新疆农业科学 ›› 2022, Vol. 59 ›› Issue (9): 2310-2317.DOI: 10.6048/j.issn.1001-4330.2022.09.027
• 畜牧兽医·农业装备工程与机械化·农业经济 • 上一篇 下一篇
范雪1,2(), 邵伟1, 赵艳坤2(), 杜晓慧1, 陈贺2, 王富兰2, 王帅2
收稿日期:
2021-02-26
出版日期:
2022-09-20
发布日期:
2023-01-16
通信作者:
赵艳坤
作者简介:
范雪(1995-),女,新疆奇台人,硕士研究生,研究方向为动物生产,(E-mail)1549418390@qq.com
基金资助:
FAN Xue1,2(), SHAO Wei1, ZHAO Yankun2(), DU Xiaohui1, CHEN He2, WANG Fulan2, WANG Shuai2
Received:
2021-02-26
Online:
2022-09-20
Published:
2023-01-16
Correspondence author:
ZHAO Yankun
Supported by:
摘要:
【目的】 研究无乳链球菌对抗菌药物的耐药性,分析无乳链球菌毒力基因与耐药基因的携带情况。【方法】 分别采用微量肉汤稀释法和普通PCR的方法,检测牛源无乳链球菌对16种抗菌药物的耐药性和相关耐药基因及毒力基因,并且采用荧光定量PCR技术对不同菌株的8种毒力基因的表达量差异进行分析。【结果】 (1)无乳链球菌对11种药物的敏感性达到了65%以上,其中敏感性最高的是氟苯尼考(92.4.%)和头孢噻呋(88.4%),对氨苄西林、红霉素、克林霉素的耐药率均达到了50%以上,对磺胺异恶唑的耐药率也达到了45%以上。(2)无乳链球菌耐药基因gyrA、sul1、ermB、ermC的检出率分别为100%、 86.67%、 93.3%、 33.3%,而ermA、sul2、sul3及parC 4种耐药基因未检出。(3)无乳链球菌毒力基因pavA、cfb、fbsA、bibA、cspA、sip、iagA、hylB的检出率为100%,rib检出率为13.3%,bca的检出率为53.3%,cyl E检出率为73.3%;未检测到bac、lmb、scp B这三种毒力基因。(4)不同菌株之间毒力基因的表达量有显著性差异(P<0.05)。【结论】 无乳链球菌对红霉素和克林霉素的耐药率较高,无乳链球菌毒力因子是宿主感染疾病的重要因素。
中图分类号:
范雪, 邵伟, 赵艳坤, 杜晓慧, 陈贺, 王富兰, 王帅. 牛源无乳链球菌耐药性与毒力基因的表达量差异分析[J]. 新疆农业科学, 2022, 59(9): 2310-2317.
FAN Xue, SHAO Wei, ZHAO Yankun, DU Xiaohui, CHEN He, WANG Fulan, WANG Shuai. Differential Expression of Drug Resistance and Virulence Genes in Streptococcus agalactiae Isolated from Cattle[J]. Xinjiang Agricultural Sciences, 2022, 59(9): 2310-2317.
基因名称 Gene name | 引物序列 Primer sequence | 目的片段 Target fragment(bp) |
---|---|---|
pavA | F:TTCCCATGATTTCAACAACAAG | 495 |
R:AACCTTTTGACCATGAATTGGTA | ||
scpB | F: AGTTGCTTCTTACAGCCCAGA | 567 |
R: GGCGCAGACATACTAGTTCCA | ||
rib | F:CAGGAAGTGCTGTTACGTTAAAC | 306 |
R: CGTCCCATTTAGGGTCTTCC | ||
bac | F:CTATTTTTGATATTGACAATGCAA | 592 |
R:GTCGTTACTTCCTTGAGATGTAAC | ||
cylE | F: TGACATTTACAAGTGACGAAG | 248 |
R: TTGCCAGGAGGAGAATAGGA | ||
bca | F:TAACAGTTATGATACTTCACAGAC | 320 |
R:ACGACTTTCTTCCGTCCACTTAGG | ||
cfb | F: GCTGTTTGAAGTGCTGCTTG | 288 |
R: GACTTCATTGCGTGCCAAC | ||
lmb | F: AGTCAGCAAACCCCAAACAG | 397 |
R: GCTTCCTCACCAGCTAAAACG | ||
hylB | F: CACCAATCCCCACTCTACTA | 346 |
R: TGTGTCAAACCATCTATCAG | ||
fbsA | F:AGAGCCAAGTAGGTCAACTTATAG | 290 |
R: TTCATTGCGTCTCAAACCG | ||
bibA | F:AACCAGAAGCCAAGCCAGCAACC | 127 |
R: AGTGGACTTGCGGCTTCACCC | ||
cspA | F: GGTCGCGATAGAGTTTCTTCCGC | 104 |
R: AACGCCTGGGGCTGATTTGGC | ||
sip | F: TCTCTCAATACAATTTCGGAAG | 867 |
R: GTGGTCATAGTGGTTGGCAGT | ||
iagA | F: CGGGATTGATCTAAGTCGCT | 459 |
R: CCATCAACATCAGTCGCTAA | ||
ermA | F: TCTAAAAAGCATGTAAAAGAA | 645 |
R: CTTCGATAGTTTATTAATATTAGT | ||
ermB | F: ATTGGAACAGGTAAAGGGC | 442 |
R: GAACATCTGTGGTATGGCG | ||
ermC | F: TCAAAACATAATATAGATAAA | 642 |
R: GCTAATATTGTTTAAATCGTCAAT | ||
sul1 | F: CATTGCCTGGTTGCTTCAT | 238 |
R: ATCCGACTCGCAGCATTT | ||
sul2 | F: CATCATTTTCGGCATCGTC | 793 |
R: TCTTGCGGTTTCTTTCAGC | ||
sul3 | F: AGATGTGATTGATTTGGGAGC | 443 |
R: TAGTTGTTTCTGGATTAGAGCCT | ||
gyrA | F: GACAAGTGAAATGAAAACGAG | 474 |
R: CGCTCCATTGACTAATAAATTAGG | ||
parC | F: CAAAACATGTCCCTTGAGGA | 568 |
R: CTAGCTTTGGGATGATCAATCAT |
表 1 无乳链球菌毒力基因与耐药基因引物序列
Table 1 Primer sequence of virulence gene and drug resistance gene of Streptococcus agalactiae
基因名称 Gene name | 引物序列 Primer sequence | 目的片段 Target fragment(bp) |
---|---|---|
pavA | F:TTCCCATGATTTCAACAACAAG | 495 |
R:AACCTTTTGACCATGAATTGGTA | ||
scpB | F: AGTTGCTTCTTACAGCCCAGA | 567 |
R: GGCGCAGACATACTAGTTCCA | ||
rib | F:CAGGAAGTGCTGTTACGTTAAAC | 306 |
R: CGTCCCATTTAGGGTCTTCC | ||
bac | F:CTATTTTTGATATTGACAATGCAA | 592 |
R:GTCGTTACTTCCTTGAGATGTAAC | ||
cylE | F: TGACATTTACAAGTGACGAAG | 248 |
R: TTGCCAGGAGGAGAATAGGA | ||
bca | F:TAACAGTTATGATACTTCACAGAC | 320 |
R:ACGACTTTCTTCCGTCCACTTAGG | ||
cfb | F: GCTGTTTGAAGTGCTGCTTG | 288 |
R: GACTTCATTGCGTGCCAAC | ||
lmb | F: AGTCAGCAAACCCCAAACAG | 397 |
R: GCTTCCTCACCAGCTAAAACG | ||
hylB | F: CACCAATCCCCACTCTACTA | 346 |
R: TGTGTCAAACCATCTATCAG | ||
fbsA | F:AGAGCCAAGTAGGTCAACTTATAG | 290 |
R: TTCATTGCGTCTCAAACCG | ||
bibA | F:AACCAGAAGCCAAGCCAGCAACC | 127 |
R: AGTGGACTTGCGGCTTCACCC | ||
cspA | F: GGTCGCGATAGAGTTTCTTCCGC | 104 |
R: AACGCCTGGGGCTGATTTGGC | ||
sip | F: TCTCTCAATACAATTTCGGAAG | 867 |
R: GTGGTCATAGTGGTTGGCAGT | ||
iagA | F: CGGGATTGATCTAAGTCGCT | 459 |
R: CCATCAACATCAGTCGCTAA | ||
ermA | F: TCTAAAAAGCATGTAAAAGAA | 645 |
R: CTTCGATAGTTTATTAATATTAGT | ||
ermB | F: ATTGGAACAGGTAAAGGGC | 442 |
R: GAACATCTGTGGTATGGCG | ||
ermC | F: TCAAAACATAATATAGATAAA | 642 |
R: GCTAATATTGTTTAAATCGTCAAT | ||
sul1 | F: CATTGCCTGGTTGCTTCAT | 238 |
R: ATCCGACTCGCAGCATTT | ||
sul2 | F: CATCATTTTCGGCATCGTC | 793 |
R: TCTTGCGGTTTCTTTCAGC | ||
sul3 | F: AGATGTGATTGATTTGGGAGC | 443 |
R: TAGTTGTTTCTGGATTAGAGCCT | ||
gyrA | F: GACAAGTGAAATGAAAACGAG | 474 |
R: CGCTCCATTGACTAATAAATTAGG | ||
parC | F: CAAAACATGTCCCTTGAGGA | 568 |
R: CTAGCTTTGGGATGATCAATCAT |
基因名称 Gene name | 引物序列 Primer sequence | 目的片段 Target fragment(bp) |
---|---|---|
GAPDH | F:CGCCATCAGTAGCCCAGT | 219 |
R:CATTCCGTCGCATCCAAA | ||
bibA | F:AACCAGAAGCCAAGCCAGCAACC | 127 |
R:AGTGGACTTGCGGCTTCACCC | ||
cspA | F:AGTTAGGTGCCGTATCTGTAC | 177 |
R:CAAGGGCTGATGACGTGTTG | ||
fbsA | F:GTATCTCAGGTTACTAATGTAGC | 163 |
R:GTCTCAAACCGCAGCGACT | ||
hylB | F:AAGCCCTTGGTGGTAATCTAG | 178 |
R:GCAACATTCGTATGGTCGATG | ||
iagA | F:GGGTTATTCCTCGAAAGGTTAG | 145 |
R:TCTGTCTCTTCACGACTCGG | ||
pavA | F:ACTACTCCTATCTGCTCATCC | 134 |
R:GAATGGTTTCAATCACTGCACC | ||
cfb | F:GACTTCATTGCGTGCCAACC | 166 |
R:CAGCTTAGTTATCCCAAATCCC | ||
sip | F:AGCAAGCTGTTAGTCAAGCAG | 160 |
R:GTTTCAGCGGCAACAGAAGC |
表 2 无乳链球菌毒力基因荧光定量引物序列
Table 2 Sequence of fluorescent quantitative primers for virulence gene of Streptococcus agalactiae
基因名称 Gene name | 引物序列 Primer sequence | 目的片段 Target fragment(bp) |
---|---|---|
GAPDH | F:CGCCATCAGTAGCCCAGT | 219 |
R:CATTCCGTCGCATCCAAA | ||
bibA | F:AACCAGAAGCCAAGCCAGCAACC | 127 |
R:AGTGGACTTGCGGCTTCACCC | ||
cspA | F:AGTTAGGTGCCGTATCTGTAC | 177 |
R:CAAGGGCTGATGACGTGTTG | ||
fbsA | F:GTATCTCAGGTTACTAATGTAGC | 163 |
R:GTCTCAAACCGCAGCGACT | ||
hylB | F:AAGCCCTTGGTGGTAATCTAG | 178 |
R:GCAACATTCGTATGGTCGATG | ||
iagA | F:GGGTTATTCCTCGAAAGGTTAG | 145 |
R:TCTGTCTCTTCACGACTCGG | ||
pavA | F:ACTACTCCTATCTGCTCATCC | 134 |
R:GAATGGTTTCAATCACTGCACC | ||
cfb | F:GACTTCATTGCGTGCCAACC | 166 |
R:CAGCTTAGTTATCCCAAATCCC | ||
sip | F:AGCAAGCTGTTAGTCAAGCAG | 160 |
R:GTTTCAGCGGCAACAGAAGC |
[1] | 黄锦炉. 罗非鱼无乳链球菌病病原学、病理学及cpsE基因的原核表达研究[D]. 成都: 四川农业大学, 2012. |
HUANG Jinlu. Study on Etiology, Pathology of Tilapia Streptococcus agalactiae disease and on the Prokaryotic Expression of cpsE Gene[D]. Chengdu: Sichuan Agricultural University, 2012. | |
[2] | 袁伟. 中国罗非鱼源无乳链球菌流行特征及耐药性研究[D]. 上海: 上海海洋大学, 2017. |
YUAN Wei. Epidemiological Characteristics and Drug Resistance of Streptococcus agalactiae Isolated from Tilapia in China[D]. Shanghai: Shanghai Ocean University, 2017. | |
[3] |
Kaczorek E, Małaczewska J, Wójcik R, et al. Biofilm Production and Other Virulence Factors in Streptococcus spp. Isolated from Clinical Cases of Bovine Mastitis in Poland[J]. BMC Veterinary Research, 2017, 13: 398.
DOI PMID |
[4] | 刘龙海. 奶牛乳房炎无乳链球菌血清型分布、耐药性及其相关基因的研究[D]. 北京: 中国农业科学院, 2017. |
LIU Longhai. Distribution of Serotypes, Antimicrobial Susceptibility and Related Genes of S. agalactiae Isolates from Mastitis Cases[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017. | |
[5] | 祝宇, 路璐, 颜兴琼, 等. 云南地区牛源无乳链球菌分离鉴定及毒力基因和耐药性的检测[J]. 中国畜牧兽医, 2018, 45(11): 3261-3270. |
ZHU Yu, LU Lu, YAN Xingqiong, et al. Isolation, Identification and Detection of Virulence Genes and Drug Resistance of Streptococcus agalactiae from Cattle in Yunnan[J]. China Animal Husbandry & Veterinary Medicine, 2018, 45(11):3261-3270. | |
[6] |
Tazi A, Gueudet T, Varon E, et al. Fluoroquinolone-Resistant Group B Streptococci in Acute Exacerbation of Chronic Bronchitis[J]. Emerging Infectious Diseases, 2008, 14(2):349-350.
DOI PMID |
[7] |
Rogues A M, Dumartin C, Amadeo B, et al. Relationship between Rates of Antimicrobial Consumption and the Incidence of Antimicrobial Resistance in Staphylococcus aureus and Pseudomonas aeruginosa Isolates from 47 French Hospitals[J]. Infection Control and Hospital Epidemiology, 2007, 28(12):1389-1395.
PMID |
[8] |
Hsueh P R, Chen W H, Luh K T. Relationships between Antimicrobial Use and Antimicrobial Resistance in Gram-negative Bacteria Causing Nosocomial Infections from 1991-2003 at a University Hospital in Taiwan[J]. International Journal of Antimicrobial Agents, 2005, 26(6):463-472.
DOI URL |
[9] | 汤雨晴, 叶倩, 郑维义. 抗生素类药物的研究现状和进展[J]. 国外医药(抗生素分册), 2019, 40(4):295-301. |
TANG Yuqing, YE Qian, ZHENG Weiyi. Research Status and Development of Antibiotics[J]. World Notes on Antibiotics, 2019, 40(4):295-301. | |
[10] | 陈程, 郭淑丽, 贾海英, 等. 乌鲁木齐市SLE患者尿路感染病原菌分布特点及无乳链球菌药敏结果分析[J]. 中国抗生素杂志, 2021, 46(1):87-89. |
CHEN Cheng, GUO Shuli, JIA Haiying, et al. Distribution Characteristics of UrinaryTract Infection Pathogens and Analysis of Drug Susceptibility of Streptococcus agalactiae in Patients with Systemic Lupus Erythemotosus in Urumqi[J]. Chinese Journal of Antibiotics, 2021, 46(1):87-89. | |
[11] |
Elikwu C, Oduyebo O, Anorlu R, et al. Antibiotic Susceptibility/Resistant Gene Profiles of Group B Streptococci Isolates from Pregnant Women in a Tertiary Institution in Nigeria[J]. Journal of Clinical Sciences, 2016, 13(3):132.
DOI URL |
[12] | Minami M, Nishiyama H, Ikegami S, et al. Trend of Invasive Streptococcus agalactiae at Tertiary Care Hospital in Japan[J]. Journal of Bioences and Medicines, 2016, 4(3):15-20. |
[13] | Sunkara B, Bheemreddy S, Lorber B, et al. Group B Streptococcus Infections in Non-pregnant Adults: the Role of Immunosuppression[J]. International Journal of Infectious Diseases, 2012, 16(3):e182-e186. |
[14] | 邓颖颖. 无乳链球菌的耐药性及耐药机制研究[D]. 广州: 南方医科大学, 2019. |
DENG Yingying. Antimicrobial Resistance and Mechanism Research of Streptococcus agalactiae[D]. Guangzhou: Southern Medical University, 2019. | |
[15] | 刘婵. 广东省罗非鱼无乳链球菌分子流行病学及耐药性研究[D]. 天津: 天津农学院, 2018. |
LIU Chan. Molecular Epidemiology and Drug Resistance of Streptococcus agalactiae Isolated in Tilapia from Guangdong Province[D]. Tianjing: Tianjin Agricultural University, 2018. | |
[16] | Cossart P, Vicente M F, Mengaud J, et al. Listeriolysin O is Essential for Virulence of Listeria Monocytogenes: Direct Evidence Obtained by Gene Complementation[J]. Infection & Immunity, 1989, 57(11):3629. |
[17] |
Kim M J, Han J, Park J S, et al. Various Enterotoxin and Other Virulence Factor Genes Widespread Among Bacillus cereus and Bacillus thuringiensis Strains[J]. Journal of Microbiology and Biotechnology, 2015, 25(6):872-879.
DOI URL |
[18] |
Burnside K, Lembo A, de los Reyes M, et al. Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase[J]. PLoS ONE, 2010, 5(6): e11071.
DOI URL |
[19] | 王爱媛, 郑立新, 蒲文渊, 等. 海南无乳链球菌的毒力基因与耐药特性分析[J]. 水产科学, 2020, 39(1):117-123. |
WANG Aiyuan, ZHENG Lixing, PU Wenyuan, et al. Virulence Genes and Drug Resistance of Pathogen Streptococcus agalactiae in Hainan[J]. Fisheries Science, 2020, 39(1):117-123. | |
[20] | 张保海, 罗梓丹, 芦彪, 等. 四川部分地区奶牛源无乳链球菌的分离鉴定、毒力基因检测及耐药性分析[J]. 西北农业学报, 2020, 29(3):327-333. |
ZHANG Baohai, LUO Zidan, LU Biao, et al. Isolation, Virulence Gene Test and Drug Resistance Analysis of Streptococcus agalactiae from Dairy Cows in Some Areas of Sichuan[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2020, 29(3):327-333. | |
[21] |
Carvalho-Castro G A, Silva J R, Paiva L V, et al. Molecular Epidemiology of Streptococcus agalactiaeIsolated from Mastitis in Brazilian Dairy Herds[J]. Brazilian Journal of Microbiology, 2017, 48(3):551-559.
DOI PMID |
[22] |
Duarte R S, Bellei B C, Miranda O P, et al. Distribution of Antimicrobial Resistance and Virulence-Related Genes among Brazilian Group B Streptococci Recovered from Bovine and Human Sources[J]. Antimicrobial Agents and Chemotherapy, 2005, 49(1):97-103.
PMID |
[23] | 苏友禄, 王宝屯, 谢云丹, 等. cel-EIIB蛋白调控罗非鱼无乳链球菌强毒,弱毒株毒力相关基因的表达模式[J]. 中国水产科学, 2020, v. 27(11):96-106. |
SU Youlu, WANG Baotun, XIE Yundan, et al. Analysis of Expression Patterns of Virulence-related Genes Regulated by the cel-EIIB Protein in Virulent and Attenuated Strains of Streptococcus agalactiae in Tilapia[J]. Journal of Fishery Sciences of China, 2020, 27(11):96-106. | |
[24] | Wang Z, Guo C, Xu Y, et al. Two Novel Functions of Hyaluronidase from Streptococcus agalactiaeAre Enhanced Intracellular Survival and Inhibition of Proinflammatory Cytokine Expression[J]. Infection & Immunity, 2014, 82(6):2615-2625. |
[1] | 唐碧徽, 张俐华, 李海英, 张冲, 蒋廷浩, 赵晓钰, 蒋腾, 丁雅文, 吴盈萍, 赵全庄. 伊犁鹅和霍尔多巴吉鹅在繁殖性能、血清激素水平和基因表达量的比较[J]. 新疆农业科学, 2023, 60(5): 1271-1280. |
[2] | 王东, 易海波, 李宏博, 秦蕾, 徐琦琦, 吴慧敏, 夏利宁. 新疆阿克苏地区猪源粪肠球菌耐药性分析及相关耐药基因的检测[J]. 新疆农业科学, 2023, 60(12): 3113-3120. |
[3] | 侯晨曦, 洪文娟, 何宗龙, 决肯·阿尼瓦什. LEF1基因在巴什拜羊不同毛色皮肤组织中的DNA甲基化及mRNA表达水平分析[J]. 新疆农业科学, 2022, 59(11): 2742-2478. |
[4] | 孙慧琴, 轩慧勇, 买占海, 杨紫嫣, 夏利宁. 猪源大肠杆菌耐药性及相关耐药基因检测[J]. 新疆农业科学, 2021, 58(1): 190-196. |
[5] | 胥兰, 刘英玉, 麦多, 朱明月, 蒋金豆, 卢炜, 朱梦含, 郑晓风, 彭斌. 新疆牛羊产业链中金黄色葡萄球菌的污染调查与毒力基因检测[J]. 新疆农业科学, 2021, 58(1): 182-189. |
[6] | 杨紫嫣, 姚晓慧, 马木尔·阿克木汉, 王舒丰, 王凯, 夏利宁. 不同动物粪源葡萄球菌耐药性调查及耐药基因检测分析[J]. 新疆农业科学, 2020, 57(5): 967-973. |
[7] | 张凌, 佟盼盼, 张毅, 马晓玉, 张萌萌, 刘璐瑶, 姚刚, 陈童锦悦, 苏战强. 羔羊STEC的耐药性、毒力基因和血清型分析[J]. 新疆农业科学, 2020, 57(10): 1921-1930. |
[8] | 杨雪梅, 范雪, 田可川, 黄锡霞, 朱桦, 阿布来提·苏来曼, 陈华峰, 何军敏, 赵冰茹, 徐新明, 付雪峰, 田月珍, 吴伟伟, 哈尼克孜·吐拉甫, 杜建文. 苏博美利奴羊胚胎期WNT2基因的组织表达及其生物信息学预测分析[J]. 新疆农业科学, 2019, 56(12): 2320-2328. |
[9] | 陈月月,王凯,王舒丰,姚晓慧,轩慧勇,马木尔,夏利宁. 新疆伊犁地区不同动物源大肠杆菌耐药性调查[J]. 新疆农业科学, 2018, 55(8): 1560-1568. |
[10] | 韩猛立, 张星星, 吴桐忠, 郭强强, 黄新, 钟发刚. 兔源巴氏杆菌分离与鉴定[J]. 新疆农业科学, 2018, 55(7): 1333-1342. |
[11] | 马木尔·阿克木汉,库尔班乃木·卡地尔,姚晓慧,王舒丰,王凯,夏利宁. 新疆焉耆县不同生长期猪不同部位携带葡萄球菌的耐药性比较[J]. 新疆农业科学, 2018, 55(2): 379-382. |
[12] | 代婧, 彭斌, 雷程红, 阿热阿依·海依拉提. 新疆部分地区牛源空肠弯曲菌分离鉴定及耐药性分析[J]. 新疆农业科学, 2017, 54(9): 1730-1736. |
[13] | 李亚东;贺双;田笑明;董雪;杨德松. 啶磺草胺对不同品种小麦生长发育的影响[J]. , 2017, 54(4): 682-693. |
[14] | 董雪;梁友;田笑明;曹媛媛;杨德松. 不同小麦品种对啶磺草胺的耐药性差异[J]. , 2014, 51(8): 1474-1481. |
[15] | 王蒴;杨学云;李建军;冉多良;王治才. 奶牛乳房炎无乳链球菌的分离鉴定与特异、快速检测方法[J]. , 2014, 51(7): 1328-1334. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||