猪霍乱沙门菌抗噬菌体菌株筛选及受体基因突变位点分析

Screening of Bacteriophage Resistant Strains of Salmonella cholerae and Analysis of Mutation Sites in Receptor Gene

【目的】筛选抗噬菌体菌株并分析其受体基因突变位点,为解析猪霍乱沙门菌噬菌体抗性突变的产生机制提供理论依据。【方法】以猪霍乱沙门菌CICC 21501及其裂解性噬菌体vB_SenS_S528(噬菌体S528)为研究对象,通过波动实验筛选自发突变的抗噬菌体菌株,观察菌落形态,并测定其对噬菌体的敏感性、吸附特性、生长曲线及对温度和pH的敏感性。通过全基因组重测序结合PCR验证定位耐受基因突变位点。【结果】成功筛选出1株抗噬菌体S528的突变菌株,命名为B6-2。B6-2菌落边缘粗糙,与野生菌相比,生长曲线无显著差异,对pH表现出敏感性,在40、50℃时表现出温度耐受性。噬菌体S528对抗噬菌体菌株B6-2的吸附率减少60%(对野生菌吸附率为93%)。通过全基因组重测序及比对分析得知,B6-2菌株有3个位点发生了突变,分别为PROKKA_04510基因中2个位点和rfbC基因中的1个位点发生突变。突变基因片段的PCR产物电泳及测序结果均表明,PROKKA_04510基因上的2个位点并未发生突变,而真正发生突变的位点位于rfbC基因的350 bp处,碱基由C突变为T。【结论】筛选到1株与受体改变有关的抗噬菌体菌株B6-2,其通过rfbC基因上的碱基突变来阻止噬菌体吸附。

【Objective】This study was aimed to screen the phage-resistant strains, and analyze the mutation sites of the receptor gene, in order to provide theoretical basis for revealing the mechanism of phage resistance mutation of Salmonella cholerae.【Method】 Salmonella cholerae CICC 21501 and its lytic phage vB_SenS_S528(phage S528) were used as the research objects, the spontaneous phage-resistant strains were screened by fluctuation experiment, and the colony morphology was observed.The sensitivity to phage, adsorption characteristics, growth curve and sensitivity to temperature and pH were measured.The whole genome resequencing and PCR were used to verify the mutation site of the potential receptor gene.【Result】 A mutant strain resistant to phage S528 named B6-2 was successfully obtained.The strain B6-2 displayed rough edge of colony different from wild-type strain.Compared with wild bacteria, the growth curves showed no significant difference, but showed sensitivity to pH and temperature tolerance at 40 and 50 ℃. The adsorption rate of phage S528 to the phage-resistant strain was reduced by 60%(93% to host strain).The whole genome resequencing and comparative analysis showed that there were 3 mutation sites of the phage-resistant strain B6-2,2 sites of PROKKA_04510 gene and 1 site of rfbC gene, respectively.The PCR product electrophoresis and sequencing results of the mutant gene fragment showed that 2 sites of PROKKA_04510 gene were not mutated, but the actual mutation site was located at 350 bp of rfbC gene, and the base was mutated from C to T.【Conclusion】 A phage-resistant strain B6-2 related to receptor changes was screened and it might prevent phage adsorption through base mutation in rfbC gene.