鸡源鼠伤寒沙门菌的分离鉴定、药敏试验与毒力基因检测

doi:10.3969/j.issn.1004-1524.20230168

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (2): 284-294.DOI: 10.3969/j.issn.1004-1524.20230168

鸡源鼠伤寒沙门菌的分离鉴定、药敏试验与毒力基因检测

郭伟娜¹^,²(), 陶晶¹, 何梦婷¹, 王紫苇¹, 马佰贺¹, 赵磊¹^,²

1.安徽科技学院动物科学学院,安徽凤阳 233100
2.动物营养调控与健康安徽省重点实验室,安徽凤阳 233100

收稿日期:2023-02-15 出版日期:2024-02-25 发布日期:2024-03-05
作者简介:郭伟娜(1982—),女,河南内黄人,博士研究生,副教授,主要从事兽医微生物的分离鉴定及致病性研究。E-mail:weina0925@126.com
基金资助:
安徽省教育厅自然科学研究重点计划(KJ2020A0087);安徽科技学院稳定人才计划(DKWD201801);安徽科技学院科研发展基金(811504);安徽科技学院大学生创新创业训练计划(S202210879189)

Isolation, identification, antimicrobial susceptibility test and virulence genes detection of Salmonella typhimurium from chicken

GUO Weina¹^,²(), TAO Jing¹, HE Mengting¹, WANG Ziwei¹, MA Baihe¹, ZHAO Lei¹^,²

1. College of Animal Science, Anhui Science and Technology University, Fengyang 233100, Anhui, China
2. Anhui Province Key Laboratory of Animal Nutrition Regulation and Health, Fengyang 233100, Anhui, China

Received:2023-02-15 Online:2024-02-25 Published:2024-03-05

摘要/Abstract

摘要：

鼠伤寒沙门菌是一种重要的人兽共患病病原,不仅影响养禽业的健康发展,还对禽类产品安全构成严重威胁,本研究主要是对鸡源鼠伤寒沙门菌进行分离鉴定、药敏试验和毒力基因的测定。首先采集病死产蛋鸡的脾脏组织进行分离培养和染色镜检,以及16S rRNA 基因的PCR鉴定,然后用圆纸片扩散法测定分离株对26种药物的敏感性,用PCR方法检测分离株的28个毒力基因。结果显示,分离株在普通营养琼脂和血琼脂培养基上形成灰白色菌落,在麦康凯培养基上为无色菌落,在BS培养基上为黑色带金属光泽的菌落;染色镜检结果显示分离株为革兰阴性的红色短杆菌;16S rRNA基因测序结果显示,分离株与鼠伤寒沙门菌OLF-FSR1-WB-Sparrow-ST-87的相似性为99.86%,与10个鼠伤寒沙门菌参考菌株的同源性介于99.5%~100%;药敏试验结果表明,分离株对环丙沙星最为敏感,抑菌圈直径达30 mm;除sseC基因未检测出外,其余27个毒力基因的测序结果与参考菌株对应毒力基因的相似性介于98.58%~100%。本研究成功从病死产蛋鸡分离鉴定出一株鼠伤寒沙门菌菌株FY2021,为深入研究鼠伤寒沙门菌的致病机理和禽源沙门菌病的防控提供参考依据。

关键词: 鼠伤寒沙门菌, 分离鉴定, 药敏试验, 毒力基因, PCR检测

Abstract:

As a critical zoonosis pathogen, Salmonella typhimurium cannot only affects the healthy development of the poultry industry, but also poses a serious threat to the safety of poultry products. The primary purpose of this study was to isolate and identify the pathogen, test the drug sensitivity and detect the virulence genes. Firstly, the spleen tissues of dead laying hens were collected for isolation, culture, microscopic examination, and PCR identification of 16S rRNA gene. Then, antimicrobial susceptibility was determined for the isolated strain to 26 drugs by circular disk diffusion method, while 28 virulence genes were detected by PCR. The isolated strain formed gray colonies on ordinary nutrient agar and blood agar, colorless colonies on MacConkey medium, and black metallic colonies on BS medium. The staining microscopy showed gram-negative, red, and short bacillus. PCR and sequencing for 16S rRNA indicated that, the similarity of the isolated strain was 99.86% compared with S. typhimurium strain OLF-FSR1-WB-Sparrow-ST-87, and the homology ranged from 99.5% to 100% compared with 10 reference strains of S. typhimurium. The susceptibility test showed the strain was highly sensitive to Ciprofloxacin with an inhibition zone of 30 mm. Furthermore, except that the gene of sseC was not be detected, the similarity of the other 27 virulence genes was between 98.58% and 100%, compared with the corresponding virulence genes of the reference strains in GenBank. In conclusion, S. typhimurium strain FY2021 was successfully isolated and identified from the dead layer, which provides a basis for the further study of the pathogenesis of S. typhimurium and the prevention and control of salmonellosis.

Key words: S. typhimurium, isolate and identify, susceptibility test, virulence gene, PCR detection

中图分类号:

S858.291

郭伟娜, 陶晶, 何梦婷, 王紫苇, 马佰贺, 赵磊. 鸡源鼠伤寒沙门菌的分离鉴定、药敏试验与毒力基因检测[J]. 浙江农业学报, 2024, 36(2): 284-294.

GUO Weina, TAO Jing, HE Mengting, WANG Ziwei, MA Baihe, ZHAO Lei. Isolation, identification, antimicrobial susceptibility test and virulence genes detection of Salmonella typhimurium from chicken[J]. Acta Agriculturae Zhejiangensis, 2024, 36(2): 284-294.

图/表 10

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基因名称 Gene name	引物序列(5'→3') Primer sequence (5'→3')	产物大小 Size/bp
invA	Up: GTGAAATTATCGCCACGTTCGGGCAA Down: TCATCGCACCGTCAAAGGAACC	284
spvR	Up: AGGAAATCGGACCTACGG Down: TAACATCGCCAGCCCTTG	473
spvA	Up: GCTAACTGTCGGGCAAAG Down: GGACAATGGCACGAACCT	432
spvB	Up: CCTGATGTTCCACCACTTTC Down: ATGCCTTATCTGGCGATGT	590
spvC	Up: AAGGTCGTTCAACAAGCC Down: CATTTCACCACCATCACG	252
spvD	Up: CCCCTGATGATGAGAAGT Down: ACAGTGGGATTAGACAGC	316
sseC	Up: ATGAATCGAATTCACAGTAA Down: TTAAGCGCGATAGCCAGCTA	1 455
sseD	Up: ATGGAAGCGAGTAACGTAGC Down: TTACCTCGTTAATGCCCGGA	588
sseE	Up: ATGGTGCAAGAAATAGAGCA Down: TTAAAAACGTCGCTGGATAA	417
sseL	Up: CTATCCTATTGGGCTTAT Down: GTTGGGTACATTGTTCTG	304
mogA	Up: ATTGGCTTAGTTTCTATCTCCG Down: CCTTCCAGCGTTTCTTTGA	419
mgtC	Up: CGACGATCATTATTCTTTGC Down: GACCGAACCTAACCCTTGT	200
bcfA	Up: CTTTGGCGGAATGTTGTC Down: CTGGCTGGTCTGAGTATCG	235
araB	Up: AGGTAGACGTGCCGATGACTT Down: CGAATGCGATGTTTGTGCT	558
invJ	Up: TCGGCAGTGGGAAAAATA Down: AAGGCGTTCGTAAAGAGG	232
sscA	Up: ATGAAAAAAGACCCGACCTA Down: TTAGCTCCTGTCAGAAAGTT	474
virK	Up: CGCCTTGAGTATGTTTGT Down: ATGGGAAGTTCAGGTATC	376
sipA	Up: TTCCCCTTTTAGCCT Down: ACCTCCACACCGTTC	243
sopA	Up: ACCTGCCGACTGGGCTAAG Down: ACGAGGGCTGTTGTTGTGT	347
ssaB	Up: ATGTCTGAGGAGGGAT Down: GTTTATGGTGATTGCG	382
misL	Up: ATGTCTGAGGAGGGAT Down: GTTTATGGTGATTGCG	458
orf319	Up: GTATCGGACAAAGAAGG Down: ATGAAAAGGGTAACAGG	323
pipC	Up: CGCCTCTTCTTCGGT Down: TATGCCATTGCCTGA	145
SPI-1	Up: ACATCGACAGACGTAAGGAGG Down: CCGCCAAACCTAAAACCAGC	979
SPI-2	Up: TTGTCCGCCAACTCCTCTTC Down: TTACGTCTTATTTTCGGCACC	440
SPI-3	Up: ATTGGGAGTTGATTTATACGC Down: TGGGATTGGCTTTCTGGGG	210
SPI-4	Up: ATTCAGTGGTTCATGGTCAGG Down: TTAAAGAACGGGTGCCATCC	282
SPI-5	Up: CCAGCTCATTAACACCCACAT Down: CGGGCATCACTATACCAACAC	550

基因名称 Gene name	引物序列(5'→3') Primer sequence (5'→3')	产物大小 Size/bp
invA	Up: GTGAAATTATCGCCACGTTCGGGCAA Down: TCATCGCACCGTCAAAGGAACC	284
spvR	Up: AGGAAATCGGACCTACGG Down: TAACATCGCCAGCCCTTG	473
spvA	Up: GCTAACTGTCGGGCAAAG Down: GGACAATGGCACGAACCT	432
spvB	Up: CCTGATGTTCCACCACTTTC Down: ATGCCTTATCTGGCGATGT	590
spvC	Up: AAGGTCGTTCAACAAGCC Down: CATTTCACCACCATCACG	252
spvD	Up: CCCCTGATGATGAGAAGT Down: ACAGTGGGATTAGACAGC	316
sseC	Up: ATGAATCGAATTCACAGTAA Down: TTAAGCGCGATAGCCAGCTA	1 455
sseD	Up: ATGGAAGCGAGTAACGTAGC Down: TTACCTCGTTAATGCCCGGA	588
sseE	Up: ATGGTGCAAGAAATAGAGCA Down: TTAAAAACGTCGCTGGATAA	417
sseL	Up: CTATCCTATTGGGCTTAT Down: GTTGGGTACATTGTTCTG	304
mogA	Up: ATTGGCTTAGTTTCTATCTCCG Down: CCTTCCAGCGTTTCTTTGA	419
mgtC	Up: CGACGATCATTATTCTTTGC Down: GACCGAACCTAACCCTTGT	200
bcfA	Up: CTTTGGCGGAATGTTGTC Down: CTGGCTGGTCTGAGTATCG	235
araB	Up: AGGTAGACGTGCCGATGACTT Down: CGAATGCGATGTTTGTGCT	558
invJ	Up: TCGGCAGTGGGAAAAATA Down: AAGGCGTTCGTAAAGAGG	232
sscA	Up: ATGAAAAAAGACCCGACCTA Down: TTAGCTCCTGTCAGAAAGTT	474
virK	Up: CGCCTTGAGTATGTTTGT Down: ATGGGAAGTTCAGGTATC	376
sipA	Up: TTCCCCTTTTAGCCT Down: ACCTCCACACCGTTC	243
sopA	Up: ACCTGCCGACTGGGCTAAG Down: ACGAGGGCTGTTGTTGTGT	347
ssaB	Up: ATGTCTGAGGAGGGAT Down: GTTTATGGTGATTGCG	382
misL	Up: ATGTCTGAGGAGGGAT Down: GTTTATGGTGATTGCG	458
orf319	Up: GTATCGGACAAAGAAGG Down: ATGAAAAGGGTAACAGG	323
pipC	Up: CGCCTCTTCTTCGGT Down: TATGCCATTGCCTGA	145
SPI-1	Up: ACATCGACAGACGTAAGGAGG Down: CCGCCAAACCTAAAACCAGC	979
SPI-2	Up: TTGTCCGCCAACTCCTCTTC Down: TTACGTCTTATTTTCGGCACC	440
SPI-3	Up: ATTGGGAGTTGATTTATACGC Down: TGGGATTGGCTTTCTGGGG	210
SPI-4	Up: ATTCAGTGGTTCATGGTCAGG Down: TTAAAGAACGGGTGCCATCC	282
SPI-5	Up: CCAGCTCATTAACACCCACAT Down: CGGGCATCACTATACCAACAC	550

GenBank登录号 GenBank accession number	菌株 Strain	地区 Country	来源 Isolation source
CP051276	OLF-FSR1-WB-Sparrow-ST-87	加拿大Canada	麻雀Sparrow
CP047323	RM13672	美国USA	人类粪便Feces of homo sapiens
MK795389	LC04	中国China	生物资源中心Biological Resource Center
MK795388	LC03	中国China	生物资源中心Biological Resource Center
MK795387	LC02	中国China	生物资源中心Biological Resource Center
MK795386	LC01	中国China	生物资源中心Biological Resource Center
CP034230	ATCC14028	美国USA	生物资源中心Biological Resource Center
CP074663	CFSAN008081	美国USA	鸡Chicken
CP028199	CFSAN018746	美国USA	生物资源中心Biological Resource Center
NR074910	LT2	美国American	生物资源中心Biological Resource Center

GenBank登录号 GenBank accession number	菌株 Strain	地区 Country	来源 Isolation source
CP051276	OLF-FSR1-WB-Sparrow-ST-87	加拿大Canada	麻雀Sparrow
CP047323	RM13672	美国USA	人类粪便Feces of homo sapiens
MK795389	LC04	中国China	生物资源中心Biological Resource Center
MK795388	LC03	中国China	生物资源中心Biological Resource Center
MK795387	LC02	中国China	生物资源中心Biological Resource Center
MK795386	LC01	中国China	生物资源中心Biological Resource Center
CP034230	ATCC14028	美国USA	生物资源中心Biological Resource Center
CP074663	CFSAN008081	美国USA	鸡Chicken
CP028199	CFSAN018746	美国USA	生物资源中心Biological Resource Center
NR074910	LT2	美国American	生物资源中心Biological Resource Center

抗菌药物 Drug		抑菌圈/敏感性 Inhibition zone/ sensitivity	抗菌药物 Drug		抑菌圈/敏感性 Inhibition zone/ sensitvity
青霉素类	氨苄西林Ampicillin	14/I	喹诺酮类	氧氟沙星Ofloxacin	28/S
Penicillins	苯唑西林Oxacillin	0/R	Quinolones	环丙沙星Ciprofloxacin	30/S
	羧苄西林Carbenicillin	25/S		诺氟沙星Norfloxacin	25/S
氨基糖苷类	新霉素Neomycin	14/I		恩诺沙星Enrofloxacin	26/S
Aminoglycosides	卡那霉素Kanamycins	18/I	氯霉素类Chloramphenicol	氟苯尼考Florfenicol	26/S
	丁胺卡那霉素Amikacin	18/S	四环素类Tetracyclines	多西环素Doxycycline	18/S
	奈替米星Netilmicin	24/S	多肽类Polypeptides	多粘菌素B Polymyxin B	12/I
头孢菌素类	头孢哌酮Cefoperazone	22/S		万古霉素Vancomycin	0/R
Cephalosporins	头孢西丁Cefoxitin	28/S	磺胺类Sulfonamides	复方新诺明Trimethoprim/sulfamethoxazole	0/R
	头孢氨苄Cephalexin	12/I	大环内酯类Macrolides	红霉素Erythromycin	0/R
	头孢噻肟Cefotaxime	28/S		麦迪霉素Midecamycin	0/R
	头孢呋辛Cefuroxime	15/I	林可酰胺类	林可霉素Lincomycin	0/R
	头孢曲松Ceftriaxone	24/S	Lincosamides	克林霉素Clindamycin	0/R

鸡源鼠伤寒沙门菌的分离鉴定、药敏试验与毒力基因检测

Isolation, identification, antimicrobial susceptibility test and virulence genes detection of Salmonella typhimurium from chicken

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基因名称 Gene name	菌株 Strain	GenBank登录号 GenBank accession number	同源性 Identity/%
invA	16A242	CP020922	98.95
spvR	FDAARGOS-711	CP055131	99.56
spvA	FDAARGOS-711	CP055131	99.08
spvB	FDAARGOS-687	CP046282	99.48
spvC	07-0715	CP053399	99.21
spvD	FDAARGOS-711	CP055131	99.06
sseE	FDAARGOS-711	CP055130	99.29
sseL	FDAARGOS-707	CP046279	100
mogA	FDAARGOS-711	CP055130	98.58
mgtC	FDAARGOS-711	CP055130	100
bcfA	FDAARGOS-711	CP055130	100
araB	FDAARGOS-711	CP055130	99.81
invJ	FDAARGOS-711	CP055130	99.57
sscA	SL7207	CP053865	99.16
sseD	16A242	CP020922	99.49
virK	16A242	CP020922	100
sipA	FDAARGOS-711	CP055130	99.18
sopA	SL7207	CP053865	99.71
ssaB	FDAARGOS-711	CP055130	99.73
misL	FDAARGOS-711	CP055130	99.77
orf319	SL7207	CP053865	98.77
pipC	FDAARGOS-711	CP055130	100
SPI-1	FDAARGOS-711	CP055130	100
SPI-2	FDAARGOS-711	CP055130	100
SPI-3	FDAARGOS-711	CP055130	99.07
SPI-4	FDAARGOS-711	CP055130	98.94
SPI-5	FDAARGOS-711	CP055130	100

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