甘肃牦牛源肺炎克雷伯菌的遗传进化、毒力基因及耐药性分析

doi:10.11843/j.issn.0366-6964.2025.06.032

Abstract

Abstract:

This study aimed to understand the epidemiological characteristics, virulence genes, resistance genes, resistant phenotype and genetic diversity of Klebsiella pneumoniae in Gansu Province. In this study, K. pneumoniae was isolated and purified from 156 yak lung samples collected from a slaughterhouse in Gansu Province. Serotypes, virulence genes and drug resistance genes of the isolates were tested by molecular biology means, and resistance profiles of the isolates were tested by K-B method. The results showed that, a total of 84 K. pneumoniae strains were isolated from 156 lung samples, the separation rate was 53.85%; Eight capsular serotypes were not detected, indicated that the isolates might belong to other serotypes; The virulence genes detection results showed that, the detection rate of pilus biosynthesis-related genes fimH (84.52%) and mrkD (95.23%), kipopolysaccharide-related gene uge (94.05%), the urease-related gene ereA (88.10%), and the iron uptake system gene ybtA (97.62%) were high, the detection rate of other virulence genes was low or not detected. The results of drug-resistance gene testing showed that, the detection rate of the bla_TEM gene of the ESBLs class (85.71%), Carbapenem VIM genes (79.76%) and acc (6')-Ib of aminoglycosides (77.38%) were high, other drug resistance genes was low or not detected. The results of the drug susceptibility test showed that the isolates were highly resistant to co-trimoxazole (98.81%), erythromycin (97.62%), vancomycin (95.24%) and clindamycin (94.05%), and have different degrees of resistance to the rest of the drugs; ERIC-PCR results showed, similarity> 60% can be classified into a cluster, divided into E 1 to E 6, where E1 was the dominant cluster, accounting for 50.00%. This paper analyzed the prevalence, virulence genes, drug resistance genes and drug resistance phenotypes of K. pneumoniae in Gansu yak, which has certain reference significance for clinical and preventive guidance.

Key words: Klebsiella pneumoniae, virulence gene, drug resistance gene, ERIC-PCR

CLC Number:

S855.12

ZHAO Enhao, SHI Hongmei, GESANG Zhuoma, SUOLANG Sizhu, GONG Ga. Genetic Evolution, Virulence Genes, and Drug Resistance Analysis of Klebsiella pneumoniae from Yak in Gansu Province[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2893-2905.

Figures/Tables 13

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引物名称 Primer name	序列(5′→3′) Sequence (5′→3′)	退火温度/℃ T_m	长度/bp Length
16S rRNA	GGTTACCTTGTTACGACTT	58	1 493
16S rRNA	AGAGTTTGATCCTGGCTCAG	58	1 493
mrkA	CCGCTCGAGTTACTGATAAGTAATTTCGTAAG	60	609
mrkA	CGCGGATCCATGAAAAAGGTTCTTCTCTCTG	60	609

引物名称 Primer name	序列(5′→3′) Sequence (5′→3′)	退火温度/℃ T_m	长度/bp Length
K1	GGTGCTCTTTACATCATTGC	53	1 383
K1	GCAATGGCCATTGCGTTAG	53	1 383
K2	GACCCGATATTCATACTTGACAGAG	54	641
K2	CCTGAAGTAAAATCGTTAAATAGATGGC	54	641
K3	AGGCAATTGACTTTAGGTG	50	547
K3	AGTGAATCAGCCTTCACCT	50	547
K5	TGGTAGTGATGCTCGCGA	56	280
K5	CCTGAACCCACCCCAATC	56	280
K16	GTGCTTAACGGAGAACTGAAC	53	922
K16	CCTCACCTGGAAGAAGTGTA	53	922
K20	CGGTGCTACAGTGCATCATT	54	741
K20	GTTATACGATGCTCAGTCGC	54	741
K54	CATTAGCTCAGTGGTTGGC	55	881
K54	GCTTGACAAACACCATAGCAG	55	881
K57	CTCAGGGCTAGAAGTGTCAT	53	547
K57	CACTAACCCAGAAAGTCGAG	53	547

类别 Category	引物名称 Primer name	序列(5′→3′) Sequence (5′→3′)	退火温度/℃ T_m	长度/bp Length
荚膜多糖合成调控基因 Genes regulating capsular polysaccharide synthesis	rmpA	ACTGGGCTACCTCTGCTTCA	52	535
	rmpA	CTTGCATGAGCCATCTTTCA	52	535
	magA	GGTGCTCTTTACATCATTGC	55	1 283
	magA	GCAATGGCCATTTGCGTTAG	55	1 283
菌毛合成相关基因 Genes involved in pilus biosynthesis	fimH	GCTCTGGCCGATACCACCACGG	55	423
	fimH	GCGAAGTAACGTGCCTGGAACGG	55	423
	mrkD	ATGAAAAAACTGACGCTTTTTATTG	58	963
	mrkD	TTAATCGTACGTCAGGTTAAAGACC	58	963
脲酶相关基因 Urease-related genes	ureA	GCTGACTTAAGAGAACGTTATG	55	337
脲酶相关基因 Urease-related genes	ureA	GATCATGGCGCTACCTCA	55	337
脂多糖相关基因 Lipopolysaccharide-related genes	uge	GATCATCCGGTCTCCCTGTA	55	534
	uge	TCTTCACGCCTTCCTTCACT	55	534
	wabG	CGGACTGGCAGATCCATATC	55	683
	wabG	ACCATCGGCCATTTGATAGA	55	683
铁摄取系统 Iron ingestion system	ybtA	ATGACGGAGTCACCGCAAAC	53	960
铁摄取系统 Iron ingestion system	ybtA	TTACATCACGCGTTTAAAGG	53	960

类别 Category	引物名称 Primer name	序列(5′→3′) Sequence (5′→3′)	退火温度/℃ T_m	长度/bp Length
超广谱β-内酰胺酶 Extended spectrum β-lactamases (ESBLs)	bla_SHV	GCCTTTATCGGCCTTCACTCAAG	55	898
	bla_SHV	TTAGCGTTGCCAGTGCTCGATCA	55	898
	bla_TEM	CAGCGGTAAGATCCTTGAGA	52	643
	bla_TEM	ACTCCCCGTCGTGTAGATAA	52	643
	bla_CTX	AACCGTCACGCTGTTGTTAG	52	766
	bla_CTX	TTGAGGCGTGGTGAAGTAAG	52	766
碳青霉烯类 Carbopenems	bla_OXA	GCGTGGTTAAGGATGAACAC	52	438
	bla_OXA	CATCAAGTTCAACCCAACCG	52	438
	VIM	GATGGTGTTTGGTCGCATA	52	390
	VIM	CGAATGCGCAGCACCAG	52	390
	NDM	GGTTTGGCGATCTGGTTTTC	55	621
	NDM	CGGAATGGCTCATCACGATC	55	621
喹诺酮类 Quinolones	qnr	ATTTCTCACGCCAGGATTTG	53	516
	qnr	GATCGGCAAAGGTTAGGTCA	53	516
	gyrA	CGCGTACTATACGCCATGAACGTA	55	420
	gyrA	ACCGTTGATCACTTCGGTCAGG	55	420
氨基糖苷类 Aminoglycoside	acc(3’)-IIa	CGGAAGGCAATAACGGAG	53	740
	acc(3’)-IIa	TCGAACAGGTAGCACTGAG	53	740
	acc(6’)-Ib	ATGACCTTGCGATGCTCTATGA	54	486
	acc(6’)-Ib	CGAATGCCTGGCGTGTTT	54	486
磺胺类 Sulfonamides	Sul 1	GTGACGGTGTTCGGCATTCT	58	779
磺胺类 Sulfonamides	Sul 1	TCCGAGAAGGTGATTGCGCT	58	779
四环素类 Tetracyclines	tetB	CCTCAGCTTCTCAACGCGTG	57	634
四环素类 Tetracyclines	tetB	GCACCTTGCTGATGACTCTT	57	634
酰胺醇类 Acetamine alcohols	flor	CACGTTGAGCCTCTATAT	52	868
酰胺醇类 Acetamine alcohols	flor	ATGCAGAAGTAGAACGCG	52	868
头孢菌素类 Cephalosporins	bla_DHA	GCCTGTTTGGTGCTCTGA	55	460
头孢菌素类 Cephalosporins	bla_DHA	GCACGGTTATACGGCTGA	55	460
多黏菌素类 Polymyxins	mcr-1	AGTCCGTTTGTTCTTGTGGC	56	320
多黏菌素类 Polymyxins	mcr-1	AGATCCTTGGTCTCGGCTTG	56	320
大环内酯类 Macrolides	ereA	GCCGGTGCTCATGAACTTGAG	57	419
大环内酯类 Macrolides	ereA	CGACTCTATTCGATCAGAGGC	57	419

药物种类 Drug types	药物名称 Drug name	药品规格/(μg·片^-1) Specifications	抑菌圈直径判断标准/mm Criteria for determining the inhibitory diameter
药物种类 Drug types	药物名称 Drug name	药品规格/(μg·片^-1) Specifications	耐药Resistent	中介Intermedia	敏感Senstive
β-内酰胺类β-lactamase	氨苄西林	10	< 13	13~17	>17
	羧苄西林	100	< 19	19~23	>23
	哌拉西林	100	< 17	17~21	>21
	头孢氨苄	30	< 14	14~18	>18
	头孢唑林	30	< 14	14~18	>18
	头孢拉定	30	< 14	14~18	>18
	头孢呋辛	30	< 14	14~18	>18
	头孢他啶	30	< 14	14~18	>18
	头孢曲松	30	< 14	14~23	>23
	头孢哌酮	30	< 15	15~21	>21
氨基糖苷类Aminoglycoside	丁胺卡那	30	< 14	14~17	>17
	庆大霉素	30	< 12	12~15	>15
	卡那霉素	10	< 13	13~18	>18
	新霉素	30	< 12	12~17	>17
四环素类Tetracyclines	四环素	30	< 14	14~19	>19
	多西环素	30	< 12	12~16	>16
	米诺环素	30	< 12	12~16	>16
大环内酯类Macrolides	红霉素	15	< 13	13~23	>23
大环内酯类Macrolides	麦迪霉素	30	< 13	13~18	>18
喹诺酮类Quinolones	诺氟沙星	10	< 12	12~17	>17
	氧氟沙星	5	< 12	12~16	>16
	环丙沙星	5	< 15	15~21	>21
糖肽类Glycopeptides	万古霉素	30	< 14	14~17	>17
糖肽类Glycopeptides	多黏菌素b	7.5	< 8	8~12	>12
磺胺类Sulfonamides	复方新诺明	23.75	< 23	23~32	>32
硝基咪唑类Nitroimidazole class	呋喃唑酮	300	< 14	14~17	>17
酰胺醇类Acetamine alcohols	氯霉素	30	< 12	12~18	>18
林可胺类Linkomide	克林霉素	2	< 14	14~21	>21

Genetic Evolution, Virulence Genes, and Drug Resistance Analysis of Klebsiella pneumoniae from Yak in Gansu Province

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项目 Item	结果 Result	项目 Item	结果 Result	项目 Item	结果 Result
葡萄糖Glucose	+	甘露醇Mannitol	+	硫化氢H₂S	-
麦芽糖Maltose	+	阿拉伯醇Arabic alcohol	+	肌醇Inositol	-
鼠李糖Rhamnose	+	山梨醇Sorbitol	+	甲基红MR	+
海藻糖Trehalose	+	柠檬酸盐Citrate	+	Vegos-Proskauer(VP)	-

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[3]	FAN Wei, LIU Xinxin, ZHAI Yilu, ZHANG Xinyu, WANG Wei, FU Jiaqi, SUN Fuliang. Isolation and Identification of Klebsiella pneumoniae of Sheep Origin and Establishment of a Method for the Extraction of Its Outer Membrane Vesicles [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 353-364.
[4]	Qilin WANG, Runlai CAO, Weiyang WANG, Bo ZHANG, Zhijie LIU, Xiaoxu WANG. Isolation, Identification and Drug Resistance Analysis of Klebsiella pneumoniae in Aborted Fetuses of Fox [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3640-3648.
[5]	SONG Yan, YUAN Yongfeng, QIAN Hongyu, LI Xincan, LUO Hongyan, WANG Zhiying, ZHOU Zuoyong. Identification and Partial Biological Characteristics Analysis of Corynebacterium pseudotuberculosis Isolated from Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 680-687.
[6]	WU Sujuan, LIN Changcheng, WAN Peng, LI Jie, LU Yixing, HU Jianxin, PENG Xianfeng, ZENG Zhenling. Synergistic Antimicrobial Effect of Isopropoxy Benzene Guanidine Combined with Colistin on Klebsiella pneumoniae in vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5792-5801.
[7]	LIU Xinhuan, YUN Jialei, MAO Li, LI Jizong, HAO Fei, HE Miaofeng, YANG Leilei, ZHANG Wenwen, CHENG Zilong, SUN Min, LIU Maojun, WANG Shaohui, BAI Juan, LI Wenliang. Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3445-3454.
[8]	ZHAO Feifei, LI Jie, HAN Ning, XIE Shiting, ZENG Zhenling. Antibacterial Drug Resistance Analysis of Klebsiella pneumoniae Isolated from Slaughterhouse [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3044-3053.
[9]	JIANG Zenghai, TENG Lin, HE Anwen, LIU Yanyan, YUE Min, HE Qigai. Genomic Analysis of Salmonella Typhimurium Isolates and Salmonella Serotype 4, [5], 12: i:- Isolates from Pig-borne Food Chain [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1199-1209.
[10]	WANG Jianing, ZHANG Ziqiang, KONG Dejing, FENG Caicai, ZHANG Feike, LIU Yumei. Isolation and Identification of Klebsiella pneumoniae in Rabbits [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5198-5206.
[11]	YANG Menglin, ZHENG Shiqi, PENG Kai, WANG Wei, HUANG Yanhua, PENG Jie. Isolation and Identification of Pigeon-derived Salmonella Typhimurium and Pathogenic Analysis [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4880-4888.
[12]	ZHANG Kaichuan, WANG Jinyu, LI Shoujun, JIA Kun. Isolation, Identification and Biological Characteristics of Klebsiella pneumoniae from Sheep in Guangdong Province [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 328-337.
[13]	WANG Xi, LI Ke, LI Tingcui, YAN Hongya, ZHAO Rong, CHANG Zhishun, LIAO Ming, SUN Minhua, XIN Aiguo. MLST Typing and Drug Resistance Analysis of 75 Salmonella Strains Isolated from Laying Hens [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1626-1631.
[14]	ZHAO Xueliang, WANG Bin, MIAO Yongqiang, ZHAO Haoyu, XIE Qingfang, WANG Juan, YANG Zengqi. Detection of Virulence Genes and Antimicrobial Resistance Analysis of Escherichia coli Isolated from Diarrhea Sheep in Shaanxi Province [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1644-1648.
[15]	LI Huaming, XIANG Wei, LU Wenbing, LIU Feng, LEI Liancheng, ZHANG Fuxian. Pathogenicity and Drug Sensitivity Analysis of a Porcine Klebsiella pneumoniae Type ST-35 [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4356-4366.