金黄色葡萄球菌小菌落突变体诱导筛选及特性研究

doi:10.13304/j.nykjdb.2022.1052

摘要/Abstract

摘要：

稳定型金黄色葡萄球菌（Staphylococcus aureus）小菌落突变体（small colony variants， SCVs）的获得，为抗SCVs药物的研究提供了重要的生物学材料。以S. aureus ATCC43300为出发菌株，利用低质量浓度（1.25~5.00 μg·mL^-1）庆大霉素二次诱导技术获得稳定型SCVs，并对其特性进行分析。结果表明，1/2×最小抑菌浓度（minimum inhibitory concentration， MIC）及2×MIC庆大霉素分别诱导6 和12 h，可获得稳定型SCVs。SCVs生长缓慢，培养24 h可见较小菌落，培养7 h至生长对数期；对庆大霉素敏感性及产生物膜能力分别提高3和1倍；传代20次后恢复突变率为0%，抗逆性、药物敏感性、抗凝血性均增强，SigB调控系统及生物膜相关基因呈上调趋势，Agr调控系统及毒力因子下调。该方法的成功建立为SCVs的获得提出新的筛选策略，并为开展SCVs致病机理研究及新型抗SCVs药物研发提供稳定型受试菌株。

关键词: 金黄色葡萄球菌, 小菌落突变体, 庆大霉素

Abstract:

To obtain the small colony variants （SCVs） of Staphylococcus aureus （S. aureus） is important biological materials for the research of anti-SCVs drugs. In this study， S. aureus ATCC43300 was used as the starting strain， the stable SCVs were obtained by secondary induction technique of low mass concentration （1.25~5.00 μg·mL^-1） gentamicin，and their characteristics were analyzed. The results showed that the stable SCVs could be obtained by secondary induction at 1/2×minimum inhibitory concentration（MIC）gentamicin 6 hours and 2×MIC gentamicin 12 hours. SCVs grew slowly， small colonies could be seen after incubated for 24 hours， even took 7 hours to reach the exponential growth phase；the sensitivity to gentamicin and the ability to produce membrane were increased 3 and 1 times；the recovery mutation rate was 0% after successive 20 passages； anti-stress， drug sensitivity and anti-coagulation were all enhanced. The SigB regulatory systems and the biofilm-related genes were up-regulated， the Agr regulatory system and the virulence factors were down-regulated. This method proposed successful new screening strategy for screening SCVs， and also provided stable test strains for pathogenesis of SCVs and the development of novel anti-SCVs drugs.

Key words: Staphylococcus aureus, small colony variants（SCVs）, gentamicin

中图分类号:

S852.611

周宇, 李佳玉, 王乐, 贾晓爽, 高思琦, 王潇, 焦健. 金黄色葡萄球菌小菌落突变体诱导筛选及特性研究[J]. 中国农业科技导报, 2023, 25(5): 147-157.

Yu ZHOU, Jiayu LI, Le WANG, Xiaoshuang JIA, Siqi GAO, Xiao WANG, Jian JIAO. Induction Screening and Characteristics of Staphylococcus aureus Small Colony Variants[J]. Journal of Agricultural Science and Technology, 2023, 25(5): 147-157.

图/表 10

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基因 Gene	引物序列（5’-3’） Primer sequence（5’-3’）	类别 Category
agrA-F	AAGCATGACCCAGTTGGTAACA	Agr调控系统相关基因 Agr regulatory system genes
agrA-R	ATCCATCGCTGCAACTTTGTAGA
agrB-F	CCCATTCCTGTGCGACTTAT
agrB-R	TTGAATGAATTGGGCAAATG
agrC-F	TAGTGACCATGATCATAATGTATTTGAG
agrC-R	CTTCTTGATTTCGTTTGTATTTCATCTC
agrD-F	AACATVGCAGCTTATAG
agrD-R	TTCGTGTAATTGTGTTA
RNAIII-F	GCATGTAAGCTATCGTAAACAACA
RNAIII-R	AGGGGCTCACGACCATATAC
sigB-F	GGTGCCATAAATAGATTCGATATGTCCTT	SigB调控系统相关基因 SigB regulatory system genes
sigB-R	CTTTTGATTTCACCGATTACAGTAGGTACT
rsbV-F	ACGAAGTTAAAGTCGGTGGAGA
rsbV-R	ATTCGACCTCCGTTCCTTCA
rsbU-F	CGCGTGAAGATGTGTTCAAGAC
rsbU-R	CTATCTCTTTATCGTGAACTTGAAG
rsbW-F	GCGAAGGTGGCCTAGGTTTA
rsbW-R	GCCATTATTTCGCACCTGCT
relQ-F	AGAAAGTGGTTACCGCTCGT	生物膜相关基因 Biofilm related genes
relQ-R	TCATCCGGATAAGCACCATCA
relP-F	TTGCCGGAATTCGCGTAGTA
relP-R	CGCGTTCTGCTAAAAAGACTGG
rsh-F	TACATCGCACTGATTGCCCA
rsh-R	TTAAATTGCCGGCTGTCGAG

基因 Gene	引物序列（5’-3’） Primer sequence（5’-3’）	类别 Category
agrA-F	AAGCATGACCCAGTTGGTAACA	Agr调控系统相关基因 Agr regulatory system genes
agrA-R	ATCCATCGCTGCAACTTTGTAGA
agrB-F	CCCATTCCTGTGCGACTTAT
agrB-R	TTGAATGAATTGGGCAAATG
agrC-F	TAGTGACCATGATCATAATGTATTTGAG
agrC-R	CTTCTTGATTTCGTTTGTATTTCATCTC
agrD-F	AACATVGCAGCTTATAG
agrD-R	TTCGTGTAATTGTGTTA
RNAIII-F	GCATGTAAGCTATCGTAAACAACA
RNAIII-R	AGGGGCTCACGACCATATAC
sigB-F	GGTGCCATAAATAGATTCGATATGTCCTT	SigB调控系统相关基因 SigB regulatory system genes
sigB-R	CTTTTGATTTCACCGATTACAGTAGGTACT
rsbV-F	ACGAAGTTAAAGTCGGTGGAGA
rsbV-R	ATTCGACCTCCGTTCCTTCA
rsbU-F	CGCGTGAAGATGTGTTCAAGAC
rsbU-R	CTATCTCTTTATCGTGAACTTGAAG
rsbW-F	GCGAAGGTGGCCTAGGTTTA
rsbW-R	GCCATTATTTCGCACCTGCT
relQ-F	AGAAAGTGGTTACCGCTCGT	生物膜相关基因 Biofilm related genes
relQ-R	TCATCCGGATAAGCACCATCA
relP-F	TTGCCGGAATTCGCGTAGTA
relP-R	CGCGTTCTGCTAAAAAGACTGG
rsh-F	TACATCGCACTGATTGCCCA
rsh-R	TTAAATTGCCGGCTGTCGAG

基因 Gene	引物序列（5’-3’） Primer sequence（5’-3’）	类别 Category
icaD-F	ATGGTCAAGCCCAGACAGAG	生物膜相关基因 Biofilm related genes
icaD-R	AGTATTTTCAATGTTTAAAGCAA
ccp-F	ATGATGTAGCAAGAGAAGCGCG
ccp-R	TAACTGATGCTATATGTGCATCC
luxS-F	GTCATTCACAAATACGACATTCG
luxS-R	CCTGTACCGAAAACATCATGCC
eno-F	ACGTGCAGCAGCTGACT
eno-R	CAACAGCATCTTCAGTACCTTC
clfA-F	ATTGGCGTGGCTTCAGTGCT
clfA-R	CGTTTCTTCCGTAGTTGCATTTG
sarA-F	ATGGCAATTACAAAAATCAATGATTGC
sarA-R	TTATAGTTCAATTTCGTTGTTTGC
pvl-F	TCTCATGAAAAAGGCTCAGGA	毒力基因 Virulence genes
pvl-R	TTGCCATAGTGTGTTGTTCTTCT
hla-F	ACCCGGTATATGGCAATCAA
hla-R	AGCGAAGTCTGGTGAAAACC
spa-F	GCGCAACACGATGAAGCTCAACAA
spa-R	ACGTTAGCACTTTGGCTTGGATCA

基因 Gene	引物序列（5’-3’） Primer sequence（5’-3’）	类别 Category
icaD-F	ATGGTCAAGCCCAGACAGAG	生物膜相关基因 Biofilm related genes
icaD-R	AGTATTTTCAATGTTTAAAGCAA
ccp-F	ATGATGTAGCAAGAGAAGCGCG
ccp-R	TAACTGATGCTATATGTGCATCC
luxS-F	GTCATTCACAAATACGACATTCG
luxS-R	CCTGTACCGAAAACATCATGCC
eno-F	ACGTGCAGCAGCTGACT
eno-R	CAACAGCATCTTCAGTACCTTC
clfA-F	ATTGGCGTGGCTTCAGTGCT
clfA-R	CGTTTCTTCCGTAGTTGCATTTG
sarA-F	ATGGCAATTACAAAAATCAATGATTGC
sarA-R	TTATAGTTCAATTTCGTTGTTTGC
pvl-F	TCTCATGAAAAAGGCTCAGGA	毒力基因 Virulence genes
pvl-R	TTGCCATAGTGTGTTGTTCTTCT
hla-F	ACCCGGTATATGGCAATCAA
hla-R	AGCGAAGTCTGGTGAAAACC
spa-F	GCGCAACACGATGAAGCTCAACAA
spa-R	ACGTTAGCACTTTGGCTTGGATCA

菌株Strain	最小抑菌浓度 MIC/（μg·mL^-1）	最小杀菌浓度 MBC/（μg·mL^-1）
S. aureus ATCC43300	2.5	5
小菌落突变体SCVs	12.5	25