Clonging And Functional Identification Of The Genes LuxS And PilA Related To Biofilm Formation Of Haemophilus Parasuis

Bacterial biofim is a thin layer of microcolonies adhering to the surface of a structure or necrotic tissue, together with the extracellular polymers that they secrete. Research on the genes related to biofilm formation is important to elucidate the mechanism of bacterial chronic infection.Although the genome sequence of Haemophilus parasuis is completed, the genes related to biofilm formation and their function of Haemophilus parasuis is unknown to date. According to the theroy, the genes related to quorum sensing signal and flagella are involved with the biofilm formation, a strain of Haemophilus parasuis with strong ability of biofilm formation was isolated and identified in the present study. The gene luxS related to quorum sensing signal and flagella gene pilA deficient mutants of the isolate were constructed, respectively, and their ability of biofilm formation was evaluated.The six strains of Haemophilus parasuis were isolated from suspected pigs with Glasser's disease in Gansu and Qinghai Province by bacterial culturing and identified by biochemical tests and 16S rRNA gene sequence analysis. One of the Haemophilus parasuis strains, designated HPS-LZ001, had the strongest ability of biofilm formation among 6 isolates and 3 reference strains according to the assays of crystal violet staining and electron microscopy.The in vitro Ciprofloxacin, Chloramphenicol and Kanamycin susceptibility tests were conducted with planktonic bacteria and biofilm of the two strains with strong ability of biofilm formation and two strains with weak ability of biofilm formation, respectively, by the assays of broth microdilution and polystyrene microtiter. It showed that antibiotic resistance was greatly increased when the biofilm of Haemophilus parasuis formed.The luxS and pilA gene were amplified with the template of HPS-LZ001 genomic DNA and designed primers. The alignment analysis showed luxS and pilA gene of HPS-LZ001 were 94% and 87% homologous with the corresponding genes of the HPS (GenBank accession no.NC 011852).The luxS gene upstream 687bp and downstream 459bp and pilA gene upstream 468bp and downstream 579bp as homologous arms of target vector were cloned respectively, and ligated by recombinant PCR technique. The resulting plasmids of pDM4-ΔluxS and pDM4-ΔpilA were obtained by ligating the recombinant PCR products into the suicide vector pDM4. The plasmids of pDM4-ΔluxS and pDM4-ApilA were transformed into E.coli S17-1λand conjugal transfer occurred when the transformants were mixed with HPS-LZ001 on TSA plates. The suicide vector was removed on the 15% sucrose TSA plate. PCR identification and Southern blotting indicated the luxS and pilA gene deficient mutants were constructed and screened successfully. The quantitative real-time PCR revealed the gene transcriptional difference between the gene deficient mutants and the wild-type strain.Compared with wild-type HPS-LZ001, the obvious decrease of the ability of biofilm formation in luxS and pilA gene deficient mutants were observed. The gene luxS and pilA could partially complete the ability of biofilm formation of the corresponding gene deficient mutants.In summary, the luxS gene deficient mutant and pilA gene deficient mutant of Haemophilus parasuis HPS-LZ001 and their corresponding gene complement mutants were constructed with homologous recombination method. The assays of their ability of biofilm formation showed the gene luxS and pilA might were involved in the biofilm formation of Haemophilus parasuis.