福建省莆田市鸭疫里默氏杆菌的喹诺酮类耐药基因检测与QRDR基因突变分析

林彬彬; 林志敏; 谢碧林; 翁汉东; 王秀祯; 程龙飞; 傅光华; 刘荣昌

doi:10.19303/j.issn.1008-0384.2022.011.005

福建省莆田市鸭疫里默氏杆菌的喹诺酮类耐药基因检测与QRDR基因突变分析

doi: 10.19303/j.issn.1008-0384.2022.011.005

1.
福建省莆田市农业科学研究所，福建　莆田　351144
2.
福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 福建省科技计划农业引导性（重点）项目（2019N0038）

详细信息

作者简介:
林彬彬（1986−），女，硕士，助理研究员，研究方向：家禽疫病的诊断与防治研究（E-mail：lbbinz@163.com）

通讯作者:
林志敏（1967−），男，副研究员，研究方向：家禽疫病的诊断与防治研究（E-mail：ptlzm@126.com）

中图分类号: S 852.61
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出版历程
- 收稿日期: 2022-06-29
- 修回日期: 2022-10-17
- 网络出版日期: 2022-12-28
- 刊出日期: 2022-11-28

Detection of Quinolone Resistance Genes and QRDR Mutation in Riemerella anatipestifer Found in Putian, Fujian

1.
Putian Institute of Agricultural Sciences, Putian, Fujian　351144, China
2.
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的了解目前福建省莆田市鸭疫里默氏杆菌菌株对喹诺酮类相关耐药基因的携带情况，探讨其耐药机制。方法采用K-B法对2019–2021年分离自福建省莆田市的55株鸭疫里默氏杆菌菌株进行6种喹诺酮类药物的药敏试验，采用PCR方法对喹诺酮耐药决定区基因（QRDR）和质粒介导的喹诺酮耐药基因（PMQR）等6种耐药基因进行检测并分析。结果在55株鸭疫里默氏杆菌菌株中，50%以上菌株对吡哌酸、诺氟沙星、恩诺沙星、环丙沙星高度耐药，且为多重耐药；90%以上菌株对氧氟沙星高度敏感。耐药基因PCR检测结果显示，未检测到qnrA、qnrB、qnrS、oqxA等4种质粒介导的喹诺酮耐药基因。QRDR突变分析发现，gyrA基因推测氨基酸序列中有6个位点发生突变，突变类型有S83R和S83I、S84P、D87A和D87G、A183V、N202E、Y211H；其中以S83I、N202E、Y211H的突变最多见，突变率分别为98.18%（54/55）、92.73%（51/55）、90.91%（50/55）；39株耐恩诺沙星菌株中gyrA基因出现S83I（38/39）和S83R（1/39）突变，S83I突变也在3株恩诺沙星敏感菌株中发现。parC基因只在1株恩诺沙星中介菌株中出现F155S突变，这在鸭疫里默氏杆菌中未见报道。结论莆田市鸭疫里默氏杆菌的喹诺酮类药物耐药严重，未发现质粒介导的喹诺酮耐药基因，gyrA基因的S83I突变可能是介导鸭疫里默氏杆菌对喹诺酮类药物耐药的主要机制之一。
- 鸭疫里默氏杆菌 /
- 喹诺酮类 /
- 耐药基因 /
- gyrA基因 /
- parC基因
Abstract: Objective Epidemiology and mechanism of quinolone-related drug resistance genes of Riemerella anatipestifer found in ducks in Putian, Fujian were investigated. Method The K-B method was applied to examine 55 strains of R. anatipestifer to determine their resistance to 6 types of quinolones. PCR was employed to detect and analyze the quinolone resistance determining region (QRDR) and plasmid mediated quinolone resistance (PMQR) genes. Result More than half of the 55 strains examined were multi-resistant and highly resistant to pipemidic acid, norfloxacin, enrofloxacin, and ciprofloxacin; and more than 90% of them highly sensitive to ofloxacin. PCR did not find the 4 PMQRs, i.e., qnrA, qnrB, qnrS, and oqxA, in the specimens. On the other hand, 6 QRDR mutations in the deduced amino acid sequences of gyrA, including S83R and S83I, S84P, D87A and D87G, A183V, N202E, and Y211H were detected. Among them, S83I, N202E, and Y211H had the highest mutation rates of 98.18% (54/55), 92.73% (51/55), and 90.91% (50/55), respectively. Of which, the S83I (38/39) and S83R (1/39) mutations were found in gyrA of 39 enrofloxacin-resistant strains, and the S83I mutations also in that of 3 enrofloxacin-sensitive strains. There was only one enrofloxacin-intermediated strain that had the F155S mutation in parC, which was not previously reported to exist in R. anatipestifer. Conclusion The quinolone-resistance of R. anatipestifer found in ducks in Putian was severe. No PMQR was detected in the samples, but the S83I mutation in gyrA of QRDR could be the major culprit that involved in the drug resistance of R. anatipestifer.
- Riemerella anatipestifer /
- quinolones /
- drug resistance gene /
- gyrA gene /
- parC gene

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表 1 引物序列及其反应参数

Table 1. Sequences and reaction parameters of primer

耐药基因 Drug resistance gene		引物序列（5′-3′） Sequence（5′-3′）	退火温度 Tm/℃	产物大小 Product length/bp	参考文献 Reference
喹诺酮耐药决定区 QRDR	gyrA	F: ATGTCCGTTATTGTATCTCG R: TGGAACGCATCTCTAACC	52	626
喹诺酮耐药决定区 QRDR	parC	F: GACCGTGCCATTCCTTCTA R: AGCCCTACACCAATACCTTCC	56	416
质粒介导的喹诺酮耐药 PMQR	qnrA	F:TCAGCAAGAGGATTTCTCA R:GGCAGCACTATTACTCCCA	52	627	[13]
	qnrB	F:GATCGTGAAAGCCAGAAAGG R:ACGATGCCTGGTAGTTGTCC	57	469	[14]
	qnrS	F:ACGACATTCGTCAACTGCAA R:TAAATTGGCACCCTGTAGGC	54	417	[14]
	oqxA	F: GATCAGTCAGTGGGATAGTTT R: TACTCGGCGTTAACTGATTA	52	670	[15]

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表 2 55株鸭疫里默氏杆菌菌株对6种喹诺酮类药物的药敏结果

Table 2. Sensitivity to 6 quinolones of 55 R. anatipestifer strains

药物名称 Drug name	耐药菌株比例 Proportion of resistant strains/%	中介菌株比例 Proportion of intermediate strains/%	敏感菌株比例 Proportion of sensitive strains/%
恩诺沙星 Enrofloxacin	70.91（39/55）	23.64（13/55）	5.45（3/55）
环丙沙星 Ciprofloxacin	56.36（31/55）	34.55（19/55）	9.09（5/55）
氧氟沙星 Ofloxacin	7.27（4/55）	1.82（1/55）	90.91（50/55）
诺氟沙星 Norfloxacin	83.64（46/55）	10.91（6/55）	5.45（3/55）
萘啶酸 Nalidixic acid	32.73（18/55）	29.09（16/55）	38.18（21/55）
吡哌酸 Pipemidic acid	96.36（53/55）	1.82（1/55）	1.82（1/55）

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表 3 55株鸭疫里默氏杆菌菌株喹诺酮类药物的多重耐药性

Table 3. Multiple drug resistance of 55 R. anatipestifer strains to quinolones

耐药类型 Drug resistance type	耐药谱 Drug resistance spectrum	耐药株 Resistance strain/ 株	耐药率 Drug resistance rate/%
1耐 1 resistance	PPA	1	1.82（1/55）
2耐 2 resistance	ENR-CIP	1	23.64（13/55）
	ENR-NOR	1
	ENR-PPA	2
	CIP-PPA	2
	NOR-PPA	7
3耐 3 resistance	ENR-CIP-PPA	1	21.82（12/55）
	ENR-NOR-PPA	5
	ENR-NA-PPA	1
	CIP-NOR-PPA	1
	NOR-NA-PPA	4
4耐 4 resistance	ENR-CIP-OFX-PPA	1	36.36（20/55）
	ENR-CIP-NOR-PPA	13
	ENR-NOR-NA-PPA	4
	CIP-OFX-NOR-PPA	2
5耐 5 resistance	ENR-CIP-NOR-NA-PPA	6	12.73（7/55）
5耐 5 resistance	ENR-CIP-OFX-NOR-NA	1	12.73（7/55）
6耐 6 resistance	ENR-CIP-OFX-NOR-NA-PPA	2	3.64（2/55）

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表 4 55株鸭疫里默氏杆菌菌株gyrA基因及推测氨基酸的突变位置

Table 4. Mutation location in gyrA and putative amino acid of 55 R. anatipestifer strains

碱基突变 Base mutation	氨基酸突变 Amino acid mutation	突变率 Mutation rate/%
AGC-AGG	S83R	1.82（1/55）
AGC-ATC	S83I	98.18（54/55）
TCT-CCT	S84P	3.64（2/55）
GAC-GCC	D87A	1.82（1/55）
GAC-GGC	D87G	1.82（1/55）
GCG-GTG	A183V	1.82（1/55）
AAT-GAG	N202E	92.73（51/55）
TAC-CAC	Y211H	90.91（50/55）
S：丝氨酸（Ser）；R：精氨酸（Arg）；I：异亮氨酸（Ile）；P：脯氨酸（Pro）；D：天冬氨酸（Asp）；A：丙氨酸（Ala）；G：甘氨酸（Gly）；V：缬氨酸（Val）；N：天冬酰胺（Asn）；E：谷氨酸（Glu）；Y：酪氨酸（Tyr）；H：组氨酸（His）。 S: Serine (Ser); R: Arginine (Arg); I: Isoleucine (Ile); P: Proline (Pro); D: Aspartate (Asp); A: Alanine (Ala); G: Glycine (Gly); V: Valine (Val); N: Asparagine (Asn); E: Glutamate (Glu); Y: Tyrosine (Tyr); H: Histidine (His).

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表 5 55株鸭疫里默氏杆菌菌株gyrA基因推测氨基酸的突变类型

Table 5. Mutation types of putative amino acid in gyrA of 55 R. anatipestifer strains

氨基酸突变 Amino acid mutation	突变率 Mutation rate/%	耐药株 Resistance strain/株	敏感株 Sensitive strain/株	中介株 Intermediary strain/株
S83I	7.27（4/55）	4	0	0
S83I-D87A-A183V-N202E-Y211H	1.82（1/55）	1	0	0
S83R-S84P-N202E-Y211H	1.82（1/55）	1	0	0
S83I-S84P-D87G-N202E	1.82（1/55）	1	0	0
S83I-N202E-Y211H	87.27（48/55）	32	3	13
S：丝氨酸（Ser）；I：异亮氨酸（Ile）；R：精氨酸（Arg）；P：脯氨酸（Pro）；D：天冬氨酸（Asp）；A：丙氨酸（Ala）；G：甘氨酸（Gly）；V：缬氨酸（Val）；N：天冬酰胺（Asn）；E：谷氨酸（Glu）；Y：酪氨酸（Tyr）；H：组氨酸（His）。 S: Serine (Ser); I: Isoleucine (Ile); R: Arginine (Arg); P: Proline (Pro); D: Aspartate (Asp); A: Alanine (Ala); G: Glycine (Gly); V: Valine (Val); N: Asparagine (Asn); E: Glutamate (Glu); Y: Tyrosine (Tyr); H: Histidine (His).

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表 6 55株鸭疫里默氏杆菌菌株parC基因及推测氨基酸的突变位置

Table 6. Mutation location of parC and putative amino acid of 55 R. anatipestifer strains

碱基突变 Base mutation	氨基酸突变 Amino acid mutation	突变率 Mutation rate/%	耐药株 Resistance strain/株	敏感株 Sensitive strain/株	中介株 Intermediary strain/株
TTT-TCC	F155S	1.82（1/55）	0	0	1
F：苯丙氨酸（Phe）；S：丝氨酸（Ser）。 F: Phenylalanine (Phe); S: Serine (Ser).

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参考文献(27)

[1]	杨永胜, 陈研, 薛亚飞, 等. 4株鹅源鸭疫里默氏杆菌生物学特性分析 [J]. 中国动物传染病学报, 2020, 28(5):62−66. YANG Y S, CHEN Y, XUE Y F, et al. Biological characteristics of four Riemerella anatipestifer isolates from geese [J]. Chinese Journal of Animal Infectious Diseases, 2020, 28(5): 62−66.(in Chinese)
[2]	胡洋, 黄书伦, 程方俊, 等. 鹅源鸭疫里默氏杆菌的分离鉴定及药敏试验 [J]. 黑龙江畜牧兽医, 2018(2):112−115,244. HU Y, HUANG S L, CHENG F J, et al. Isolation, identification and drug sensitivity test of Riemerella anatipestifer from goose and duck [J]. Heilongjiang Animal Science and Veterinary Medicine, 2018(2): 112−115,244.(in Chinese)
[3]	LOZICA L, MAZIĆ M, GOTTSTEIN Ž. A case study of a Riemerella anatipestifer infection on a commercial turkey farm in Croatia [J]. European Poultry Science, 2021, 85: 1−7.
[4]	吴彩艳, 廖申权, 戚南山, 等. 广东省鸭疫里默氏杆菌流行病学监测及遗传进化关系 [J]. 华南农业大学学报, 2022, 43(2):1−10. WU C Y, LIAO S Q, QI N S, et al. Epidemiological surveillance and genetic evolution of Riemerella anatipestifer in Guangdong Province [J]. Journal of South China Agricultural University, 2022, 43(2): 1−10.(in Chinese)
[5]	朱元军, 王小莺, 杨德鸿, 等. 我国南方部分地区鸭疫里氏杆菌的分离鉴定及耐药情况调查 [J]. 畜牧与兽医, 2019, 51(12):106−111. ZHU Y J, WANG X Y, YANG D H, et al. Identification and antibiotic resistance analysis of Riemerella anatipestifer isolated from diseased ducks in South China [J]. Animal Husbandry & Veterinary Medicine, 2019, 51(12): 106−111.(in Chinese)
[6]	金龙翔, 程龙飞, 陈红梅, 等. 鸭疫里默氏菌对氟喹诺酮药物的主动外排作用 [J]. 福建农业学报, 2014, 29(5):413−416. JIN L X, CHENG L F, CHEN H M, et al. The active efflux of Riemerella anatipestifer for fluoroquinolone antibiotics [J]. Fujian Journal of Agricultural Sciences, 2014, 29(5): 413−416.(in Chinese)
[7]	邵莉萍, 张继瑜. 喹诺酮类药物的抗菌活性与细菌耐药性研究进展 [J]. 中国畜牧兽医, 2017, 44(9):2773−2782. SHAO L P, ZHANG J Y. Research advances on antibacterial activity and bacterial resistance of quinolones [J]. China Animal Husbandry & Veterinary Medicine, 2017, 44(9): 2773−2782.(in Chinese)
[8]	LI Y F, JIANG H X, XIANG R, et al. Effects of two efflux pump inhibitors on the drug susceptibility of Riemerella anatipestifer isolates from China [J]. Journal of Integrative Agriculture, 2016, 15(4): 929−933. doi: 10.1016/S2095-3119(15)61031-0
[9]	王美珍, 陈昌福, 刘振兴, 等. 嗜水气单胞菌对四环素类和氟喹诺酮类药物的耐药性研究 [J]. 华中农业大学学报, 2011, 30(1):89−93. WANG M Z, CHEN C F, LIU Z X, et al. In vitro study on drug-resistance characteristics of Aeromonas hydrophila to tetracyclines and fluoroquinolones [J]. Journal of Huazhong Agricultural University, 2011, 30(1): 89−93.(in Chinese)
[10]	马叶本. 我国部分地区科瓦利斯沙门菌分离鉴定、耐药性及PFGE分子分型研究[D]. 广州: 华南农业大学, 2019. MA Y B. Isolation, identification, drug resistance and PFGE molecular typing of Salmonella corvallis in some areas of China[D]. Guangzhou: South China Agricultural University, 2019. (in Chinese)
[11]	姜芹, 孙冰清, 顾欣, 等. 动物源大肠埃希菌gyrA基因突变与氟喹诺酮耐药相关性分析 [J]. 动物医学进展, 2020, 41(4):63−66. JIANG Q, SUN B Q, GU X, et al. Analysis of mutations in gyrA gene associated with fluoroquinolone resistance in Escherichia coli isolates from animals [J]. Progress in Veterinary Medicine, 2020, 41(4): 63−66.(in Chinese)
[12]	陈杨, 刘理慧, 吴翠蓉, 等. 喹诺酮类耐药基因qnr的研究进展 [J]. 国外医药(抗生素分册), 2021, 42(4):193−203. CHEN Y, LIU L H, WU C R, et al. Progress in the mechanism of quinolone resistance of qnr family [J]. World Notes on Antibiotics, 2021, 42(4): 193−203.(in Chinese)
[13]	GUERRA B, HELMUTH R, THOMAS K, et al. Plasmid-mediated quinolone resistance determinants in Salmonella spp. isolates from reptiles in Germany [J]. Journal of Antimicrobial Chemotherapy, 2010, 65(9): 2043−2045. doi: 10.1093/jac/dkq242
[14]	GAY K, ROBICSEK A, STRAHILEVITZ J, et al. Plasmid-mediated quinolone resistance in non-typhi serotypes of Salmonella enterica [J]. Clinical Infectious Diseases, 2006, 43(3): 297−304. doi: 10.1086/505397
[15]	HANSEN L H, SØRENSEN S J, JØRGENSEN H S, et al. The prevalence of the OqxAB multidrug efflux pump amongst olaquindox-resistant Escherichia coli in pigs [J]. Microbial Drug Resistance (Larchmont, N Y), 2005, 11(4): 378−382. doi: 10.1089/mdr.2005.11.378
[16]	张亚楠. 广东省鸭疫里氏杆菌耐药性调查及floR传播分子特征研究[D]. 广州: 华南农业大学, 2016. ZHANG Y N. Monitoring of antibiotic resistance and dissemination of FloR among Riemerella anatipestifer isolated from ducks and geese in guangdong[D]. Guangzhou: South China Agricultural University, 2016. (in Chinese)
[17]	郭晶晶, 侯思梦, 刘连杰, 等. 猪源大肠埃希菌中质粒介导喹诺酮耐药(PMQR)基因的多样性研究 [J]. 西北农业学报, 2021, 30(11):1611−1617. doi: 10.7606/j.issn.1004-1389.2021.11.003 GUO J J, HOU S M, LIU L J, et al. Diversity of plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli form swine [J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2021, 30(11): 1611−1617.(in Chinese) doi: 10.7606/j.issn.1004-1389.2021.11.003
[18]	CHEN Q W, GONG X W, ZHENG F Y, et al. Interplay between the phenotype and genotype, and efflux pumps in drug-resistant strains of Riemerella anatipestifer [J]. Frontiers in Microbiology, 2018, 9: 2136. doi: 10.3389/fmicb.2018.02136
[19]	杨芳芳. 鸭疫里默氏杆菌氨基糖苷类和喹诺酮类耐药基因的检测与序列分析[D]. 泰安: 山东农业大学, 2013. YANG F F. Detection and sequence analysis of aminoglycoside and quinolones resistance genes in Riemerella anatipestfer[D]. Taian: Shandong Agricultural University, 2013. (in Chinese)
[20]	王婧, 刁小龙, 陈晓兰, 等. 猪源大肠埃希菌喹诺酮类耐药基因的检测及分析 [J]. 甘肃农业大学学报, 2020, 55(2):9−15. WANG J, DIAO X L, CHEN X L, et al. Detection and analysis of resistance genes in quinolones-resistant Escherichia coli isolated from swine [J]. Journal of Gansu Agricultural University, 2020, 55(2): 9−15.(in Chinese)
[21]	郑来煜, 刘敏, 王占黎, 等. 氟喹诺酮耐药肠球菌gyrA和parC基因突变特征及其与环丙沙星耐药相关性分析 [J]. 现代预防医学, 2020, 47(14):2672−2676. ZHENG L Y, LIU M, WANG Z L, et al. Mutations and characterization in gyrA and parC genes among fluoroquinolone-resistant Enterococcus associated with ciprofloxacin resistance [J]. Modern Preventive Medicine, 2020, 47(14): 2672−2676.(in Chinese)
[22]	张友春, 王志平. 肺炎克雷伯菌gyrA和parC基因突变与喹诺酮耐药相关性研究 [J]. 浙江医学教育, 2013, 12(3):48−50. doi: 10.3969/j.issn.1672-0024.2013.03.018 ZHANG Y C, WANG Z P. The relationship between gyrA and parC gene mutations of Klebsiella pneumoniae and quinolone resistance [J]. Zhejiang Medical Education, 2013, 12(3): 48−50.(in Chinese) doi: 10.3969/j.issn.1672-0024.2013.03.018
[23]	SUN N, LIU J H, YANG F, et al. Molecular characterization of the antimicrobial resistance of Riemerella anatipestifer isolated from ducks [J]. Veterinary Microbiology, 2012, 158(3/4): 376−383.
[24]	金龙翔. 鸭疫里默氏菌对氟喹诺酮类药物耐药机制的研究[D]. 南昌: 江西农业大学, 2014. JIN L X. The mechanism of quinolone resistance in Riemerella anatipestifer[D]. Nanchang: Jiangxi Agricultural University, 2014. (in Chinese)
[25]	孙亚妮. GN52株鸭疫里默氏杆菌的传代培养和鸭疫里默氏杆菌分离株耐药性的研究[D]. 泰安: 山东农业大学, 2008. SUN Y N. The serial subcultivation of GN52 and the research of drug resistance of Riemerella anatipestifer isolates[D]. Taian: Shandong Agricultural University, 2008. (in Chinese)
[26]	刘颖, 苏敬良, 刘文华. 鸭疫里默氏菌的gyrA基因的检测与变异位点的分析 [J]. 中国兽医杂志, 2007, 43(1):12−14. doi: 10.3969/j.issn.0529-6005.2007.01.004 LIU Y, SU J L, LIU W H. Detection of gyrA gene of Riemerella anatipestifer and analysis of mutation sites [J]. Chinese Journal of Veterinary Medicine, 2007, 43(1): 12−14.(in Chinese) doi: 10.3969/j.issn.0529-6005.2007.01.004
[27]	ZHU D K, ZHENG M Y, XU J G, et al. Prevalence of fluoroquinolone resistance and mutations in the gyrA, parC and parE genes of Riemerella anatipestifer isolated from ducks in China [J]. BMC Microbiology, 2019, 19(1): 271. doi: 10.1186/s12866-019-1659-4