Application of antifungal metabolites from Streptomyces philanthi RL-1-178 for maize grain coating formulations and their efficacy as biofungicide during storage.

The major safety risk of maize grain is contamination with mycotoxins. In this study, a maize-coating formulation containing freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 (DCF RL-1-178) was developed and evaluated to prevent the growth of mycotoxins during maize grain storage. In vitro studies using confrontation tests on PDA plates indicated that S. philanthi RL-1-178 inhibited the growth of Aspergillus parasiticus TISTR 3276 (89.0%) and A. flavus PSRDC-4 (95.0%). The maize grain coating formulations containing the DCF RL-1-178 (0, 5, 10, and 15% (v/v)) and the polymer polyvinylpyrrolidone (PVP-K90, 4.0% (w/v)) were tested for their efficacy in In vitro and during 5 months storage. In In vitro assay, maize coating formular containing the optimum concentration (15.0%, v/v) of the DCF RL-1-178 exhibited 54.80% and 54.17% inhibition on the growth of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 respectively. The inhibition was also illustrated by the microstructures of interactions between the coated maize grains with or without the DCF RL-1-178 and the fungal pathogens observed under microscope and SEM. Incorporating the DCF RL-1-178 or fungicidal Metalaxyl® into the polymer PVP-K90 maize grains coating resulted in the complete inhibition of the production of aflatoxin B1 (analysed by HPLC) by the two aflatoxigenic pathogens after 5 months storage at room temperature. However, the shelf-life was shortened to only 3 months during storage at room temperature with 90% relative humidity. Overall, the application of the 10-15% DCF RL-1-178 into the maize grain coating formular provides a new alternative measure to control the mycotoxins during storage for at least 5 months. The In vitro cell cytotoxicity study showed that a concentration of 15% (v/v) or 1000 µg/mL of the DCF RL-1-178 had a strong cytotoxic effect on Vero cells. These findings indicate that DCF RL-1-178 is a potential biofungicide for controlling mycotoxins contamination in maize seed storage for planting, but not maize grain storage for animal feed.

Knema retusa is antibacterial and antibiofilm against antibiotic resistant Staphylococcus aureus and S. haemolyticus isolated in bovine mastitis.

This study aimed to assess antibacterial activity of Knema retusa wood extract (KRe) against antibiotic resistant staphylococci which are causative agents of bovine mastitis. From 75 cases of intramammary infections in dairy cows, 66 staphylococcal isolates were collected, including 11 Staphylococcus aureus isolates (17%) and 55 coagulase-negative staphylococci (83%). Sixty isolates (91%) formed strong biofilms. KRe had minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against the isolates ranging 32-256 ug/mL and 64-512 ug/mL, respectively. Two-hour KRe exposures at 4×MIC, viabilities of S. aureus and S. haemolyticus decreased by 3 log10 compared to the control. Scanning EM (SEM) showed that KRe disrupted the bacterial cells of both species. KRe at 1/16×MIC significantly inhibited biofilm formation (P < 0.05) in both S. aureus and S. haemolyticus. At 1/2×MIC, S. aureus and S. haemolyticus biofilm inhibition ranged from 75 to 99%. Cells within established biofilms were disrupted 66-83% by KRe at 32×MIC. Moreover, 1/2×MIC KRe reduced bacterial adhesion to glass surfaces observed by SEM. According to GC-MS analysis, the major compound in KRe was endo-2-hydroxy-9,9-(ethylenedioxy)-1-carbethoxy bicyclo [3.3.1] nonane (E2N). Molecular docking analysis of E2N has a high affinity for staphylococcal accessory regulator A (SarA), binding free-energy - 6.40kcal/mol. The results suggested that KRe may have medicinal benefits by inhibiting the growth, biofilm, and adhesion of antibiotic resistant staphylococci isolated from bovine mastitis.

Anti-Acanthamoeba activity of a semi-synthetic mangostin derivative and its ability in removal of Acanthamoeba triangularis WU19001 on contact lens.

Garcinia mangostana L., also known as the mangosteen tree, is a native medicinal plant in Southeast Asia having a wide variety of pharmacologically active compounds, including xanthonoid mangostin. In this study, we examined the pharmacological activities of the selected semi-synthetic mangostin derivative, namely, amoebicidal activity, encystation inhibition, excystation activity, and removal capacity of adhesive Acanthamoeba from the surface of contact lens (CL). Among the three derivatives, C1 exhibited promising anti-Acanthamoeba activity against Acanthamoeba triangularis WU19001 trophozoites and cysts. SEM images displayed morphological changes in Acanthamoeba trophozoites, including the loss of acanthopodia, pore formation in the cell membrane, and membrane damage. In addition, the treated cyst was shrunken and adopted an irregular flat cyst shape. Under a fluorescence microscope, acridine orange and propidium iodide (AO/PI) staining revealed C1 induced condensation of cytoplasm and chromatin with the loss of cell volume in the treated trophozoites, while calcofluor white staining demonstrated the leakage of cell wall in treated cysts, leading to cell death. Interestingly, at the concentration ranges in which C1 showed the anti-Acanthamoeba effects (IC50 values ranging from 0.035-0.056 mg/mL), they were not toxic to Vero cells. C1 displayed the highest inhibitory effect on A. triangularis encystation at 1/16×MIC value (0.004 mg/mL). While C1 demonstrated the excystation activity at 1/128×MIC value with a high rate of 89.47%. Furthermore, C1 exhibited the removal capacity of adhesive Acanthamoeba from the surface of CL comparable with commercial multipurpose solutions (MPSs). Based on the results obtained, C1 may be a promising lead agent to develop a therapeutic for the treatment of Acanthamoeba infections and disinfectant solutions for CL.

Robusta coffee extracts inhibit quorum sensing activity in Chromobacterium violaceum and reduce biofilms against Bacillus cereus and Staphylococcus aureus.

Background and Aim: Bacillus cereus and Staphylococcus aureus cause foodborne intoxication in humans and animals. Pathogens can produce biofilms controlled by the quorum sensing system. The study aimed to investigate the antibacterial, antibiofilm, and anti-quorum sensing activities of Coffea canephora P. ex Fr. (Robusta coffee) extracts against B. cereus and S. aureus. Materials and Methods: Ethanol extracts of fruit peels and seeds of Robusta coffee were tested for antibacterial activity against B. cereus and S. aureus using a broth microdilution assay. Reduction of the biofilm formation and elimination of the viability of mature biofilm-grown cells of B. cereus and S. aureus were determined. Inhibition of quorum sensing activity in Chromobacterium violaceum by the extracts was investigated using the disk diffusion method and flask incubation assay. Results: Fresh fruit peel extract showed the strongest antibacterial activity against B. cereus and S. aureus with minimum inhibitory concentration (MIC) values of 2 and 4 mg/mL, respectively. However, the extracts did not inhibit Escherichia coli, avian pathogenic E. coli, and Pseudomonas aeruginosa at 8 mg/mL. Significant inhibition of biofilm formation at 1/2 × MIC of the fresh peel extract was detected in B. cereus (56.37%) and S. aureus (39.69 %), respectively. At 8 × MIC of the fresh peel extract, a significant elimination of the mature biofilm viability was detected in B. cereus (92.48%) and S. aureus (74.49%), respectively. The results showed that fresh and dried peel fruit extracts at 1/2 × MIC significantly reduced violacein production with the highest percentage inhibition ranging from 44.53 to 47.48% at 24 h (p ≤ 0.05). Conclusion: The results of the present study suggest the potential therapeutic benefits of Robusta coffee extracts in inhibiting the growth, biofilm, and quorum sensing of both B. cereus and S. aureus. The results put forward an alternative strategy to control the foodborne intoxications caused by both pathogens.

Conserved Candidate Antigens and Nanoparticles to Develop Vaccine against Giardia intestinalis.

Giardia intestinalis (Giardia lambia, Giardia duodenalis) infections in humans may be asymptomatic or symptomatic and associated with diarrhea (without blood), abdominal cramps, bloating, flatulence, and weight loss. The protozoan Giardia is the third most common cause of diarrhea and death in children under five, preceded only by rotavirus and by Cryptosporidium parvum and C. hominis infections. Antimicrobial drugs, particularly 5-nitroimidazole (5-NIs), are used to treat giardiasis in humans. Immunologically naive or immunocompromised host are more vulnerable to Giardia infection, whereas a degree of resistance to this protozoan is present in humans living in endemic areas. This suggests that vaccination may be a potential and appropriate means to control this parasitic disease outbreak and protect the human population. This review discusses Giardia antigens related to vaccine development. Additionally, based on the latest development of nanoparticle technology, a combination of methods for future research and development is proposed for the design of the next generation of powerful immunogens and an effective vaccine against Giardia.

Targeting Acanthamoeba proteins interaction with flavonoids of Propolis extract by in vitro and in silico studies for promising therapeutic effects.

Background : Propolis is a natural resinous mixture produced by bees. It provides beneficial effects on human health in the treatment/management of many diseases. The present study was performed to demonstrate the anti- Acanthamoeba activity of ethanolic extracts of Propolis samples from Iran. The interactions of the compounds and essential proteins of Acanthamoeba were also visualized through docking simulation. Methods: The minimal inhibitory concentrations (MICs) of Propolis extract against Acanthamoeba trophozoites and cysts was determined in vitro. In addition, two-fold dilutions of each of agents were tested for encystment, excystment and adhesion inhibitions. Three major compounds of Propolis extract such as chrysin, tectochrysin and pinocembrin have been selected in molecular docking approach to predict the compounds that might be responsible for encystment, excystment and adhesion inhibitions of A. castellanii. Furthermore, to confirm the docking results, molecular dynamics (MD) simulations were also carried out for the most promising two ligand-pocket complexes from docking studies. Results : The minimal inhibitory concentrations (MICs) 62.5 and 125 µg/mL of the most active Propolis extract were assessed in trophozoites stage of Acanthamoeba castellanii ATCC30010 and ATCC50739, respectively. At concentrations lower than their MICs values (1/16 MIC), Propolis extract revealed inhibition of encystation. However, at 1/2 MIC, it showed a potential inhibition of excystation and anti-adhesion. The molecular docking and dynamic simulation revealed the potential capability of Pinocembrin to form hydrogen bonds with A. castellanii Sir2 family protein (AcSir2), an encystation protein of high relevance for this process in Acanthamoeba. Conclusions : The results provided a candidate for the development of therapeutic drugs against Acanthamoeba infection. In vivo experiments and clinical trials are necessary to support this claim.

Antibacterial, antibiofilm, and anti-adhesion activities of Piper betle leaf extract against Avian pathogenic Escherichia coli.

Piper betle leaves have traditionally been used to treat many diseases, including bacterial infections. The present study aimed to investigate the antibacterial, antibiofilm, and anti-adhesion activities of P. betle extract against avian pathogenic Escherichia coli (APEC). The ethanol extract of P. betle leaves demonstrated strong antibacterial activity against clinical isolates of APEC with MIC and MBC values ranging from 0.5 to 1.0 mg/mL as compared with 1% DMSO, a negative control. Disruption and breakdown of the bacterial cells were detected when the cells were challenged with the extract at 2 × MIC. Bacterial cells treated with the extract demonstrated longer cells without a septum, compared to the control. The extract at 1/8, 1/4, and 1/2 × MIC significantly inhibited the formation of the bacterial biofilm of all the tested isolates except the isolate CH10 (P < 0.05) without inhibiting growth. At 1/2 × MIC, 55% of the biofilm inhibition was detected in APEC CH09, a strong biofilm producer. At 32 × MIC, 88% of the inhibition of viable cells embedded in the mature biofilm was detected in APEC CH09. Reduction in the bacterial adhesion to surfaces was shown when APEC were treated with sub-MICs of the extract as observed by SEM. Hydroxychavicol was found to be the major compound presented in the leaf extract as detected by GC-MS analysis. The information suggested potential medicinal benefits of P. betle extract to inhibit the growth, biofilm, and adhesion of avian pathogenic E. coli.

Peganum harmala Extract Has Antiamoebic Activity to Acanthamoeba triangularis Trophozoites and Changes Expression of Autophagy-Related Genes.

Peganum harmala, a well-known medicinal plant, has been used for several therapeutic purposes as it contains numerous pharmacological active compounds. Our study reported an anti-parasitic activity of P. harmala seed extract against Acanthamoeba triangularis. The stress induced by the extract on the surviving trophozoites for Acanthamoeba encystation and vacuolization was examined by microscopy, and transcriptional expression of Acanthamoeba autophagy-related genes was investigated by quantitative PCR. Our results showed that the surviving trophozoites were not transformed into cysts, and the number of trophozoites with enlarged vacuoles were not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of tested AcATG genes, i.e., ATG3, ATG8b, and ATG16, was at a basal level along the treatment. However, upregulation of AcATG16 at 24 h post treatment was observed, which may indicate an autophagic activity of this protein in response to the stress. Altogether, these data revealed the anti-Acanthamoeba activity of P. harmala extract and indicated the association of autophagy mRNA expression and cyst formation under the extract stress, representing a promising plant for future drug development. However, further identification of an active compound and a study of autophagy at the protein level are needed.

Approach toward enhancement of halophilic protease production by Halobacterium sp. strain LBU50301 using statistical design response surface methodology.

A new potent halophilic protease producer, Halobacterium sp. strain LBU50301 was isolated from salt-fermented fish samples (budu) and identified by phenotypic analysis, and 16S rDNA gene sequencing. Thereafter, sequential statistical strategy was used to optimize halophilic protease production from Halobacterium sp. strain LBU50301 by shake-flask fermentation. The classical one-factor-at-a-time (OFAT) approach determined gelatin was the best nitrogen source. Based on Plackett-Burman (PB) experimental design; gelatin, MgSO4·7H2O, NaCl and pH significantly influenced the halophilic protease production. Central composite design (CCD) determined the optimum level of medium components. Subsequently, an 8.78-fold increase in corresponding halophilic protease yield (156.22 U/mL) was obtained, compared with that produced in the original medium (17.80 U/mL). Validation experiments proved the adequacy and accuracy of model, and the results showed the predicted value agreed well with the experimental values. An overall 13-fold increase in halophilic protease yield was achieved using a 3 L laboratory fermenter and optimized medium (231.33 U/mL).