Identification And Distribution Of The Insertion Sequence In Riemerella Anatipestifer And The Effect Of The Efflux Pump Gene AcrB

Riemerella anatipestifer(R.anatipestifer)is a Gram-negative bacterium of the genus Flavobacterium,which occurs and spreads around the world and brings huge economic losses to the duck industry.Antibiotics,as an effective means of preventive treatment,have developed strong multi-drug resistance due to clinical abuse.Insertion sequence(IS)is a mobile genetic factor that is widely present in the bacteria genome.It moves horizontally within the genome or between genomes through different transposition mechanisms to induce gene mutations,deletions,inversions.The bioinformatics method was used to predict the insertion sequence of 28 strains of R.anatipestifer genome and its effect on the genome,and was verified the prediction by constructing the corresponding mutain strain,complementation strain and phenotypic experiment.The main results are as follows: 1.Re-splicing of draft genomes and resequencing of strain RCAD0133The 18 draft genomes were spliced with the strain RA-CH-1 as the reference sequence.All the draft genomic strains had good collinearity with RA-CH-1,and there was no deletion or rearrangement of large plates.The strain RCAD0133 was re-sequenced by third-generation sequencing technology platform.The results showed that the genome consisted of a circular chromosome with a total length of 2,509,140 bp and an average GC content of 34.85%.2.Number and classification of insertion sequences and effects on the genome in RAA total of 513 IS were found in 28 RA,including 442 ISRa1,54 ISRan1,13 partial insertions of the IS1 family,IS613,IS150,IS186 A and IS3.In-depth study of 304 IS in 10 completed genomes revealed that 233 was the unique IS and 71 were the shared IS.The shared insert site AT content was higher,while the unique insert site did not.obvious base bias.110 ISs inserted into the non-coding region of the completed genome,68 ISs inserted into the gene,and 126 ISs resulted in genes loss or increase.IS inserted into the non-coding region affects 107 genes;IS inserted into the gene interrupts the efflux pump gene acrB;in addition,IS added 87 exogenous blocks to the genome,bringing 253 unique genes and some virulence genes,drug resistance genes,regulatory factors,and resulting in 17 gene rearrangements and 8 gene losses.3.Construction of acrB gene deletion strain and replenishant strain and determination of MIC valueThe IS in strain RCAD0133 interrupted the acrB gene.In order to study the effect of IS on genomic resistance,the mutain strain,complementation strain were constructed in strains ATCC 11845,RA-CH-2,RCAD0122 and RA-CH-1,respectively.The complementation plasmid was also introduced into the strain RCAD0133.Finally,the MIC values of 10 antibacterial compounds against wild-type strains,mutain strains and complementation strains of different strains were detected by 96-well dilution method.The results showed that the efflux pump gene acrB mediates RA resistance to streptomycin,trimethoprim,rifampicin,ceftiofur and chloramphenicol and the IS affected the function of the acrB gene.