Study On The Effect Of Baicalein In Vitro On Biofilm Inhibition Of APEC

Avian pathogenic Escherichia coli(APEC)is widely distributed in the natural environment,and can cause a series of poultry diseases,such as peritonitis,gasocystitis,sepsis,pericarditis,etc,to bring great harm to the breeding industry.Bacterial biofilm can increase the drug resistance of bacteria by 600-1000 times,leading to the difficulty of killing pathogenic bacteria with drugs.Once bacteria from biofilm in the feed trough,and equipment of the farm,it will be difficult to remove,and constantly release pathogenic bacteria,bringing lasting harm to the farm.It has become a research hotspot for searching for drugs than can effectively inhibit the formation or destruction of mature bacterial biofilm.In this paper,the inhibition of baicalein in the formation of APEC biofilm was studied,and the adhesion stage and aggregation stage of the biofilm were tested respectively,to explore the specific stage of baicalein in the formation of APEC biofilm.The main research work and achievements completed are summarized as follows:1)Drug screening for inhibiting the formation of APEC biofilmIn this paper,the MIC of seven traditional Chinese medicine extracts of andrographolide,matrine,quercetin,emodin,berberine hydrate hydrochloride,magnolol,baicalein,glyceryl monolaurate on APEC were determined by the micro broth dilution method.The results showed that the MIC of quercetin,emodin,glyceryl monolaurate,and magnolol on APEC was >256 μg/m L.The MIC of baicalein was 256 μg/m L.MIC of matrine and andrographolide was >1024 μg/m L.The MIC of berberine hydrochloride hydrate was >512 μg/m L.The eight drugs were screened for their inhibitory effects on the APEC biofilm,the results show that the eight kinds of drugs for APEC have inhibitory effect on bacterial biofilm,and present a dose-dependent.When the concentrations of andrographolide,matrine,quercetin,emodin,berberine hydrochloride hydrate,magnolol,baicalein,and glyceryl monolaurate glyceride were 4 μg/m L,8 μg/m L,1 μg/m L,2 μg/m L,8 μg/m L,4 μg/m L,1 μg/m L,8 μg/m L,respectively,the inhibition rates of baicalein and quercetin were 13.65% and 7.32%,respectively.When andrographolide concentration ranged from 512 μg/m L to 1024 μg/m L,matrine concentration ranged from 128 μg/m L to256 μg/m L,and glyceryl monolaurate ranged from 16 μg/m L to 32 μg/m L,the inhibition of APEC biofilm formation reached half.When the concentration of quercetin,emodin,berberine hydrochloride hydrate,magnolol,and baicalein reached 64 μg/m L,16 μg/m L,32μg/m L,64 μg/m L,and 2 μg/m L,respectively,the inhibition of the formation of APEC biofilm reached half.The inhibition rates were 57.69%,50.37%,56.83%,55.98% and54.51%,respectively.By comprehensive comparison,baicalein had the best inhibitory effect,so baicalein was selected as a follow-up drug.2)In vitro inhibition of APEC biofilm by baicaleinThe influence of sub-MIC baicalein on the growth curve of APEC was determined first,and then the growth curve of APEC biofilm was determined by crystal violet staining.The effects of baicalein on the adhesion and aggregation of APEC biofilm were studied by crystal violet staining,optical microscopy,laser confocal microscopy,and scanning electron microscopy.The results showed that baicalein had no effect on THE growth of APEC when the concentration was 64 μg/m L or below.The growth curve of biofilm shows that APEC is in the adhesion stage at 0-12 h when a small number of bacteria are attached to the surface of the carrier.APEC is in the aggregation stage at 12-24 h,when bacteria from microcolonies on the surface of the carrier and generate extracellular matrix.APEC is in the biofilm maturity stage at 24-48 h.The bacteria proliferate and produce a lot of extracellular matrices.Biofilm inhibition test showed that radix Baicalein element of APEC biofilm formation of adhesion,aggregation,and biofilm formation has a significant inhibitory effect,different concentrations of Baicalein,inhibition rate of adhesion stages were higher than the accumulation phase of inhibition rate,the inhibition of biofilm formation rate is similar to inhibition of adhesion phase,It was concluded that baicalein inhibited biofilm formation mainly by inhibiting the adhesion stage of APEC biofilm formation.