Characterization And Application Of Aeromonas Hydrophila Biofilm

Aeromonas hydrophila is an aquatic organism that could produce cytotoxins and enterotoxins associated with acute gastroenteritis and wound infections in human and hemorrhagic septicaemia of fish, reptiles and amphibians. Aeromonas hydrophila can form biofilms which are ubiquitous and play a multitude of important roles in different environments. In this work, characterization and application of Aeromonas hydrophila.biofilm were studiedAeromonas hydrophila (Ah) biofilm(BF) model in vitro was established and antibiotic effects of 11 antimicrobials on BF and free-cell(FC) bacteria were tested. It was found that the ratio of MBC between BF and FC bacteria was even more than 32 tol . The result suggested that BF enabled Ah to have strong resistance to antimicrobials and the potential influence of BF should be highly considered.The BF formings of Ah J-1, N-1,W-1 and 4332 strains were detected by AgNO3 solution staining and scanning electron microscopy(SEM). The pathogenic strain Ah J-1 and N-1 could form BF, whereas the non-pathogenic strain Ah W-1 and 4332 could not. Ah J-1 BF bacteria survived from being treated respectively with low temperature and 0.01% Benzalkonium bromide solution while Ah J-1 FC bacteria hardly survived under the above conditions. The biofilm-relative gene, transcriptional regular (AhyR) gene of Aeromonas species, was positively amplified from strain Ah J-l and N-1.genomic DNA via PCR, but not from strain Ah W-1 and 4332. The nucleotide sequence of Ah J-l strain showed 99% homology to that of Ah A1 AhyR gene registered in Genbank. It was suggested that the AhyR gene was not only connected with virulence, but also with BF formation.The mice and swordtail fish (Xiphophorus helleri) were fed with the Aeromonas hydrophila J-1 biofilm(BF)and free cell(FC) vaccine inactivated by heat, which were mixed with ingredients of feed. The relative percentage survival(RPS), the antibody titres, transformation of blood lymphocyte in mice and the slgA secretion of intestinal in mice were studied. It was suggested that the BF vaccine could enhance the immunological response offish and mice,especially the gut-associated lymphoid tissue.The reduced virulent hemolysins-deficient mutant of Aeromonas hydrophila J-1 by Tn916 insertions was screened and the mice were fed with the mutant.The antibody level reached higher 5 weeks after the vaccination. After vaccinated and unvaccinated mice werechallenged with the parent J-1 strain, survival ability of the former was higher than that of the latter. It was suggested that the hemolysins-deficient mutant strain TJ-1 remained immunogenicity.The vaccine of Aeromonas hydrophila J-l strain was inactivated with formalin and immunized to 60 Crucian carps (gynogenetic Carassiu auratus gibelio) by bath.These Crucian carps were divided into three groups. One group was immunized with the vaccine plus adjuvant IMS 1312, another group with the vaccine alone and the rest group was served as control group. Agglutinating antibody tilers in fish sera and the relative percentage survival were detected 35 days after immunization. It was suggested that bath immunization of Aeromonas hydrophila vaccine plus adjuvant IMS 1312 could significantly enhance the immunological response of fish.