| The aquaculture industry has suffered huge losses due to frequent bacterial diseases in recent years.Antibiotics are often the first choice when dealing with bacterial diseases,and their misuse can lead to widespread bacterial resistance.To investigate the possible effects of irrational drug use,bacteria isolated from eastern coastal areas in China were identified and tested for drug resistance.Meanwhile,based on Aeromonas hydrophila(A.hydrophila)’s extensive geographical distribution and zoonosis,this study further analyzed the drug resistance and evolution of Aeromonas hydrophila by whole genome sequencing.The specific research contents are as follows:The results showed that Aeromonas spp.,Vibrio spp.and Pseudomonas spp accounted for most of all strains,while the number of other strains was relatively small.The drug sensitivity of 83 strains was identified,and the selected tested drugs were enrofloxacin,neomycin sulfate,florfenicol,doxycycline and sulfamonomethoxine.The identification results showed that: All strains were resistant to at least one antibiotic commonly used in aquaculture,and most strains had multiple resistance,and among these strains,the proportion of strains resistant to sulfonamides,aminoglycosides and flubenicol was high,accounting for 65%,61%,and 63%,respectively.Enrofloxacin,a quinolone drug,had a better inhibitory effect,and the proportion of drug resistance to enrofloxacin was relatively low,only 17%.Unlike the proportion of strains resistant to enrofloxacin in all strains,the proportion of 20 A.hydrophila strains resistant to enrofloxacin was high,reaching 30%,but the proportion of A.hydrophila resistant to florfenicol was lower than that of the overall strains.2.The whole genome sequencing method was used to construct the genomic framework of the three isolated strains of A.hydrophila.The results showed that the genomic sizes of A.hydrophila 104,117 and A058 were 5Mb,4.7MB,and 4.8MB,respectively,and the GC% ranged from 61.41 to 61.49,which was very similar.Of the three strains,strain 104 was predicted to carry the most genes(4,836),while strain117 was relatively few(4,355).The results of COG functional classification showed that COG C,COG E,and COG K had more classifications.KEGG metabolic pathway analysis divided the genes of the three strains into 6 categories and 33 secondary classifications.KEGG results showed that the metabolic function of A.hydrophila was rich in diversity.The genomes of 118 strains of A.hydrophila had published in the NCBI database were re-annotated,and combine the genomes of the three strains of A.hydrophila self-tested to compare and analyze,and the results showed that the genomes of A.hydrophila were highly variable,and even if the strains were closely related to each other,the genomes could still be very different.3.In this study,the phylogenetic and evolutionary analysis of A.hydrophila was carried out using tandem proteins,and all genomes were annotated with drug resistance genes and virulence factors using CARD resistance gene database and VFDB virulence factor database,and analyzed by Bioinformatics method.The results showed that A.hydrophila was not conservative in infesting hosts and geographical distribution,and the strains from other isolated sources were more dispersed compared to the aquatic origin isolates,and strains from different sources showed dispersed distribution throughout the phylogenetic tree.The A.hydrophila genome shared seven resistance genes(including resistance genes against carbapenem,cephalosporin,diaminopyrimidine,fluoroquinolone,penam,phenicol,and tetracycline)and ten virulence factors(including LOS,Rpo S,Csr A,AI-2,Polar flagella,Tap type IV pili,MSHA type IV pili,Exe T2 SS,Type I pili,Flagella);the resistance genes and some virulence genes are shared among A.hydrophila from different sources,and the reason for this phenomenon,it is likely that the frequent genes exchange between strains due to the unconservative host and geographical distribution of A.hydrophila have facilitated the rapid spread of drug resistance genes and virulence factor-related genes throughout the species,so that despite the large genomic variation among strains,there is no significant difference in the distribution of drug resistance genes and some virulence factors throughout the species. |
