生牛乳中金黄色葡萄球菌异质性耐药及机制研究

doi:10.11843/j.issn.0366-6964.2025.04.040

Abstract

Abstract:

This experiment was conducted to study the drug resistances of Staphylococcus aureus isolated from raw milk, the prevalence of heteroresistance of Staphylococcus aureus and explore the mechanism of heteroresistance. Thirty-six Staphylococcus aureus isolates were isolated from 179 batches of raw milk collected from Northeast, North China, East China, South China, Northwest and Southwest. The sensitivity and resistance of Staphylococcus aureus were identified using broth microdilution method. The combination of disk diffusion method and E-test method was used to screen suspected heteroresistant strains from strains with sensitive phenotypes, and then the suspected heteroresistant strains were confirmed by Population analysis profiling(PAP). The stability of the heteroresistant phenotype after serial passage was measured. Finally, whole-genome sequencing and whole genome re-sequencing analysis were used to determine the heteroresistant mechanisms. The results showed that 36 strains of Staphylococcus aureus were resistant to β-lactam antibiotics, the resistant rate to ranges from 11.11% to 61.11%, and had high sensitivity to tetracycline and aminoglycoside antibiotics. 19 combinations of Staphylococcus aureus and antibiotics with heteroresistant were identified through initial screening, and it was confirmed that the 3 combinations exhibited heteroresistant phenotypes, of which only amoxicillin-clavulanate-J23 heteroresistant stabled after serial passage. This study demonstrates that S. aureus of raw milk origin is high resistant to β-lactam and quinolone antimicrobials. Heteroresistant mechanisms to amoxicillin-clavulanic acid are associated with blaZ and metal β-lactamase expression. Heteroresistant mechanisms to tetracyclines is associated with tetracycline efflux pumps and resistance proteins.

Key words: raw milk, S. aureus, heteroresistant, resistant subpopulations, whole-genome sequencing

CLC Number:

S852.611

HE Xiaolan, ZHAO Yankun, MENG Lu, LIU Huimin, GAO Jiaojiao, ZHENG Nan. Investigation of Heteroresistance and Its Mechanism of Staphylococcus aureus in Raw Milk[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1934-1946.

Figures/Tables 12

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References 32

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序号No.	地理区域Geographical region	牧场地址Pasture address
1	东北地区	黑龙江省齐齐哈尔市克山县
2		黑龙江省齐齐哈尔市甘南县
3		吉林省白城市镇赉县
4	华北地区	内蒙古自治区呼和浩特市和林格尔县
5		内蒙古自治区呼和浩特市赛罕区
6		河北省张家口市塞北管理区
7		河北省保定市满城区
8		河北省保定市徐水区
9		天津市滨海新区
10		北京市昌平区
11	华东地区	山东省淄博市高青县
12		江苏省盐城市大丰区
13		浙江省杭州市建德市
14		福建省南平市延平区
15	华南地区	广东省清远市连州市
16		广东省湛江市遂溪县
17	西北地区	陕西省渭南市大荔县
18		甘肃省金昌市金川区
19		甘肃省嘉峪关市
20	西南地区	贵州省贵阳市修文县
21		云南省曲靖市陆良县
22		四川省达州市宣汉县

抗菌药物类别 Types of antibiotics	抗菌药物名称 Antibiotic	药敏类型 Types of drug resistance	菌株MIC/(μg·mL^-1) Strain MIC	菌株数 Strain number	比率 Rate
β-内酰胺类 β-Lactams	青霉素	S	0.125	18	50.00%
		I	/	0	0.00%
		R	0.25~32	18	50.00%
	氨苄西林	S	0.125	14	38.89%
		I	/	0	0.00%
		R	0.5~64	22	61.11%
	苯唑西林	S	0.25~2	28	77.78%
		I	/	0	0.00%
		R	4~128	8	22.22%
	阿莫西林/克拉维酸	S	0.25/0.12	14	38.89%
		I	0.5/0.25	9	25.00%
		R	1/0.5~16/8	13	36.11%
	头孢噻吩	S	0.25~1	29	80.56%
		I	4	1	2.78%
		R	16~128	6	16.67%
	头孢噻呋	S	0.25~2	32	88.89%
		I	/	0	0.00%
		R	16~128	4	11.11%
大环内酯类 Macrolide	红霉素	S	0.25~0.5	28	77.78%
		I	1	1	2.78%
		R	16~128	7	19.44%
林可霉素类 Lincosamides	克林霉素	S	0.125~0.25	30	83.33%
		I	1~2	3	8.33%
		R	4~64	3	8.33%
氨基糖苷类 Aminoglycoside	庆大霉素	S	0.25~2	34	94.44%
		I	8	2	5.56%
		R	/	0	0.00%
四环素类 Tetracycline	多西环素	S	0.25~4	33	91.67%
		I	8	1	2.78%
		R	16	2	5.56%
	四环素	S	0.25~2	31	86.11%
		I	8	1	2.78%
		R	32~64	4	11.11%
氯霉素类 Chloramphenicols	氟苯尼考	S	1~4	30	83.33%
		I	8	3	8.33%
		R	16~128	3	8.33%
喹诺酮类 Quinolones	环丙沙星	S	0.125~1	28	77.78%
		I	/	0	0.00%
		R	4~64	8	22.22%
磺胺类 Sulfonamides	复方新诺明	S	0.12/2.4~1/19	35	97.22%
		I	/	0	0.00%
		R	32/608	1	2.78%

菌株 Strain	疑似异质性耐药抗菌药物 Antimicrobial agents with suspected heteroresistance
J6	克林霉素、庆大霉素
J16	苯唑西林
J23	阿莫西林/克拉维酸
J26	苯唑西林

菌株 Strain	疑似异质性耐药抗菌药物 Antimicrobial agents with suspected heteroresistance
J6	克林霉素、庆大霉素、阿莫西林/克拉维酸、环丙沙星、复方新诺明
J16	苯唑西林、多西环素
J19	复方新诺明、四环素、庆大霉素
J22	克林霉素、多西环素、四环素、复方新诺明
J23	阿莫西林/克拉维酸、复方新诺明
J26	苯唑西林、克林霉素、多西环素

抗菌药物-菌株 Antimicrobial-Strain	初始MIC值/(μg·mL^－1) Initial MIC	菌株类型 Strain type	异质性耐药亚群发生频率 Frequency of heteroresistant subpopulation
阿莫西林克拉维酸-J23 Amoxicillin Clavulanic Acid-J23	0.25/0.12	Heteroresistance	3.26×10^-7
四环素-J22 Tetracycline-J22	0.25	Heteroresistance	9.41×10^-6
多西环素-J22 Doxycycline-J22	0.25	Heteroresistance	1.69×10^-7

Investigation of Heteroresistance and Its Mechanism of Staphylococcus aureus in Raw Milk

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抗菌药物-菌株 Antimicrobial-Strain	第1代MIC MIC of the first passage	第15代MIC MIC of the 15th passage
阿莫西林克拉维酸J23 Amoxicillin Clavulanic Acid-J23	2/1(R)	2/1(R)
四环素J22 Tetracycline-J22	1(S)	2(S)
多西环素J22 Doxycycline-J22	0.25(S)	0.25(S)

基因 Gene	同义突变 Synonymous	非同义突变 Nonsynonymous	插入 Insertion	缺失 Deletion
J2GL000299	11	3	0	0
J2GL000618	14	0	0	0
J2GL000650	14	2	0	0
J2GL001192	51	17	7	7
J2GL001826	15	4	0	1

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基因 Gene	同义突变 Synonymous	非同义突变 Nonsynonymous	插入 Insertion	缺失 Deletion
J2GL000296	28	14	4	5
J2GL000464	6	1	0	0
J2GL000585	23	1	0	0
J2GL000597	0	0	0	0
J2GL001006	4	1
J2GL001140	15	4	0	0
J2GL001141	15	3	0	0
J2GL001382	2	1	0	0
J2GL001394	0	0	0	0
J2GL001383	5	3	0	0
J2GL001384	5		0	0
J2GL001385	7	7	0	0
J2GL001386	27	10	1	1