基于银纳米棒的表面增强拉曼基底探究土霉素对植物乳杆菌的影响

Effect of oxytetracycline on Lactobacillus plantarum on surface enhanced Raman substrate based on Ag NRs

摘要:

[背景] 土霉素属四环素类抗生素，在医疗制药和动物养殖行业应用广泛。抗生素的滥用与残留极易导致环境中的益生菌产生耐药，各类益生菌通过食物进入机体后，可将耐药基因转移给致病菌。目前的耐药菌株筛选方法耗时较长，最小抑菌浓度测定结果也可能存在偏差，而且无法在抗菌机理及耐药机制方面提供线索。[目的] 采用表面增强拉曼光谱（surface enhanced Raman spectroscopy，SERS）技术探究土霉素与植物乳杆菌之间的相互作用，以期为微生物耐药菌株的快速筛选提供理论基础，并为研究抗菌机理及耐药机制提供思路及方法。[方法] 组装银纳米棒与石英后得到一种具有良好、稳定的SERS增强效应的固相基底；在植物乳杆菌菌液中加入不同浓度土霉素后各自分别孵育30、60和90 min，利用组装好的固相基底对细菌细胞进行SERS信号测定，拉曼光谱的出峰位置和峰强度变化可以体现不同土霉素浓度及作用时间条件下植物乳杆菌细胞成分的差异，可进一步推断土霉素对植物乳杆菌生长的影响。[结果] 0.5×MIC土霉素会造成SERS光谱强度的降低；1.0×MIC土霉素作用90 min后，SERS在1 612、1 630 cm^-1处出现2个分别代表酪氨酸和酰胺Ⅰ的新峰，推测植物乳杆菌细胞壁被破坏；2.0×MIC土霉素作用60 min便可对受试菌体细胞壁造成较大破坏。[结论] 通过拉曼光谱检测可以反映细菌细胞状态的改变、胞内胞外成分的变化情况，在研究抗菌药物对细菌的作用方式及耐药机制等方面都具有重要意义，为微生物耐药性菌株的快速筛选提供了良好的技术基础。

Abstract:

[Background] As a kind of tetracycline, oxytetracycline (OTC) is used widely in pharmacy making and animal breeding. Abuse and residues of antibiotics can easily lead to drug resistance of probiotics in the environment, and all kinds of probiotics can transfer drug-resistant genes to pathogenic bacteria after entering the body through food. The current screening methods for drug-resistant strains are time-consuming, and the results of the minimum inhibitory concentration determination may be biased, and the results do not directly show the antibacterial mechanism and drug resistance mechanism. [Objective] In this study, surface enhanced Raman spectroscopy (SERS) technology was used to explore the interaction between oxytetracycline and Lactobacillus plantarum, aiming to provide a theoretical basis for the rapid screening of microbial drug-resistant strains and provide ideas and methods for the study of antimicrobial mechanism and drug-resistant mechanism.[Methods] In this experiment, a kind of solid phase substrate with good and stable SERS enhanced effect was obtained after assembling silver nanorods and quartz. After adding oxytetracycline at different concentrations, Lactobacillus planatum was incubated for 30 min, 60 min and 90 min respectively. The assembled solid substrate was used to measure the SERS signal of the bacteria cells. The changes of the peak location and peak intensity of Raman spectrum could reflect the differences in the cell components of Lactobacillus planatum under different oxytetracycline concentrations and action time. The effect of oxytetracycline on the growth of Lactobacillus plantarum can be further inferred.[Results] The results showed that 0.5×MIC oxytetracycline mainly caused the decrease of SERS spectrum intensity. 1.0×MIC oxytetracycline at 90 min may lead to the destruction of the bacterial cell wall, the SERS spectra in, 1612 cm^-1and 1630 cm^-1 appear two peaks represent the tyrosine and amideⅠ. The cell wall was damaged by 2.0×MIC oxytetracycline at 60 min. [Conclusion] Raman spectroscopy detection can reflect the changes of bacterial cell state and intracellular and extracellular components, which is of great significance for the study of antimicrobial agents on bacteria and the mechanism of drug resistance, and also provides a good technical basis for the rapid screening of microbial drug-resistant strains.