The bacteriostatic effect and mechanism of berberine on Methicillin resistant Staphylococcus aureus in vitro

Abstract

Background: To observe the bacteriostatic effect of berberine (BBR) and BBR combined with gentamicin (GEN), levofloxacin (LEV) and amikacin (AMI) on Methicillin resistant Staphylococcus aureus (MRSA), while also exploring the bacteriostatic mechanism of BBR on MRSA. Methods: The minimal inhibitory concentration (MIC) of BBR, GEN, LEV and AMI on 26 clinical MRSA strains was determined by broth microdilution, while the MICs of BBR combined with GEN, LEV and AMI against MRSA were determined using a microdilution checkerboard. Time-killing curves were used to determine the kinetics of BBR combined with antibiotics for MRSA. We used conductivity tests to assess the changes in membrane permeability in response to BBR on MRSA, while also investigating the changes in MRSA morphology by transmission electron microscopy. RNA-sequencing was used to analyze the expression of differentially expressed genes in reference strain USA300 after its treatment with BBR at different concentrations.Results: The MICs range of BBR on 26 strains of MRSA was 32-256 µg/mL. BBR combined with GEN, LEV and AMI had obvious bacteriostatic effect on MASA. After co-culturing MRSA with BBR at 512 ug/mL, 64 ug/mL and 8 ug/mL, respectively, the electrical conductivity increased, compared with the control group, by 8.14%, 13.08% and 12.01%, respectively. Using transmission electron microscopy, we found that low concentration of BBR (8 ug/mL) had no significant effect on MRSA structure (keeping intact), medium concentration of BBR (64 ug/mL) thinned the cell wall of MRSA, while high concentration of BBR (512 ug/mL) induced the destruction and dissolution of MRSA cell wall structure and the leakage of bacterial contents, leading to bacterial lysis. RNA-sequencing results showed that there were 754 differentially expressed genes in the high concentration group compared with the normal control group. Compared with the low concentration group, there were 590 differentially expressed genes in the high concentration group. Compared with the control group, only 19 genes were differentially expressed in the low concentration group. The up-regulated genes are mainly related to the cell wall hydrolysis regulatory genes, while the down-regulated genes are mainly related to the serine protease family.Conclusions: BBR displayed an excellent bacteriostatic effect on MRSA. BBR combined with GEN and AMI significantly enhanced the bacteriostatic effect on MRSA, while BBR combined with LEV showed no significant change in the bacteriostatic effect on MRSA. BBR inhibited bacteria by destroying and dissolving the structure of MRSA cell wall. RNA-sequencing results further demonstrated that the expression of cell wall hydrolysis genes ssaA, lytM and virulence factor serine protease genes were significantly differentially expressed when high concentration BBR treated on MRSA.