Comparative Genomic Analysis of Virulent Vibrio (Listonella) anguillarum Serotypes Revealed Genetic Diversity and Genomic Signatures in the O-Antigen Biosynthesis Gene Cluster.

Vibrio anguillarum is the most frequent pathogen affecting fish worldwide. The only known virulent strains of V. anguillarum are serotypes O1, O2, and O3. Genetic differences between the serotypes that could shed insight on the evolution and serotype differences of this marine pathogen are unknown. Here, we fully sequenced and characterized a strain of V. anguillarum O1 (J382) isolated from winter steelhead trout (Oncorhynchus mykiss irideus) in British Columbia, Canada. Koch's postulates using the O1 strain were replicated in naïve lumpfish (Cyclopterus lumpus) and compared to O2. Phenotypic and genotypic comparisons were conducted for serotypes O1, O2, and O3, using biochemical tests and bioinformatic tools, respectively. The genome of V. anguillarum O1 (J382) contains two chromosomes (3.13 Mb and 1.03 Mb) and two typical pJM1-like plasmids (65,573 and 76,959 bp). Furthermore, V. anguillarum O1 (J382) displayed resistance to colistin sulphate, which differs from serotype O2 and could be attributed to the presence of the ugd gene. Comparative genomic analysis, among the serotypes, showed that intra-species evolution is driven by insertion sequences, bacteriophages, and a different repertoire of putative ncRNAs. Genetic heterogeneity in the O-antigen biosynthesis gene cluster is characterized by the absence or the presence of unique genes, which could result in differences in the immune evasion mechanisms employed by the respective serotypes. This study contributes to understanding the genetic differences among V. anguillarum serovars and their evolution.

Multi-Organ Transcriptome Response of Lumpfish (Cyclopterus lumpus) to Aeromonas salmonicida Subspecies salmonicida Systemic Infection.

Lumpfish is utilized as a cleaner fish to biocontrol sealice infestations in Atlantic salmon farms. Aeromonas salmonicida, a Gram-negative facultative intracellular pathogen, is the causative agent of furunculosis in several fish species, including lumpfish. In this study, lumpfish were intraperitoneally injected with different doses of A. salmonicida to calculate the LD50. Samples of blood, head-kidney, spleen, and liver were collected at different time points to determine the infection kinetics. We determined that A. salmonicida LD50 is 102 CFU per dose. We found that the lumpfish head-kidney is the primary target organ of A. salmonicida. Triplicate biological samples were collected from head-kidney, spleen, and liver pre-infection and at 3- and 10-days post-infection for RNA-sequencing. The reference genome-guided transcriptome assembly resulted in 6246 differentially expressed genes. The de novo assembly resulted in 403,204 transcripts, which added 1307 novel genes not identified by the reference genome-guided transcriptome. Differential gene expression and gene ontology enrichment analyses suggested that A. salmonicida induces lethal infection in lumpfish by uncontrolled and detrimental blood coagulation, complement activation, inflammation, DNA damage, suppression of the adaptive immune system, and prevention of cytoskeleton formation.

CD45 in ocular tissues during larval and juvenile stages and early stages of V. anguillarum infection in young lumpfish (Cyclopterus lumpus).

Immune responses to infectious diseases impacting lumpfish (Cyclopterus lumpus) eye tissue are only starting to be studied at a molecular and histopathological level. In this study, we extend our understanding of lumpfish sensory organ anatomy, of components of the lumpfish nasal and ocular immune system and the nature of the intraocular response to Vibrio anguillarum infection. We have evaluated the expression of cluster of differentiation (CD) 45 protein, a tyrosine phosphatase, in larval and juvenile lumpfish tissues in order to spatially survey ocular and related head structures that may participate in early stages of intraocular immune responses. We provide here a histological mapping of the larval lumpfish nasal chamber system since its connectively with the eye though mucosal epithelia have not been explored. These results build upon our growing understanding of the lumpfish intraocular immune response to pathogens, exemplified herein by experimental nasally delivered V. anguillarum infection. CD45 is developmentally regulated in lumpfish eyes and periocular anatomy with early expression appearing in larvae in corneal epithelium and in nasal structures adjacent to the eye. Normal juvenile and adult lumpfish eyes express CD45 in the corneal epithelium, in leukocyte cells within blood vessel lumens of the rete mirabile, choroid body and choriocapillaris vasculatures. Experimental nasally delivered V. anguillarum infection led to qualitative and quantitative changes in CD45 expression in head kidney renal tubule tissues by 7 days post infection (dpi). The same animals showed redistribution and upregulation of corneal epithelial CD45 expression, corneal epithelial dysplasia and an increased frequency of CD45+ cells in ocular vasculature. Interestingly, while CD45 upregulation and/or CD45+ cell infiltration into inner ocular and retinal tissues was not observed under this experimental scenario, subtle neural retinal changes were observed in infected fish. This work provides new fundamental knowledge on North Atlantic teleost visual systems and vision biology in general.

Characterization of Xanthomonas arboricola pv. juglandis Bacteriophages against Bacterial Walnut Blight and Field Evaluation.

Xanthomonas arboricola pv. juglandis (hereafter X. juglandis) is the etiological agent of walnut blight, the most important bacterial disease affecting walnut production worldwide. Currently, the disease is treated mainly with copper-derived compounds (e.g., CuSO4) despite the evidence of genetic resistance in these strains. Regarding the effectiveness and sustainability, the use of a bacteriophage appears to be a biocontrol alternative to reduce X. juglandis load and symptomatology of walnut blight. Here, the phages f20-Xaj, f29-Xaj, and f30-Xaj were characterized, and their effectiveness in walnut orchards against walnut blight was determined. These bacteriophages showed a specific lytic infection in X. juglandis strains isolated from Chile and France. Phylogenetic analysis of the complete genome of f20-Xaj and f30-Xaj indicates that these phages belong to the Pradovirus genus. In the field, the cocktail of these bacteriophages showed similar effectivity to CuSO4 in the reduction of incidence and severity in walnut tissue. Moreover, the bacterial load of X. juglandis was significantly reduced in the presence of bacteriophages in contrast to a CuSO4 treatment. These results show that the use of bacteriophages can be an alternative to combat the symptoms of walnut blight caused by X. juglandis.

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.

A Novel Marine Pathogen Isolated from Wild Cunners (Tautogolabrus adspersus): Comparative Genomics and Transcriptome Profiling of Pseudomonas sp. Strain J380.

Cunner (Tautogolabrus adspersus) is a cleaner fish being considered for utilized in the North Atlantic salmon (Salmo salar) aquaculture industry to biocontrol sea lice infestations. However, bacterial diseases due to natural infections in wild cunners have yet to be described. This study reports the isolation of Pseudomonas sp. J380 from infected wild cunners and its phenotypic, genomic, and transcriptomic characterization. This Gram-negative motile rod-shaped bacterium showed a mesophilic (4-28 °C) and halotolerant growth. Under iron-limited conditions, Pseudomonas sp. J380 produced pyoverdine-type fluorescent siderophore. Koch's postulates were verified in wild cunners by intraperitoneally (i.p.) injecting Pseudomonas sp. J380 at 4 × 103, 4 × 105, and 4 × 107 colony forming units (CFU)/dose. Host-range and comparative virulence were also investigated in lumpfish and Atlantic salmon i.p. injected with ~106 CFU/dose. Lumpfish were more susceptible compared to cunners, and Atlantic salmon was resistant to Pseudomonas sp. J380 infection. Cunner tissues were heavily colonized by Pseudomonas sp. J380 compared to lumpfish and Atlantic salmon suggesting that it might be an opportunistic pathogen in cunners. The genome of Pseudomonas sp. J380 was 6.26 megabases (Mb) with a guanine-cytosine (GC) content of 59.7%. Biochemical profiles, as well as comparative and phylogenomic analyses, suggested that Pseudomonas sp. J380 belongs to the P. fluorescens species complex. Transcriptome profiling under iron-limited vs. iron-enriched conditions identified 1159 differentially expressed genes (DEGs). Cellular metabolic processes, such as ribosomal and energy production, and protein synthesis, were impeded by iron limitation. In contrast, genes involved in environmental adaptation mechanisms including two-component systems, histidine catabolism, and redox balance were transcriptionally up-regulated. Furthermore, iron limitation triggered the differential expression of genes encoding proteins associated with iron homeostasis. As the first report on a bacterial infection in cunners, the current study provides an overview of a new marine pathogen, Pseudomonas sp. J380.

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes.

Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A. salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A. salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A. salmonicida have an intraerythrocytic phase. Here, we evaluate whether A. salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A. salmonicida did not kill primary S. salar erythrocytes, even in the presence of high bacterial loads, but A. salmonicida invaded the S. salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A. salmonicida showed bacteraemia 5 days post-infection and the presence of intraerythrocytic A. salmonicida. Our results reveal a novel intraerythrocytic phase during A. salmonicida infection.

Core non-coding RNAs of Piscirickettsia salmonis.

Piscirickettsia salmonis, a fastidious Gram-negative intracellular facultative bacterium, is the causative agent o Piscirickettsiosis. P. salmonis has broad host range with a nearly worldwide distribution, causing significant mortality. The molecular regulatory mechanisms of P. salmonis pathogenesis are relatively unknown, mainly due to its difficult in vitro culture and genomic differences between genogroups. Bacterial non-coding RNAs (ncRNAs) are important post-transcriptional regulators of bacterial physiology and virulence that are predominantly transcribed from intergenic regions (trans-acting) or antisense strand of open reading frames (cis-acting). The repertoire of ncRNAs present in the genome of P. salmonis and its possible role in bacterial physiology and pathogenesis are unknown. Here, we predicted and analyzed the core ncRNAs of P. salmonis base on structure and correlate this prediction to RNA sequencing data. We identified a total of 69 ncRNA classes related to tRNAs, rRNA, thermoregulators, antitoxins, ribozymes, riboswitches, miRNAs and antisense-RNAs. Among these ncRNAs, 29 classes of ncRNAs are shared between all P. salmonis genomes, constituting the core ncRNAs of P. salmonis. The ncRNA core of P. salmonis could serve to develop diagnostic tools and explore the role of ncRNA in fish pathogenesis.

Draft Genome Sequence of Xanthomonas arboricola pv. juglandis J303, Isolated from Infected Walnut Trees in Southern Chile.

Here, we report the draft genome sequence of Xanthomonas arboricola pv. juglandis J303, isolated from infected walnut trees in southern Chile. The size of the genome is 5,066,424 bp with a G+C content of 65.4%. X. arboricola pv. juglandis J303 has several genes related to virulence, antibiotic resistance, and copper resistance.

Draft Genome Sequence of Epilithonimonas sp. FP211-J200, Isolated from an Outbreak Episode on a Rainbow Trout (Oncorhynchus mykiss) Farm.

Here, we report the draft genome sequence of Epilithonimonas sp. FP211-J200, isolated from rainbow trout head kidney cells. The size of the genome is 4,110,772 bp, with a G+C content of 37.1%. The Epilithonimonas sp. FP211-J200 genome has genes related to tetracycline and ß-lactam resistance. This is the first reported Epilithonimonas species genome isolated from a fish host.

Colibacillosis in a New Zealand white rabbit (Oryctolagus cuniculus).

Colibacillosis is a disease caused by Escherichia coli in a variety of animals, including humans. Rabbit colibacillosis is infrequent or with an incipient description in Chile. Here, we describe an E. coli case in a white New Zealand rabbit at an animal facility in Santiago, Chile. Necropsy, histology, bacteriology, and 16S sequencing indicated an E. coli systemic infection. Phylogenetic analysis suggested that this E. coli J305 isolate is closely related to Shigella spp.

Complete genome sequence of the salmonella enterica serovar enteritidis bacteriophages fSE1C and fSE4C isolated from food matrices.

Salmonella enterica serovar Enteritidis is one of the most common causes of Salmonellosis worldwide. Utilization of bacteriophages as prophylactic agents is a practical solution to prevent Salmonellosis in ready-to-eat products. Shelf stability is one of the desirable properties for prophylactic bacteriophages. Here, we describe the phenotype, genome, and phylogeny of fSE1C and fSE4S Salmonella bacteriophages. fSE1C and fSE4S were previously isolated from pickle sauce and ground beef respectively and selected for their significant shelf stability. fSE1C and fSE4S showed a broad S. enterica serovar range, infecting several Salmonella serovars. The viral particles showed an icosahedral head structure and flexible tail, a typical morphology of the Siphoviridae family. fSE1C and fSE4C genomes consists of dsDNA of 41,720 bp and 41,768 bp with 49.73% and 49.78% G + C, respectively. Comparative genomic analysis reveals a mosaic relationship between S. enterica serovar Enteritidis phages isolated from Valparaiso, Chile.

A functional ferric uptake regulator (Fur) protein in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis, a Gram-negative fastidious facultative intracellular pathogen, is the causative agent of the salmonid rickettsial septicemia (SRS). The P. salmonis iron acquisition mechanisms and its molecular regulation are unknown. Iron is an essential element for bacterial pathogenesis. Typically, genes that encode for the iron acquisition machinery are regulated by the ferric uptake regulator (Fur) protein. P. salmonis fur sequence database reveals a diversity of fur genes without functional verification. Due to the fastidious nature of this bacterium, we evaluated the functionality of P. salmonis fur in the Salmonella Δfur heterologous system. Although P. salmonis fur gene strongly differed from the common Fur sequences, it restored the regulatory mechanisms of iron acquisition in Salmonella. We concluded that P. salmonis LF-89 has a conserved functional Fur protein, which reinforces the importance of iron during fish infection. [Int Microbiol 2016; 49-55].

Complete Genome Sequences of Lytic Bacteriophages of Xanthomonas arboricola pv. juglandis.

Three bacteriophages, f20-Xaj, f29-Xaj, and f30-Xaj, with lytic activity against Xanthomonas arboricola pv. juglandis were isolated from walnut trees (VIII Bío Bío Region, Chile). These lytic bacteriophages have double-stranded DNA (dsDNA) genomes of 43,851 bp, 41,865 bp, and 44,262 bp, respectively. These are the first described bacteriophages with lytic activity against X. arboricola pv. juglandis that can be utilized as biocontrol agents.

Influence of lipopolysaccharide outer-core in the intrinsic resistance to antimicrobial peptides and virulence in Edwardsiella ictaluri.

The genus Edwardsiella consists of bacteria with an intrinsic resistance to cyclic cationic antimicrobial peptides (CAMPs). Edwardsiella ictaluri, a pathogen of the catfish (Ictalurus punctatus) and the causative agent of a systemic infection, is highly resistant to CAMPs. Previously, we determined that the oligo-polysaccharide (O-PS) of the lipopolysaccharide (LPS) does not play a role in the E. ictaluri CAMP resistance and an intact core-lipid A structure is necessary for CAMPs resistance. Here, we evaluated the influence of the outer-core in the CAMPs resistance and fish virulence. E. ictaluri wabG, a gene that encodes for the UDP-glucuronic acid transferase that links the lipid A-inner-core to the outer-core-oligopolysaccharides, was deleted. Deletion of ΔwabG caused a pleiotropic effect, influencing LPS synthesis, CAMPs resistance, growth, and biofilm formation. E. ictaluri ΔwabG was attenuated in zebrafish indicating the important role of LPS during fish pathogenesis. Also, we evaluated the inflammatory effects of wabG LPS in catfish ligated loop model, showing a decreased inflammatory effect at the gut level respects to the E. ictaluri wild type. We conclude that E. ictaluri CAMPs resistance is related to the molecules present in the LPS outer-core and that fish gut inflammation triggered by E. ictaluri is LPS dependent, reinforcing the hypothesis that fish gut recognizes LPS in an O-PS dependent fashion.

A functional ferric uptake regulator (Fur) protein in the fish pathogen Piscirickettsia salmonis

Piscirickettsia salmonis, a Gram-negative fastidious facultative intracellular pathogen, is the causative agent of the salmonid rickettsial septicemia (SRS). The P. salmonis iron acquisition mechanisms and its molecular regulation are unknown. Iron is an essential element for bacterial pathogenesis. Typically, genes that encode for the iron acquisition machinery are regulated by the ferric uptake regulator (Fur) protein. P. salmonis fur sequence database reveals a diversity of fur genes without functional verification. Due to the fastidious nature of this bacterium, we evaluated the functionality of P. salmonis fur in the Salmonella Δfur heterologous system. Although P. salmonis fur gene strongly differed from the common Fur sequences, it restored the regulatory mechanisms of iron acquisition in Salmonella. We concluded that P. salmonis LF-89 has a conserved functional Fur protein, which reinforces the importance of iron during fish infection (AU)

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The expression of heterologous MAM-7 in Lactobacillus rhamnosus reduces its intrinsic capacity to inhibit colonization of pathogen Vibrio parahaemolyticus in vitro.

BACKGROUND: Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative, halophilic bacterium recognized as one of the most important foodborne pathogen. When ingested, V. parahaemolyticus causes a self-limiting illness (Vibriosis), characterized mainly by watery diarrhoea. Treatment is usually oral rehydration and/or antibiotics in complicated cases. Since 1996, the pathogenic and pandemic V. parahaemolyticus O3:K6 serotype has spread worldwide, increasing the reported number of vibriosis cases. Thus, the design of new strategies for pathogen control and illness prevention is necessary. Lactobacillus sp. grouped Gram positive innocuous bacteria, part of normal intestinal microbiota and usually used as oral vaccines for several diarrheic diseases. Recombinants strains of Lactobacillus (RL) expressing pathogen antigens can be used as part of an anti-adhesion strategy where RL block the pathogen union sites in host cells. Thus, we aimed to express MAM-7 V. parahaemolyticus adhesion protein in Lactobacillus sp. to generate an RL that prevents pathogen colonization. RESULTS: We cloned the MAM-7 gene from V. parahaemolyticus RIMD 2210633 in Lactobacillus expression vectors. Recombinant strains (Lactobacillus rhamnosus pSEC-MAM7 and L. rhamnosus pCWA-MAM7) adhered to CaCo-2 cells and competed with the pathogen. However, the L. rhamnosus wild type strain showed the best capacity to inhibit pathogen colonization in vitro. In addition, LDH-assay showed that recombinant strains were cytotoxic compared with the wild type isogenic strain. CONCLUSIONS: MAM-7 expression in lactobacilli reduces the intrinsic inhibitory capacity of L. rhamnosus against V. parahaemolyticus.

The expression of heterologous MAM-7 in Lactobacillus rhamnosus reduces its intrinsic capacity to inhibit colonization of pathogen Vibrio parahaemolyticus in vitro

BACKGROUND: Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative, halophilic bacterium recognized as one of the most important foodborne pathogen. When ingested, V. parahaemolyticus causes a self-limiting illness (Vibriosis), characterized mainly by watery diarrhoea. Treatment is usually oral rehydration and/or antibiotics in complicated cases. Since 1996, the pathogenic and pandemic V. parahaemolyticus O3:K6 serotype has spread worldwide, increasing the reported number of vibriosis cases. Thus, the design of new strategies for pathogen control and illness prevention is necessary. Lactobacillus sp. grouped Gram positive innocuous bacteria, part of normal intestinal microbiota and usually used as oral vaccines for several diarrheic diseases. Recombinants strains of Lactobacillus (RL) expressing pathogen antigens can be used as part of an anti-adhesion strategy where RL block the pathogen union sites in host cells. Thus, we aimed to express MAM-7 V. parahaemolyticus adhesion protein in Lactobacillus sp. to generate an RL that prevents pathogen colonization RESULTS: We cloned the MAM-7 gene from V. parahaemolyticus RIMD 2210633 in Lactobacillus expression vectors. Recombinant strains (Lactobacillus rhamnosus pSEC-MAM7 and L. rhamnosus pCWA-MAM7) adhered to CaCo-2 cells and competed with the pathogen. However, the L. rhamnosus wild type strain showed the best capacity to inhibit pathogen colonization in vitro. In addition, LDH-assay showed that recombinant strains were cytotoxic compared with the wild type isogenic strain CONCLUSIONS: MAM-7 expression in lactobacilli reduces the intrinsic inhibitory capacity of L. rhamnosus against V. parahaemolyticus.

Complete Genome Sequence of Salmonella enterica Serovar Enteritidis Bacteriophage f18SE, Isolated in Chile.

Bacteriophage f18SE was isolated from poultry sewage in Olmue, Chile, and lytic activity was demonstrated against Salmonella enterica serovar Enteritidis and serovar Pullorum strains. This bacteriophage has a 41,868-bp double-stranded DNA (ds-DNA) genome encoding 53 coding sequences (CDSs) and belongs to the family Siphoviridae, subfamily Jerseyvirinae.

Genome Sequence of Vibrio VPAP30, Isolated from an Episode of Massive Mortality of Reared Larvae of the Scallop Argopecten purpuratus.

We report here the 5.167-Mbp draft genome sequence of Vibrio VPAP30, isolated from an Argopecten purpuratus larval culture. Vibrio VPAP30 is the etiological agent of a vibriosis outbreak causing a complete collapse of a larval culture of the scallop A. purpuratus, which occurred in a commercial hatchery in Chile.