关键词: 副猪嗜血杆菌, lpxM基因, 生物被膜, 抗血清杀菌, 细胞毒性, 抗生素耐药性

Abstract:

【Objective】The effects of lpxM, a gene related to lipopolysaccharide synthesis of Haemophilus parasuis, on its some biological characteristics such as growth, biofilm formation ability, antibacterial ability against 50% porcine serum, virulence to macrophage and antibiotic susceptibility were studied, which laid a certain theory for revealing the pathogenesis of HPS and the construction of lpxM gene deletion vaccine, and also provided a basis for the selection of drugs for the prevention and treatment of HPS on pig farms.【Method】The highly pathogenic serotype 5 of HPS local isolate H45 was used as the research object, and the suicide plasmid PK18mobsacB was used as a vector. The constructed recombinant plasmid was transformed into H45 by natural transformation method, and homologous recombination occurred under the pressure of antibiotics. Finally, the lpxM gene deleted strain H45-ΔlpxM was obtained by antibiotic screening, and verified by PCR and sequencing. The differences in biological characteristics between the two were compared. The OD600-t curve of the two was used to compare the growth conditions. The ability of the two to form biofilm formation after 24 hours of culture was compared by crystal violet staining. The survival rate of the two in 50% porcine serum was compared, and the antiserum complements of the two were compared. The two both simultaneously stimulated mouse monocyte macrophage RAW264.7 cells for 6, 12 and 24h, and detected the release of LDH in cell culture supernatant for comparing the virulence of macrophages. The difference in antibiotic susceptibility was studied by KB diffusion method. Antibiotics included 13 antibiotics in clinical practice, such as ampicillin and polymyxin B antibiotics. The sensitivity to antibiotics was determined by measuring the diameter of the inhibition zone according to the resistance standard. 【Result】 The lpxM gene deletion strain H45-ΔlpxM was successfully constructed. The growth of the deleted strain was found to be slower than the wild-type strain in the early growth stage, but they were consistent after 8 hours. The results showed that the lpxM gene deletion could inhibit the growth of H45 to some extent. The two both could form biofilm, but the deleted strain was weaker than the wild strain. The wild strain had a survival rate of 16.1% in 50% porcine serum, while the deletion strain was only 0.71%, and the deletion strain was significantly lower than the wild strain. The two both could cause macrophage death. The lethal rate of wild-type plants was 6.63%, 10.86% and 22.17%, after 6, 12 and 24h, respectively, while the lethal rate of the deleted strain was 2.62%, 6.35% and 18.01%, respectively. With the prolongation of action time, the virulence effect was more obvious, and had a certain time dependence. At each time point, the virulence of the deleted strains was lower than that of the wild strain. The antibiotic sensitivity results showed sensitive to ten antibiotics such as thiophene and showed resistance to enrofloxacin. But the resistance to amoxicillin clavulanic acid, sulfamethoxazole and ampicillin changed greatly. Amoxicillin clavulanic acid and sulfamethoxazole became sensitive from resistance and ampicillin also changed from intermediate to sensitive. The results showed that lpxM gene deletion had certain influence on some antibiotic sensitivity of H45.【Conclusion】 The deletion of lpxM gene could inhibit growth of HPS to a certain extent, reduce its biofilm formation ability, anti-serum bactericidal ability and virulence to macrophages, and increase sensitivity to some commonly used antibiotics, revealing that lpxM gene might be the virulence gene of HPS and be closely related to the pathogenic ability of HPS, but the specific mechanism needed further study.

Key words: Haemophilus parasuis, lpxM gene, biofilm, anti-serum bactericidal, cytotoxicity, antibiotic resistance