SSRI类化合物对单核细胞增生李斯特菌多重耐药的逆转影响研究

Novel antimicrobial drugs are lacking, however, so-called “non-antibiotics” may act as 19 antibiotics and/or add in restoring the antibiotic sensitivity of antibiotic resistant bacteria.Listeria monocytogenes (LM) is the main pathogen of zoonosis listerellosis. It is critical to investigate the mechanism and the method to control the antibiotic resistance of LM. In this study, we study the inhibitory effect and the mechanism of multidrug resistance reversal effect of selective serotonin reuptake inhibitors (SSRIs) on LM. The antibacterial activities andmultidrug resistance reversal activities of SSRIs are detected by drug susceptibility test. The effect of SSRIs on the three-dimensional morphology ofthe surface of LM cell membrane is observed by means of cell biology, such as transmission electron microscopy, and the indirect influence on the cell membraneis measured by intracellular oxidation indexes. The influence of SSRIs on thelevel of LM transcriptome is analyzed by high-throughput sequencing technology.Further, the related protein and metabolic pathways are determined. Finally, wefurther analyze themechanism of multidrug resistance reversal of SSRIs on LM by main related gene knockout with lambda - red restructuring system. The aim of thisstudy is to explore the mechanism of antibacterial and drug resistance reversalactivities of this kind of the compounds from gene level, which is safe to peopleand animal and also with high efficiency. This study will provide theoreticalbasis and technical basis for generating more choices of treatment methods in caseof people or animals infected with multidrug-resistant bacteria, forscientifically application of antibiotics in animal husbandry and microecology,and to control and solve the problem of multi-drug resistance of microorganisms.

单核细胞增生李斯特菌（Listeria monocytogenes,LM）是典型的人畜共患食源性病原体，其耐药机制及抑制技术的研究对保障人畜安全具有重大意义。本课题以食源性多重耐药LM为对象，探讨SSRI类化合物对LM的抑制及多重耐药性逆转作用机理。在前期研究利用药敏检测SSRI类化合物体外抑制LM多种耐药株基础上，借助透射电镜等细胞学手段观测细胞膜表面三维形貌影响，通过测定胞内氧化指标，间接考察其对细胞膜代谢的影响；利用高通量测序技术分析其对LM转录组水平的影响，确定相关作用蛋白及其代谢途径，用λ-red重组系统敲除假设的耐药性作用基因，深入研究SSRI类化合物对LM多重耐药性的逆转作用机理，从基因水平探索出对人畜安全又能高效抑菌且逆转耐药活性的机制。在人畜受到多重耐药菌感染时，增加可选择的处理手段，为控制和解决微生物多重耐药问题提供理论依据和技术基础。

本项目探究了舍曲林对单核细胞增生李斯特菌的固有体外抑菌活性，验证了舍曲林作为辅助化合物有望解决细菌耐药性问题，并初步证明了舍曲林的抑菌机制与外排泵无关，与细菌细胞膜及毒力系统有关。通过研究舍曲林对细菌的体外抑菌活性，证明舍曲林的最小抑菌浓度和最小杀菌浓度分别为16和32μg/mL，表明舍曲林对细菌具有固有体外抑菌活性。场发射扫描电镜结果表明，舍曲林可以呈浓度依赖性地破坏细菌细胞的表面形态和细胞间的聚集程度，引起严重的细胞损伤和皱缩；基于细菌产生抗生素耐药性的现象，验证了舍曲林作为“辅助化合物”发挥抑菌性能的潜力，发现曲林可提高卡那霉素、链霉素、四环素、红霉素、利福平和头孢噻肟六种抗生素的抗微生物活性，利用结晶紫染色实验证明舍曲林可以呈浓度依赖性地抑制细菌在非生物表面形成生物被膜的能力，64 μg/mL的药物可以使其下降67.9%-86%，并通过接合转移实验结果发现舍曲林能够抑制耐药性质粒RP4在不同细菌间的接合效率，8μg/mL舍曲林可以导致其下降90.8%。；而外排泵基因PCR结果证明了舍曲林的抑菌机制与外排泵无关。细胞膜相关实验结果证明了舍曲林可以呈浓度依赖性的破坏细菌细胞膜，导致细胞膜功能障碍、通透性增大，进一步导致细胞死亡。具体表现为细胞内ATP浓度下降了76%-96%，细胞内环境pH由中性变为偏酸性。. 基于以上内容表明舍曲林及其结构类似物存在良好的抑菌潜力，既可以单独发挥抑菌活性，也可以作为辅助化合物提高抗生素的抑菌能力。因此，舍曲林有望在食品加工及养殖环境中作为辅助消毒剂消除单核细胞增生李斯特菌的耐药性及污染。同时为舍曲林对细菌细胞膜、生物被膜、毒力系统以及接合效率的作用效果为进一步探究舍曲林对革兰氏阳性菌的抑菌机制提供了思路。