Oral administration of transgenic biosafe microorganism containing antimicrobial peptide enhances the survival of tilapia fry infected bacterial pathogen
| Autor: | Chen-Han Shih, Cheng-Yung Lin, Bing-Chang Lee, Huai-Jen Tsai, Chun-Wei Hung |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Oreochromis mossambicus food.ingredient Administration Oral Aquaculture Bacillus subtilis Aquatic Science medicine.disease_cause Microbiology Fish Diseases 03 medical and health sciences chemistry.chemical_compound Plasmid food Lactoferricin medicine Animals Environmental Chemistry Escherichia coli Disease Resistance Expression vector Bacteria Organisms Genetically Modified biology Probiotics Edwardsiella tarda Tilapia 04 agricultural and veterinary sciences General Medicine biology.organism_classification 030104 developmental biology chemistry 040102 fisheries 0401 agriculture forestry and fisheries Antimicrobial Cationic Peptides |
| Zdroj: | Fish & Shellfish Immunology. 95:606-616 |
| ISSN: | 1050-4648 |
| DOI: | 10.1016/j.fsi.2019.10.052 |
| Popis: | To develop an alternative to conventional antibiotics used in the aquaculture and livestock industries, we employed Bacillus subtilis, considered a biosafe microorganism, to express the degradable antimicrobial peptide lactoferricin. An expression plasmid pP43-6LFBII-GFP, in which reporter GFP cDNA was fused downstream of lactoferricin cDNA driven by an endogenous constitutive P43 promoter was electroporated into B. subtilis, followed by regeneration and cultivation. The putative colonies harboring plasmids were primarily screened by PCR-amplification of lactoferricin cDNA. Four transformants which were stable inheritance of plasmid containing lactoferricin cDNA included strains T1, T4, T7 and T13. Based on Western blot and Southern blot analyses, we found that transgenic strains T1 and T13 not only highly expressed exogenous recombinant lactoferricin, but also exhibited more stable inheritance of plasmids with 931 and 647 copies per cell, respectively. In the antibacterial in vitro experiment, the bactericidal activity of each microliter of cell lysate from transgenic strains T1 and T13 (5 × 108 CFU) for Escherichia coli was equivalent to 56 and 53 ng of Ampicillin dosage, respectively, while for Staphylococcus epidermidis, the equivalency T1 and T13 was 154 and 130 ng of Ampicillin dosage, respectively. Equivalencies of bacterial activity for Vibrio parahaemolyticus and Edwardsiella tarda followed suit. In the antibacterial in vivo experiment, we oral-in-tube fed tilapia fry (Oreochromis mossambicus X O. niloticus) with cell lysate from transgenic strain T1 and T13 individually. After 1-h of incubation, we immersed these treated fish fry in a water tank containing E. tarda (5 × 1011 CFU) for a 5-hr bacterial challenge. After one month cultivation, an average survival rate of 63 and 67% was observed after having fed the fish fry with transgenic strains T1 and T13, respectively. However, the average survival rate of fish fry fed with B. subtilis WT strain and transgenic strain T19 without expressing recombinant lactoferricin reached only 5 and 9%, respectively. These data indicate that the survival of fish fry infected by the intestinal pathogen tested could be significantly enhanced by feeding transgenic B. subtilis containing antibacterial peptide. Therefore, we suggest that this strategy could be applied to both aquaculture and livestock industries to (i) reduce the dependency on conventional antibiotics during seasonal outbreaks and (ii) eliminate the problem of antibiotic resistance. |
| Databáze: | OpenAIRE |
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