Evaluation For The Permeability Of Bacterial Biofilms To Antibiotics And Its Application In The Screening Of Active Compounds

Bacterial biofilm is a systematic and organized multicellular community attached to the biotic or abiotic surface and surrounded by the extracellular matrix.The formation of biofilm enables microorganisms to survive in the adverse environment and to produce multiple anti-adversity phenotypes,therefore,it is highly correlated to the drug resistance and pathogensis in bacterial pathogens.While the resistance of the biofilm is caused by a variety of mechanisms,the extracellular matrix of the biofilm is the 'first line of defense' against antibiotic invasion as a physical barrier.Agar disk diffusion assay has been commonly used to measure the ability of antibiotics to penetrate bacterial biofilm.However,there are several problems in the method,e.g.,the difference between the biofilm used in the method versus the one formed by pathogenic bacteria in clinical practice,time and labor-consuming of the test,and the relatively low throughput for a large-scale screening of active compounds.In view of these deficiencies,we established an improved method employing submerged bacterial biofilm.Escherichia coli K12 as the common standard strain for drug screening,and two important pathogenic bacteria causing biofilm related infection,i.e.,Gram-positive Acinetobacter baumannii ATCC19606 and Gram-negative Streptococcus mutans UA159,were employed in the study.In order to increase the throughput of method,bacterial submerged biofilm were cultured on the filter membrane of Transwell system using optimized media.Comparing with the biofilm cultured on agar disc,the submerged biofilm is more similar to infectious biofilm in clinical practice.Moreover,the improved MHA-N medium was prepared,which made the edge of the indicated bacteriostatic circle more easier to be read,be automatically detected by using an software.Based on these improvement,we established a method for the test of the permeability of submerged biofilms to antibiotics(TPSBA).Using the submerged biofilm,the sensitivities of the cells in the biofilm to different antibiotics were analyzed,and the results are consistent with the previous finding that the bacterial cells in biofilm are in the state of multi-drug resistance or even pan drug resistance.In addition,the dense biofilm formed by S.mutans hindering the permeability of antibiotics is one of the important mechanisms for its high level of drug resistance.Different representative antibiotics were employed to evaluate the TPSBA method,which showed good feasibility to measure the penetration of antibiotics into biofilms.The comparative analysis indicated that the permeability of antibiotics in the biofilm formed on agar disc was significantly higher than that by TPSBA,which suggested that the agar-biofilm is a weaker physical barrier.Furthermore,it was revealed that the possible role of osmotic adjuvant in the killing of bacterial biofilm by antibiotics,and the results of MBEC90,MBEC100,and permeability should be all taken into account when analyzing the activity against biofilm.Finally,the bio-activities of 32 thiazole-derived compounds were screened and evaluated,which might be used in the prevention and treatment of oral caries.The results showed that compounds 12c and 17e had inhibitory and killing activities on S mutans UA159 cells,and showed good biological activities against UA159 biofilm suppling with DMSO.At a specific concentration,compounds 12c and 17e only inhibited the growth of UA159,but had no significant activity on the growth of normal oral bacteria,i.e.,Streptococcus mitis ATCC6249,Streptococcus salivarius ATCC7073 and Lactobacillus delbrueckii ATCC9649.