Inhibition And Eradicaion Effects Of Baicalin On Biofilm Produced By Swine Extraintestinal Pathogenic Escherichia Coli In Vitro

Swine extraintestinal Escherichia coli?Ex PEC?is a zoonotic pathogen that colonizes in the extraintestinal organizations of host and often infects swines with other pathogens.The multiple drug resistance of porcine Ex PEC results in increasing difficult of antibacterial treatment and the high mortality rate of sick swines,which seriously restricts the healthy development in the swine industry and brings potential risks to veterinary public health.The biofilm formation is one of the important mechanisms of drug resistance for porcine Ex PEC.Searching for an effective drug to inhibit or clear biofilm has become a hot topic in antibacterial infection research.Studies have shown that baicalin,the effective component of Scutellaria baicalensis,can inhibit or clear the biofilm of Streptococcus mutans,Pseudomonas aeruginosa and Extraintestinal Escherichia coli isolated from patient.However,the impact of baicalin on porcine Ex PEC biofilm is unclear.Therefor,in this paper,three self-isolated porcine Ex PEC were used as the research object,the optimum biofilm-formation characteristics of these isolates were selected,and the their biofilm characteristics were analyzed.On the basis,the effect of baicalin on biofilm matrix,metabolic activity of bacteria into biofilm,biofilm structure and biofilm-forming gene expression at different stages was investigated.The main research contents and results are summarized as follows:1)Aanalysis of biofilm formation characteristics of the porcine Ex PEC isolates.Here,the optimum conditions for biofilm formation of isolates were optimized by microplate crystal violet staining combined with orthogonal experiments.The biofilm-forming ability,dynamic process and biofilm structure of each isolate were checked under optimal biofilm-forming conditions.The results showed below:?i?the optimal biofilm-forming conditions of SE-1 strain were that the initial bacterial suspension at OD₆₀₀of 0.5 was inoculated into TSB medium and cultured at 37°C for24 h.The best conditions for SE-2 strain were inoculated in nutrition broth with a initial concentration OD₆₀₀of 0.3 and cultured at 34°C for 24 h.The optimal biofilm-forming conditions of SE-3 strain were inoculated in BHI medium with a initial concentration OD₆₀₀of 0.5 and cultured at 34°C for 24 h;?ii?all isolates had a strong ability to form biofilm,but the time to enter the stage of adhesion,aggregation and maturity was not exactly the same;?iii?the SE-1 strain could forme a lamellar biofilm,while both SE-2 and SE-3 isolates formed a multilayer biofilm.These results suggested that all isolates had strong ability to form biofilm,but the optimal conditions,dynamic process and structure of biofilm were not completely identical.2)Effect of baicalin on biofilm formation of porcine Ex PEC isolates.Here,the effect of baicalin on biofilm formation were examined by crystal violet staining,TTC staining and scanning electron microscopy.The results showed below:?i?compared with the pre-treatment group,the matrices of biofilm at both adhesion and aggregation stages was significantly discreased after treated with baicalin?P<0.05 or 0.01?,and the inhibitory effect of baicalin was best when its concentration was 1/2 MIC.?ii?compared with the pre-treatment group,the metabolic activity of the bacteria into biofilm at the adhesion and aggregation stages was significantly decreased after treated with baicalin?P<0.05 or 0.01?,and the bacteriostatic effect of baicalin was best when its concentration was 1/2 MIC.?iii?after treatment with 1/2MIC of baicalin,isolated strains at both adhesion and aggregation stages didi not form or form a small amount of biofilm observed under a scanning electron microscopy.These results indicated that 1/2MIC concentration of baicalin had a strong inhibitory effect on the biofilm formation of the porcine Ex PEC isolates.3)Cleaning effect analysis of baicalin on the biofilm of porcine Ex PEC isolates.Here,the cleaning effect of baicalin on mature biofilm of isolates was examined as described above.The results showed below:?i?compared with the pre-treatment group,the matrices of mature biofilm was significantly decreased after treated with baicalin?P<0.05 or 0.01?,and the cleaning effect of baicalin was best when its concentration was 2MIC.?ii?compared with the pre-treatment group,the metabolic activity of bacteria into mature biofilm was significantly decreased after treated with baicalin?P<0.05 or 0.01?,and the bacteriostatic effect of baicalin was best when its concentration was 2MIC.?iii?after treatment with 2MIC of baicalin,The biofilm of each isolate was almost completely cleared observed under a scanning electron microscopy.These results indicated that the baicalin with 2MIC concentration had a better clearance effect on the biofilm of porcine Ex PEC isolates.4)Effect of baicalin on expression of biofilm-forming genes from porcine Ex PEC isolates.Here,RT-q PCR was performed to detected the transcriptional expression levels of biofilm-forming genes in each isolates before and after treatment with baicalin.The results showed below:compared with the pre-treatment group,the lux S gene and glm U gene were significantly down-regulated after treated with 2MIC of baicalin?P<0.05?,and the iha gene were significantly upregulated,suggesting that the baicalin with concentration of 2MIC could significantly change the transcriptional expression levels of biofilm-related genes from porcine Ex PEC isolates.In summary,the optimum biofilm-formation characteristics.The baicalin with concentration of 1/2 MIC had a strong inhibitory effect on the biofilm formation,while the baicalin with concentration of 2MIC had a better cleaning effect.The baicalin with concentration of 2MIC could significantly change the transcriptional expression levels of three biofilm-forming genes from porcine Ex PEC isolates.Our results lay a good foundation for the prevention and control of porcine Ex PEC infection from the perspective of biofilm.