Aims: Metarhizium anisopliae is an entomopathogenic fungus (EPF) that exists naturally in the environment and potentially be used as a biological control agent against many insect pests. This study aims to evaluate the effect of nutrient additives on the yield and viability of M. anisopliae spore and to determine the optimum incubation period for maximum spore production. Methodology and results: In this study, M. anisopliae was cultivated by solid-state fermentation using rice as a growth medium. Three different nutrient additives were examined which aimed to maximize the production of M. anisopliae spores. Among the three nutrient additives evaluated, yeast (1.84 ± 0.04 g) supported better growth and spore production than molasses (0.58 ± 0.04 g) and palm oil (0.47 ± 0.09 g). The incubation period between 2-6 weeks produced higher spore yield (0.97 ± 0.02 g spores) at week 4 with a better spore viability (86.30 ± 0.45%) at week 2. Conclusion, significance and impact of study Hence, it is suggested that the optimum incubation period is between 2 and 6 weeks after inoculation, and M. anisopliae could be mass produced in large quantities on rice substrate with the addition of yeast as the nutrient additives.
Biological Control Agents ; Microbial Viability ; Metarhizium

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

PURPOSE: Direct application of atmospheric-pressure plasma jets (APPJs) has been established as an effective method of microbial decontamination. This study aimed to investigate the bactericidal effect of direct application of an APPJ using helium gas (He-APPJ) on Porphyromonas gingivalis biofilms on sandblasted and acid-etched (SLA) titanium discs. METHODS: On the SLA discs covered by P. gingivalis biofilms, an APPJ with helium (He) as a discharge gas was applied at 3 different time intervals (0, 3, and 5 minutes). To evaluate the effect of the plasma itself, the He gas–only group was used as the control group. The bactericidal effect of the He-APPJ was determined by the number of colony-forming units. Bacterial viability was observed by confocal laser scanning microscopy (CLSM), and bacterial morphology was examined by scanning electron microscopy (SEM). RESULTS: As the plasma treatment time increased, the amount of P. gingivalis decreased, and the difference was statistically significant. In the SEM images, compared to the control group, the bacterial biofilm structure on SLA discs treated by the He-APPJ for more than 3 minutes was destroyed. In addition, the CLSM images showed consistent results. Even in sites distant from the area of direct He-APPJ exposure, decontamination effects were observed in both SEM and CLSM images. CONCLUSIONS: He-APPJ application was effective in removing P. gingivalis biofilm on SLA titanium discs in an in vitro experiment.
Bacterial Load ; Biofilms ; Decontamination ; Helium ; In Vitro Techniques ; Methods ; Microbial Viability ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Plasma Gases ; Plasma ; Porphyromonas gingivalis ; Porphyromonas ; Stem Cells ; Titanium

BACKGROUND: Pen-based devices have emerged as useful tools for measuring pH and glucose, and for fabricating microchannels and microarrays. Pen-based devices take advantage of flexible patterning, inexpensive costs, and small volumes, thereby saving time and increasing efficiency. We have developed a gradient nib marker pen device that generated simultaneously different antibiotic concentrations in bacteria antibiotic susceptibility testing (AST). METHODS: The device can deposit on the target surface with the antibiotic gradient. The designed polyester fiber nibs are a highly uniform porosity with unidirectional orientation and produce a visible gradient pattern. RESULTS: We have demonstrated and quantitatively analyzed bacterial growth after antibiotic marking. The antibiotic marking produces an inhibition zone of bacterial growth. The inhibition zones of bacterial growth are captured and converted to 8-bit grayscale images, and then quantified by gray values using the Image J program. A profile of the inhibition zone showed different gray values in response to bacterial viability. CONCLUSION: The gradient nib marker pen device can be used to determine the quantitative antibiotic concentration based on the relationship between gray values and bacterial density conveniently without requiring a series of dilution tubes, including nutrient medium, and diversely diluted antibiotics.
Anti-Bacterial Agents ; Bacteria ; Clothing ; Glucose ; Hydrogen-Ion Concentration ; Microbial Viability ; Polyesters ; Porosity

Dental caries is the most common chronic disease in the dental field. Streptococcus mutans (S. mutans) is the most important bacteria in the formation of dental plaque and dental caries. In a previous study, we confirmed that the essential oil of Chrysanthemum boreale has antibacterial activity against S. mutans. Alpha-pinene is one of the major chemical components of Chrysanthemum boreale essential oil. In the present study, we investigated the inhibitory effects of α-pinene on cariogenic properties such as growth, acid production, biofilm formation, and bactericidal activity on S. mutans. Alpha-pinene at a concentration range of 0.25-0.5 mg/mL significantly inhibited the growth of S. mutans and acid production of S. mutans. Biofilm formation was significantly inhibited at < 0.0625 mg/mL α-pinene, similar to the data from scanning electronic microscopy. Under confocal laser scanning microscopy, the bacterial viability was decreased by α-pinene in a dose-dependent manner. These results suggested that α-pinene may be a useful agent for inhibiting the cariogenic properties of S. mutans.
Bacteria ; Biofilms ; Chronic Disease ; Chrysanthemum ; Dental Caries ; Dental Plaque ; Microbial Viability ; Microscopy ; Microscopy, Confocal ; Plants* ; Streptococcus mutans

PURPOSE: The current live attenuated rabies vaccine must be replaced with a safer vaccine based on the ERAGS strain to prevent rabies in South Korea. We evaluated the safety and immunogenicity of a new strain in dogs and cattle. MATERIALS AND METHODS: The ERAGS strain, featuring two mutations altering two amino acids in a glycoprotein of rabies virus, was propagated in NG108-15 cells. We lyophilized the virus in the presence of two different stabilizers to evaluate the utilities of such preparations as novel rabies vaccines for animals. To explore safety and immunogenicity, dogs and cattle were inoculated with the vaccine at various doses via different routes and observed daily for 8 weeks post-inoculation (WPI). Immunogenicity was evaluated using a fluorescent antibody virus neutralization test or enzyme-linked immunosorbent assay. RESULTS: The two different stabilizers did not differ greatly in terms of maintenance of virus viability in accelerated stability testing. No clinical signs of rabies developed in dogs or cattle inoculated with the vaccines (10(7.0) FAID₅₀/mL). Dogs and cattle inoculated intramuscularly with 10(5.0) FAID₅₀/mL exhibited virus neutralization assay titers of 4.6 IU/mL and 1.5 to 0.87 IU/mL at 4 WPI, respectively. All control animals remained rabies virus–seronegative throughout, confirming that no contact transmission occurred between vaccinated and control animals. CONCLUSION: Our findings indicate that the new rabies vaccine is safe and immunogenic in dogs and cattle.
Amino Acids ; Animals ; Cattle* ; Dogs* ; Enzyme-Linked Immunosorbent Assay ; Glycoproteins ; Korea ; Microbial Viability ; Neutralization Tests ; Rabies Vaccines* ; Rabies virus ; Rabies* ; Vaccines

Newcastle disease viruses (NDVs) cause systemic diseases in chickens with high mortality. However, little is known about persistence of NDVs in contaminated tissues from infected birds. In this study, we examined viral replication in the feather pulp of chickens inoculated with viscerotropic velogenic NDV (vvNDV) genotype VII. Reverse transcription real-time PCR and immunohistochemistry were used to investigate viral persistence in the samples. vvNDV was detected in the oropharynx and cloaca and viral antigens were detected in the feathers, suggesting that feathers act as sources of viral transmission.
Animals ; Antigens, Viral/analysis ; Chickens ; Cloaca/virology ; Feathers/*virology ; Microbial Viability ; Newcastle Disease/transmission/*virology ; Newcastle disease virus/isolation & purification/*physiology ; Oropharynx/virology ; Poultry Diseases/transmission/*virology ; Virus Replication/*physiology

PURPOSE: The purpose of this study was to evaluate the effect of photodynamic therapy (PDT) using erythrosine and a green light emitting diode (LED) light source on biofilms of Aggregatibacter actinomycetemcomitans attached to resorbable blasted media (RBM) and sandblasted, large-grit, acid-etched (SLA) titanium surfaces in vitro. METHODS: RBM and SLA disks were subdivided into four groups, including one control group and three test groups (referred to as E0, E30, E60), in order to evaluate the effect of PDT on each surface. The E0 group was put into 500 microL of 20 microM erythrosine for 60 seconds without irradiation, the E30 group was put into erythrosine for 60 seconds and was then irradiated with a LED for 30 seconds, and the E60 group was put into erythrosine for 60 seconds and then irradiated with a LED for 60 seconds. After PDT, sonication was performed in order to detach the bacteria, the plates were incubated under anaerobic conditions on brucella blood agar plates for 72 hours at 37degrees C, and the number of colony-forming units (CFUs) was determined. RESULTS: Significant differences were found between the control group and the E30 and E60 groups (P<0.05). A significantly lower quantity of CFU/mL was found in the E30 and E60 groups on both titanium disk surfaces. In confocal scanning laser microscopy images, increased bacterial death was observed when disks were irradiated for a longer period of time. CONCLUSIONS: These findings suggest that PDT using erythrosine and a green LED is effective in reducing the viability of A. actinomycetemcomitans attached to surface-modified titanium in vitro.
Agar ; Aggregatibacter actinomycetemcomitans* ; Bacteria ; Biofilms ; Brucella ; Erythrosine ; Microbial Viability ; Microscopy, Confocal ; Photochemotherapy* ; Sonication ; Stem Cells ; Titanium*

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