Identification And Characterization Of Biofilm-associated Genes Of Aeromonas Hydrophila

Bacteria often exist as biofilms in nature, which is bacterial communities attached to a surface and surrounded by extracellular polymer substances (EPS). Recently, a lot of work had demonstrated that most bacteria can form biofilm. Compared to planktonic bacteria, bacteria living in biofilm often not only possess the virulence of the planktonic, but also appear resistance to the host immune system and many antibacterial agents, so generate even up to1000 times more resistant to antibacterial compounds than planktonic bacteria, leading that it is difficult to claerd up the infection and it caused serious damages.So studing of the pathogenic bacteria biofilm become a hot topic.The characteristics of biofilm formation of pathogenic Aeromanas hydrophila B11 was investigated by the method of modified microtiter-plate test. The results indicated that: A. hydrophila B11 strain can form biofilm on the surface of polystyrene microtiter plate, the bacterial biofilm formation was affected by different environmental factors such as temperature, bacterial concentration, pH, Mg²⁺ and Ca²⁺ concentration and so on; muscle extract of eel promoted the biofilm formation.Based on the results above, transposon tagging method that was performed by using the suicide plasmid pLOF/Km,carring a transposon mini-Tn10,was transferred to the recipient A. hydrophila. to generate an insertional mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOFKm) and the recipient A. hydrophila. B11 by filter mating.214 mutants were screened and two mutants stably exhibited defective in biofilm formation.To make sure the mutant phenotype was associated with only a single insertion, two pair of primers designed according to the known kanamycin resistance gene sequence used as tag. One pair was used to PCR using the genome of the mutants as template and the other pair was used to cloning probe sequence for Southern blot analysis by PCR.And the results indicate that the mutations were caused by the insertion of the tranposon and the insertion site was single. Genes tagged by mini-Tn10 insertions were amplified by thermal asymmetric interlaced PCR(Tail-PCR) and modified thermal asymmetric interlaced PCR(Actail-PCR). The sequences blasting results indicated that the gene sequence derived from B188 showed the identity of 92% to cytochrome c4 gene of A.hydrophila subsp. hydrophila ATCC 7966 and 88% to A.salmonicida subsp. salmonicida A449.To the B212,the partial flanking sequence blasting results showed the identity of 90% to cytochrome c4 gene of A.hydrophila subsp. hydrophila ATCC 7966 and 88% to A.salmonicida subsp. salmonicida A449.The biological characteristics of the mutants including growth,early stage of adhesion,intracellular survival were determined.The results displayed that B188 were greatly impaired in all the three characteristics examined when comparied with the wild strain B11. The mutant strain B212 exhibited no difference in growth, but impairied in adhesion ability and intracellular survival.The results indicated pathogenic A.hydrophila can form apparent biofilm, both the cytochrome c4(Cyt c4)and the MshQ genes were associated with the biofilm formation.The mutation of the two genes caused the biologic characteristics including the growth, adherenc and intracellular survival and so on varied in different degree and all these characteristics were related directly to the toxicity, so it indicated that the formation of biofilm may be one of the toxic factors.