牛源无乳链球菌耐药性与毒力基因的表达量差异分析

doi:10.6048/j.issn.1001-4330.2022.09.027

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

牛源无乳链球菌耐药性与毒力基因的表达量差异分析

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

1.新疆农业大学动物科学学院/新疆肉乳用草食动物营养实验室，乌鲁木齐 830052
2.新疆农业科学院农业质量标准与检测技术研究所/农业农村部农产品质量安全风险评估实验室/新疆农产品质量安全实验室，乌鲁木齐 830091

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:
新疆维吾尔自治区自然科学基金“hylB介导无乳链球菌对乳腺上皮细胞泌乳功能的调控机制研究”(2019D01B32);自治区公益性科研院所基本科研业务经费项目“无乳链球菌VBNC状态诱导与分子检测技术研究”(KY2019031);自治区重点实验室开放课题“新疆地区生鲜乳中无乳链球菌的耐药性及其机制研究”(2018D04016);自治区天山青年计划“新疆地区生鲜乳中嗜冷菌分离鉴定及污染源分析”(2018Q087);自治区重大科技专项“乳品质量风险分析与预警技术研究应用”(2020A01001-3);新疆维吾尔自治区研究生创新项目“新疆北疆规模化牛场牛源无乳链球菌耐药表型与耐药基因相关性调查分析”(XJ2020G158)

Differential Expression of Drug Resistance and Virulence Genes in Streptococcus agalactiae Isolated from Cattle

FAN Xue¹^,²(), SHAO Wei¹, ZHAO Yankun²(), DU Xiaohui¹, CHEN He², WANG Fulan², WANG Shuai²

1. College of Animal Science， Xinjiang Agricultural University， Xinjiang Key Laboratory of Meat Milk Production Herbivore Nutrition， Urumqi 830052， China
2. Institute of Quality Standard and Testing Technology for Agro-Products， Xinjiang Academy of Agriculture Science， Ministry of Agriculture Laboratory of Quality and Safety Risk Assessment for Agro-Products， Key Laboratory of Agro-Products Quality and Safety of Xinjiang， Urumqi 830091， China

Received:2021-02-26 Online:2022-09-20 Published:2023-01-16
Correspondence author: ZHAO Yankun
Supported by:
National Natural Science Foundation of China “Regulatory mechanism of hylB mediated Streptococcus agalactiae on lactation function of breast epithelial cells”(2019D01B32);The basic scientific research funds of public welfare scientific research institutes of the Autonomous Region “Study on VBNC induction and molecular detection of Streptococcus agalactiae”(KY2019031);Open project of Key Laboratory in Xinjiang “Drug resistance and mechanism of Streptococcus agalactiae isolated from fresh milk in Xinjiang”(2018D04016);Tianshan youth program “Isolation and identification of psychrophilic bacteria from fresh milk and analysis of pollution sources in Xinjiang”(2018Q087);Major science and technology projects of the Autonomous Region “Research and application of dairy quality risk analysis and early warning technology”(2020A01001-3);Postgraduate innovation project of Xinjiang Uygur Autonomous Region “Investigation and Analysis on the correlation between drug resistance phenotype and drug resistance gene of Streptococcus agalactiae isolated from cattle in large scale cattle farms in Northern Xinjiang”(XJ2020G158)

摘要/Abstract

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。

关键词: 无乳链球菌; 毒力基因; 耐药基因; 耐药性; 表达量

Abstract:

【Objective】 The purpose of this study was to investigate the drug resistance of Streptococcus agalactiae to antibacterial agents and determine the carrying status of virulence gene and drug resistance gene of Streptococcus agalactiae.【Methods】 In this study， microdilution broth method and common PCR were used to detect the resistance of 16 kinds of antibacterial drugs and related drug resistance genes and virulence genes of Streptococcus agalactiae from bovine. Moreover， the expression levels of 8 virulence genes in different strains were analyzed by fluorescence quantitative PCR. 【Results】 （1）The sensitivity of Streptococcus agalactiae to 11 drugs was more than 65%， among which the highest sensitivity was florfenicol （92.4%） and ceftiofur （88.4%）. The resistance rate of Streptococcus agalactiae to ampicillin， erythromycin and clindamycin was more than 50%， and the resistance rate of Streptococcus agalactiae to sulfagan was more than 45%. （2） The detection rates of gyrA， sul1， ermb and ermc were 100%， 86.67%， 93.3% and 33.3% respectively， while erma， sul2， sul3 and parc were not detected. （3） The detection rates of virulence genes pavA， cfb， fbsA， bibA， cspA， sip， iagA， hylB， rib， bca and cylE were 100%. The detection rates of rib， bca and cylE were 13.3%， 53.3% and 73.3%， respectively. However， the bac， lmb and scpB were not detected. （4） Fluorescence quantitative analysis showed that the expression levels of virulence gene was significantly different among different strains （P<0.05）. 【Conclusion】 The resistance rate of Streptococcus agalactiae to erythromycin and clindamycin is high. The virulence factor of Streptococcus agalactiae is an important factor of host infection disease.

Key words: Streptococcus agalactiae; virulence gene; drug resistance gene; drug resistance; expression level

中图分类号:

S815

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.

图/表 7

参考文献 24

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基因名称 Gene name	引物序列 Primer sequence	目的片段 Target fragment（bp）
pavA	F：TTCCCATGATTTCAACAACAAG	495
pavA	R：AACCTTTTGACCATGAATTGGTA	495
scpB	F： AGTTGCTTCTTACAGCCCAGA	567
scpB	R： GGCGCAGACATACTAGTTCCA	567
rib	F：CAGGAAGTGCTGTTACGTTAAAC	306
rib	R： CGTCCCATTTAGGGTCTTCC	306
bac	F：CTATTTTTGATATTGACAATGCAA	592
bac	R：GTCGTTACTTCCTTGAGATGTAAC	592
cylE	F： TGACATTTACAAGTGACGAAG	248
cylE	R： TTGCCAGGAGGAGAATAGGA	248
bca	F：TAACAGTTATGATACTTCACAGAC	320
bca	R：ACGACTTTCTTCCGTCCACTTAGG	320
cfb	F： GCTGTTTGAAGTGCTGCTTG	288
cfb	R： GACTTCATTGCGTGCCAAC	288
lmb	F： AGTCAGCAAACCCCAAACAG	397
lmb	R： GCTTCCTCACCAGCTAAAACG	397
hylB	F： CACCAATCCCCACTCTACTA	346
hylB	R： TGTGTCAAACCATCTATCAG	346
fbsA	F：AGAGCCAAGTAGGTCAACTTATAG	290
fbsA	R： TTCATTGCGTCTCAAACCG	290
bibA	F：AACCAGAAGCCAAGCCAGCAACC	127
bibA	R： AGTGGACTTGCGGCTTCACCC	127
cspA	F： GGTCGCGATAGAGTTTCTTCCGC	104
cspA	R： AACGCCTGGGGCTGATTTGGC	104
sip	F： TCTCTCAATACAATTTCGGAAG	867
sip	R： GTGGTCATAGTGGTTGGCAGT	867
iagA	F： CGGGATTGATCTAAGTCGCT	459
iagA	R： CCATCAACATCAGTCGCTAA	459
ermA	F： TCTAAAAAGCATGTAAAAGAA	645
ermA	R： CTTCGATAGTTTATTAATATTAGT	645
ermB	F： ATTGGAACAGGTAAAGGGC	442
ermB	R： GAACATCTGTGGTATGGCG	442
ermC	F： TCAAAACATAATATAGATAAA	642
ermC	R： GCTAATATTGTTTAAATCGTCAAT	642
sul1	F： CATTGCCTGGTTGCTTCAT	238
sul1	R： ATCCGACTCGCAGCATTT	238
sul2	F： CATCATTTTCGGCATCGTC	793
sul2	R： TCTTGCGGTTTCTTTCAGC	793
sul3	F： AGATGTGATTGATTTGGGAGC	443
sul3	R： TAGTTGTTTCTGGATTAGAGCCT	443
gyrA	F： GACAAGTGAAATGAAAACGAG	474
gyrA	R： CGCTCCATTGACTAATAAATTAGG	474
parC	F： CAAAACATGTCCCTTGAGGA	568
parC	R： CTAGCTTTGGGATGATCAATCAT	568

基因名称 Gene name	引物序列 Primer sequence	目的片段 Target fragment（bp）
pavA	F：TTCCCATGATTTCAACAACAAG	495
pavA	R：AACCTTTTGACCATGAATTGGTA	495
scpB	F： AGTTGCTTCTTACAGCCCAGA	567
scpB	R： GGCGCAGACATACTAGTTCCA	567
rib	F：CAGGAAGTGCTGTTACGTTAAAC	306
rib	R： CGTCCCATTTAGGGTCTTCC	306
bac	F：CTATTTTTGATATTGACAATGCAA	592
bac	R：GTCGTTACTTCCTTGAGATGTAAC	592
cylE	F： TGACATTTACAAGTGACGAAG	248
cylE	R： TTGCCAGGAGGAGAATAGGA	248
bca	F：TAACAGTTATGATACTTCACAGAC	320
bca	R：ACGACTTTCTTCCGTCCACTTAGG	320
cfb	F： GCTGTTTGAAGTGCTGCTTG	288
cfb	R： GACTTCATTGCGTGCCAAC	288
lmb	F： AGTCAGCAAACCCCAAACAG	397
lmb	R： GCTTCCTCACCAGCTAAAACG	397
hylB	F： CACCAATCCCCACTCTACTA	346
hylB	R： TGTGTCAAACCATCTATCAG	346
fbsA	F：AGAGCCAAGTAGGTCAACTTATAG	290
fbsA	R： TTCATTGCGTCTCAAACCG	290
bibA	F：AACCAGAAGCCAAGCCAGCAACC	127
bibA	R： AGTGGACTTGCGGCTTCACCC	127
cspA	F： GGTCGCGATAGAGTTTCTTCCGC	104
cspA	R： AACGCCTGGGGCTGATTTGGC	104
sip	F： TCTCTCAATACAATTTCGGAAG	867
sip	R： GTGGTCATAGTGGTTGGCAGT	867
iagA	F： CGGGATTGATCTAAGTCGCT	459
iagA	R： CCATCAACATCAGTCGCTAA	459
ermA	F： TCTAAAAAGCATGTAAAAGAA	645
ermA	R： CTTCGATAGTTTATTAATATTAGT	645
ermB	F： ATTGGAACAGGTAAAGGGC	442
ermB	R： GAACATCTGTGGTATGGCG	442
ermC	F： TCAAAACATAATATAGATAAA	642
ermC	R： GCTAATATTGTTTAAATCGTCAAT	642
sul1	F： CATTGCCTGGTTGCTTCAT	238
sul1	R： ATCCGACTCGCAGCATTT	238
sul2	F： CATCATTTTCGGCATCGTC	793
sul2	R： TCTTGCGGTTTCTTTCAGC	793
sul3	F： AGATGTGATTGATTTGGGAGC	443
sul3	R： TAGTTGTTTCTGGATTAGAGCCT	443
gyrA	F： GACAAGTGAAATGAAAACGAG	474
gyrA	R： CGCTCCATTGACTAATAAATTAGG	474
parC	F： CAAAACATGTCCCTTGAGGA	568
parC	R： CTAGCTTTGGGATGATCAATCAT	568

基因名称 Gene name	引物序列 Primer sequence	目的片段 Target fragment（bp）
GAPDH	F：CGCCATCAGTAGCCCAGT	219
GAPDH	R：CATTCCGTCGCATCCAAA	219
bibA	F：AACCAGAAGCCAAGCCAGCAACC	127
bibA	R：AGTGGACTTGCGGCTTCACCC	127
cspA	F：AGTTAGGTGCCGTATCTGTAC	177
cspA	R：CAAGGGCTGATGACGTGTTG	177
fbsA	F：GTATCTCAGGTTACTAATGTAGC	163
fbsA	R：GTCTCAAACCGCAGCGACT	163
hylB	F：AAGCCCTTGGTGGTAATCTAG	178
hylB	R：GCAACATTCGTATGGTCGATG	178
iagA	F：GGGTTATTCCTCGAAAGGTTAG	145
iagA	R：TCTGTCTCTTCACGACTCGG	145
pavA	F：ACTACTCCTATCTGCTCATCC	134
pavA	R：GAATGGTTTCAATCACTGCACC	134
cfb	F：GACTTCATTGCGTGCCAACC	166
cfb	R：CAGCTTAGTTATCCCAAATCCC	166
sip	F：AGCAAGCTGTTAGTCAAGCAG	160
sip	R：GTTTCAGCGGCAACAGAAGC	160

基因名称 Gene name	引物序列 Primer sequence	目的片段 Target fragment（bp）
GAPDH	F：CGCCATCAGTAGCCCAGT	219
GAPDH	R：CATTCCGTCGCATCCAAA	219
bibA	F：AACCAGAAGCCAAGCCAGCAACC	127
bibA	R：AGTGGACTTGCGGCTTCACCC	127
cspA	F：AGTTAGGTGCCGTATCTGTAC	177
cspA	R：CAAGGGCTGATGACGTGTTG	177
fbsA	F：GTATCTCAGGTTACTAATGTAGC	163
fbsA	R：GTCTCAAACCGCAGCGACT	163
hylB	F：AAGCCCTTGGTGGTAATCTAG	178
hylB	R：GCAACATTCGTATGGTCGATG	178
iagA	F：GGGTTATTCCTCGAAAGGTTAG	145
iagA	R：TCTGTCTCTTCACGACTCGG	145
pavA	F：ACTACTCCTATCTGCTCATCC	134
pavA	R：GAATGGTTTCAATCACTGCACC	134
cfb	F：GACTTCATTGCGTGCCAACC	166
cfb	R：CAGCTTAGTTATCCCAAATCCC	166
sip	F：AGCAAGCTGTTAGTCAAGCAG	160
sip	R：GTTTCAGCGGCAACAGAAGC	160

牛源无乳链球菌耐药性与毒力基因的表达量差异分析

Differential Expression of Drug Resistance and Virulence Genes in Streptococcus agalactiae Isolated from Cattle

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[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.