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

摘要/Abstract

摘要：

旨在了解甘肃牦牛肺炎克雷伯菌(Klebsiella pneumoniae, Kp)的流行特征、毒力基因、耐药基因、耐药表型以及遗传多样性等情况。本研究从甘肃某屠宰场采集牦牛肺样本156份，通过分离纯化、染色镜检等对肺炎克雷伯菌进行分离鉴定，运用分子生物学手段对分离株的血清型、毒力基因和耐药基因进行检测，并用纸片扩散法对分离株的耐药谱进行检测。结果显示，从156份肺组织样品中分离出84株肺炎克雷伯菌，分离率为53.85%；8种荚膜血清型均未检出，可能是分属其他血清型；毒力基因检测结果显示，菌毛合成相关基因fimH(84.52%)和mrkD(95.23%)、脂多糖相关基因uge(94.05%)、脲酶相关基因ereA(88.10%)、铁摄取系统基因ybtA(97.62%)的检出率较高，其余毒力基因检出率低或未检出；耐药基因检测结果显示，ESBLs类的bla_TEM基因(85.71%)、碳青霉烯类VIM基因(79.76%)以及氨基糖苷类的acc(6’)-Ib(77.38%)的检出率较高，其余耐药基因检出率较低或未检出；药敏试验结果显示，分离株对复方新诺明(98.81%)、红霉素(97.62%)、万古霉素(95.24%)和克林霉素(94.05%)高度耐药，对其余药物均有不同程度的耐药；ERIC-PCR结果显示，相似度>60%的可分为一簇，分为E1~E6，其中E1为优势簇，占比50.00%。本研究对甘肃牦牛肺炎克雷伯菌的流行情况、毒力基因、耐药基因、耐药表型等进行了分析研究，对临床和预防的指导用药有一定的参考意义。

关键词: 肺炎克雷伯菌, 毒力基因, 耐药基因, ERIC-PCR

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

中图分类号:

S855.12

赵恩浩, 石红梅, 格桑卓玛, 索朗斯珠, 贡嘎. 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[J]. 畜牧兽医学报, 2025, 56(6): 2893-2905.

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.

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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