鹅源鼠伤寒沙门菌crp、hfq基因缺失株的构建及生物学特性分析

Construction and biological characteristics analysis of the crp and hfq deletion mutants of Salmonella typhimurium isolated from geese

为构建鹅鼠伤寒沙门菌单基因缺失株并鉴定其生物学特性,本研究以广东地区鹅源鼠伤寒沙门菌流行株A29为研究对象,采用λ-Red同源重组技术,分别构建crp、hfq基因缺失株(A29Δcrp和A29Δhfq)及相应基因回补株,并分别采用相应引物经PCR鉴定。通过细菌培养,对各菌株生物学特性进行比较;通过PCR鉴定各菌株的遗传稳定性;采用K-B纸片扩散法检测各菌株的药物敏感性;将各菌株纯培养物经腹腔注射4周龄小鼠,测定各菌株对小鼠的毒力,并观察感染后小鼠的临床症状及各组织剖检病变与组织病理变化。PCR和测序结果表明,正确构建各基因缺失株和相应回补株。生物学特性试验结果显示,与亲本株相比,A29Δcrp菌落直径极显著变小(P<0.001),不产酸、不产生H_(2)S,菌体极显著变短(P<0.001);A29Δhfq菌落边缘不整齐,不产生H_(2)S,菌体极显著变长(P<0.001),表明crp、hfq基因影响细菌的菌落形态、生化特性和形态特征。菌株遗传稳定试验结果显示,菌株传至60代时,基因缺失株仍能够稳定缺失。药敏试验结果显示,与亲本株比较,A29Δcrp对氨基糖苷类、碳青霉烯类、环丙沙星、甲氧苄啶、阿奇霉素等药物的敏感性降低,对四环素类药物的敏感性增强,A29Δhfq对除青霉素类、头孢唑啉以外的12种药物的敏感性增强。小鼠毒力试验结果显示,与亲本株的LD_(50)(2.479×10^(4)cfu)相比,A29Δcrp的LD_(50)(5.379×10^(5) cfu)和A29Δhfq的LD_(50)(5.012×106 cfu)均明显上升。各菌株感染小鼠后的临床症状及各组织剖检病变基本一致;组织病理变化观察显示,与亲本株相比,A29Δcrp组小鼠肝组织可见多量大小不一的坏死灶,脾组织红髓面积增大、白髓面积减小,A29Δhfq对小鼠脾脏、肝脏及脑组织损伤均较小。本研究为探究鼠伤寒沙门菌crp和hfq基因功能奠定了基础,同时也为进一步研发鹅沙门菌病疫苗提供了参考依据。

In order to develop a single gene deletion strain of Salmonella typhimurium and verify its biological characteristics,this study took the prevalent strain A29 of Salmonella typhimurium from geese in Guangdong province as the object,and usedλ-Red homologous recombination technology to construct crp and hfq single-gene strains(A29Δcrp and A29Δhfq)and the gene revertants were identified by PCR with corresponding primers.The bacterial culture were prepared to compare biological characteristics of each strain,PCR to test the genetic stability of the strain,K-B disk diffusion method to test drug sensitivity.The pure bacterial culture of the strains were injected into 4-week-old mice,and then the virulence of the strains to mice was determined,and the clinical symptoms,pathological changes and histopathological changes of each tissue were observed after infection.The results of PCR and sequencing showed that the gene deletion strains and corresponding complement strains were correctly constructed.The results of biological characteristics test showed that compared with the parent strain,the diameters of the A29Δcrp colonies decreased significantly(P<0.001),no acid and H_(2)S production was detected,and the length of the cells decreased significantly(P<0.001).The edge of A29Δhfq colonies was irregular,no H_(2)S was produced,and the length of the bacterial cell was significantly longer(P<0.001),showing that crp and hfq affect the bacterial colony formation,biochemical characteristics and morphological characteristics.The result of PCR identification of the strain showed that when the strain was passed to 60 generations,the gene deletion strain could still be stably inherited.The results of drug sensitivity test showed that compared with the parent strain,the sensitivity of A29Δcrp to aminoglycosides,carbapenems,ciprofloxacin,trimethoprim,azithromycin and other drugs decreased,and the sensitivity to tetracycline drugs increased.A29Δhfq was more sensitive to 12 drugs except penicillin and cefazolin.The results of mice virulence test showed that the LD_(50)(5.379×10^(5)cfu)of A29Δcrp and LD_(50)(5.012×106cfu)of A29Δhfq decreased significantly,compared with the LD_(50)(2.479×10^(4)cfu)of the parent strain.The clinical symptoms and pathological changes of all tissues of mice infected by different strains were the same.Histopathological observation showed that compared with the parent strain,in A29Δcrp group there were many necrotic foci of different sizes in liver tissue,and the area of red pulp increased and the area of white pulp decreased in spleen tissue.A29Δhfq displayed less damage to spleen,liver and brain tissue of mice,but stronger affinity to brain tissue.This study laid a foundation for exploring the functions of the crp and hfq genes of Salmonella typhimurium,and also provided a reference basis for further research and development of Salmonella goose disease vaccine strains.