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

doi:10.3969/j.issn.1004-1524.20230168

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (2): 284-294.DOI: 10.3969/j.issn.1004-1524.20230168

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S858.291

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.

Figures/Tables 10

References 34

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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

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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