety has attracted significant research interest. The aim of this study was to select non-dairy origin LAB strains with anti-Listeria activity and to characterise their industrially relevant properties for potential roles as biopreservative adjunct cultures for use in cheese production. A total of 897 LAB isolated from herbs, fruits and vegetables were screened for their anti-Listeria activity using an MRS agar overlay assay. Fourteen isolates with strong activity were identified by 16S rRNA gene sequencing as Lactococcus lactis and all contained the nisin gene cluster. Of these fourteen isolates, four strains also showed detectable anti-Listeria activity in a milk medium. These wild Lc. lactis strains possessed slow lactose utilisation and acidification capacity in milk despite exhibiting high growth rates. This was likely due to the adoption of the mixed acid fermentation pathway since ethanol was a major metabolic end product. Analysis of volatiles in milk and laboratory scale cheese fermented with wild and industrial Lc. lactis strain combination using headspace solid phase microextraction (SPME) GC-MS identified up to a 300–400 fold increase in the production of 3-methylbutanal, an important cheese odorant with a malty flavour. Whole genome sequencing of four wild strains provided some genetic insights into their phenotypes. The wild Lc. lactis lack the lactose utilisation operon (lacABCDFEG) commonly found in industrial strains, but contain genes encoding a putative β-galactosidase and a previously characterised alternative lactose assimilation pathway with slow lactose uptake. Wild strains also contain genes encoding α-keto-acid decarboxylase (kdcA) required for 3-methylbutanal production. These results provide insight into technological properties of wild non-dairy Lc. lactis strains which could be potentially used as biopreservative adjunct cultures to control the growth of L. monocytogenes in cheese.

Potential for Biocontrol of Hairy Root Disease by a Paenibacillus Clade.

Rhizogenic Agrobacterium biovar 1 is the causative agent of hairy root disease (HRD) in the hydroponic cultivation of tomato and cucumber causing significant losses in marketable yield. In order to prevent and control the disease chemical disinfectants such as hydrogen peroxide or hypochlorite are generally applied to sanitize the hydroponic system and/or hydroponic solution. However, effective control of HRD sometimes requires high disinfectant doses that may have phytotoxic effects. Moreover, several of these chemicals may be converted to unwanted by-products with human health hazards. Here we explored the potential of beneficial bacteria as a sustainable means to control HRD. A large collection of diverse bacterial genera was screened for antagonistic activity against rhizogenic Agrobacterium biovar 1 using the agar overlay assay. Out of more than 150 strains tested, only closely related Paenibacillus strains belonging to a particular clade showed antagonistic activity, representing the species P. illinoisensis, P. pabuli, P. taichungensis, P. tundrae, P. tylopili, P. xylanexedens, and P. xylanilyticus. Assessment of the spectrum of activity revealed that some strains were able to inhibit the growth of all 35 rhizogenic agrobacteria strains tested, while others were only active against part of the collection, suggesting a different mode of action. Preliminary characterization of the compounds involved in the antagonistic activity of two closely related Paenibacillus strains, tentatively identified as P. xylanexedens, revealed that they are water-soluble and have low molecular weight. Application of a combination of these strains in greenhouse conditions resulted in a significant reduction of HRD, indicating the great potential of these strains to control HRD.

Phytochemical Analysis, Identification and Quantification of Antibacterial Active Compounds in Betel Leaves, Piper betle Methanolic Extract.

The problems of bacterial diseases in aquaculture are primarily controlled by antibiotics. Medicinal plants and herbs which are seemed to be candidates of replacements for conventional antibiotics have therefore gained increasing interest. Current study was performed to investigate the presence of phytochemical constituents, antibacterial activities and composition of antibacterial active compounds in methanolic extract of local herb, Piper betle . Qualitative phytochemical analysis was firstly carried out to determine the possible active compounds in P. betle leaves methanolic extract. The antibacterial activities of major compounds from this extract against nine fish pathogenic bacteria were then assessed using TLC-bioautography agar overlay assay and their quantity were determined simultaneously by HPLC method. The use of methanol has proved to be successful in extracting numerous bioactive compounds including antibacterial compounds. The TLC-bioautography assay revealed the inhibitory action of two compounds which were identified as hydroxychavicol and eugenol. The $-caryophyllene however was totally inactive against all the tested bacterial species. In this study, the concentration of hydroxychavicol in extract was found to be 374.72±2.79 mg g-1, while eugenol was 49.67±0.16 mg g-1. Based on these findings, it could be concluded that hydroxychavicol and eugenol were the responsible compounds for the promising antibacterial activity of P. betle leaves methanolic extract. This inhibitory action has significantly correlated with the amount of the compounds in extract. Due to its potential, the extract of P. betle leaves or it compounds can be alternative source of potent natural antibacterial agents for aquaculture disease management.

Removal efficacy and cytotoxicity of a calcium hydroxide paste using N-2-methyl-pyrrolidone as a vehicle.

ObjectivesThis study investigated the removal efficacy and cytotoxicity of a newly developed calcium hydroxide paste (cleaniCal, Maruchi) using N-2-methyl-pyrrolidone (NMP) as a vehicle in comparison with ApexCal (Ivoclar Vivadent) and Calcipex II (Nishika), which use different vehicles such as polyethylene glycol and propylene glycol, respectively.Materials and MethodsThirty maxillary premolars with oval-shaped canals were divided into 3 groups and the teeth were filled with one of the pastes. After removal of the paste, micro-computed tomographic (μ-CT) imaging was obtained to assess the volume of residual paste in the root canal of each tooth. The teeth were then split longitudinally and the area of the paste-coated surface was evaluated by stereomicroscopy. The cytotoxicity of each product was assessed using an agar overlay assay. The effect of each vehicle on cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The data were analyzed using one-way analysis of variance and Tukey's tests to detect any significance (p < 0.05).ResultsIn the μ-CT and stereomicroscopic analysis, cleaniCal exhibited less remnants of medicament than ApexCal and Calcipex. cleaniCal showed a higher cytotoxicity than the other pastes in the agar overlay assay. Furthermore, NMP exhibited lower cell viability compared to the other vehicles.ConclusionscleaniCal showed better removal efficacy compared to the other products. However, clinicians should be aware of the higher cytotoxicity of the NMP-based material and consider its possible adverse effects on periradicular tissue when it is overfilled.

Probiotic Lactobacillus reuteri has antifungal effects on oral Candida species in vitro

ABSTRACTBackground: An alternative approach for managing Candida infections in the oral cavity by modulating the oral microbiota with probiotic bacteria has been proposed.Objective: The aim was to investigate the antifungal potential of the probiotic bacterium Lactobacillus reuteri (DSM 17938 and ATCC PTA 5289) against six oral Candida species (C. albicans, C. glabrata, C. krusei, C. tropicalis, C. dubliniensis, and C. parapsilosis).Design: The lactobacilli were tested for their ability to co-aggregate with and inhibit the growth of the yeasts assessed by spectrophotometry and the agar overlay inhibition assay. Additionally, the pH was evaluated with microsensors, and the production of hydrogen peroxide (H2O2) by the lactobacilli was verified.Results: Both L. reuteri strains showed co-aggregation abilities with the yeasts. The lactobacilli almost completely inhibited the growth of C. albicans and C. parapsilosis, but did not affect C. krusei. Statistically significant differences in co-aggregation and growth inhibition capacities between the two L. reuteri strains were observed (p<0.001). The pH measurements suggested that C. krusei can resist the acids produced by the lactobacilli.Conclusions: L. reuteri exhibited antifungal properties against five of the six most common oral Candida species. Further, the results reconfirms that the probiotic capacity of L. reuteri is strain specific.

Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model.

There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar-overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited.