Comparative Genomics of Typical and Atypical Aeromonas salmonicida Complete Genomes Revealed New Insights into Pathogenesis Evolution

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes, masoucida, smithia, and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55 ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies’ taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.

The Early Immune Response of Lymphoid and Myeloid Head-Kidney Cells of Rainbow Trout (Oncorhynchus mykiss) Stimulated with Aeromonas salmonicida

The teleost head kidney is a highly relevant immune organ, and myeloid cells play a major role in this organ’s innate and adaptive immune responses. Because of their complexity, the early phases of the innate immune reaction of fish against bacteria are still poorly understood. In this study, naïve rainbow trout were stimulated with inactivated A. salmonicida and sampled at 12 h, 24 h and 7 d poststimulation. Cells from the head kidney were magnetically sorted with a monoclonal antibody mAB21 to obtain one (MAb21-positive) fraction enriched with myeloid cells and one (MAb21-negative) fraction enriched with lymphocytes and thrombocytes. The gene expression pattern of the resulting cell subpopulations was analysed using a panel of 43 immune-related genes. The results show an overall downregulation of the complement pathway and cytokine production at the considered time points. Some of the selected genes may be considered as parameters for diagnosing bacterial furunculosis of rainbow trout.

CD38 Defines a Subset of B Cells in Rainbow Trout Kidney With High IgM Secreting Capacities

CD38 is a multifunctional molecule that functions both as a transmembrane signaling receptor and as an ectoenzyme with important roles in cell adhesion, calcium regulation and signal transduction. Within the B cell linage, CD38 is expressed in diverse murine B cell subsets, with highest levels in innate B cell subpopulations such as marginal zone (MZ) B cells or B1 cells. In humans, however, CD38 is transiently expressed on early lymphocyte precursors, is lost on mature B cells and is consistently expressed on terminally differentiated plasma cells. In the present work, we have identified two homologues of mammalian CD38 in rainbow trout (Oncorhynchus mykiss), designating them as CD38A and CD38B. Although constitutively transcribed throughout different tissues in homeostasis, both CD38A and CD38B mRNA levels were significantly up-regulated in head kidney (HK) in response to a viral infection. In this organ, after the generation of a specific monoclonal antibody (mAb) against CD38A, the presence of CD38A+ populations among IgM+ B cells and IgM- leukocytes was investigated by flow cytometry. Interestingly, the percentage of IgM+CD38A+ B cells increased in response to an in vitro stimulation with inactivated Aeromonas salmonicida. Finally, we demonstrated that HK IgM+CD38A+ B cells had an increased IgM secreting capacity than that of cells lacking CD38A on the cell surface, also showing increased transcription levels of genes associated with B cell differentiation. This study strongly suggests a role for CD38 on the B cell differentiation process in teleosts, and provides us with novel tools to discern between B cell subsets in these species.

A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

Evaluation of the Adhesive Potential of Bacteria Isolated from Meat-Related Sources

Microbial adhesion constitutes the transition of microorganisms from a planktonic mode to a static one. It promotes the formation of biofilm which is responsible for spoilage, foodborne diseases, and corrosion in the food processing industry. In this study, the adhesive potential of fourteen meat-borne bacterial isolates belonging to seven different genera was investigated. All strains were found able to colonize polystyrene surfaces with different levels of firmness. Significant variations were determined in assays of bacterial hydrophobicity and motility. Among the 14 strains, Pseudomonas fragi, Aeromonas salmonicida II, Serratia liquefaciens, Citrobacter braakii, Pseudomonas putida, and Aeromonas veronii had a strong hydrophobic force, while the isolates of Lactobacillus genus showed the most hydrophilic property. In terms of motility, Citrobacter braakii and Escherichia coli exhibited exceptional swarming and swimming abilities, whilst conservatively weak performances were observed in the Lactobacillus strains. Furthermore, the majority of the isolates were predominantly electron donors and weak electron acceptors. Overall, a high level of correlation was observed between biofilm-forming ability with cell surface hydrophobicity and Lewis acid–base properties, whereas the contribution of motility in bacterial adhesion could not be confirmed. Research on the adhesive performance of foodborne bacteria is potentially conducive to developing novel control strategies, such as food processing equipment with specific surfaces, not facilitating attachment.

Genomic Perspectives on Aeromonas salmonicida subsp. salmonicida Strain 890054 as a Model System for Pathogenicity Studies and Mitigation of Fish Infections

Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that causes furunculosis, a fish disease claiming substantial economic losses in the aquaculture industry. Major challenges exist in monitoring and controlling fish infections in aquaculture farms. Development of management practices to improve the sustainability of fish farming with disease prevention necessitates studies using well-defined systems and well-characterized bacterial isolates. Even though several A. salmonicida subsp. salmonicida genomes have been completely assembled and thoroughly annotated, in vivo pathogenicity data are lacking. Here we present A. salmonicida subsp. salmonicida 890054 as a prototype strain for standardized furunculosis challenges with survival data. Computational analysis of sequencing results provided a complete circular genome with annotations of plasmids carrying virulence factors, antimicrobial resistance, and secondary metabolite coding genes. The analysis also revealed the presence of an IncU plasmid distinct from other IncU plasmids previously associated with Aeromonas.