副猪嗜血杆菌lpxM基因缺失株构建及生物学特性分析

【目的】研究副猪嗜血杆菌脂寡糖合成相关基因lpxM对其生长、生物被膜形成能力、抗50%猪血清杀菌能力、对巨噬细胞毒力和抗生素敏感性部分生物特性的影响,为揭示HPS致病机制,lpxM基因缺失疫苗的构建奠定理论基础,为猪场防治HPS进行药物选择时提供依据。【方法】以高致病性血清5型HPS地方分离株H45为研究对象,自杀性质粒PK18mobsacB为载体,通过自然转化法将构建好的重组质粒转进H45,使其在抗生素的压力下发生同源重组,最终经过抗生素筛选并通过PCR和测序验证得到lpxM基因缺失株H45-△lpxM,比较两者之间部分生物学特性的差异。测定两者的OD₆₀₀-t关系曲线比较生长情况;用结晶紫染色法比较两者在培养24h后生物被膜形成的能力;测定两者在50%猪血清中存活率,比较两者的抗血清补体杀菌能力;将两者同时刺激小鼠单核巨噬细胞RAW264.7细胞,作用时间为6、12和24h,检测细胞培养上清液中LDH的释放量,比较两者对巨噬细胞毒力影响;利用K-B 纸片扩散法研究两者对氨苄西林等临床上常用13种抗生素和多粘菌素B抗生素敏感性,通过测定抑菌圈直径来并参照耐药标准判断对抗生素的敏感性。【结果】成功构建lpxM基因缺失株H45-△lpxM,生长情况比较发现菌株生长前期缺失株慢于野生株,8h后保持一致,结果表明lpxM基因缺失能在一定程度上抑制H45的生长;两者均能形成生物被膜,但是缺失株弱于野生株;野生菌株在50%猪血清中存活率为16.1%,而缺失株只有0.71%,缺失株明显低于野生菌株;两者均能引起巨噬细胞的死亡,作用6、12和24h后,野生株对细胞的致死率分别为6.63%、10.86%和22.17%,而缺失株对细胞的致死率为2.62%、6.35%和18.01%,结果表明随着作用时间的延长,两者对细胞的毒力作用越来越大,具有一定的时间依赖性,在各个时间点,缺失株的毒力作用均低于野生株;抗生素敏感性结果发现,两者对头孢噻吩等10种抗生素均表现敏感,对恩诺沙星均表现抗性,但对阿莫西林克拉维酸、磺胺二甲异噁唑和氨苄西林三种抗生素的耐药性发生了较大的差异,阿莫西林克拉维酸和磺胺二甲异噁唑两种抗生素由耐药变成了敏感,氨苄西林也从中介变成敏感,结果表明lpxM基因缺失对H45抗生素敏感性具有一定影响。【结论】lpxM基因缺失能一定程度地抑制HPS生长,降低其生物被膜形成能力、抗血清杀菌能力和对巨噬细胞的毒力作用,增大对临床上多数常用抗生素的敏感性,揭示lpxM基因可能是HPS毒力基因,与HPS的致病能力密切有关,但具体机制还需要进一步研究。

Construction of lpxM Gene Deletion Strain of Haemophilus parasuis and It's Some Biological Characteristics

【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.