Genomic diversity in flavobacterial pathogens of aquatic origin.

Flavobacterium species are considered important fish pathogens in wild and cultured fish throughout the world. They can cause acute, subacute, and chronic infections, which are mainly characterized by gill damage, skin lesions, and deep necrotic ulcerations. Primarily, three Flavobacterium species, F. branchiophilum, F. columnare, and F. psychrophilum, have been reported to cause substantial losses to freshwater fish. In this study, we evaluated genomes of 86 Flavobacterium species isolated from aquatic hosts (mainly fish) to identify their unique and shared genome features. Our results showed that F. columnare genomes cluster into four different genetic groups. In silico secretion system analysis identified that all genomes carry type I (T1SS) and type IX (T9SS) secretion systems, but the number of type I secretion system genes shows diversity between species. F. branchiophilum, F. araucananum, F. chilense, F. spartansii, and F. tructae genomes have full type VI secretion system (T6SS). F. columnare, F. hydatis, and F. plurextorum carry partial T6SS with some of the T6SS genes missing. F. columnare, F. araucananum, F. chilense, F. spartansii, F. araucananum, F. tructae, Flavobacterium sp., F. crassostreae, F. succinicans, F. hydatis, and F. plurextorum carry most of the type IV secretion system genes (T4SS). F. columnare genetic groups 1 and 2, Flavobacterium sp., and F. crassostreae encode the least number of antibiotic resistance elements. F. hydatis, F. chilense, and F. plurextorum encode the greatest number of antibiotic resistance genes. Additionally, F. spartansii, F. araucananum, and chilense encode the greatest number of virulence genes while Flavobacterium sp. and F. crassostreae encode the least number of virulence genes. In conclusion, comparative genomics of Flavobacterium species of aquatic origin will help our understanding of Flavobacterium pathogenesis.

Tad pili contribute to the virulence and biofilm formation of virulent Aeromonas hydrophila.

Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila, a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170 A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections.

Comparative genomics of Aeromonas veronii: Identification of a pathotype impacting aquaculture globally.

Aeromonas veronii is a gram-negative species abundant in aquatic environments that causes disease in humans as well as terrestrial and aquatic animals. In the current study, 41 publicly available A. veronii genomes were compared to investigate distribution of putative virulence genes, global dissemination of pathotypes, and potential mechanisms of virulence. The complete genome of A. veronii strain ML09-123 from an outbreak of motile aeromonas septicemia in farm-raised catfish in the southeastern United States was included. Dissemination of A. veronii strain types was discovered in dispersed geographical locations. Isolate ML09-123 is highly similar to Chinese isolate TH0426, suggesting the two strains have a common origin and may represent a pathotype impacting aquaculture in both countries. Virulence of strain ML09-123 in catfish in a dose-dependent manner was confirmed experimentally. Subsystem category disposition showed the majority of genomes exhibit similar distribution of genomic elements. The type I secretion system (T1SS), type II secretion system (T2SS), type 4 pilus (T4P), and flagellum core elements are conserved in all A. veronii genomes, whereas the type III secretion system (T3SS), type V secretion system (T5SS), type VI secretion system (T6SS), and tight adherence (TAD) system demonstrate variable dispersal. Distribution of mobile elements is dependent on host and geographic origin, suggesting this species has undergone considerable genetic exchange. The data presented here lends insight into the genomic variation of A. veronii and identifies a pathotype impacting aquaculture globally.

Comparative Genomics of Aeromonas hydrophila Secretion Systems and Mutational Analysis of hcp1 and vgrG1 Genes From T6SS.

Virulent Aeromonas hydrophila causes severe motile Aeromonas septicemia in warmwater fishes. In recent years, channel catfish farming in the U.S.A. and carp farming in China have been affected by virulent A. hydrophila, and genome comparisons revealed that these virulent A. hydrophila strains belong to the same clonal group. Bacterial secretion systems are often important virulence factors; in the current study, we investigated whether secretion systems contribute to the virulent phenotype of these strains. Thus, we conducted comparative secretion system analysis using 55 A. hydrophila genomes, including virulent A. hydrophila strains from U.S.A. and China. Interestingly, tight adherence (TaD) system is consistently encoded in all the vAh strains. The majority of U.S.A. isolates do not possess a complete type VI secretion system, but three core elements [tssD (hcp), tssH, and tssI (vgrG)] are encoded. On the other hand, Chinese isolates have a complete type VI secretion system operon. None of the virulent A. hydrophila isolates have a type III secretion system. Deletion of two genes encoding type VI secretion system proteins (hcp1 and vgrG1) from virulent A. hydrophila isolate ML09-119 reduced virulence 2.24-fold in catfish fingerlings compared to the parent strain ML09-119. By determining the distribution of genes encoding secretion systems in A. hydrophila strains, our study clarifies which systems may contribute to core A. hydrophila functions and which may contribute to more specialized adaptations such as virulence. Our study also clarifies the role of type VI secretion system in A. hydrophila virulence.

Draft Genome Sequence of Fish Pathogen Aeromonas bestiarum GA97-22.

Aeromonas bestiarum is a Gram-negative mesophilic motile bacterium causing acute hemorrhagic septicemia or chronic skin ulcers in fish. Here, we report the draft genome sequence of A. bestiarum strain GA97-22, which was isolated from rainbow trout in 1997. This genome sequence will improve our understanding of the complex taxonomy of motile aeromonads.

Comparative Analysis of the Flavobacterium columnare Genomovar I and II Genomes.

Columnaris disease caused by Gram-negative rod Flavobacterium columnare is one of the most common diseases of catfish. F. columnare is also a common problem in other cultured fish species worldwide. F. columnare has three major genomovars; we have sequenced a representative strain from genomovar I (ATCC 49512, which is avirulent in catfish) and genomovar II (94-081, which is highly pathogenic in catfish). Here, we present a comparative analysis of the two genomes. Interestingly, F. columnare ATCC 49512 and 94-081 meet criteria to be considered different species based on the Average Nucleotide Identity (90.71% similar) and DNA-DNA Hybridization (42.6% similar). Genome alignment indicated the two genomes have a large number of rearrangements. However, function-based comparative genomics analysis indicated that the two strains have similar functional capabilities with 2,263 conserved orthologous clusters; strain ATCC 49512 has 290 unique orthologous clusters while strain 94-081 has 391. Both strains carry type I secretion system, type VI secretion system, and type IX secretion system. The two genomes also have similar CRISPR capacities. The F. columnare strain ATCC 49512 genome contains a higher number of insertion sequence families and phage regions, while the F. columnare strain 94-081 genome has more genomic islands and more regulatory gene capacity. Transposon mutagenesis using Tn4351 in pathogenic strain 94-081 yielded six mutants, and experimental infections of fish showed hemolysin and glycine cleavage protein mutants had 15 and 10% mortalities, respectively, while the wild-type strain caused 100% mortalities. Our comparative and mutational analysis yielded important information on classification of genomovars I and II F. columnare as well as potential virulence genes in F. columnare strain 94-081.

Draft Genome Sequences of Three Aeromonas hydrophila Isolates from Catfish and Tilapia.

Aeromonas hydrophila is a Gram-negative bacterium that is particularly adapted to freshwater environments and can cause severe infections in fish and humans. Here, we report the draft genomes of three A. hydrophila catfish and tilapia isolates.

Genome Sequence of the Fish Pathogen Flavobacterium columnare Genomovar II Strain 94-081.

Flavobacterium columnare causes columnaris disease in fresh and brackish water worldwide. F. columnare strain 94-081 was isolated from a diseased channel catfish in 1994; its genome sequence is the first completed genomovar II sequence.

Draft Genome Sequences of Four Virulent Aeromonas hydrophila Strains from Catfish Aquaculture.

Since 2009, a clonal group of virulent Aeromonas hydrophila strains has been causing severe disease in the catfish aquaculture industry in the southeastern United States. Here, we report draft genomes of four A. hydrophila isolates from catfish aquaculture that represent this clonal group.

Draft Genome Sequence of Aeromonas hydrophila TN97-08.

Aeromonas hydrophila is an opportunistic pathogen residing in freshwater environments that causes infection in fish and mammals. Here, we report the draft genome sequence of A. hydrophila strain TN97-08 isolated from a diseased bluegill (Lepomis macrochirus) in 1997.