奶牛乳腺炎源粪肠球菌生物学特性研究

doi:10.11843/j.issn.0366-6964.2025.05.030

摘要/Abstract

摘要：

旨在了解奶牛乳腺炎源粪肠球菌的耐药特征和毒力情况，本研究对107株患乳腺炎源粪肠球菌进行药物敏感性试验、耐药基因和毒力基因的检测；选取粪肠球菌F2为代表菌株，对该菌株进行全基因组测序和生物学特性研究，进一步用感染复数(MOI)为1 000的肠球菌JH2-2和粪肠球菌F2感染奶牛乳腺上皮细胞(BMECs)，检测F2菌株对BMECs的黏附、入侵能力和菌株对BMECs增殖和损伤的影响；最后建立大蜡螟幼虫感染模型，通过幼虫生存率和病理组织切片两个方面验证该菌株对幼虫组织损伤的病理学特征。结果表明，乳腺炎源粪肠球菌对林可霉素、四环素、红霉素耐药率最高，为100%~90%；分离菌株中有95株粪肠球菌检测出携带耐药基因，84株粪肠球菌检测出携带毒力基因，耐药基因携带率最高的是ermB(91.6%)，毒力基因的携带率最高的是esp(78.6%)。对F2菌株全基因测序结果显示该菌株对林可酰胺类、大环内酯类、喹诺酮类等23类抗生素耐药，存在3个携带耐药基因的接合转移元件(ICE)和efaA、gelE、ace、AS等91个相关毒力因子。F2菌株具有较强的环境耐受性，在高温、酸性和碱性环境下存活率较高，具有运动能力和较强的生物膜形成能力(OD_{570 nm}值＞2 ODc)，细菌侵染BMECs 6 h后，F2菌株对BMECs的黏附、入侵能力极显著(P＜0.001)高于对照组，随着时间的推移F2菌株对BMECs的毒性增加，细胞损伤严重、死亡数量增加；F2感染大蜡螟幼虫1 d后，幼虫死亡率为100%，幼虫体内有大量炎性细胞浸润且体腔破坏严重。本研究显示，乳腺炎源粪肠球菌耐药性严重，存在多重耐药现象，可引起BMECs的损伤；该试验结果为后续研究乳源粪肠球菌耐药性和致病性的传播提供实验基础。

关键词: 粪肠球菌, 奶牛乳腺炎, 生物学特性, 全基因组分析

Abstract:

This study conducted drug susceptibility tests, as well as resistance and virulence gene detection, on 107 Enterococcus faecalis strains isolated from mastitis cases to understand the drug-resistance characteristics and virulence of E. faecalis from bovine mastitis. E. faecalis strain F2 was selected as a representative strain for whole-genome sequencing and biological characterization. Further, bovine mammary epithelial cells (BMECs) were infected with Enterococcus JH2-2 and E. faecalis F2 at a multiplicity of infection (MOI) of 1 000 to assess the adhesive and invasive abilities of strain F2, as well as its impact on BMECs damage and proliferation. A larval infection model using Galleria mellonella was established to verify the pathological features of tissue damage caused by this strain through larval survival rates and histopathological analyses. The results indicated that E. faecalis strains from bovine mastitis exhibited the highest resistance rates to lincomycin, tetracycline, and erythromycin, ranging between 90% and 100%. Among the isolates, 95 strains carried resistance genes, and 84 strains carried virulence genes. The most frequently detected resistance gene was ermB (91.6%), and the most common virulence gene was esp (78.6%). Whole-genome sequencing of strain F2 revealed resistance to 23 classes of antibiotics, including lincosamides, macrolides, and quinolones. Additionally, three integrative and conjugative elements (ICEs) carrying resistance genes and 91 associated virulence factors, such as efaA, gelE, ace, and AS, were identified. Strain F2 demonstrated strong environmental tolerance, surviving under high temperatures, acidic, and alkaline conditions, and exhibited motility and robust biofilm formation capabilities (OD_{570 nm}>2 ODc). Six hours post-infection of BMECs, strain F2 showed significantly greater adhesion and invasion abilities (P < 0.001) compared to the control group. Over time, strain F2 exhibited increased toxicity to BMECs, leading to severe cell damage and death. Following infection of G. mellonella larvae with strain F2 for one day, larval mortality reached 100%, accompanied by extensive inflammatory cell infiltration and severe disruption of the larval body cavity. This study reveals that E. faecalis isolates from bovine mastitis exhibit severe multidrug resistance, harbor multiple ICEs carrying resistance genes, and possess numerous virulence factors, contributing to BMECs damage. These findings provide an experimental foundation for further research on the transmission of antimicrobial resistance and pathogenicity of E. faecalis from bovine sources.

Key words: Enterococcus faecalis, mastitis in dairy cows, biological characteristics, whole genome analysis

中图分类号:

S852.611

乔亚蕊, 苗宇航, 黄倩, 周学章. 奶牛乳腺炎源粪肠球菌生物学特性研究[J]. 畜牧兽医学报, 2025, 56(5): 2325-2339.

QIAO Yarui, MIAO Yuhang, HUANG Qian, ZHOU Xuezhang. Research on the Biological Characteristics of Enterococcus faecalis in Dairy Cow Mastitis in Ningxia[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2325-2339.

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基因种类 Type of genes	基因 Genes	引物序列(5′→ 3′) Sequence	扩增长度/bp Fragments size	退火温度/℃ Annealing temperature	参考文献 Reference
耐药基因 Drug resistance gene	tetM	F-GTGGACAAAGGTACAACGAG	406	56	[17]
		R-CGGTAAAGTTCGTCACACAC
	tetA	F-GCTACATCCTGCTTGCCTTC	210	64
		R-CATAGATCGCCGTGAAGAGG
	tetB	F-GCCCAGTGCTGTTGTTGTCAT	617	56
		R-CGTTTTTTCGCCCCATTTAGT
	tetC	F-CTTGAGAGCCTTCAACCCAG	418	55
		R-ATGGTCGTCATCTACCTGCC
	tetX4	F-CTGATTCGTGTGACATCATCTTTTG	204	56
		R-GTTAAATTTCCCATTGGTCAGATTA
	tetX5	F-GGTATCAACATTTCAATGCTTG	265	56
		R-CGATTCGTCCTGCGTATCTTTTG
	mecA	F-TCCAGATTACAACTTCACCAGG	162	54
		R-CCACTTCATATCTTGTAACG
	ermA	F-GGATCAGGAAAAGGACATTTTAC	412	54
		R-GTTCAAGAACAATCAATACAGAG
	ermB	F-GAAATTGGAACAGGTAAAGGGC	359	56
		R-GAATCGAGACTTGAGTGTGC
	ermC	F-GGATCAGGAAAAGGACATTTTAC	572	55
		R-GCTAATATTGTTTAAATCGTCAATTCC
	lsaA	F-GGCAATCGCTTGTGTTTTAGCG	1 200	61	[18]
		R-GTGAATCCCATGATGTTGATACC
	lsaE	F-TGTCAAATGGTGAGCAAACG	496	54	[19]
		R-TGTAAAACGGCTTCCTGATG
	lnuA	F-GGTGGCTGGGGGGTAGATGTATTAACTGG	323	54	[20]
		R-GCTCTCTTTGAAATACATGGTATTTTTCGATC
	lnuG	F-AAGGAACTAATGGCAAGGGT	706	53	[21]
		R-CCATAACGAGAAGCAAAGAG
毒力基因 Virulence genes	cylA	F-GACTCGGGGATTGATAGGC	688	56	[22]
毒力基因 Virulence genes		R-GCTGCTAAAGCTGCGCTTAC
	esp	F-TTGCTAATGCTAGTCCACGACC	932	56
		R-GCGTCAACACTTGCATTGCCGA
	asa1	F-CCAGCCAACTATGGCGGAATC	529	56
		R-CCTGTCGCAAGATCGACTGTA
	efaA	F-GCCAATTGGGACAGACCCTC	688	58
		R-CGCCTTCTGTTCCTTCTTTGGC
	gelE	F-ACCCCGTATCATTGGTTT	405	56
		R-ACGCATTGCTTTTCCATC
	ace	F-GGAATGACCGAGAACGATGGC	616	56
		R-GCTTGATGTTGGCCTGCTTCCG
	agg	F- AAGAAAAAGAAGTAGACCAAC	1 553	58
		R-AAACGGCAAGACAAGTAAATA
	hyl	F-ACAGAAGAGCAGGAAATG	276	56
		R-GACTGACGTCCAAGTTTCCAA

基因 Genes	引物序列(5′→ 3′) Sequence	扩增长度/bp Product size	退火温度/℃ Annealing temperatures
adk	F-GAACCTCATTTTAATGGGG	437	50
	R-TGATGTTGATAGCCAGACG
atpA	F-CGGTTCATACGGAATGGCACA	556
	R-AAGTTCACGATAAGC CACGG
ddl	F-GAGACATTGAATATGCCTTATG	465
	R-AAAAAGAAATCGCACCG
gyd	F-CAAACTGCTTAGCTCCAAGGC	395
	R-CATTTCGTTGTCATACCAAGC
gdh	F-GGCGCACTAAAAGATATGGT	530
	R-CCAAGATTGGGCAACTTCGTC
purk	F-CAGATTGGCACATTGAAAG	492
	R-TTCATTCACATATAGCCCG
pstS	F-TTGAGCCAAGTCGAAGCTGGA	583
	R-CGTGATCACGTTCTACTTCC

抗生素种类 Types of antibiotics	敏感 Susceptible		中介 Mediator		耐药 Resistant
抗生素种类 Types of antibiotics	菌株数 Number of strains	检出率/% Detection rate	菌株数 Number of strains	检出率/% Detection rate	菌株数 Number of strains	检出率/% Detection rate
青霉素 Penicillin	87	81.3	-	-	20	18.7
庆大霉素 Gentamicin	77	72.0	6	5.6	24	22.4
四环素 Tetracycline	101	94.4	2	1.9	4	3.7
红霉素 Erythromycin	98	91.6	2	1.9	7	6.5
林可霉素 Lincomycin	107	100	-	-	-	-
恩诺沙星 Enrofloxacin	73	68.2	8	7.5	26	24.3
头孢唑林 Cephazolin	81	75.7	10	9.3	16	15.0

耐药基因 Resistance genes	菌株数 Number of strains	检出率/% Detection rate
ermA	49	51.6
ermB	87	91.6
ermC	27	28.4
lsaA	14	14.7
lsaE	68	71.6
lnuA	21	22.1
lnuB	36	37.9
tetM	81	75.7

菌株 Sample	抗生素耐药性类型 Type of antibiotic resistance
F2	Lincosamide	Macrolide	Gentamincin-b
	Streptogramin-b	Fluoroquinolone	Amikacin
	Spectomycin	Streptomycin	Paromomycin
	Tetracycline	Tobramycin	Ribostamycin
	Chloramphenicol	Sisomicin	Butirosin
	Lincomycin	Dibekacin	Lividomycin
	Amikacin	Neomycin	Isepamicin
	Netilmicin	Kanamycin