致脑膜炎猪链球菌2型的分子分型鉴定及其生物学特性

doi:10.11843/j.issn.0366-6964.2024.03.031

摘要/Abstract

摘要： 旨在了解猪源致脑膜炎猪链球菌2型的分子分型和致病性及其生物学特性。本研究从患病猪的脑组织中分离到1株猪链球菌PX0923，通过电镜观察、16S rRNA基因测序、多重PCR、PCR-RFLP和多位点序列分析（MLST）等方法对其进行血清型和分子分型鉴定，毒力基因、生物被膜形成能力、耐药基因检测、小鼠感染试验、抗生素和中药药敏试验分析分离株PX0923的致病性和药物敏感性。结果表明，分离菌株PX0923为革兰阳性菌，电镜下可见连续排列成链状的卵圆形菌体；PCR和16S rRNA基因系统发育树分析确定其属于猪链球菌2型；序列分型为ST7，与ST1型猪链球菌聚为一支；携带epf⁺/mrp⁺/sly⁺/gapdh⁺/fbps⁺/orf2⁺/sao⁺ 7种毒力基因，呈现α型溶血活性；感染小鼠可导致小鼠肺、脾和肝等多个器官不同程度的病变，出现了小鼠脑膜炎等与病猪类似的临床症状；半数致死量（LD₅₀）为1.08×10⁹ CFU/小鼠，提示 PX0923可引起小鼠脑膜炎。分离菌株对庆大霉素、卡那霉素、新霉素、阿米卡星等12种药物耐药，携带耐药基因：aadA1 和bla-TEM，对中药全蝎和三七敏感。本研究分离到猪源猪链球菌PX0923为血清2型，MLST分型为ST7，其携带多种毒力基因和耐药基因，感染小鼠可引起脑膜炎等多组织损伤；对多种抗生素和中药全蝎、三七敏感。研究结果为猪链球菌的流行和溯源分析提供了分子生物学基础，对致脑膜炎猪链球菌感染的防控提供参考依据。

关键词: 猪链球菌2型, 分子分型, ST7型, 生物学特性, 耐药性, 中药

Abstract: The study was design to understand the molecular typing, pathogenicity and biological characteristics of Streptococcus suis meningitidis type 2 from swine. In this study, a strain of Streptococcus suis PX0923 was isolated from the brain tissue of diseased pigs, and its Serotype and molecular typing were identified by electron microscopy, 16S rRNA gene sequencing, multiplex PCR, PCR-RFLP and multiple site sequence analysis (MLST), the pathogenicity and drug sensitivity of the isolated strain were analyzed by virulence gene, biofilm formation ability, drug resistance gene detection, mouse infection test, antibiotic and Chinese medicine susceptibility test. The results showed that the isolated strain PX0923 belongs to Gram-positive bacteria. Under electron microscopy, continuous arrangement of oval shaped mycelium can be seen in chains. In terms of 16S rRNA gene sequence analysis and phylogenetic tree construction, the isolated strain was identified as S. suis type 2. MLST (multilocus sequence typing) analysis confirmed that it is type ST7 and merged with type ST1. The isolated strain PX0923 carried seven virulence genes, epf, mrp, sly, gapdh, fbps, orf2 and sao, and showed α hemolytic activity. After artificial infection of PX0923, it was found that the infected mice presented meningitis and other symptoms similar to the natural diseases, such as bleeding in the lung, spleen and liver of the infected mice. The LD₅₀ of the isolated strain was 1.08×10⁹ CFU per mice, these findings suggest that PX0923 may cause meningitis in infected animals. PX0923 carrys resistant genes aadA1 and bla-TEM. PX0923 is resistant to 12 drugs such as Gentamicin, kanamycin and tetracycline, and sensitive to traditional Chinese medicine scorpion and panax notoginseng. In summary, Streptococcus suis PX0923 from pigs was isolated as serotype 2, and MLST was classified as ST7, and carries a variety of virulence genes and drug resistance genes. Artificial infection of mice can cause multiple tissue damage such as meningitis in mice. It is sensitive to various antibiotics and traditional Chinese medicine scorpion, Panax notoginseng. The research results provide a reference basis for the prevention and treatment of streptococci and vaccine research. The results of this study provide a molecular biological basis for the epidemic and source analysis of S. suis, and provide a reference for the prevention and control of S. suis Meningitidis type 2 infection

Key words: Streptococcus suis type 2, molecular subtyp, ST-7, serotype, resistance, traditional Chinese medicine

中图分类号:

S852.611

李芃绪, 李世景, 孙骏, 项维, 赵苗苗, 侯天牧, 李华明, 广敏, 陈瑞格, 徐梦然, 吴晓敏, 姜合祥, 雷连成, 张付贤. 致脑膜炎猪链球菌2型的分子分型鉴定及其生物学特性[J]. 畜牧兽医学报, 2024, 55(3): 1192-1207.

LI Pengxu, LI Shijing, SUN Jun, XIANG Wei, ZHAO Miaomiao, HOU Tianmu, LI Huaming, GUANG Min, CHEN Ruige, XU Mengran, WU Xiaomin, JIANG Hexiang, LEI Liancheng, ZHANG Fuxian. Molecular Subtyping and Identification of Streptococcus suis Meningitidis Type 2 and Its Biological Characteristics[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1192-1207.

参考文献

[1] DUTKIEWICZ J, SROKA J, ZAJ[XCAA.TIF;S*4, JZ] C V, et al. Streptococcus suis:a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part I-Epidemiology[J]. Ann Agric Environ Med, 2017, 24(4):683-695.
[2] OKWUMABUA O, WILLIAMSON C H D, PEARSON T R, et al. Draft genome sequence of a Streptococcus suis isolate from a case of cattle meningitis[J]. Microbiol Resour Announc, 2020, 9(19):e00153-20.
[3] SEGURA M. Streptococcus suis research:progress and challenges[J]. Pathogens, 2020, 9(9):707.
[4] GOYETTE-DESJARDINS G, AUGER J P, XU J G, et al. Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing[J]. Emerg Microbes Infect, 2014, 3(6):e45.
[5] DONG X X, CHAO Y J, ZHOU Y, et al. The global emergence of a novel Streptococcus suis clade associated with human infections[J]. EMBO Mol Med, 2021, 13(7):e13810.
[6] SEGURA M, ARAGON V, BROCKMEIER S, et al. Update on Streptococcus suis research and prevention in the era of antimicrobial restriction:4^th international workshop on S. suis[J]. Pathogens, 2020, 9(5):374.
[7] HATRONGJIT R, FITTIPALDI N, GOTTSCHALK M, et al. Tools for molecular epidemiology of Streptococcus suis[J]. Pathogens, 2020, 9(2):81.
[8] SEGURA M, FITTIPALDI N, CALZAS C, et al. Critical Streptococcus suis virulence factors:are they all really critical?[J]. Trends Microbiol, 2017, 25(7):585-599.
[9] GOTTSCHALK M, SEGURA M. The pathogenesis of the meningitis caused by Streptococcus suis:the unresolved questions[J]. Vet Microbiol, 2000, 76(3):259-272.
[10] 赵仲艳, 赵二义, 刘涛, 等. 6例猪链球菌脑膜炎临床特征[J]. 中国热带医学, 2020, 20(5):469-472.
ZHAO Z Y, ZHAO E Y, LIU T, et al. Clinical analysis of 6 cases of Streptococcus suis meningitis[J]. China Tropical Medicine, 2020, 20(5):469-472. (in Chinese)
[11] 王海峰, 李亚飞, 张白帆, 等. 河南省5起人感染猪链球菌病疫情特征分析[J]. 河南预防医学杂志, 2022, 33(9):647-649, 677.
WANG H F, LI Y F, ZHANG B F, et al. Incidence characteristics of five events of human infection with s Streptococcus suis in Henan province[J]. Henan Journal of Preventive Medicine, 2022, 33(9):647-649, 677. (in Chinese)
[12] 梁娟. 猪呼吸系统病原菌五重PCR检测方法的建立与应用[D]. 重庆:西南大学, 2020.
LIANG J. Establishment and application of 5-plex PCR method for detection of porcine respiratory pathogenic bacteria[D]. Chongqing:Southwest University, 2020. (in Chinese)
[13] 汤细彪. 猪源多杀性巴氏杆菌的分子流行病学与致病性研究[D]. 武汉:华中农业大学, 2010.
TANG X B. Study on the molecular epidemiology and pathogenicity of Pasteurella multocida from swine[D]. Wuhan:Huazhong Agricultural University, 2010. (in Chinese)
[14] ISHIDA S, TIEN L H T, OSAWA R, et al. Development of an appropriate PCR system for the reclassification of Streptococcus suis[J]. J Microbiol Methods, 2014, 107:66-70.
[15] 张秀亮, 沈琴芳, 郑新添, 等. 猪链球菌的鉴定及其PCR分型方法的建立[J]. 黑龙江畜牧兽医, 2005(9):86-87.
ZHANG X L, SHEN Q F, ZHENG X T, et al. The isolation and identification of serotype the Streptocouis suis in the hyperpyrexia of swine[J]. Heilongjiang Animal Science and Veterinary Medicine, 2005(9):86-87. (in Chinese)
[16] OKWUMABUA O, O'CONNOR M, SHULL E. A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase[J]. FEMS Microbiol Lett, 2003, 218(1):79-84.
[17] MATIASOVIC J, ZOUHAROVA M, NEDBALCOVA K, et al. Resolution of Streptococcus suis serotypes 1/2 versus 2 and 1 versus 14 by PCR-restriction fragment length polymorphism method[J]. J Clin Microbiol, 2020, 58(7):e00480-20.
[18] KING S J, LEIGH J A, HEATH P J, et al. Development of a multilocus sequence typing scheme for the pig pathogen Streptococcus suis:identification of virulent clones and potential capsular serotype exchange[J]. J Clin Microbiol, 2002, 40(10):3671-3680.
[19] 潘建刚, 张华伟, 陈波, 等. 猪链球菌2型的分离鉴定及致病性试验[J]. 中国兽药杂志, 2020, 54(8):14-19.
PAN J G, ZHANG H W, CHEN B, et al. Isolation, identification and pathogenicity of Streptococcus suis type 2[J]. Chinese Journal of Veterinary Drug, 2020, 54(8):14-19. (in Chinese)
[20] SMITH H E, BUIJS H, WISSELINK H J, et al. Selection of virulence-associated determinants of Streptococcus suis serotype 2 by in vivo complementation[J]. Infect Immun, 2001, 69(3):1961-1966.
[21] 张宇航. 猪链球菌2型强毒株特有基因簇nmt功能研究[D]. 南京:南京农业大学, 2019.
ZHANG Y H. Research on the role of the unique gene cluster nmt in Streptococcus suis serotype 2 virulent strains[D]. Nanjing:Nanjing Agricultural University, 2019. (in Chinese)
[22] 李翠萍, 吴民耀, 王宏元. 3种半数致死浓度计算方法之比较[J]. 动物医学进展, 2012, 33(9):89-92.
LI C P, WU M Y, WANG H Y. LC50 caculated by kochi, probit analysis and linear regression methods[J]. Progress in Veterinary Medicine, 2012, 33(9):89-92. (in Chinese)
[23] KAISER T D L, PEREIRA E M, DOS SANTOS K R N, et al. Modification of the Congo red agar method to detect biofilm production by Staphylococcus epidermidis[J]. Diagn Microbiol Infect Dis, 2013, 75(3):235-239.
[24] DALLENNE C, DA COSTA A, DECRÉ D, et al. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae[J]. J Antimicrob Chemother, 2010, 65(3):490-495.
[25] FREIRE S, GRILO T, NORDMANN P, et al. Multiplex PCR for detection of acquired plasmid-borne fosfomycin resistance fos genes in Escherichia coli[J]. Diagn Microbiol Infect Dis, 2023, 105(2):115864.
[26] HATRONGJIT R, KERDSIN A, GOTTSCHALK M, et al. First human case report of sepsis due to infection with Streptococcus suis serotype 31 in Thailand[J]. BMC Infect Dis, 2015, 15(1):392.
[27] LIANG P J, WANG M L, GOTTSCHALK M, et al. Genomic and pathogenic investigations of Streptococcus suis serotype 7 population derived from a human patient and pigs[J]. Emerg Microbes Infect, 2021, 10(1):1960-1974.
[28] JIANG F, GUO J J, CHENG C, et al. Human infection caused by Streptococcus suis serotype 2 in China:report of two cases and epidemic distribution based on sequence type[J]. BMC Infect Dis, 2020, 20(1):223.
[29] 廖虹瑜, 罗隆泽, 曾林子, 等. 2006-2016年四川省猪链球菌血清2型多位点序列分型分析[J]. 疾病监测, 2017, 32(7):544-547.
LIAO H Y, LUO L Z, ZENG L Z, et al. Multi locus sequence typing of Streptococcus suis serotype 2 isolated in Sichuan province, 2006-2016[J]. Disease Surveillance, 2017, 32(7):544-547. (in Chinese)
[30] 张桓桥, 商宝娣, 张效平, 等. 31种中草药及其复方对维氏气单胞菌体外抑菌研究[J]. 淡水渔业, 2022, 52(3):74-81.
ZHANG H Q, SHANG B D, ZHANG X P, et al. The antibacterial effect of 31 kinds of Chinese herbal medicines and their compounds on Aeromonas vernoii in vitro[J]. Freshwater Fisheries, 2022, 52(3):74-81. (in Chinese)
[31] SCHERRER S, ROSATO G, SPOERRY SERRANO N, et al. Population structure, genetic diversity and pathotypes of Streptococcus suis isolated during the last 13 years from diseased pigs in Switzerland[J]. Vet Res, 2020, 51(1):85.
[32] KERDSIN A, HATRONGJIT R, GOTTSCHALK M, et al. Emergence of Streptococcus suis serotype 9 infection in humans[J]. J Microbiol Immunol Infect, 2017, 50(4):545-546.
[33] 熊衍峰, 王建平, 李建华, 等. 江西省赣州市首例人感染ST7型猪链球菌病例的病原学分析[J]. 中国卫生检验杂志, 2022, 32(3):257-260.
XIONG Y F, WANG J P, LI J H, et al. Pathogenologic analysis of first case of human sequence type 7 Streptococcus suis infection in Ganzhou city, Jiangxi province[J]. Chinese Journal of Health Laboratory Technology, 2022, 32(3):257-260. (in Chinese)
[34] DUTKIEWICZ J, ZAJ[XCAA.TIF, JZ] C V, SROKA J, et al. Streptococcus suis:a re-emerging pathogen associated with occupational exposure to pigs or pork products. Part II-Pathogenesis[J]. Ann Agric Environ Med, 2018, 25(1):186-203.
[35] 林岚. 猪链球菌2型激活炎性小体致中毒样休克综合征分子机制的研究[D]. 武汉:华中农业大学, 2019.
LIN L. Research on the molecular mechanism of toxic-shock-like syndrome induced by Streptococcus suis serotype 2-activated inflammasome[D]. Wuhan:Huazhong Agricultural University, 2019. (in Chinese)
[36] O'DEA M A, LAIRD T, ABRAHAM R, et al. Examination of Australian Streptococcus suis isolates from clinically affected pigs in a global context and the genomic characterisation of ST1 as a predictor of virulence[J]. Vet Microbiol, 2018, 226:31-40.
[37] 吴秋云, 黄琳, 皮真, 等. 中草药抑菌作用及其机制研究进展[J]. 中兽医医药杂志, 2018, 37(1):25-29.
WU Q Y, HUANG L, PI Z, et al. Research progress of antibacterial activity and mechanism of Chinese herbal medicine[J]. Journal of Traditional Chinese Veterinary Medicine, 2018, 37(1):25-29. (in Chinese)
[38] 王旭东, 胡海波, 王振山, 等. 中药对细菌生物被膜作用机制的研究进展[J]. 现代药物与临床, 2019, 34(7):2248-2252.
WANG X D, HU H B, WANG Z S, et al. Research progress on mechanism of traditional Chinese medicine on bacterial biofilm[J]. Drugs and Clinic, 2019, 34(7):2248-2252. (in Chinese)
[39] LI Y H, ZHOU Y H, REN Y Z, et al. Inhibition of Streptococcus suis adhesion and biofilm formation in vitro by water extracts of Rhizoma Coptidis[J]. Front Pharmacol, 2018, 9:371.
[40] 冯沙沙, 宋鹏琰, 翟福展, 等. 42种常见中药对猪链球菌的体外抑菌试验[J]. 黑龙江畜牧兽医, 2017(12):131-133.
FENG S S, SONG P Y, ZHAI F Z, et al. Anitibacterial activity of 42 kinds of common tratidional Chinese medicine against Strepotoccus suis in vitro[J]. Heilongjiang Animal Science and Veterinary Medicine, 2017(12):131-133. (in Chinese)
[41] 陈俭清, 周永辉, 杨艳北, 等. 中药水提物对体外猪链球菌生物被膜作用的试验[J]. 中国兽医杂志, 2016, 52(11):14-16, 19.
CHEN J Q, ZHOU Y H, YANG Y B, et al. Screening of Chinese herbal extracts against Streptococcus suis biofilm formation[J]. Chinese Journal of Veterinary Medicine, 2016, 52(11):14-16, 19. (in Chinese)
[42] 詹佳飞, 徐魁, 张磊, 等. 毛蕊花糖苷抑制2型猪链球菌的溶血素蛋白活性而降低其小鼠致病性[J]. 浙江农业学报, 2022, 34(8):1609-1616.
ZHAN J F, XU K, ZHANG L, et al. Verbascoside lowers Streptococcus suis serotype 2 pathogenicity in mice by inhibiting hemolytic activity of suilysin[J]. Acta Agriculturae Zhejiangensis, 2022, 34(8):1609-1616. (in Chinese)
[43] 唐阳. 药用大黄药效成分提取及抑制猪链球菌活性研究[D]. 哈尔滨:东北农业大学, 2021.
TANG Y. Study on extraction of effective components from Rheum officinale Baill, and its activity against Streptococcus suis[J]. Harbin:Northeast Agricultural University, 2021. (in Chinese)