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

Abstract

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

CLC Number:

S852.611

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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References 34

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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

Research on the Biological Characteristics of Enterococcus faecalis in Dairy Cow Mastitis in Ningxia

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菌株 Sample	毒力因子 Virulence factors
F2	DnaK	GAPDH	KatA	MgtBC
	degP	Ybt	Lap	LysA
	SlrA	T6SS-Ⅱ	ClpB	PDH-B
	PEB1	AllB/C/D	YbtQ	SigA
	ML1683	Hyaluronidase	HtrA	Hemolysin Ⅲ
	RegX	LPS	VctC/D	UgpC
	carB/A	AgrA/C	SrtA	Lsp
	PurM	STP	PiuA	HA
	GelE	ClpC	Fss1/2	Lgt
	ENO	MprAB	EfaA	TTSS
	HemH	Lmb	EF	SprE
	EF-Tu	AdsA	ArgK	RelA
	GspG	RegX3	FagC	EPS
	SitC	FBPs	Ebp pili	GS
	IlpA	AS	CbrC	RmlC
	PsaA	FlmH	HitABC	Wzt2
	GlnA1	LOS	GalE	SSA
	Capsule	AI-2	T4SS	BopD
	DltA	Ace	ctpV	FbpABC
	LplA1	VirR/S	AatC	LytR
	BSH	Hemolysin	Nuc	GroEL
	SodB	NDKs	PgdA	GtcA
	RopA	LisR/K	ClpE/P	CdsA

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