Biofilm Formation And Its Related Regulation Genes In Avian Pathogenic Escherichia Coli

Avian Pathogenicity Escherichia coli?APEC?can induce poultry disease,including pericarditis,perihepatitis,peritonitis,airbag and sepsis and other symptoms,seriously restricting the healthy development of poultry industry.Prevention and control research of the disease have important economic significance.At present,the treatment of the disease is mainly through antibiotics,but because of APEC extensive drug resistance led to drug prevention and control of the situation,which is very grim.APEC biofilm formation is one of the important factors of drug resistance.Bacterial biofilm is a bacterial adhesion to the surface of the material,and throu gh the extracellular matrix wrapped with a tightly structured multi-cell population m orphology,its formation influenced by the nutrition,temperature,incubated time and other environmental factors,in addition to bacterial second messenger cyclic di-G MP?c-di-GMP?and LuxS/AI-2 quorum sensing system?Quorum Sensing,QS?als o play an important role in the regulation of bacterial biofilm formation.In this study,APEC biofilm formation and regulatory mechanism provide a new i dea for further prevention and control of APEC biofilm.1.Establishment of DE17 Biofilm Model and Its RegulationDE17 is O₂ serotype of APEC strain originated from our laboratory.In this study,DE17 biofilm formation under different culture conditions was studied by the meth od of crystal violet staining.The results showed that DE17 incubated in M9?Mini mal 9?supplement with hydrates?Except sucrose?at 37?,which can significantly promote the biofilm formation?p<0.05?;In the LB liquid culture medium,the biof ilm can be promoted under the culture condition of 25??p<0.05?,while DE17 i ncubated in DMEM medium at 37?or 25?can not promote the biofilm forma tion.The effect of different carbohydrates on the formation of DE17 biofilm showed t hat the biofilm formation was significantly enhanced at 37?under the culture con ditions of M9.Then the biofilm cultured in M9 containing saccharides was stained by SYTO9/PI dye.The dynamic process of biofilm formation was studied.The r esults showed that DE17 was adhered surfuce before 6 h,and Gradually proliferatin g before 12 h,accompanied by the demise of bacteria was eliminated.The bacteria s showed a large number of proliferation at 30 h,and died at 48-60 h.In order to further study the morphological structure of the biofilm,DE17 was c ultured at 37?for 30h.The results showed that the biofilm structure contained v arious channel structures with a large amount of extracellular matrix.Further studies have shown that the DE17 biofilm-forming is encapsulated with a thick extracellul ar matrix and a complete channel structure compared to other sugars in the presenc e of glucose,indicating that APEC biofilm formation in M9 supplement with 0.2%glucose at 37°C characteristics of strong.The biofilm formation of 49 strains of isolated O1,O2,O78 serotypes showed that 19 strains?OD>4 ODc?were strong,accounting for 38.78%.And medium formation ability of 18 strains?2ODc<OD<4ODc?,accounting for 36.73%.It can be seen that clinical isolates have strong biof ilm formation ability,but do not depend on serotype distribution.In order to study the environmental factors and signal molecular effect on DE17 bi ofilm formation,c-di-GMP was added to the M9 medium containing 0.2%glucose,and the results showed that c-di-GMP?>0.01?mol/L?significantly inhibited the f ormation of biofilm.In addition,the effect of osmotic pressure on the formation of APEC biofilm showed that the glucose concentration reached a maximum at 0.4%.2.Effects of luxS gene deletion on the biological characteristics of avian pathogenic Escherichia coli O1,O2 and O78 serotypesThe signal molecule AI-2 produced by the LuxS/AI-2 quorum sensing system i s involved in the regulation of numerous physiological functions of bacteria.LuxS i s involved in the generation of AI-2.In order to investigate the effect of luxS on t he biological characteristics of different serotypes of APEC,the APECO1?O1 serot ype?,DE17?O2 serotype?,E940?O78 serotype?and its corresponding luxS mutant strains were studied in this study.The growth characteristics,biofilm formation,rdar?red,dry and rough?morphology,motility and drug resistance of wild and mutant strains were analyzed.The results showed that the deletion of luxS gene,eliminatin g AI-2-producing,did not affect the growth characteristics.However,the deletion of luxS gene significantly reduced the biofilm formation of APECO1 and E940?p<0.05?,the biofilm formation of DE17 did not change significantly.The rdar morpholo gy and motility of each strain showed that the deletion of luxS gene changed the r dar morphology of APECO1 and had no effect on DE17 and E940.The motility of APECO1 and DE17 were significantly reduced and had no effect on E940.The tra nscriptional levels of fliG and fliI,flagella genes relating to bacterial motility,were significantly decreased in APECO1,DE17 and E940?p<0.05?.In addition,the res ults of drug resistance test of each strain showed that the lack of luxS gene resulte d in APECO1 becoming more sensitive to cefepime and amikacin by resistance,an d the same as E940 to chloramphenicol from high sensitivity to resistance,but ther e was no significant change in resistance to DE17.This study shows that luxS has an important conditioning effect on the biological properties of APEC,and this re gulation depends on strain specificity.3.yaiC and rtn gene deletion and biological characteristics of the studyThe bacterial second messenger c-di-GMP can regulate the physiological and bioc hemical characteristics of bacterial biofilm,motility,virulence and cell growth.Its i ntracellular level is determined by two guanylate cyclases containing GGDEF and E AL conserved sequence?DGC?and phosphodiesterase?PDE?.In this study,yaiC?D GC?and rtn?PDE?gene were knocked out from DE17 genome.The wild strain an d deletion strain Growth characteristics,biofilm formation,rdar morphology,exercise,drug resistance,LD50 and other characteristics of the analysis.The results show:y aiC and rtn do not affect the growth characteristics;Motility of the mutant strains are significantly reduced;yaiC gene deletion caused a significant decline in biofilm,and rtn gene deletion result in a significant increase in biofilm;rtn deletion turned rdar morphological into pink,while yaiC deletion Strain did not change;otherwise through the drug resistance test,rtn gene deletion led to DE17 on amikacin from resistance to high sensitivity,the gentamicin from high sensitivity into moderate sen sitivity.The deletion of yaiC gene has changed from cefotaxime,cefuroxime and lo mefloxacin to high sensitivity.The results showed that yaiC and rtn had a significa nt effect on the physiological characteristics of APEC biofilm.In this study,APEC biofilm research model was established by analyzing the for mation factors of APEC biofilm,which provided a new idea for further prevention and control of APEC biofilm.