Genomic characteristics analysis on Vibrio parahaemolyticus isolated from food-borne diseases outbreaks in Fuzhou
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摘要:
目的 获得四起暴发事件分离的副溶血弧菌序列分布特点，并分析不同菌株间遗传关系。 方法 对19株副溶血弧菌进行全基因组序列测定，使用相应的生物信息学软件对测序数据进行基因组装和基因预测，借助相关数据库获得不同菌株的多位点序列分型、耐药基因和毒力基因情况，采取最大似然法构建系统发育树。 结果 19株副溶血弧菌依据7个管家基因分型，得到 4种ST型，其中 ST3为优势型，1株菌暂定为Unknown型。 全部菌株均携带β-内酰胺类和四环素类抗生素耐药基因，同时携带影响宿主细胞致病力的重要毒力基因。 2种喹诺酮类耐药基因和trh毒力基因仅在F265菌株中检出。 全基因组系统发育树进化分析结果显示，暴发菌株被分成3个进化分支，E2019、E2020-1和E2020-2的菌株主要集中在Lineage B进化分支，E2018的菌株均在Lineage C 进化分支。 结论 四起暴发事件由ST3克隆复合体主导，并伴随其他ST型别菌株的影响。 同起暴发事件分离菌株具有遗传多样性， 菌株测序数据为暴发事件溯源提供技术支持。 Abstract:Objective To obtain the sequence distribution characteristics of Vibrio parahaemolyticus strains isolated from four outbreaks in Fuzhou, Fujian province, and analyze genetic correlations among different strains. Methods The 19 strains of V. parahaemolyticus were sequenced with whole genome sequencing (WGS). Genome assembly and gene prediction for sequenced strains were performed using corresponding bioinformatics software. Multilocus sequence typing, antibiotic resistance genes and virulence genes of the V. parahaemolyticus strains were discovered using relevant databases. The phylogenetic tree was constructed by the maximum likelihood method. Results The 19 V. parahaemolyticus strains were classified into four STs according to seven housekeeping genes, ST3 was the predominant STs and there was 1 strain belonged to an unknown ST. All the strains contained β -lactam and tetracycline antibiotic resistance genes, and carried important virulence genes causing pathogenicity of host cells. Two quinolone resistance genes and 1 trh+ virulence gene were detected only in F265 strain. The phylogenetic tree evolution analysis showed that the strains isolated in the outbreaks could be divided into three evolutionary clades, with strains E2019, E2020-1, and E2020-2 mainly in lineage B evolutionary branch and strains E2018 all in lineage C evolutionary branch. Conclusion The V. parahaemolyticus causing the four outbreaks were predominated by ST3 clonal complex accompanied by other STs. The strains isolated from the same outbreak showed genetic diversity. The sequencing data provided technical support for source tracing of the outbreaks. -
图 1 19株副溶血弧菌重要毒力基因情况
Figure 1. Important virulence genes of 19 V. parahaemolyticus strains
图 2 副溶血弧菌基于核心基因组单核苷酸多态性构建系统发育树
Figure 2. Phylogenetic tree constructed based on cgSNP of V. parahaemolyticus strains
图 3 对B和C进化分支菌株基于核心基因组单核苷酸多态性再构建系统发育树
注： B. 以 RIMD2210633菌株为参考；C. 以F51菌株为参考
Figure 3. Phylogenetic trees of strains in lineage B and C based on cgSNP
表 1 34株不同地区、不同来源的基因组信息
Table 1. Genomic information of 34 strains from different areas and different sources
菌株名称/Assembly 数量（个） 基因组大小（bp） G＋C含量 （%） N50 时间（年） 分离来源 分离地区 F51 79 5037175 45.20 450170 2018 患者 中国 福州 F52 85 5036994 45.20 470715 2018 患者 中国 福州 F53 86 5029249 45.20 470143 2018 患者 中国 福州 F54 89 5036179 45.20 407656 2018 患者 中国 福州 F163 171 5170524 45.31 137129 2019 患者 中国 福州 F164 143 5029092 45.40 107667 2019 患者 中国 福州 F165 159 5131277 45.28 190945 2019 患者 中国 福州 F166 246 5143272 45.27 61822 2019 患者 中国 福州 F167 142 5009235 45.33 170889 2019 患者 中国 福州 F263 113 5071890 45.27 300721 2020 患者 中国 福州 F264 127 5104710 45.27 312516 2020 患者 中国 福州 F265 94 5185725 45.15 349269 2020 患者 中国 福州 F266 124 5074847 45.27 266436 2020 患者 中国 福州 F267 118 5104903 45.27 425351 2020 患者 中国 福州 F268 125 5094814 45.27 261977 2020 患者 中国 福州 F312 125 5120085 45.26 312920 2020 患者 中国 福州 F313 157 5136990 45.23 103162 2020 患者 中国 福州 F314 132 5166814 45.23 182672 2020 患者 中国 福州 F315 221 5140170 45.26 58430 2020 患者 中国 福州 SRR11357164 33 5165064 45.28 1101425 2015 虾 中国 乌鲁木齐 SRR11430677 47 5020255 45.34 725933 2013 罗非鱼 中国 韶关 SRR13023686 52 5200959 45.19 535131 2019 海水 中国 厦门 SRR14373893 42 5050048 45.25 471159 2017 鸟粪 中国 宁波 SRR14684349 46 4986426 45.35 400689 2017 南美白对虾 中国 唐山 SRR15218671 53 5182621 45.26 461872 2015 患者 中国 深圳 SRR15239739 52 5094216 45.29 311606 2018 患者 中国 深圳 SRR15239894 37 4947484 45.35 723841 2017 环境 中国 深圳 SRR15239901 49 5038492 45.22 557944 2017 环境 中国 深圳 SRR15239917 49 5091468 45.29 461882 2016 患者 中国 深圳 SRR18507851 81 5302555 45.16 211308 2019 虾 美国 纽约 SRR6476836 43 5094140 45.4 279707 2014 患者 美国 SRR7588481 39 5032859 45.37 402283 2018 蟹肉块 委内瑞拉 SRR8103884 50 5297954 45.16 581757 2016 患者 秘鲁 RIMD 2210633 - 5165770 45.40 3288558 1996 患者 日本 注：SRR开头菌株号来源NCBI数据库，运用于本研究系统发育树构建，RIMD 2210633为参考菌株 
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表 2 19株副溶血弧菌耐药基因测序结果
Table 2. Sequencing results of antibiotic resistance genes of 19 V. parahaemolyticus
strains 暴发事件 年份 包括菌株 分型情况 耐药基因 E2018 2018 F51～F54 ST2516 blaCARB-20，tet（34），tet（35） E2019 2019 F164 ST990（6） blaCARB-26，tet（34），tet（35） F167 ST1353 blaCARB-21，tet（34），tet（35） F163，F165，F166 ST3 blaCARB-22，tet（34），tet（35） E2020-2 2020 F312～F315 ST3 blaCARB-22，tet（34），tet（35） E2020-1 2020 F263，F264，F266～F268 ST3 blaCARB-22，tet（34），tet（35） F265 ST1750 blaCARB-29，blaCARB-33，blaCARB-41，
tet（34），tet（35） ，qnrS4，qnrS5注：blaCARB基因属于β-内酰胺类抗生素，tet类基因属于四环素类抗生素，F164与ST990型别的6个等位基因信息完全匹配
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