抗生素的发现使人们不再为治疗感染性疾病而苦恼，但滥用及不合理使用抗生素却又将人类推入“耐药菌”的漩涡，使人类面临抗生素枯竭的现状。耐药菌通常以产生外排泵、水解酶、钝化酶、改变作用靶点、改变膜通透性等方式逃避抗生素的攻击，这使得原有抗生素不再起治疗作用。耐甲氧西林金黄色葡萄球菌（Methicillin-Resistant Staphylococcus aureus，MRSA）是一种重要的人畜共患革兰氏阳性菌，严重危害人类和牲畜的健康。2019年全国细菌耐药监测报告显示金黄色葡萄球菌（Staphylococcus aureus，S. aureus）的检出率居革兰氏阳性菌之首，其中MRSA的全国平均检出率为30.2%，仍处于较高水平，解决微生物耐药问题刻不容缓。

目前，针对细菌耐药问题主要有两种策略，一种是开发新抗生素，一种是寻找有效的抗生素增敏剂，该方法快捷、经济，且不易引起耐药。实验室前期的工作发现D-Ser是一种良好的抗生素增敏剂，其与β-内酰胺类抗生素联用可使药物对MRSA的MIC值最高下降128倍，在体内也可显著提升MRSA全身感染小鼠的存活率、降低MRSA腿部感染小鼠的组织持菌量。机制研究初步结果显示D-Ser/苯唑西林诱导耐药突变株alaS基因有突变，提示D-Ser的增敏作用可能与alaS基因有关。在体内D-Ser可被D-氨基酸氧化酶（DAAO）氧化代谢，不仅会降低D-Ser的增敏效果，还会引起肾毒性反应，基于以上背景我们开展了如下工作：

论文第一部分以加入DAAO特异性抑制剂的策略研究了β-内酰胺类抗生素、D-Ser和CBIO三药联用抗MRSA感染。在体外，肉汤微稀释法的结果显示CBIO单用并无抑菌作用，三药联用可使苯唑西林和美罗培南对MRSA的MIC值明显降低，但相比抗生素与D-Ser联用时稍有升高，提示CBIO可能对两种抗生素的抗菌活性有轻度拮抗作用。结晶紫染色法评价了联用对细菌生物被膜的影响，结果显示D-Ser和CBIO各自单独使用时对MRSA N315所产生的生物被膜并无明显的抑制和清除作用，但与抗生素联用时可表现出明显的抑制和清除生物被膜的效果。在体内，建立了小鼠全身感染模型和大鼠腿部感染模型，以生存率和组织匀浆菌落计数为指标评价联用的效果，三种药物联用相比单用抗生素以及抗生素和D-Ser两药联用，可一定程度上提升小鼠存活率、降低大鼠腿部肌肉组织持菌量。以小鼠和大鼠皮下给药的方式评价药物联用的安全性及对肾毒性的改善作用，结果显示大剂量的D-Ser会使大鼠肾功能指标异常，出现短时间内血清肌酐值上升等急性肾毒性反应。CBIO在体内安全性良好，与D-Ser联用时可使大鼠肾功能指标恢复正常，消除D-Ser所造成的肾毒性。

论文第二部分首先利用λ-red构建胞嘧啶甲基化（dcm）基因缺失的E. coli DH10B，用于提高MRSA N315的电转化效率。之后利用大肠杆菌-金黄色葡萄球菌穿梭质粒pIMAY*探索构建alaS突变株的方法，为研究alaS与D-Ser的增敏机制提供基础。结果显示，成功构建了dcm基因缺失的E. coli DH10B（E. coli DC10B），但通过穿梭质粒pIMAY*系统难以在S. aureus RN4220和MRSA N315中构建alaS突变株，可能原因是alaS基因是细菌的必需基因，其突变后可能对细菌影响比较大，筛选得到突变株的几率低，后续考虑用CRISPR/Cas9基因敲除技术进行突变株构建以及通过其他方法研究D-Ser增敏机制。

The discovery of antibiotics makes people no longer worry about the treatment of infectious diseases, but the abuse and unreasonable use of antibiotics has pushed humans into the vortex of “drug-resistant bacteria”, making humans face the current situation of antibiotic depletion. Drug-resistant bacteria usually evade the attack of antibiotics by producing efflux pumps, hydrolytic enzymes and modifying enzymes, or changing of the targets and membrane permeability, which makes the original antibiotics no longer active. Methicillin-Resistant Staphylococcus aureus is an important Gram-positive bacteria responsible for infections in humans and livestock that seriously endangers the health of them. The 2019 National Bacterial Resistance Surveillance Report shows that the detection rate of Staphylococcus aureus is the highest among Gram-positive bacteria, and the average detection rate of MRSA is 30.2% in the country, which is still at a high level. It is urgent to solve the problem of microbial resistance.

At present, there are two main strategies for the problem of bacterial resistance. One is to develop new antibiotics. And the other is to find effective antibiotic sensitizers, which is fast, economical, and not easy to cause drug resistance. Previous study of our laboratory found that D-Ser is a good antibiotic sensitizer. The combination of D-Ser with β-lactam antibiotics can reduce the MIC values of the drugs against MRSA by up to 128 times. In vivo, it can also significantly increase the survival rates in mouse systemic infection model induced by MRSA and reduce thigh bacteria counts in mouse thigh infection model induced by MRSA. The preliminary results of mechanism study showed that D-Ser +oxacillin induced mutant had a mutation in alaS gene, suggesting that the sensitization effect of D-Ser may be related to alaS gene. In vivo, D-Ser can be oxidized and metabolized by D-amino acid oxidase (DAAO), which not only reduces the sensitization effect of D-Ser, but also cause nephrotoxic effect. Based on the above background, we carried out our research work as follows:

In the first part, we studied the combination of β-lactam antibiotics, D-Ser and CBIO (a DAAO specific inhibitor) against MRSA infections. In vitro, the results of the broth microdilution method showed that CBIO has no bacteriostatic effect. The three-drug combination can significantly reduce the MIC values of oxacillin and meropenem against MRSA, but the MICs were slightly higher comparing with the two drug combinations (antibiotics and D-Ser), suggesting that CBIO has a slight antagonistic effect on the antibacterial activity of the two antibiotics. The crystal violet staining method evaluated the effect of the combinations on the bacterial biofilm. The results showed that when D-Ser and CBIO are used alone, they had no obvious inhibitory and clearing effects on the biofilms produced by MRSA N315, but when combined with antibiotics, they can show a significant inhibitory and clearing effect. In vivo, we established a mouse systemic infection model and a rat thigh infection model using survival rates and the bacterial colony counts of the tissue homogenates as indicators to evaluate the effect of the combination. Compared with antibiotics alone and the combination of antibiotics and D-Ser, the combination of the three drugs can improve the survival rates of mice to a certain extent in mouse systemic infection model and reduce the bacterial colony counts of the thigh homogenates in rat thigh infection model. The safety and the effect on kidney toxicity of the drug combination were evaluated by subcutaneous administration in mice and rats. The results showed that high-doses of D-Ser can cause abnormal renal function in rats, manifested by acute nephrotoxic effects such as increased serum creatinine values in a short time. CBIO has good in vivo safety, and when combined with D-Ser, it can restore the renal function of the rats and eliminate the nephrotoxicity caused by D-Ser.

In the second part, we used λ-red to construct E. coli DH10B which deletes cytosine methylation gene to improve the electroporation efficiency of MRSA N315. Then we used the Escherichia coli-Staphylococcus aureus shuttle plasmid pIMAY* to explore the method of constructing alaS mutant strains, hoping to provide a basis for studying the relationship of sensitization mechanism of D-Ser and alaS. The results showed that we successfully construct E. coli DH10B (E. coli DC10B) which delete dcm gene. However, it is difficult to construct alaS mutant strains in S. aureus RN4220 and MRSA N315 through the shuttle plasmid pIMAY* system. The possible reason is that alaS is an essential gene of bacteria, and its mutation may have a great impact on the bacteria. The chance of screening for mutant strains is low. We will consider using CRISPR/Cas9 gene knockout technology to construct mutant strains as well as studying the sensitization mechanism through other routes.