Complete genome sequence of the marine fish pathogen Vibrio anguillarum and genome-wide transposon mutagenesis analysis of genes essential for in vivo infection.
Vibrio anguillarum is a notorious bacterial pathogen that causes vibriosis in various marine farmed fish species. The highly pathogenic V. anguillarum strain MVM425 has been isolated from moribund turbot in the China Yellow Sea, but the genetic basis for its pathogenesis is undefined. Complete genome sequencing uncovered that the bacterium encodes 3985 protein-coding sequences (CDSs) on two chromosomes and 62 CDSs on the virulence plasmid pEIB1. To investigate the genes that are essential for its in vitro and in vivo growth, we applied transposon insertion sequencing technology (Tn-seq) to screen a highly saturated transposon insertion mutant library grown in Luria-Bertani broth with 2% NaCl (LB20) and in its natural host turbot. Tn-seq identified 473 and ∼173 putative genes as essential for its growth in rich medium and the in vivo infection process, respectively. Additionally, our analysis revealed that the genes are associated to various fitness levels in the liver, spleen and kidney, suggesting different genetic requirements for colonization in these organs. Furthermore, we validated our Tn-seq data using gene knockout mutants and in vivo infection experiments. Comprehensive functional genomics analysis highlighted the conditionally essential genes for important pathways involved in energy metabolism, nitrogen metabolism, nucleotide synthesis, amino acid synthesis, cofactors, siderophore synthesis, secretion (T1SS, T2SS, and T6SS), quorum sensing, and flagellum biosynthesis. This study provides a wealth of information about V. anguillarum genes related to infection in fish and will facilitate the understanding of its pathogenesis as well as the development of diagnostics and vaccines against the pathogen.
Guanhua Y
，Wang C
，Wang X
，Ma R
，Zheng H
，Liu Q
，Zhang Y
，Ma Y
，Wang Q
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Identification, characterization and complete genome analysis of a Vibrio anguillarum isolated from Sebastes schlegelii.
Vibrio anguillarum is an important fish pathogen in mariculture, which can infect fish with great economic losses. In this study, a Vibrio anguillarum isolated from Sebastes schlegelii was named VA1 and was identified and characterized from aspects of morphology, physiological and biochemical characteristics, 16SRNA, virulence genes, drug sensitivity, and extracellular enzyme activity. At the same time, The VA1 was investigated at the genomic level. The results showed that a Gram-negative was isolated from the diseased fish. The VA1 was characterized with uneven surface and visible flagella wrapped in a sheath and microbubble structures. The VA1 was identified as Vibrio anguillarum based on the 16S RNA sequence and physiological and biochemical characteristics. The VA1 carried most of the virulence genes (24/29) and was resistant to penicillin, oxacillin, ampicillin, cefradine, neomycin, pipemidic acid, ofloxacin, and norfloxacin. The pathogenicity of the isolated strain was confirmed by an experimental analysis, and its LD50 was 6.43 × 106 CFU/ml. The VA1 had the ability to secrete gelatinase, protease, and amylase, and it had α-hemolysis. The whole genome size of the VA1 was 4232328bp and the G + C content was 44.95 %, consisting of two circular chromosomes, Chromosome1 and Chromosome2, with no plasmid. There were 1006 predicted protein coding sequences (CDSs). A total of 526 genes were predicted as virulence-related genes which could be classified as type IV pili, flagella, hemolysin, siderophore, and type VI secretion system. Virulence genes and correlation data were supported with the histopathological examination of the affected organs and tissues. 194 genes were predicted as antibiotic resistance genes, including fluoroquinolone antibiotic, aminoglycoside antibiotic, and beta-lactam resistant genes, which agreed with the results of the above drug sensitivity, indicating VA1 to be a multidrug-resistant bacterium. This study provided a theoretical basis for a better understanding of pathogenicity and antibiotic resistance, which might contribute to the prevention of V. anguillarum in the future.
Li Z
，Sun Y
，Tan R
，Gao Y
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Unveiling the pathogenic and multidrug-resistant profiles of Vibrio alfacsensis: A potential identified threat in turbot (Scophthalmus maximus) aquaculture.
Vibrio alfacsensis is traditionally seen as an environmental symbiont within its genus, with no detailedly documented pathogenicity in marine aquaculture to date. This study delves into the largely unexplored pathogenic potential and emerging antibiotic resistance of V. alfacsensis. The VA-1 strain, isolated from recirculating aquaculture system (RAS) effluent of cultured turbot (Scophthalmus maximus), underwent comprehensive analysis including biochemical identification, antibiotic susceptibility testing and reinfection trials. The results confirmed VA-1's pathogenicity and significant multiple antibiotic resistance. VA-1 could induce systemic infection in turbot, with symptoms like kidney enlargement, exhibiting virulence comparable to known Vibrio pathogens, with an LD50 around 2.36 × 106 CFU/fish. VA-1's remarkable resistance phenotype (14/22) suggested potential for genetic exchange and resistance factor acquisition in aquaculture environments. Phylogenetic analysis based on 16S rDNA sequences and whole-genome sequencing has firmly placed VA-1 within the V. alfacsensis clade, while genome-wide analysis highlights its similarity and diversity in relation to strains from across the globe. VA-1 contained numerous replicons, indicating the possibility for the spread of resistance and virulence genes. This study suggests V. alfacsensis may acquire and transfer pathogenic and resistant traits through horizontal gene transfer, a likelihood intensified by changing environmental and aquaculture conditions, highlighting the need for vigilant pathogen monitoring and new non-antibiotic treatments.
Hu RG
，Yang L
，Wang LY
，Yang YL
，Li HJ
，Yang BT
，Kang YH
，Liang ZL
，Cong W
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First report of isolation and complete genome of Vibrio rotiferianus strain SSVR1601 from cage-cultured black rockfish (Sebastes schlegelii) associated with skin ulcer.
Vibrio rotiferianus is an important marine pathogen of various aquatic organisms and can be found widely distributed in the marine environment. To further characterize this pathogen, the pathogenic properties and genome of V. rotiferianus SSVR1601 isolated from Sebastes schlegelii with skin ulcer were analysed. SSVR1601 was shown to be short rod-shaped cell with a single polar flagellum. Different degrees of pathological changes in fish kidney, intestine, gills and liver were observed after SSVR1601 challenge. The SSVR1601 genome consists of two chromosomes and two plasmids with a total of 5,717,113 bp, 42.04%-44.93% GC content, 5,269 predicted CDSs, 134 tRNAs and 40 rRNAs. The common virulence factors including OMPs, haemolysin, flagellin, DNase, entF, algU, tcpI, acfB and rfaD were found in strain SSVR1601. Furthermore, factors responsible for iron uptake (fur, fepC and ccmC) and types II, IV and VI secretion systems were detected, which are likely responsible for the pathogenicity of SSVR1601. The antimicrobial resistance genes, bacA, tet34 and norM, were detected based on Antibiotic Resistance Genes Database. The phylogenetic analysis revealed SSVR1601 to be most closely related to V. rotiferianus strains CAIM577 and B64D1.
Zhang Z
，Yu YX
，Jiang Y
，Wang YG
，Liao MJ
，Rong XJ
，Wang K
，Zhang H
，Chen J
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ResearchGate
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