Antibacterial Activity Screening And Mechanism Research Of Macrolide Derivatives

In recent years, with extensive use of antimicrobial agents, especially the abuse of antibiotics, the bacterial resistance showed an increasing trend, and create great difficulties for the treatment of infectious diseases. Therefore, the growing problem of bacterial resistance promoted the screening and mechanism research of active compounds of anti-drug-resistant.In this study, we screened a number of macrolide derivatives, which are all provided by Department of Medicinal Chemistry of Shangdong University. Three compounds with high activity against resistant strains were screened, and then we evaluated the pharmacodynamics and in vivo activity of them, and studied the mechanisms further.In the screening experiment, the Minimal inhibitory concentration (MIC) was adopted as indicators to evaluate the antibacterial activity of the compounds under test. A broth microdilution method in accordance with the Clinical and laboratory standards institute (CLSI) guidelines was used to determine the MICs. Through a hundred of compounds, we got three active compounds, which have the MIC between0.06～2μg/ml against S. pneumoniae resistant strains, and also better activity against S.aureus and S.pyogenes than control group.The further pharmacodynamics study of early selected compounds. Firstly, using the clinical newly isolated eight S.pneumoniae resistance strains to screen the antimicrobial activity of the compounds, including MIC and MBC, and confirming the resistant mechanisms of the eightstrains. Secondly, investigating the variation of the values of the MIC influenced by the changes of the culture conditions, including the PH and the bacterialload. Then, drawing the antibacterial curve. The experience results show:1.The three selected compounds have a good antibacterial effect on the clinical newly isolated S.pneumoniae resistance strains, all values of the MIC are less than2μg/ml, All the three compounds show the sterilization effect at high enough concentrations, about4-32times MIC.2.When the.bacterialload increases from103CFU/ml to107CFU/ml, the value of the MIC increases about4-64times. The value of the MIC decreases about32-258times as the pH of the culture medium increased to9from5.The results show that the.bacterialload has certain effect on the MIC value, while the pH value has a larger effect. When the pH value comes to9, the MIC value reaches the minimum, which shows the compound has a stronger antibacterial effect under the alkaline conditions.3.Drawing the killing curve of compound F7. The results indicate that after fostering24h, there exist a portion of living bacteria of the S.aureus and S.pyogenes, but can stabilize the bacteria number, thus show the antibacterial role. When reaching the4xMIC, the S.pyogenes almost has no living bacterium, thus shows the sterilization effect.After that we evaluated the in vivo activity of compound F7. Firstly, repeatedly continuous vaccination, isolation to ascension pathogenicity of the S.pyogenes through the body, then confirm the100%minimum lethal dose to do the experimental research. Randomly dividing the mice into some groups, each has20, male and female half. The trials divided into high dose of drugs, medium dose of drugs, low dose of drugs, erythromycin control and blank control group. Using the certain minimum lethal dose to infected mice through intraperitoneal injection. Immediately injecting drugs through vein after infection and dose once again in6h after first dose. Observing5-7days after dosing, recording the number of living mice in each group, then analysising and calculating the ED50of the drug. Data showed that compound F7which have a ED50value about24.83mg/kg can protect mouce infected at certain concentration.To evaluate the effect of compound F7on the viability of liver cells HL-7702, MTT assay was done next. TC50was calculated according to the Reed-Muench method which values17.04μg/ml.The last part of the study is the mechanism research of F7.1. The effect of compound F7on external morphology and ultrastructure of S.pneumoniae were observed by scanning and transmission electron microscopy. Through these photos it can be saw that, the external morphology of the bacteria were seriously distorted irregular, surface uplift or depression. TEM observation showed us the bacterial cell wall and membrane were part of defect and the cell core electron density was greatly reduced.2. The effect of compound F7on the mRNA level of ermB and mefA was tested by RT-PCR. The strain used in this study is S. pneumoniae resistant strain express both ermB and mefA. The total RNA of the strain was extracted, and we designed specific primers to amplify ermB and mefA gene. The disparity of mRNA level of ermB and mefA gene between different groups was tested by RT-PCR using16S rRNA gene as inter reference. Data showed that, there is no difference among experimental groups in the expression of the inter reference gene16S rRNA, but, compound F7can inhibit the mRNA level of ermB and mefA gene significantly.3. The in vivo DNA and protein content after dose of F7were tested at last. The results showed that there is no significant different in DNA content between control group and dose group12h after dose of F7. But the protein content decreased about19%after dose of F7, which showed a significant difference.In summary, this study focused on the in vitro and in vivo antibacterial activity and mechanism of macrolide derivatives. F7is one of the derivatives which have high activity against resistant strains both in vitro and in vivo. The preliminary mechanism study confirmed that the possible antibacterial mechanism of F7maybe:destroy the ultrastructure of the bacteria and inhibit the protein expression of bacteria on the one hand, and inhibit the expression of resistance genes of bacteria at the same time.