Biological Characterization Of Citrobacter Koseri And Functional Studies On Its Flagellin Gene FliC

Bovine mastitis is a common bovine disease that causes huge economic losses to the dairy industry worldwide.The main causes of bovine ma stitis are the intramammary infection(IMI)of mastitis bacterial pathogens.Until now,over 150 pathogens have been reported as mastitis pathogens,Citrobacter koseri(C.koseri)is a pathogen of environmental conditions,which was first thought to be the normal colony of the intestinal flora of dairy cows.In recent years,many countries have isolated the C.koseri from milk samples of bovine mastitis.However,it is still not clear that the biological characteristics of C.koseri and the function of the flagellin gene fliC.In this experiment,isolation,identification and biological characteristics of C.koseri were performed on bovine mastitis samples;The mutant strain of flagellin gene fliC was constructed and the function of flagellin gene fliC was verified.The main findings are as follows:1.A strain of Gram-negative bacteria was isolated from bovine mastitis,after morphological examination,biochemical identification,16 S rRNA sequence determination and phylogenetic analysis,the strain was identified as C.koseri.Therefore,the C.koseri isolates was named as CKO-WT99.2.CKO-WT99 caused death of mice and mastitis in mice,which was the pathogenic bacteria of bovine mastitis.CKO-WT99 was resistant to penicillin and ampicillin,and sensitive to other antibiotics.CKO-WT99 accepted blaCTX-M-55 as a receptor bacterium and transfered blaCTX-M-55 to wild-type E.coli by conjugative transfer;CKO-WT99 was motility and contained the flagellin gene fliC;CKO-WT99 biofilm was mainly composed of curli fimbriae and cellulose.3.In this experiment,the CKO-WT99 genome was used as a template,and the upper and lower homologous arms of fliC gene were fused by overlapping PCR.The fusion fragment was cloned into the suicide plasmid pRE112 to construct a recomb inant suicide plasmid pRE?fliC.E.coli X7213(containing recombinant suicide plasmid pRE?fliC)was used as donor bacteria,CKO-WT99 was used as co-culture for recipient bacteria,and the deletion strain ?fliC was screened by the method of double-crossoverr allelic.The complementary strain ?fliC-C was also constructed by suicide plasmid pRE112.4.There are almost no difference in biochemical characteristics and growth rate among CKO-WT99,?fliC deletion mutant and ?fliC-C complementary strain.The results showed that compared with CKO-WT99,the biofilm formation ability of ?fliC deletion mutant strain attenuated,lost motility,did not produce flagella,and reduced virulence,but ?fliC-C complementary strain was able to recover these abilities,which indicated that the flagellin gene fliC was a structural gene and a virulence gene of C.koseri.