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

doi:10.16431/j.cnki.1671-7236.2022.11.041

摘要/Abstract

摘要： 【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。

关键词: 猪霍乱沙门菌; 噬菌体抗性; rfbC基因

Abstract: 【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.

Key words: Salmonella cholerae; phage resistance; rfbC gene

中图分类号:

S859.79

李晓宇, 张慧君, 迟新月, 李根, 王丽丽, 魏炳栋, 孙晓雯, 李纪彬, 徐永平. 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[J]. 中国畜牧兽医, 2022, 49(11): 4495-4502.

LI Xiaoyu, ZHANG Huijun, CHI Xinyue, LI Gen, WANG Lili, WEI Bingdong, SUN Xiaowen, LI Jibin, XU Yongping. Screening of Bacteriophage Resistant Strains of Salmonella cholerae and Analysis of Mutation Sites in Receptor Gene[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4495-4502.

参考文献

[1] KONGSTED H, TOFT N, NIELSEN J P.Risk factors and epidemiological characteristics of new neonatal porcine diarrhoea syndrome in four Danish herds[J].BMC Veterinary Research, 2014, 10(1):151.
[2] 徐引弟, 郭爱珍, 陈焕春.猪霍乱沙门氏菌的危害与防制[J].中国畜牧兽医, 2008, 35(3):115-117. XU Y D, GUO A Z, CHEN H C.Harm and control of Salmonella choleraesuis[J].China Animal Husbandry & Veterinary Medicine, 2008, 35(3):115-117.(in Chinese)
[3] 孙景昱, 刘思洁, 赵薇, 等.2011~2018年吉林省食品中沙门氏菌的污染监测及血清型别分布[J].食品安全质量检测学报, 2020, 11(24):9377-9382. SUN J Y, LIU S J, ZHAO W, et al.Contamination monitoring and serotype distribution of Salmonella in food in Jilin province from 2011 to 2018[J].Journal of Food Safety and Quality, 2020, 11(24):9377-9382.(in Chinese)
[4] 邱颖, 王宇卓, 张丽敏, 等.2015-2017年河北省廊坊市食品中沙门氏菌污染状况及血清型监测分析[J].医学动物防制, 2019, 35(2):134-136. QIU Y, WANG Y Z, ZHANG L M, et al.Monitoring and analysis of Salmonella contamination and serotypes in food in Langfang city, Hebei province from 2015 to 2017[J].Journal of Medical Pest Control, 2019, 35(2):134-136.(in Chinese)
[5] PAN Y L, FANG Y, FENG Y Q, et al.Discovery of mcr-3.1 gene carried by a prophage located in a conjugative IncA/C2 plasmid from a Salmonella choleraesuis clinical isolate[J].Journal of Infection, 2021, 82(3):414-451.
[6] 刘艳红, 李颖, 吕淑霞.沙门氏菌和大肠杆菌耐药性的研究进展[J].黑龙江畜牧兽医, 2016, 10:88-90. LIU Y H, LI Y, LYU S X.Research progress on drug resistance of Salmonella and Escherichia coli[J].Heilongjiang Animal Science and Veterinary Medicine, 2016, 10:88-90.(in Chinese)
[7] ROHWER F, SEGALL A M.A century of phage lessons[J].Nature, 2015, 528:46-47.
[8] DALMASSO M, HILL C, ROSS R P.Exploiting gut bacteriophages for human health[J].Trends in Microbiology, 2014, 22(7):399-405.
[9] CHAN B K, ABEDON S T, LOC-CARRILLO C.Phage cocktails and the future of phage therapy[J].Future Microbiology, 2013, 8(6):769-783.
[10] KUTATELADZE M, ADAMIA R.Bacteriophages as potential new therapeutics to replace or supplement antibiotics[J].Trends in Biotechnology, 2010, 28(12):591-595.
[11] GE H J, HU M Z, ZHAO G, et al.The "fighting wisdom and bravery" of tailed phage and host in the process of adsorption[J].Microbiological Research, 2020, 230:126344.
[12] RAKHUBA D V, KOLOMIETS E I, DEY E S, et al.Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell[J].Polish Journal of Microbiology, 2010, 59(3):145-155.
[13] SILVA J B, STORMS Z, SAUVAGEAU D.Host receptors for bacteriophage adsorption[J].FEMS Microbiology Letters, 2016, 363(4):fnw002.
[14] SHIN H, LEE J H, KIM H, et al.Receptor diversity and host interaction of bacteriophages infecting Salmonella enterica Serovar Typhimurium[J].PLoS One, 2012, 7(8):e43392.
[15] 蒋依倩, 齐宇, 徐凤宇.猪霍乱沙门菌裂解性噬菌体的分离鉴定及生物学特性研究[J].中国兽药杂志, 2015, 49(11):5-10. JIANG Y Q, QI Y, XU F Y.Isolation, Identification and biological characteristics study of lytic Salmonella choleraesuis bacteriophage[J].Chinese Journal of Veterinary Drug, 2015, 49(11):5-10.(in Chinese)
[16] 张玉宇, 张敏, 黄赞, 等.1株跨属感染猪霍乱沙门菌和大肠杆菌烈性噬菌体的分离及其生物学特性[J].畜牧兽医学报, 2020, 51(8):2032-2038. ZHANG Y Y, ZHANG M, HUANG Z, et al.Isolation and characterization of a lytic polyvalent bacteriophage infecting Salmonella choleraesuis and Escherichia coli[J].Acta Veterinaria et Zootechnica Sinica, 2020, 51(8):2032-2038.(in Chinese)
[17] PARRA B, ROBESON J.Selection of polyvalent bacteriophages infecting Salmonella enterica serovar Choleraesuis[J]. Electronic Journal of Biotechnology, 2016, 21:72-76.
[18] TAN D M, ZHANG Y Y, QIN J H, et al.A frameshift mutation in wcaJ associated with phage resistance in Klebsiella pneumoniae[J].Microorganisms, 2020, 8:378.
[19] KIM H, KIM M, BAI J, et al.Colanic acid is a novel phage receptor of Pectobacterium carotovorum subsp.carotovorum phage POP72[J].Frontiers in Microbiology, 2019, 10:143.
[20] 包红朵, 朱树娇, 张辉, 等.沙门菌噬菌体抗性菌的筛选鉴定及致病力研究[J].微生物学报, 2020, 60(4):759-768. BAO H D, ZHU S J, ZHANG H, et al.Screening, identification and pathogenicity of phage-resistant strain of Salmonella[J].Acta Microbiologica Sinica, 2020, 60(4):759-768.(in Chinese)
[21] 毛普加, 冯梦蝶, 洪愉, 等.抗噬菌体甲型副伤寒沙门菌突变菌的构建及其生物学特性[J].吉林大学学报(医学版), 2015, 41(2):275-281. MAO P J, FENG M D, HONG Y, et al.Construction of anti-phage Salmonella paratyphi A mutant strains and its biological characteristics[J].Journal of Jilin University (Medicine Edition), 2015, 41(2):275-281.(in Chinese)
[22] AZAM A H, TANJI Y.Bacteriophage-host arm race:An update on the mechanism of phage resistance in bacteria and revenge of the phage with the perspective for phage therapy[J].Applied Microbiology and Biotechnology, 2019, 103(5):2121-2131.
[23] ROSTØL J T, MARRAFFINI L.(Ph)ighting phages:How bacteria resist their parasites[J].Cell Host & Microbe, 2019, 25(2):184-194.
[24] KIM M, KIM S, PARK B, et al.Core lipopolysaccharide-specific phage SSU5 as an auxiliary component of a phage cocktail for Salmonella biocontrol[J].Applied and Environmental Microbiology, 2014, 80(3):1026-1034.
[25] SHORNIKOV A, TRAN H, MACIAS J, et al.Structure of the Bacillus anthracis dTDP-L-rhamnose-biosynthetic enzyme dTDP-4-dehydrorhamnose 3, 5-epimerase (RfbC)[J].Acta Crystallographica, 2017, F73:664-671.
[26] GIRAUD M F, LEONARD G A, FIELD R A, et al.RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase[J].Nature Structural Biology, 2000, 7:398-402.
[27] 韩生义.沙门氏菌的噬菌体试验性治疗及噬菌体抗性机制研究[D].兰州:甘肃农业大学, 2020. HAN S Y.The experimental treatment of Salmonella phage and study on the mechanism of phage resistance[D].Lanzhou:Gansu Agricultural University, 2020.(in Chinese)