Etiology And Molecular Epidemiological Characteristics Of Pathogenic Aeromonas

Aeromonas belongs to Gram-negative bacteria,which consists of two zoonotic pathogens Aeromonas hydrophila(Ah)and Aeromonas salmonicida.In this study,A.hydrophila strain A1310 isolated from the kidney tissue of the common grass carp with typical symptoms.Meanwhile,two A.salmonicida strains AS-17 and AS-18 were isolated from the lesions of the diseased guppies.Whole genome sequencing was then conducted and confirmed the bacterial identification of three strains.Three strains were sequenced using MiSeq 150-bp paired-end sequencing in a multiplexed format.The average coverage depth for all genomes was 67,with the lowest coverage depth of45.The reads were assembled de novo.The genome size ranged from 4.9 Mb to 5.4 Mb.In order to further understand the molecular epidemiological characteristics and virulence gene profiles of these strains,62 strains of A.hydrophila were selected from the Genbank.Multi-locus sequence typing(MLST)was used to delineate the evolutionary relationships among the A.hydrophila strains.MLST analysis divided A.hydrophila into four clusters.The results showed that strain A1310 was clustered with four strains(S15-242,S15-458,S15-591,S15-700)obtained from American channel catfish with the closest relationship and located on cluster II.Two sequenced strains AS-17 and AS-18 were combined with 35 A.salmonicida strains selected from NCBI for MLST analysis.The results showed that 37 A.salmonicida strains can be divided into six clusters;strains AS-17 and AS-18 isolated from the fish farms in Anshan were clustered at an independent branch and had highly genetic homology.Genomic features including clusters of regularly spaced short palindromic repeats(CRISPR),prophage,Insertion Sequences,and virulence factors were analyzed A.hydrophila and A.salmonicida,respectively.CRISPR-finder was used to predict the CRISPR sequence of three sequenced strains.Results showed that A.hydrophila strain A1310,two A.salmonicida strains AS-17 and AS-18 all harbored zero theoretically reliable CRISPR.A total of six Insertion Sequences(IS)were predicted for A1310 by IS-Finder,including ISAs13,ISAve2,ISAs4,ISAeme7,ISKpn31,and ISAs19.In contrast,prophage analysis by program Phaster revealed that there were two prophage regions in the genome of A1310,of which phage 1 was incomplete and phage 2 was intact.AS-17 predicted a total of 12 suspected CRISPR sequences,and AS-18 predicted a total of9 suspected CRISPR sequences,of which the theoretically credible CRISPR was 0.AS-17 and AS-18 predicted the same amount of insertion sequence with 4 inserts of Score(>500).There are three phage regions in the genome of AS-17 and AS-18,all of which are intact.Analysis of the correlations of MLST typing with the profiles of CRISPR,prophage,andinsertion sequences of the A hydrophila and A.salmonicida strains reveled the consistency of phylogenetic evolution of house-keeping genes with the genetic elements of CRISPR,prophage,and insertion sequences.Therefore,it is reasonable to speculate that CRISPR,prophage,and insertion sequences were co-evolved with the evolution for the most strains which lead to the evolutionary consistency.The results of co-analysis with other Aeromonas sp.showed that the CRISPRs of A.salmonicida was seriously degraded,and Chinese isolates carried more insertion sequences,which may be related to the abuse of domestic antibiotics.Of the 37 strains of A.salmonicida,most strains contain Type 1 phage.A total of 154 virulence factors were predicted using the VFDB online website.A total of154 and 122 virulence factors were predicted for A1310 and AS-17(AS-18).The virulence factor identified in A1310 includes flp type IV pili,tap type IV pili,polar flagella,Type II secretion system(T2SS),Type VI secretion system(T6SS),aerolysin,extracellular hemolysin,heat-stable cytotonic enterotoxin,hemolysin,thermostable hemolysin,capsule.Moreover,AS-17 and AS-18 consist of type IV pili,polar flagella,T2 SS,aerolysin,extracellular hemolysin,thermostable hemolysin,capsular polysaccharide,capsule.This study compared the genomic evolution and genomic diversity of A hydrophila and A.salmonicida.These findings have extended our understanding of the genomic diversity and genome evolution of A hydrophila and A.salmonicida,particularly its relationship with CRISPR evolution and prophage variation,which would facilitate the disease control and prevention of Aeromonas-associated infection.