Acute Hepatopancreatic Necrosis Disease (AHPND) is a widespread disease targeting cultured shrimps, which results in heavy losses in many regions around the world including Vietnam. The causative agent of the disease is Vibrio paraheamolyticus which is a Gram-negative bacterium present ubiquitously in marine environment. However, this normal floral organism once acquired a plasmid containing Pir- toxin encoding gene (AHPND plasmid) can transform into a highly toxic strain (AHPND strain) with the ability to kill a large number of cultured shrimps in a short period. To understand more on the virulence of V. parahaemolyticus, 17 different V. parahaemolyticus isolates collected from different locations in Southern Vietnam have been analyzed for the presence of AHPND plasmid using PCR method and their extracellular enzyme profile using agar- based method. Seven enzymes which are known to be important virulence factors of the bacterium were studied including caseinase, gelatinase, lecithinase, hemolysin, elastase, lipase and chitinase. Results showed that among 17 studied isolates, six were detected to have AHPND plasmid. Enzymatic activity was observed for caseinase, gelatinase and lecithinase while undetectable for others. The ability to produce these three enzymes varied among different V. parahaemolyticus isolates. Lecithinase appeared in all isolates while the presence of caseinase and gelatinase varied. This indicated that lecithinase seems to be core enzyme of V. paraheamolyticus and the extracellular enzymes do not correlate with the presence of AHPND plasmid. In conclusion, our data suggested that the virulence of Vibrio parahaemolyticus mostly depends on the presence of AHPND plasmid other than extracellular enzymes.
This research demonstrated antibacterial activity of wood-decaying fungi to treat the aquaculture pathogenic bacterium, Vibrio parahaemolyticus (V. parahaemolyticus). Three wild wood-decaying fungi (WDF) collected in Southern Vietnam including Flavodon flavus (F. flavus), Physisporinus vitreis (P. vitreis), and Schizophyllum commune (S. commune) were tested to treat V. parahaemolyticus presented in aquaculture wastewater. Fungal adaptation to different salt concentrations at 0, 10, 15, 20, and 30‰ was tested for the assessment of application potential of these fungi in the aquaculture farming. Fungal mycelium could adapt differently to saline conditions. All three strains grew well at low salt concentration (0-10‰), but only F. flavus survived up to 20‰ NaCl. For the antibacterial ability toward V. parahaemolyticus, two experiments using mycelium culture broth and mycofiltration were performed. Results revealed that all the three species of WDF effectively inhibited V. parahaemolyticus. Thus, mycelium culture broth of P. vitreis, S. commune, and F. flavus could inhibit 98.7, 96.5 and 97.8% V. parahaemolyticus, respectively after 8 hours. Even higher inhibitory effects were observed in the mycofiltration experiment, the efficiency reached 100% for P. vitreis, and 98% for F. flavus and S. commune after 8 hours. The obtained results showed very good application potential of myco-remediation technique using WDF in the treatment of V. parahaemolyticus in aquaculture farming. Mycofilter can be used as a new approach for controlling aquaculture disease, specifically Vibrio sp. treatment based on the antibiotic capability of these WDF. The reduction in pH of the mycelium broth can illustrated for the acidification during the mycelium development and could link with the bactericide capacity of these WDF.
The antibacterial activity of twelve different wood-decay fungal isolated in Southern Vietnam was investigated to find out the new potential antibiotics produced by macro fungi. The antibacterial activity of the collected fungal isolates was tested by three screening methods against seven different strains of bacteria: Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, Vibrio cholerae, and Salmonella typhi. The screening test were performed at first with mycelium grew on surface of agar medium similar to a disc diffusion test, next by an extracellular metabolite test by flood-plate method, and finally with an intracellular metabolite test using grounded mycelium fluid. To determine the activity of metabolites produced in the mycelium, the fungal isolates were grown on culture substrates to promote mycelium formation. Antibacterial activity of the selected fungi was confirmed by disc diffusion method using crude extract of the mycelium harvested from the substrate cultures. Four fungal species identified with strong antibacterial compounds produced by their mycelium including Pycnoporus sanguineus, Flavodon flavus, Fomitopsis ostreiformis and Meripilus giganteus. Crude extract of Pycnoporus sanguineus showed the strongest antibacterial activity, with 7/7 bacterial strains inhibited. Fomitopsis ostreiformis antibacterial activity was recognized with the ability to inhibit 6/7 bacterial strains. Flavodon flavus and Meripilus giganteus showed weaker activity than other two species, with only 4/7 strains inhibited. The fungal isolates identified with antibacterial activity discovered in this study can be potential candidates for the new source of antibiotic to fight against new generations of antibiotic resistance bacteria in the future.
Polysaccharide monooxygenases (PMOs) and Glycoside Hydrolases (GH) play a crucial role in breaking down chitin and cellulose. Research on PMOs has mainly focused on their role in the production of bioethanol from biomass. Still, the potential of PMOs as virulence factors in microorganisms is not been fully studied. Some pathogenic bacteria secrete PMOs, which have been identified as virulence factors. The ability of Vibrio species to metabolize chitin is well-known, but the question of which Vibrio species contains the PMO gene remains unanswered. Materials and methods: Black tiger shrimp Penaeus monodon was collected from a culture pond in Long An, Vietnam. Thiosulfate-citrate-bile salts-sucrose and CHROMagar Vibrio agar were used to isolate, Vibrio bacteria from the shrimp samples. Mixed bases primers were designed to detect PMOs gene from Vibrio strains. Results: Total of 35 different strains of bacteria were isolated from the guts and pancreas of the shrimp sample. Based on biochemical tests, there were a total of 20 different Vibrio strains identified among 35 isolated strains. The PCR results showed that six isolates could have the PMOs gene. The partial 16S rDNA fragments of six isolate Vibrio strains were sequenced. Comparing 16S rDNA sequences with sequences available in NCBI databases revealed that all the sequences are Vibrio spp. 16S rDNA sequences.
Polysaccharide monooxygenases (PMOs) cleave glycoside linkages in polysaccharides, which has great potential in manufacturing biofuel from polysaccharide biomasses. Therefore, heterologous production of PMOs is required for the development of their application. We aim to clone PMO genes into the plasmid pEX2B to express PMOs in Aspergillus oryzae, a filamentous fungus widely used for industrial enzyme production.Here we cloned the NCU08746 gene, an AA13 PMO from Neurospora crassa, into pEX2B. We successfully designed primers and amplified the NCU08746 gene from the N. crassa genome. NCU08746 was then cloned into the pEX2B vector. The recombinant plasmid pEX2B-NCU08746 was subsequently transferred into A. tumefaciens AGL1 prior to being transferred into A. oryzae. This work provides the foundation for further study on PMOs expression in A. oryzae.
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