前噬菌体对猪链球菌毒力、环境适应性、耐药性及代谢活动的影响

doi:10.11843/j.issn.0366-6964.2022.01.029

摘要/Abstract

摘要： 旨在了解和掌握前噬菌体与猪链球菌（Streptococcus suis，SS）毒力、环境适应性、耐药性及代谢活动之间的关系，本研究对前噬菌体阳性菌株（简称阳性菌）与前噬菌体阴性菌株（简称阴性菌）进行了致病性试验、LD₅₀的测定、组织荷菌数的测定及病理组织学观察、生物被膜（BF）形成能力的测定、药敏试验和转录组测序。结果显示：39株阳性菌和7株阴性菌中，各有11株和2株菌对小鼠致死率≥80.0%，11株阳性菌LD₅₀为8.4×10⁸~2.36×10⁹CFU·只^-1，2株阴性菌LD₅₀分别为1.88×10⁹、2.72×10⁹CFU·只^-1，阳性菌与阴性菌之间LD₅₀差异不显著（P>0.05）。LD₅₀为8.4×10⁸CFU·只^-1的阳性菌攻毒后小鼠各脏器（肺、肝、脾、肾、心、脑）组织荷菌数最多，病理变化最明显，LD₅₀为2.72×10⁹ CFU·只^-1的阴性菌攻毒后小鼠各脏器组织荷菌数最少，病理变化最轻微；BF形成阳性率阳性菌为97.4%，阴性菌为100.0%；阳性菌和阴性菌均对四环素、红霉素、克林霉素、阿奇霉素、头孢曲松、多西环素呈现多重耐药，二者的耐药性无显著差异（P>0.05）；相较于阳性菌，毒力相关基因、BF形成相关基因、耐药基因、脂肪酸生物合成通路以及精氨酸和脯氨酸代谢通路中所涉及到的相关基因在阴性菌中多为上调表达。综上表明：前噬菌体存在与否与SS的毒力增强或减弱、耐药性的高或低以及BF的形成能力之间未有明显相关性；但前噬菌体的存在会引致与SS脂肪酸生物合成以及精氨酸和脯氨酸代谢过程中相关联基因的表达下调，从而造成脂肪酸和氨基酸代谢水平降低。

关键词: 猪链球菌, 前噬菌体, 毒力, 耐药性, 环境适应性

Abstract: To understand and master the relationship between prophage and virulence, environmental adaptability, drug resistance and metabolic activity of Streptococcus suis (SS), in this study, the pathogenicity test, LD₅₀ test, the number of bacteria loaded in tissue, histopathological observation, biofilm formation(BF) ability test, drug sensitivity test and transcriptome sequencing were carried out for the prophage positive strains (positive bacteria for short) and prophage negative strain(negative bacteria for short). The results showed that among 39 strains of positive bacteria and 7 strains of negative bacteria, 11 strains and 2 strains of bacteria had a lethal rate of more than 80.0%, The LD₅₀ of 11 positive bacteria ranged from 8.4×10⁸ to 2.36×10⁹CFU·piece^-1, and that of 2 negative bacteria were 1.88×10⁹ and 2.72×10⁹ CFU·piece^-1, respectively. There was no significant difference in LD₅₀ between positive and negative bacteria (P>0.05). After being challenged with positive bacteria with LD₅₀ of 8.4×10⁸CFU·piece^-1, the number of bacteria in the organs (lung, liver, spleen, kidney, heart and brain) of mice was the most, and the pathological changes were the most obvious, after being challenged with the negative bacteria with LD₅₀ of 2.72×10⁹CFU·piece^-1, the number of bacteria in each organ tissue of mice was the least, and the pathological changes were the least; The positive rate of BF formation was 97.4% for positive bacteria and 100.0% for negative bacteria; Both positive and negative bacteria showed multiple drug resistance to tetracycline, erythromycin, clindamycin, azithromycin, ceftriaxone and doxycycline, and there was no significant difference in drug resistance between them (P>0.05); Compared with positive bacteria, virulence related genes, BF formation related genes, drug resistance genes, fatty acid biosynthesis pathway and arginine and proline metabolism pathway related genes were up-regulated in negative bacteria. the results showed that there was no significant correlation between the presence or absence of prophage and the increase or decrease of SS virulence, high or low drug resistance and BF formation ability; However, the presence of prophage will lead to the down-regulation of genes associated with SS fatty acid biosynthesis and arginine and proline metabolism, resulting in the reduction of fatty acid and amino acid metabolism.

Key words: Streptococcus suis, prophage, virulence, drug resistance, environmental adaptability

中图分类号:

Q939.48

韩雪姣, 李亮, 占松鹤, 段倩倩, 崔丽荣, 刘雪兰, 孙裴, 魏建忠, 李郁. 前噬菌体对猪链球菌毒力、环境适应性、耐药性及代谢活动的影响[J]. 畜牧兽医学报, 2022, 53(1): 290-303.

HAN Xuejiao, LI Liang, ZHAN Songhe, DUAN Qianqian, CUI Lirong, LIU Xuelan, SUN Pei, WEI Jianzhong, LI Yu. Effects of Prophage on Virulence, Environmental Adaptability, Drug Resistance and Metabolic Activity of Streptococcus suis[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 290-303.

参考文献 21

[1]	陆承平, 吴宗福. 猪链球菌病[M]. 北京:中国农业出版社, 2015.LU C P, WU Z F. Swine streptococcosis[M]. Beijing:China Agriculture Press, 2015. (in Chinese)
[2]	汪洋. 猪链球菌生物被膜形成及致病机理研究[D]. 南京:南京农业大学, 2011.WANG Y. Study on the mechanism of biofilm formation and molecular pathogenesis of Streptococcus suis[D]. Nanjing:Nanjing Agricultural University, 2011. (in Chinese)
[3]	RONCOLATO E C, CAMPOS L B, PESSENDA G, et al. Phage display as a novel promising antivenom therapy:a review[J]. Toxicon, 2015, 93:79-84.
[4]	FORTIER L C, SEKULOVIC O. Importance of prophages to evolution and virulence of bacterial pathogens[J]. Virulence, 2013, 4(5):354-365.
[5]	PALMIERI C, PRINCIVALLI M S, BRENCIANI A, et al. Different genetic elements carrying the tet(W) gene in two human clinical isolates of Streptococcus suis[J]. Antimicrob Agents Chemother, 2011, 55(2):631-636.
[6]	SHEN M Y, YANG Y H, SHEN W, et al. A linear plasmid-like prophage of Actinomyces odontolyticus promotes biofilm assembly[J]. Appl Environ Microbiol, 2018, 84(17):e01263-18.
[7]	STEVENS R H, ZHANG H M, SEDGLEY C, et al. The prevalence and impact of lysogeny among oral isolates of Enterococcus faecalis[J]. J Oral Microbiol, 2019, 11(1):1643207.
[8]	吴迪. 猪链球菌2型分离鉴定及其对免疫抑制小鼠的致病力比较[D]. 杭州:浙江大学, 2009.WU D. Identification of Streptococcus suis type 2 and their pathogenicity in immuno-compromised mice[D]. Hangzhou:Zhejiang University, 2009. (in Chinese)
[9]	吴全忠. 猪链球菌2型在模型动物豚鼠体内的动态分布及基因免疫[D]. 南京:南京农业大学, 2001.WU Q Z. The dynamic distribution and gene immunization of Streptococcus suis type 2 in guinea pig model[D]. Nanjing:Nanjing Agricultural University, 2001. (in Chinese)
[10]	孙智远, 丁文傒, 陈军, 等. 猪链球菌9型ICR小鼠感染模型的建立及评分[J]. 中国兽医科学, 2018, 48(8):1024-1030.SUN Z Y, DING W X, CHEN J, et al. Establishment of animal models of ICR mice infected with Streptococcus suis type 9 using scoring method[J]. Chinese Veterinary Science, 2018, 48(8):1024-1030. (in Chinese)
[11]	JIN H, ZHOU R, KANG M S, et al. Biofilm formation by field isolates and reference strains of Haemophilus parasuis[J]. Vet Microbiol, 2006, 118(1-2):117-123.
[12]	STEPANOVIĆ S, VUKOVIĆ D, HOLA V, et al. Quantification of biofilm in microtiter plates:overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci[J]. APMIS, 2007, 115(8):891-899.
[13]	PHAM T D, NGUYEN T H, IWASHITA H, et al. Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia[J]. Microbiol Immunol, 2018, 62(10):635-650.
[14]	WANG X X, KIM Y, MA Q, et al. Cryptic prophages help bacteria cope with adverse environments[J]. Nat Commun, 2010, 1:147.
[15]	杜宝中, 曾蔚, 陶传敏, 等. 溶原性噬菌体对铜绿假单胞菌生物被膜形成的影响[J]. 四川大学学报:医学版, 2008, 39(6):886-889.DU B Z, ZENG W, TAO C M, et al. The influence of lysogenic phage on biofilm formation of Pseudomonas aeruginosa[J]. Journal of Sichuan University:Medical Science Edition, 2008, 39(6):886-889. (in Chinese)
[16]	MATOS R C, LAPAQUE N, RIGOTTIER-GOIS L, et al. Enterococcus faecalis prophage dynamics and contributions to pathogenic traits[J]. PLoS Genet, 2013, 9(6):e1003539.
[17]	彭子欣, 张思雨, 闫韶飞, 等. 食源性希拉肠球菌R17基因组中前噬菌体的结构特征及其与宿主菌的相互影响[J]. 中国食品卫生杂志, 2017, 29(4):393-399.PENG Z X, ZHANG S Y, YAN S F, et al. The structure characteristics of prophages of foodborne Enterococcus hirae R17 and their interaction relationships with host bacterium[J]. Chinese Journal of Food Hygiene, 2017, 29(4):393-399. (in Chinese)
[18]	VESES-GARCIA M, LIU X, RIGDEN D J, et al. Transcriptomic analysis of Shiga-toxigenic bacteriophage carriage reveals a profound regulatory effect on acid resistance in Escherichia coli[J]. Appl Environ Microbiol, 2015, 81(23):8118-8125.
[19]	HAREL J, MARTINEZ G, NASSAR A, et al. Identification of an inducible bacteriophage in a virulent strain of Streptococcus suis serotype 2[J]. Infect Immunity, 2003, 71(10):6104-6108.
[20]	杜德超. 猪链球菌2型不同毒力菌株的鉴别及Pan-genome分析[D]. 南京:南京农业大学, 2015.DU D C. Virulence evaluation of different strains and pan-genome analysis of Streptococcus suis serotype 2[D]. Nanjing:Nanjing Agricultural University, 2015. (in Chinese)
[21]	虞莉, 陈绵绵, 辛思培, 等. 猪链球菌2型前噬菌体基因的检测及其与毒力基因分布的关系[J]. 畜牧与兽医, 2016, 48(5):26-31.YU L, CHEN M M, XIN S P, et al. Detection of the prophage genes in Streptococcus suis serotype 2 and its relationship with the distribution of virulent genes[J]. Animal Husbandry & Veterinary Medicine, 2016, 48(5):26-31. (in Chinese)