Study On Antimicrobial Resistance,Resistance Genes And Molecular Typing Of16Yersinia Ruckeri Strains

Yersinia ruckeri is a worldwide important pathogenic bacterium that can infect a variety of cold-water fishes,and cause severe economic losses to the world aquaculture industry.This study has been conducted to investigate the resistant phenotype,drug resistance genes prevalence and genotype difference and relationship of Y.ruckeri in some areas of Sichuan province.Primarily,rupA gene of Y.ruckeri was targeted through PCR for its identification.After that morphological,physiological,biochemical testing,Multilocus sequence typing(MLST)and Pulsed field gel electrophoresis(PFGE)were used to confirmed genotypes and molecular characteristic diversity of 16 Y.ruckeri strains isolated from sturgeon,Ietalurus Punetaus,and Pelteobagrus fulvidraco between 2015 and 2018 in Sichuanprovince.The results revealed that 16 strains of Y.ruckeri were identified as biotype 1.This study used K-B method to check the sensitivity of 20 clinical commonly used antibiotics sensitivity of 16 Y.ruckeri strains according to CLSI guidelines,and use the SmartChip~TMM Real-Time PCR System to detect 6 classes 96 ARGs(Beta-lactamase,Aminoglycoside,Tetracycline,Chloramphenicol,Quinolone and sulfonamides).The results revealed that 16 strains were completely resistant to ampicillin.The drug resistance rates of kanamycin,sulfamethoxazole,amoxicillin and tetracycline were between 37.5%and 75%and drug resistance rates of neomycin,norfloxacin and gentamicin were between 12.5%and 25%.These all isolates showed multiple drug resistance.A total of 39 ARGs were detected in the 16 Y.ruckeri strains,in which aac(6')-Ib(aka aacA4)-02?mphA-01?blaCMY2-01?tetC-02?blaTEM?sul1 were more prevalent,and the detection rates were 100%,93.75%,93.75%,87.5%,81.25%and 50%,respectively.The detection results of ARGs of isolated strains with different host sources and geographical distribution were different,and there was a certain correlation between the detection results of ARGs and resistance phenotypes,but no absolute consistency.MLST results showed a total of 5 STs(ST-5,ST-7,ST-14,ST-18,ST-28)and 1 clonal group(CC-1),in which ST-14 was the main creator of CC-1,and there was one or more site difference between ST-14 and other sequences.The distribution of each STs was concentrated in 16 Y.ruckeri strains with low degree of genetic variation and the high similarity.PFGE results showed that UPGMA cluster analysis was divided into 6(cluster A-F)and 5(cluster A-E)banding patterns by using two different restriction enzymes(Sep I and Xba I)with more than 85%of the similarity.The similarity between the lineages was higher,showing a close affinity and a relatively concentrated distribution with the host as the unit.