Thecomparative Analysis Of Aeromonas Hydrophila And The Research Of Drug Resistance

Aeromonas hydrophila widely exists in aquaculture,and its pathogenic throughout the world.Septicemia,enteritis and skin ulceration caused by A.hydrophila brought great economic losses to aquaculture,and have a great impact to our country and even the world’s farmers.The diseases that caused by A.hydrophila were often treated with antibiotics,however,in actual production,there is not a relatively sound system for the detection of the disease,which leads to the serious abuse of antibiotics.Due to a large number of antibiotics applied to the environment,lead to serious antibiotic residues in aquaculture water environment,the resistance of A.hydrophila increase gradually,which brings more difficulties to the prevention and control.To explore the mechanism of drug resistance of A.hydrophila,providing theoretical basis for practicalproduction,this study induced A.hydrophila resistance to doxycycline,sequencing the genome and comparative analysis the genome to carry out the following work:(1)we have sequenced the whole genome of A.hydrophilaAH10(CCTCC,AB2014155)to fully understand the whole genome information structure and function of AH10.Extract genomic DNA from AH10 and the library was constructed andfollowed by sequencing;and quality assessment the raw data and assemble them,then genome annotation and comparison analysis after assembly.Results shows that: The genome full length of AH10 was 4.91 Mb,in which G+C % was 61.1% and 4570 predicted genes were included.3351 genes with a clear biological function.2592 genes was found in cluster of groups orthologous(COG),1281 genes related to metabolic pathway,and 31 genome island.Comparative genomics research showed that: AH10 has 102481 SNPs,402 insertion and deletion,and the linear genomic organization with A.hydrophilaYL17 was the best.AH10 has a close genetic relationship with A.hydrophila ATCC 7966,the standard strain of U S A.The pathogenicity of AH10 closely related to Hemolysin gene(hly)、Aerolysin gene(aer)、Major adhesion gene(ahal)、 Serine protease gene(ahp)、 Cytotonic enterotoxin gene(alt).These genes are highly homologous in different strains.The Autoinducer-2(AI-2)QS andahyI/AhyR QS have found in AH10,but there is no type III secretion system(TTSS).(2)Five typical antibiotics drugs representing the five major categories of antibiotics were selected to induce drug-resistant A.hydrophila strain AH10(CCTCC number: AB2014155)on agar plate suppled with a concentr ation of mutant prevention concentration(MPC)for each specific antibioti cs.Through determining the minimum inhibitory concentration(MIC)ofdifferent antibiotics on all the identified drug-resistant mutant bacterial,the cross drug-resistance map was drawn in this report.The results showed that induced drug-resistant bacterial demonstrated an over 100 times-higher MIC value in comparison to the wild type of A.hydrophila strain AH10.Individual strain resistance decreased after storage at 4゜C in one month.Under the pressure of drug screening,the resistance of the strains increased with the increase of passages.Resistant mutants induced from antibiotics belonging to the same class drug-resistant correlation is higher;while,resistant mutants induced from antibiotics belonging to different classes demonstrated different resistant map.(3)We sequenced all 5 strains and performed comparative genomic analysis of these draft genomes with 9 A.hydrophila complete genomes from Gen Bank and AH10 pan-genome analysis revealed that the size of the pan-genome was 4730 genes,and there are 3056 genes(core)shared among the 14 strains,compared with AH10 thereis no specific single nucleotide polymorphisms(SNP)and insertion-deletion(INDEL)wereiden tified in any functional gene locus,but significant copy number variation(CNV)and structure variation(SV)were identified among the genome of AH10 mutated strains.Detection the drug resistance gene we found that:a variety of drug resistance genes,bl1 fox only ecists in 4AK4.Among all of the 5 doxycycline resistance strains,tetl only existed in AH104,comparativeanalysis found that original AH10 also don’t have this gene.In summary: A.hydrophila was easy resistance to antibiotics once produced,the resistance will be difficult to disappear.If the strains resistance,the cross resistance is serious.The resistant mutant was less and they all not in coding region,it shows the formation of drug resistance of A.hydrophila may not mainly be caused by gene mutation.There is much CNV and SV,have a large effect on genetic structure,have a great relationship with the formation of drug resistance.Horizontal gene transfer is also involved in the process of the formation of drug resistance.There is multiple drug resistance mechanisms during the process of the formation of drug resistance.Different strains with different resistance mechanisms.The resistance mechanism is complicated.