Study On Effects Of Sub-MIC β-lactam Antibiotics On Pseudomonas Aeruginosa Biofilms Formation

Objective:To investigate effects of antibiotics at subminimal inhibitory concentration on bacterial biofilm formation, providing theoretical and experimental evidence for mechanism study of bacterial biofilm formation and new drug targets exploitation.Method:Clinically isolated Pseudomonas aeruginosa strains, PA11, PA12 and PA14, and standard strains, PAO1 and ATCC27853, were selected as the experimental strains. The broth microdilution was adopted to determine the minimum inhibitory concentration (MIC) of imipenem and cilastatin sodium (IM/CI), ceftazidime (CAZ), piperacillin and sulbactam sodium for injection (P/S), ceftriaxone sodium (CRO), cefotaxime (CTX), and amoxicillin and flucloxacillin sodium (A/F). The experimental PA strains were treated with antibiotics mentioned above with 1/2M3C,1/4MIC and 1/8MIC respectively, and the adhesion test was used to study the effects of antibiotics on bacterial adhesion. The effects of antibiotics at 1/4MIC on PA.12 biofilm formation were focused.through the motility test, plate count, crystal violet staining and confocal laser scanning microscope. The biofilm related genes, algC and alg D were analysed through RT-PCR.Result:1. Adhesion test showed that:(1) Adhesion of PA.11 can be enhanced by IM/CI, CAZ, P/S, CRO and CAZ at 1/2MIC; adhesion of PAO1 can be enhanced by CAZ and CTX at 1/2MIC and adhesion of all strains the experiment involoved can be enhanced by A/F at 1/2MIC(p<0.05). (2)Except CAZ at 1/4MIC on PA.14, the rest of antibiotics at 1/4 or 1/8 MIC have induced effects on adhesion force of PA (P<0.05).2. After PA.12 being treated by antibiotics at 1/4MIC, compared with control group. (1)Motility test showed that IM/CI, CRO and CTX can enhance the swimming ability of PA.12 at 1/4MIC (p<0.05) and that IM/CI, CAZ, P/S, CRO and A/F can enhance the swarming motility of PA.12 at 1/4MIC (p<0.05). (2)Bacteria count showed that live bacteria have increased significantly in the groups of IM/CI, P/S, CRO and CTX at 1/4MIC (p<0.05). (3) Crystal violet staining showed that the bacterial biofilm formation in the groups of IM/CI, P/S, CRO and CTX at 1/4 MIC have increased (p≤0.05). (4)CLSM observation showed that the bacterial biofilm formation in the groups of IM/CI, P/S, CRO and CTX at 1/4MIC increased, and that the thickness of biofilm also increased. (5)RT-PCR showed that all 6 kinds antibiotics can induce algC gene expression (p≤0.05) while IM/CI and P/S at 1/4MIC can induce algD gene expression (p<0.05).Conclusion:At concentration of 1/4MIC, IM/CI, P/S, CRO and CTX can induce the bacterial biofilm formation of PA.12 while CAZ and A/F had no effects on the formation of bacterial biofilm; IM/CI, CAZ, P/S, CRO, CTX and A/F have regulating effects on quorum sensing system of pseudomonas aeruginosa at 1/4MIC.