The Anti-MRSA Effect Of ?-lactam Antibiotics, D-Ser And DAAO Inhibitor Combination And The Construction Method Of AlaS Mutants Were Explored

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enzymes and modifying enzymes,or changing of the targets and membrane permeability,which makes the original antibiotics no longer active.Methicillin-Resistant Staphylococcus aureus is an important Gram-positive bacteria responsible for infections in humans and livestock that seriously endangers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 P-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 ?-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 DSer 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 ?-red to construct E.coli DH10B which deletes cytosine methylation gene to improve the electroporation efficiency of MRSAN315.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 alaSis 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.