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In this study, we identified and analyzed a novel genomic island (GI), named pddGI-1, located on chromosome II of certain strains of the marine pathogen Photobacterium damselae subsp. damselae (Pdd). This GI shares structural similarities with other GIs found in Vibrio species, such as the Vibrio seventh pandemic island-II (VSP-II) of V. cholerae. The pddGI-1 island is a mosaic of gene blocks that encode functions related to ROS defense, anaerobic energy metabolism, and restriction-modification (RM) systems. Notably, pddGI-1 also includes a complete vibrioferrin siderophore system, enabling the bacteria to thrive in low-iron environments. Vibrioferrin was chemically identified from cell-free supernatants of Pdd RG91. Additionally, a pvsD mutant deficient in vibrioferrin biosynthesis was generated and analyzed. The results suggest that Pdd strains harbouring pddGI-1 gain a distinct growth advantage under iron-limited conditions. These findings, along with previous research, highlight the significant heterogeneity in iron assimilation systems among Pdd strains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Facultative marine bacterial pathogens sense environmental signals so that the expression of virulence factors is upregulated on entry into hosts and downregulated during the free-living lifestyle in the environment. In this study, we utilized transcriptome sequencing to compare the transcriptional profiles of Photobacterium damselae subsp. damselae , a generalist pathogen that causes disease in diverse marine animals and fatal infections in humans at NaCl concentrations that mimic the free-living lifestyle or host internal milieu, respectively. We here show that NaCl concentration constitutes a major regulatory signal that shapes the transcriptome and uncover 1,808 differentially expressed genes (888 upregulated and 920 downregulated in response to low-salt conditions). Growth at 3% NaCl, a salinity that mimics the free-living lifestyle, upregulated genes involved in energy production, nitrogen metabolism, transport of compatible solutes, utilization of trehalose and fructose, and carbohydrate and amino acid metabolism with strong upregulation of the arginine deiminase system (ADS). In addition, we observed a marked increase in resistance to antibiotics at 3% NaCl. On the contrary, the low salinity conditions (1% NaCl) that mimic those encountered in the host triggered a virulence gene expression profile that maximized the production of the type 2 secretion system (T2SS)-dependent cytotoxins damselysin, phobalysin P, and a putative PirAB-like toxin, observations that were corroborated by the analysis of the secretome. Low salinity also upregulated the expression of iron-acquisition systems, efflux pumps, and other functions related to stress response and virulence. The results of this study greatly expand our knowledge of the salinity-responsive adaptations of a generalist and versatile marine pathogen. IMPORTANCE Pathogenic Vibrionaceae species experience continuous shifts of NaCl concentration in their life cycles. However, the impact of salinity changes in gene regulation has been studied in a small number of Vibrio species. In this study, we analyzed the transcriptional response of Photobacterium damselae subsp. damselae ( Pdd ), a generalist and facultative pathogen, to changes in salinity, and demonstrate that growth at 1% NaCl in comparison to 3% NaCl triggers a virulence program of gene expression, with a major impact in the T2SS-dependent secretome. The decrease in NaCl concentration encountered by bacteria on entry into a host is proposed to constitute a regulatory signal that upregulates a genetic program involved in host invasion and tissue damage, nutrient scavenging (notably iron), and stress responses. This study will surely inspire new research on Pdd pathobiology, as well as on other important pathogens of the family Vibrionaceae and related taxa whose salinity regulons still await investigation.

The marine bacterium Photobacterium damselae subsp. piscicida ( Pdp ) causes photobacteriosis in fish and important financial losses in aquaculture, but knowledge of its virulence factors is still scarce. We here demonstrate that an unstable plasmid (pPHDPT3) that encodes a type III secretion system (T3SS) is highly prevalent in Pdp strains from different geographical origins and fish host species. We found that pPHDPT3 undergoes curing upon in vitro cultivation, and this instability constitutes a generalized feature of pPHDPT3-like plasmids in Pdp strains. pPHDPT3 markers were detected in tissues of naturally-infected moribund fish and in the Pdp colonies grown directly from the fish tissues but were undetectable in a fraction of the colonies produced upon the first passage of the primeval colonies on agar plates. Notably, cured strains exhibited a marked reduction in virulence for fish, demonstrating that pPHDPT3 is a major virulence factor of Pdp . The attempts to stabilize pPHDPT3 by insertion of antibiotic resistance markers by allelic exchange caused an even greater reduction in virulence. We hypothesize that the existence of a high pressure to shed pPHDPT3 plasmid in vitro caused the selection of clones with off-target mutations and gene rearrangements during the process of genetic modification. Collectively, these results show that pPHDPT3 constitutes a novel, hitherto unreported virulence factor of Pdp that shows a high instability in vitro and warn that the picture of Pdp virulence genes has been historically underestimated, since the loss of the T3SS and other plasmid-borne genes may have occurred systematically in laboratories for decades.

The marine bacterium Photobacterium damselae subsp. damselae (Pdd) causes disease in marine animals and humans. Previous studies demonstrated that mutation of the two-component system RstAB strongly impacts virulence of this pathogen, but the RstAB regulon has not been thoroughly elucidated. We here compared the transcriptomes of Pdd RM-71 and ΔrstA and ΔrstB derivatives using RNA-seq. In accordance with previous studies, RstAB positively regulated cytotoxins Dly, PhlyP and PhlyC. This analysis also demonstrated a positive regulation of outer membrane proteins, resistance against antimicrobials and potential virulence factors by this system. Remarkably, RstAB positively regulated two hitherto uncharacterised gene clusters involved in the synthesis of a polysaccharide capsule. Presence of a capsular layer in wild-type cells was confirmed by transmission electron microscopy, whereas rstA and rstB mutants were non-capsulated. Mutants for capsule synthesis genes, wza and wzc exhibited acapsular phenotypes, were impaired in resistance against the bactericidal action of fish serum and mucus, and were strongly impaired in virulence for fish, indicating a major role of capsule in virulence. Collectively, this study demonstrates that RstAB is a major positive regulator of key virulence factors including a polysaccharide capsule essential for full virulence in a pathogenic Photobacterium., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida ( Phdp ), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA ( P hotobacteriumN lpC-like p rotein A ) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-d-glutamyl- meso -diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida , a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition., (Copyright © 2021 Lisboa et al.)

The ability to metabolize sucrose is a variable trait within the family Vibrionaceae. The marine bacterium Photobacterium damselae subsp. damselae (Pdd), pathogenic for marine animals and humans, is generally described as negative for sucrose utilization (Scr - ). Previous studies have reported sucrose-utilizing isolates (Scr + ), but the genetic basis of this variable phenotype remains uncharacterized. Here, we carried out the genome sequencing of five Scr + and two Scr - Pdd isolates and conducted a comparative genomics analysis with sixteen additional Pdd genomes sequenced in previous studies. We identified two different versions of a four-gene cluster (scr cluster) exclusive of Scr + isolates encoding a PTS system sucrose-specific IIBC component (scrA), a fructokinase (scrK), a sucrose-6-phosphate hydrolase (scrB), and a sucrose operon repressor (scrR). A scrA deletion mutant did not ferment sucrose and was impaired for growth with sucrose as carbon source. Comparative genomics analyses suggested that scr clusters were acquired by horizontal transfer by different lineages of Pdd and were inserted into a recombination hot-spot in the Pdd genome. The incongruence of phylogenies based on housekeeping genes and on scr genes revealed that phylogenetically diverse gene clusters for sucrose utilization have undergone extensive horizontal transfer among species of Vibrio and Photobacterium., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The marine bacterium Photobacterium damselae subsp. damselae is a pathogen that causes disease in diverse marine animals, and is also a serious opportunistic human pathogen that can cause fatal infections. Strains of this pathogen isolated from diseased European sea bass in aquaculture facilities in the Turkish coast of the Black Sea were found to exhibit reduced sensitivity to multiple antimicrobials. Selected representative strains were subjected to complete genome sequencing and plasmid characterization. It was found that multidrug resistant (MDR) isolates harboured large conjugative plasmids sharing part of their sequence backbone with pAQU-group plasmids, hitherto reported exclusively in China and Japan. Four new pAQU-group versions of plasmids were identified in the present study, containing distinct combinations of the resistance determinants tetB, floR, sul2, qnrVC, dfrA and strAB. Conjugative transfer of pPHDD2-OG2, a representative plasmid of 170,998 bp, occurred at high frequencies (2.2 × 10 -2 transconjugants per donor cell), to E. coli and to pathogenic P. damselae subsp. damselae and subsp. piscicida strains. Upon transfer, pPHDD2-OG2 conferred reduced susceptibility to a number of antimicrobials to the recipient strains. Comparative genomics analysis of host strains suggested that these MDR plasmids of the pAQU-group were acquired by different genetic lineages of Pdd. This study provides evidence that P. damselae subsp. damselae isolated from diseased fish constitute a reservoir for conjugative MDR pAQU-group plasmids in the Mediterranean basin, and have the potential to spread to diverse bacterial species.

Photobacterium damselae subsp. damselae ( Pdd ), an important pathogen for marine animals, is also an opportunistic human pathogen that can cause fatal necrotizing fasciitis. The regulatory changes triggered by the temperature shift experienced by this marine pathogen upon entering the human body, are completely unknown. Here we report an RNA-seq approach combined with phenotypical assays to study the response of Pdd to cultivation at 37°C in comparison to 25°C. We found that cultivation of a Pdd highly virulent strain for fish and mice, RM-71, at 37°C, initially enhanced bacterial growth in comparison to 25°C as evidenced by the increase in optical density. However, cells were found to undergo a progressive loss of viability after 6 h cultivation at 37°C, and no viable cells could be detected from 30 h cultures at 37°C. In contrast, at 25°C, viable cell counts achieved the highest values at 30 h cultivation. Cells grown at 25°C showed normal rod morphology by scanning electron microscopy analysis whereas cells grown at 37°C exhibited chain-like structures and aberrant long shapes suggesting a defect in daughter cell separation and in septum formation. Cells grown at 37°C also exhibited reduced tolerance to benzylpenicillin. Using a RNA-seq approach we discovered that growth at 37°C triggered a heat-shock response, whereas genes involved in motility and virulence were repressed including iron acquisition systems, the type two secretion system, and damselysin toxin, a major virulence factor of Pdd . Human isolates did not exhibit advantage growing at 37°C compared to fish isolates, and comparative genomics did not reveal gene markers specific of human isolates, suggesting that any Pdd genotype existing in the marine environment might potentially cause disease in humans. Altogether, these data indicate that the potential of Pdd to cause disease in humans is an accidental condition rather than a selected trait, and that human body temperature constitutes a stressful condition for Pdd. This study provides the first transcriptome profile of Pdd exposed at human body temperature, and unveils a number of candidate molecular targets for prevention and control of human infections caused by this pathogen., (Copyright © 2020 Matanza and Osorio.)

Here, we report the draft genome sequences of two strains of the fish pathogen Photobacterium damselae subsp. piscicida , isolated from Salmo salar (SNW-8.1) and Seriola quinqueradiata (PP3). The identification of a type III secretion system in the two genomes furthers our understanding of the pathobiology of this subspecies., (Copyright © 2019 Abushattal et al.)

The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC) cytotoxins in the fish and human pathogen Photobacterium damselae subsp. damselae , a marine bacterium of the family Vibrionaceae . However, the function of the predicted cognate response regulator RstA has not been studied so far, and the role of the RstAB system in other cell functions and phenotypes remain uninvestigated. Here, we analyzed the effect of rstA and rstB mutations in cell fitness and in diverse virulence-related features. Both rstA and rstB mutants were severely impaired in virulence for sea bream and sea bass fish. Mutants in rstA and rstB genes were impaired in hemolysis and in Dly-dependent phospholipase activity but had intact PlpV-dependent phospholipase and ColP-dependent gelatinase activities. rstA and rstB mutants grown at 0.5% NaCl exhibited impaired swimming motility, enlarged cell size and impaired ability to separate after cell division, whereas at 1% NaCl the mutants exhibited normal phenotypes. Mutation of any of the two genes also impacted tolerance to benzylpenicillin. Notably, rstA and rstB mutants showed impaired secretion of a number of type II secretion system (T2SS)-dependent proteins, which included the three major cytotoxins Dly, PhlyP and PhlyC, as well as a putative delta-endotoxin and three additional uncharacterized proteins which might constitute novel virulence factors of this pathogenic bacterium. The analysis of the T2SS-dependent secretome of P. damselae subsp. damselae also led to the identification of RstAB-independent potential virulence factors as lipoproteins, sialidases and proteases. The RstAB regulon included plasmid, chromosome I and chromosome II-encoded genes that showed a differential distribution among isolates of this subspecies. This study establishes RstAB as a major regulator of virulence and diverse cellular functions in P. damselae subsp. damselae .

The marine bacterium Photobacterium damselae subsp. damselae (Pdd) is a generalist and facultative pathogen that causes disease in a wide range of marine animals including fish species of importance in aquaculture. Disease outbreaks in fish farms have been correlated with an increased water temperature during summer months. In this study, we have used RNA sequencing to analyze the transcriptome of Pdd RM-71 cultured at two different temperatures, which simulated temperature conditions experienced during free swimming lifestyle at mid latitudes in winter months (15°C) and during outbreaks in aquaculture in warm summer months (25°C). The enhanced bacterial growth of Pdd observed at 25°C in comparison to 15°C suggests that an elevated seawater temperature contributes to the build-up of a sufficient bacterial population to cause disease. In comparison to growth at 15°C, growth at 25°C resulted in the upregulation of genes involved in DNA synthesis, nutrient uptake, chemotaxis, flagellar motility, secretion systems and antimicrobial resistance. Plasmid-encoded virulence factors, which include a putative adhesin/invasin OmpU, a transferrin receptor and a serum resistance protein, were also upregulated. Transcription factor RpoS, genes involved in cold shock response, modulation of cell envelope and amino acid metabolism, as well as genes of yet unknown function were downregulated at 25°C. Notably, the gene encoding damselysin cytotoxin (Dly) was among the most highly transcribed genes at the two assayed temperatures, at levels comparable to the most highly expressed housekeeping genes. This study contributes to our understanding of the regulatory networks and biology of a generalist marine bacterial pathogen, and provides evidence that temperature regulates multiple physiological and virulence-related functions in Pdd., Competing Interests: The authors have declared that no competing interests exist.

Photobacterium damselae subsp. damselae ( Pdd ) is an emerging pathogen of marine animals that sometimes causes serious infections in humans. Two related pore forming toxins, phobalysins P and C, and damselysin, a phospholipase D, confer strong virulence of Pdd in mice. Because infections by Pdd are typically caused following exposure of wounds to sea water we investigated how salinity impacts toxin activity, swimming, and association of Pdd with epithelial cells. These activities were low when bacteria were pre-cultured in media with 3.5% NaCl, the global average salinity of sea water. In contrast, lower salinity increased swimming of wild type Pdd peaking at 2% NaCl, hemolysis, and association with epithelial cells peaking at 1-1.5%. Previously, we have found that hemolysin genes enhance the association of Pdd with epithelial cells, but the underlying mechanisms have remained ill-defined. We here searched for potential links between hemolysin-production, chemotaxis and association of Pdd with target cells at varying salt concentrations. Unexpectedly, disruption of chemotaxis regulator cheA not only affected bacterial swimming and association with epithelial cells at intermediate to low salinity, but also reduced the production of plasmid-encoded phobalysin (PhlyP). The results thus reveal unforeseen links between chemotaxis regulators, a pore forming toxin and the association of a marine bacterium with target cells.

The marine bacterium Photobacterium damselae subsp. damselae is a pathogen for a variety of marine animals, as well as for humans, and is nowadays considered an emerging pathogen for fish of importance in marine aquaculture. Recent studies have suggested that outbreaks in fish farms are caused by multiclonal populations of this subspecies that exist in the environment. Here, we report the study of a collection of 31 strains isolated during the course of disease outbreaks in marine rainbow trout farms in Denmark in 1994, 1995, and 2006, respectively. A phylogenetic analysis based on the toxR gene sequence, and the screening of virulence-related genes uncovered a high genetic heterogeneity, even among strains isolated from the same fish farm at the same time. Moreover, comparative analysis of the whole genome sequences of four selected strains revealed a large number of differentially occurring genes, which included virulence genes, pPHDD1 plasmid, polysaccharide synthesis gene clusters, CRISPR-Cas systems and putative new mobile genetic elements. This study provides sound evidence that P. damselae subsp. damselae outbreaks in Danish rainbow trout farms were caused by multiclonal populations and that horizontal gene transfer constitutes a strong driving force in the generation of intraspecific diversity in this pathogen.

Photobacterium damselae subsp. damselae causes vibriosis in a variety of marine animals, including fish species of importance in aquaculture. It also may cause wound infections in humans that can progress into a fatal outcome. Two major virulence factors are encoded within the large conjugative plasmid pPHDD1: the phospholipase-D damselysin (Dly) and the pore-forming toxin Phobalysin P (PhlyP). The two toxins exert hemolytic and cytolytic activity in a synergistic manner. Albeit PhlyP has close homologues in many Vibrio species, it has unique features that differentiate it from related toxins. Dly phospholipase constitutes a singular trait of P. damselae subsp. damselae among the Vibrionaceae , although related toxins are found in members of the Aeromonadaceae Fish farm outbreaks can also be caused by plasmidless strains. Such observation led to the characterization of two ubiquitous, chromosome-encoded toxins with lesser cytolytic activity: the pore forming-toxin Phobalysin C (PhlyC) and the phospholipase-hemolysin PlpV. Special attention deserves the high genetic diversity of this pathogen, with a number of strain-specific features including the cell envelope polysaccharide synthesis clusters. Fish outbreaks are likely caused by multiclonal populations which contain both plasmidless and pPHDD1-harbouring isolates, and not by well-adapted clonal complexes. Still, among such a genetic heterogeneity, it is feasible to identify conserved weak points in the biology of this bacterium: the two-component regulatory system RstAB (CarSR) was found to be necessary for maximal production of virulence factors and its inactivation severely impaired virulence., (Copyright © 2018 American Society for Microbiology.)

AIP56 (apoptosis-inducing protein of 56 kDa) is a key virulence factor of Photobacterium damselae subsp. piscicida (Phdp), the causative agent of a septicaemia affecting warm water marine fish species. Phdp -associated pathology is triggered by AIP56, a short trip AB toxin with a metalloprotease A domain that cleaves the p65 subunit of NF-κB, an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes and plays a central role in host responses to infection. During infection by Phdp , AIP56 is systemically disseminated and induces apoptosis of macrophages and neutrophils, compromising the host phagocytic defence and contributing to the genesis of pathology. Although it is well established that the secretion of AIP56 is crucial for Phdp pathogenicity, the protein secretion systems operating in Phdp and the mechanism responsible for the extracellular release of the toxin remain unknown. Here, we report that Phdp encodes a type II secretion system (T2SS) and show that mutation of the EpsL component of this system impairs AIP56 secretion. This work demonstrates that Phdp has a functional T2SS that mediates secretion of its key virulence factor AIP56., Competing Interests: The authors declare no conflict of interest.

Photobacterium damselae subsp. piscicida (Pdp) is an intracellular fish pathogen that causes photobacteriosis, a disease proven deadly in farmed fish worldwide. This work focuses on the analysis of genome sequences, chromosomes structure and gene contents of two strains from Sparus aurata (DI21) and Solea senegalensis (L091106-03H), isolated on the Spanish Atlantic coast. The comparative genomic analysis revealed that DI21 and L091106-03H share 98% of their genomes, including two virulence plasmids: pPHDP70 encoding siderophore piscibactin synthesis and pPHDP10 encoding the apoptotic toxin AIP56. Both genomes harbour a surprisingly large number of IS elements accounting for 12-17% of the total genome, representing an IS density of 0.15 elements per kb, one of the highest IS density values in a bacterial pathogen. This massive proliferation of ISs is responsible for the generation of a high number of pseudogenes that caused extensive loss of biological functions. Pseudogene formation is one of the main features of Pdp genome that explains most of the ecological and phenotypic differences with respect to its sibling subspecies P. damselae subsp. damselae and to other Vibrionaceae. Evidence was also found proving the existence of two chromosomal configurations depending on the origin of the strains: an European and an Asian/American types of genome organisation, reinforcing the idea of the existence of two geographically-linked clonal lineages in Pdp. In short, our study suggests that the host-dependent lifestyle of Pdp allowed massive IS proliferation and gene decay processes, which are major evolutionary forces in the shaping of the Pdp genome., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Photobacterium damselae subsp damselae ( Pdd ) is a Vibrionaceae that has a wide pathogenic potential against many marine animals and also against humans. Some strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there are virulent strains that do not produce vibrioferrin, but they still give a strong positive reaction in the CAS test for siderophore production. In an in silico search on the genome sequences of this type of strains we could not find any ORF which could be related to a siderophore system. To identify genes that could encode a siderophore-mediated iron acquisition system we used a mini-Tn 10 transposon random mutagenesis approach. From more than 1,400 mutants examined, we could isolate a mutant (BP53) that showed a strong CAS reaction independently of the iron levels of the medium. In this mutant the transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase that participates in the tricarboxylic acid cycle. The mutant did not show any growth impairment in rich or minimal media, but it accumulated a noticeable amount of citrate (around 7 mM) in the culture medium, irrespective of the iron levels. The parental strain accumulated citrate, but in an iron-regulated fashion, being citrate levels 5-6 times higher under iron restricted conditions. In addition, a null mutant deficient in citrate synthase showed an impairment for growth at high concentrations of iron chelators, and showed almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the iron-restricted culture supernatants resulted in a CAS-positive fraction with biological activity as siderophore. HPLC purification of that fraction yielded a pure compound which was identified as citrate from its MS and NMR spectral data. Although the production of another citrate-based compound with siderophore activity cannot be ruled out, our results suggest that Pdd secretes endogenous citrate and use it for iron scavenging from the cell environment.

Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity. IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen., (Copyright © 2017 American Society for Microbiology.)

The marine pathogenic bacterium Photobacterium damselae subsp. damselae causes septicemia in marine animals and in humans. The pPHDD1 plasmid-encoded hemolysins damselysin (Dly) and phobalysin P (PhlyP), and the chromosome-encoded hemolysin phobalysin C (PhlyC) constitute its main virulence factors. However, the mechanisms by which expression of these three hemolysins is regulated remain unknown. Here we report the isolation of a mini-Tn 10 transposon mutant which showed a strong impairment in its hemolytic activity. The transposon disrupted a putative sensor histidine kinase gene vda_000600 ( rstB ), which together with vda_000601 ( rstA ) is predicted to encode a putative two-component regulatory system. This system showed to be homologous to the Vibrio cholerae CarSR/VprAB and Escherichia coli RstAB systems. Reconstruction of the mutant by allelic exchange of rstB showed equal impairment in hemolysis, and complementation with a plasmid expressing rstAB restored hemolysis to wild-type levels. Remarkably, we demonstrated by promoter expression analyses that the reduced hemolysis in the rstB mutant was accompanied by a strong decrease in transcription activities of the three hemolysin genes dly (damselysin), hlyA pl (phobalysin P) and hlyA ch (phobalysin C). Thus, RstB, encoded in the small chromosome, regulates plasmid and chromosomal virulence genes. We also found that reduced expression of the three virulence genes correlated with a strong decrease in virulence in a sea bass model, demonstrating that RstB constitutes a master regulator of the three P. damselae subsp. damselae hemolysins and plays critical roles in the pathogenicity of this bacterium. This study represents the first evidence of a direct role of a RstAB-like system in the regulation of bacterial toxins.

Photobacterium damselae subsp. damselae (Pdd) is a marine bacterium that can infect numerous species of marine fish as well as other species including humans. Low iron availability is one of the signs that bacterial pathogens can detect in order to begin colonizing their host, and the reduction of iron levels is a nonspecific host defense strategy that prevents bacterial proliferation. In this work a proteomic approach was used to study the gene expression adaptations of a Pdd strain in response to iron availability. A comparative analysis of induced proteins in both high- and low-iron conditions showed profound cellular metabolic adaptations that result, for instance, in amino acid requirement. It also provided important information about the changes that occur in the energetic metabolism induced by the surrounding iron levels, allowing for the identification of novel potential virulence factors. Among others, genes involved in the synthesis and transport of a vibrioferrin-like siderophore were identified for the first time. In addition to plasmid pPHDD1-encoded Dly and HlyA hemolysins, a pPHDD1-borne operon, which may encode a transferrin receptor, was also found. This operon identification suggests that this virulence plasmid could encode so-far unknown additional virulence factors other than hemolysins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in several fish species, produces acinetobactin and amonabactin as siderophores. In a previous study, we chemically characterized these siderophores and proposed a biosynthetic pathway based on genetic analysis. However, the internalization mechanisms of ferric-acinetobactin and ferric-amonabactin remain largely unknown. In the present study, we demonstrate that the outer membrane protein FstB is the ferric-acinetobactin receptor in A. salmonicida since an fstB defective mutant is unable to grow under iron limitation and does not use acinetobactin as an iron source. In order to study the effect that structural changes in acinetobactin have on its siderophore activity, a collection of acinetobactin-based analogues was synthesized, including its enantiomer and four demethylated derivatives. The biological activity of these analogues on an fstB(+) strain compared to an fstB(-) strain allowed structure-activity relationships to be elucidated. We found a lack of enantiomer preference on the siderophore activity of acinetobactin over A. salmonicida or on the molecular recognition by FstB protein receptor. In addition, it was observed that A. salmonicida could not use acinetobactin analogues when imidazole or a similar heterocyclic ring was absent from the structure. Surprisingly, removal of the methyl group at the isoxazolidinone ring induced a higher biological activity, thus suggesting alternative route(s) of entry into the cell that must be further investigated. It is proposed that some of the synthetic acinetobactin analogues described here could be used as starting points in the development of novel drugs against A. salmonicida and probably against other acinetobactin producers like the human pathogen Acinetobacter baumannii.

The characterization of antibiotic-resistant vibrios isolated from shellfish aquaculture is necessary to elucidate the potential transfer of resistance and to establish effective strategies against vibriosis. With this aim, we analyzed a collection of bacterial isolates obtained from 15 failed hatchery larval cultures that, for the most part, had been treated experimentally with chloramphenicol to prevent vibriosis. Isolates were obtained during a 2-year study from experimental cultures of five different clam species. Among a total of 121 Vibrio isolates studied, 28 were found to be chloramphenicol resistant, suggesting that the shellfish hatchery had been using a sublethal concentration of the antibiotic. Interestingly, chloramphenicol-resistant vibrios showed also resistance to tetracycline and amoxicillin (group A; n = 19) or to streptomycin (group B; n = 9). Chloramphenicol-resistant vibrios were subjected to a PCR amplification and DNA sequencing of the chloramphenicol acetyltransferase genes (cat), and the same approach was followed to study the tetracycline resistance markers (tet). 16S ribosomal RNA (rRNA) gene sequencing revealed that chloramphenicol-resistant vibrios pertained mostly to the Splendidus clade. Conjugation assays demonstrated that various R-plasmids which harbored the cat II/tet(D) genes and cat III gene in groups A and B respectively, were transferred to E. coli and bivalve pathogenic vibrios. Most interestingly, transconjugants exhibited the antibiotic resistance patterns of the donors, despite having been selected only on the basis of chloramphenicol resistance. This is the first report carried out in a bivalve hatchery elucidating the persistence of resistant vibrios, the mechanisms of antibiotic resistance, and the transfer of different R-plasmids.

Integrating conjugative elements (ICEs) of the SXT/R391 family have been identified in fish-isolated bacterial strains collected from marine aquaculture environments of the northwestern Iberian Peninsula. Here we analysed the variable regions of two ICEs, one preliminarily characterised in a previous study (ICEVscSpa3) and one newly identified (ICEPspSpa1). Bacterial strains harboring these ICEs were phylogenetically assigned to Vibrio scophthalmi and Pseudoalteromonas sp., thus constituting the first evidence of SXT/R391-like ICEs in the genus Pseudoalteromonas to date. Variable DNA regions, which confer element-specific properties to ICEs of this family, were characterised. Interestingly, the two ICEs contained 29 genes not found in variable DNA insertions of previously described ICEs. Most notably, variable gene content for ICEVscSpa3 showed similarity to genes potentially involved in housekeeping functions of replication, nucleotide metabolism and transcription. For these genes, closest homologues were found clustered in the genome of Pseudomonas psychrotolerans L19, suggesting a transfer as a block to ICEVscSpa3. Genes encoding antibiotic resistance, restriction modification systems and toxin/antitoxin systems were absent from hotspots of ICEVscSpa3. In contrast, the variable gene content of ICEPspSpa1 included genes involved in restriction/modification functions in two different hotspots and genes related to ICE maintenance. The present study unveils a relatively large number of novel genes in SXT/R391-ICEs, and demonstrates the major role of ICE elements as contributors to horizontal gene transfer.

Unlabelled: Photobacterium damselae subsp. damselae is considered to be an emerging pathogen of marine fish of importance in aquaculture, with a notable increase in its geographical distribution during the last several years. In this study, we carried out for the first time to our knowledge a genetic and pathobiological characterization of 14 strains isolated from sea bass (Dicentrarchus labrax) reared in the Southeastern Black Sea, where high mortalities were observed at two aquaculture farms during the summer and autumn of 2011. Heterogeneity was evidenced among strains in phenotypical traits, such as sucrose fermentation, motility, and hemolysis. Although 11 of 14 isolates were hemolytic, we found that all of the isolates lacked the pPHDD1 virulence plasmid that encodes the phospholipase-D damselysin (Dly) and the pore-forming toxin PhlyP, two hemolysins previously reported to constitute major virulence factors for turbot. Subsequent PCR and sequencing analyses demonstrated that the 11 hemolytic isolates harbored a complete hlyAch gene, a chromosome I-borne gene that encodes HlyAch hemolysin, whereas the three nonhemolytic isolates contained hlyAch pseudogenes caused by insertion sequence elements. Virulence challenges with two representative strains revealed that, albeit less virulent than the pPHDD1-harboring strain RM-71, the plasmidless hlyAch-positive and hlyAch-negative Black Sea isolates were pathogenic for sea bass. A phylogenetic analysis based on the toxR gene sequence uncovered a greater diversity in the isolates, indicating that the presence of this pathogen in the Black Sea was not caused by the introduction and spread of a single virulent clone but by the proliferation of different clones., Importance: The geographical distribution of marine bacterial pathogens is undergoing a worldwide increase. In particular, bacteria of the group vibrios are increasingly being isolated as the causative agents of disease in novel species of cultivated fish in areas where they had not been previously reported. Here we characterize for the first time to our knowledge a collection of isolates of the fish and human pathogen Photobacterium damselae subsp. damselae from diseased sea bass reared in the Black Sea. We uncovered great genetic diversity in the Black Sea isolates of this pathogen, suggesting a multiclonal origin. We also demonstrate for the first time that these isolates bear pathogenic potential for sea bass cultures by virulence challenges., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Edwardsiella tarda has long been known as a pathogen that causes severe economic losses in aquaculture industry. Insights gained on E. tarda pathogenesis may prove useful in the development of new methods for the treatment of infections as well as preventive measures against future outbreaks. In this report, we have established the correlation between the presence of virulence genes, related with three aspects typically involved in bacterial pathogenesis (chondroitinase activity, quorum sensing and siderophore-mediated ferric uptake systems), in the genome of E. tarda strains isolated from turbot in Europe and their phenotypic traits. A total of 8 genes were tested by PCR for their presence in 73 E. tarda isolates. High homogeneity was observed in the presence/absence pattern of all the strains. Positive results in the amplification of virulence-related genes were correlated with the detection of chondroitinase activity in agar plates, in vivo AHL production during fish infection and determination of type of siderophore produced by E. tarda. To the best of our knowledge, this is the first study carried out with European strains on potential virulence factors. Furthermore, we demonstrated for the first time that E. tarda produces the siderophore vibrioferrin., (© 2015 John Wiley & Sons Ltd.)

The iron uptake mechanisms based on siderophore synthesis used by the fish pathogen Aeromonas salmonicida subsp. salmonicida are still not completely understood, and the precise structure of the siderophore(s) is unknown. The analysis of genome sequences revealed that this bacterium possesses two gene clusters putatively involved in the synthesis of siderophores. One cluster is a candidate to encode the synthesis of acinetobactin, the siderophore of the human pathogen Acinetobacter baumannii, while the second cluster shows high similarity to the genes encoding amonabactin synthesis in Aeromonas hydrophila. Using a combination of genomic analysis, mutagenesis, biological assays, chemical purification, and structural determination procedures, here we demonstrate that most A. salmonicida subsp. salmonicida strains produce simultaneously the two siderophores, acinetobactin and amonabactin. Interestingly, the synthesis of both siderophores relies on a single copy of the genes encoding the synthesis of the catechol moiety (2,3-dihydroxybenzoic acid) and on one encoding a phosphopantetheinyl transferase. These genes are present only in the amonabactin cluster, and a single mutation in any of them abolishes production of both siderophores. We could also demonstrate that some strains, isolated from fish raised in seawater, produce only acinetobactin since they present a deletion in the amonabactin biosynthesis gene amoG. Our study represents the first evidence of simultaneous production of acinetobactin and amonabactin by a bacterial pathogen and reveals the plasticity of bacterial genomes and biosynthetic pathways. The fact that the same siderophore is produced by unrelated pathogens highlights the importance of these systems and their interchangeability between different bacteria.

Photobacterium damselae subsp. damselae, an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized the hlyA gene product, a putative small β-pore-forming toxin, and termed it phobalysin P (PhlyP), for "photobacterial lysin encoded on a plasmid." PhlyP formed stable oligomers and small membrane pores, causing efflux of K(+), with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the related Vibrio cholerae cytolysin. Attack by PhlyP provoked a loss of cellular ATP, attenuated translation, and caused profound morphological changes in epithelial cells. In coculture experiments with epithelial cells, Photobacterium damselae subsp. damselae led to rapid hemolysin-dependent membrane permeabilization. Unexpectedly, hemolysins also promoted the association of P. damselae subsp. damselae with epithelial cells. The collective observations of this study suggest that membrane-damaging toxins commonly enhance bacterial adherence., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The fish pathogen Photobacterium damselae subsp. piscicida produces the siderophore piscibactin. A gene cluster that resembles the Yersinia high-pathogenicity island (HPI) encodes piscibactin biosynthesis. Here, we report that this HPI-like cluster is part of a hitherto-uncharacterized 68-kb plasmid dubbed pPHDP70. This plasmid lacks homologs of genes that mediate conjugation, but we found that it could be transferred at low frequencies from P. damselae subsp. piscicida to a mollusk pathogenic Vibrio alginolyticus strain and to other Gram-negative bacteria, likely dependent on the conjugative functions of the coresident plasmid pPHDP60. Following its conjugative transfer, pPHDP70 restored the capacity of a vibrioferrin mutant of V. alginolyticus to grow under low-iron conditions, and piscibactin became detectable in its supernatant. Thus, pPHDP70 appears to harbor all the genes required for piscibactin biosynthesis and transport. P. damselae subsp. piscicida strains cured of pPHDP70 no longer produced piscibactin, had impaired growth under iron-limited conditions, and exhibited markedly decreased virulence in fish. Collectively, our findings highlight the importance of pPHDP70, with its capacity for piscibactin-mediated iron acquisition, in the virulence of P. damselae subsp. piscicida. Horizontal transmission of this plasmid-borne piscibactin synthesis gene cluster in the marine environment may facilitate the emergence of new pathogens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security.

Photobacterium damselae subsp. damselae is a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10 transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion in epsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement of epsL in HlyAch secretion. In addition, mutation of epsL in a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating that epsL plays a major role in secretion of the plasmid-encoded HlyApl and Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase gene pilD severely affected hemolysis, which dropped at levels inferior to those of epsL mutants. Promoter expression analyses suggested that impairment of hemolysin secretion in epsL and pilD mutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, single epsL and pilD mutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS and pilD in P. damselae subsp. damselae virulence., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Photobacterium damselae subsp. damselae, a marine bacterium that causes infections in marine animals and in humans, produces up to three different haemolysins involved in virulence, which include the pPHDD1 plasmid-encoded damselysin (Dly) and HlyApl , and the chromosome-encoded HlyAch . We screened 45 isolates from different origins, and found a correlation between their haemolytic phenotypes and the differential haemolysin gene content. All highly and medium haemolytic strains harboured pPHDD1, with amino acid substitutions in HlyApl and HlyAch being the cause of the medium haemolytic phenotypes in some pPHDD1-harbouring strains. Weakly haemolytic strains contained only hlyAch , whereas nonhaemolytic isolates, in addition to lacking pPHDD1, either lacked hlyAch or contained a hlyAch pseudogene. Sequence analysis of the genomic context of hlyAch uncovered an unexpected genetic diversity, suggesting that hlyAch is located in an unstable chromosomal region. Phylogenetic analysis suggested that hlyApl and hlyAch originated by gene duplication within P. damselae subsp. damselae following acquisition by horizontal transfer. These observations together with the differential distribution of pPHDD1 plasmid among strains suggest that horizontal gene transfer has played a main role in shaping the haemolysin gene baggage in this pathogen., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Photobacterium damselae subsp. damselae (formerly Vibrio damsela) is a pathogen of a variety of marine animals including fish, crustaceans, molluscs, and cetaceans. In humans, it can cause opportunistic infections that may evolve into necrotizing fasciitis with fatal outcome. Although the genetic basis of virulence in this bacterium is not completely elucidated, recent findings demonstrate that the phospholipase-D Dly (damselysin) and the pore-forming toxins HlyApl and HlyAch play a main role in virulence for homeotherms and poikilotherms. The acquisition of the virulence plasmid pPHDD1 that encodes Dly and HlyApl has likely constituted a main driving force in the evolution of a highly hemolytic lineage within the subspecies. Interestingly, strains that naturally lack pPHDD1 show a strong pathogenic potential for a variety of fish species, indicating the existence of yet uncharacterized virulence factors. Future and deep analysis of the complete genome sequence of Photobacterium damselae subsp. damselae will surely provide a clearer picture of the virulence factors employed by this bacterium to cause disease in such a varied range of hosts.

Photobacterium damselae subsp. damselae causes infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and plasmid-encoded HlyA (HlyA(pl)). These hemolysins are encoded by plasmid pPHDD1 and contribute to hemolysis and virulence for fish and mice. In this study, we report that all the hemolytic strains produce a hitherto uncharacterized chromosome-encoded HlyA (HlyAch). Hemolysis was completely abolished in a single hlyAch mutant of a plasmidless strain and in a dly hlyApl hlyAch triple mutant. We found that Dly, HlyA(pl), and HlyAch are needed for full hemolytic values in strains harboring pPHDD1, and these values are the result of the additive effects between HlyApl and HlyAch, on the one hand, and of the synergistic effect of Dly with HlyApl and HlyAch, on the other hand. Interestingly, Dly-producing strains produced synergistic effects with strains lacking Dly production but secreting HlyA, constituting a case of the CAMP (Christie, Atkins, and Munch-Petersen) reaction. Environmental factors such as iron starvation and salt concentration were found to regulate the expression of the three hemolysins. We found that the contributions, in terms of the individual and combined effects, of the three hemolysins to hemolysis and virulence varied depending on the animal species tested. While Dly and HlyApl were found to be main contributors in the virulence for mice, we observed that the contribution of hemolysins to virulence for fish was mainly based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects.

A new plasmid designated pPHDP60 from a strain of the marine bacterium Photobacterium damselae subsp. piscicida isolated from diseased seabream has been characterised. pPHDP60 consists of 59,731bp, has a G+C content of 37.2% and encodes 63 predicted open-reading frames (ORFs). The plasmid backbone sequence includes, among other genes, 15 ORFs homologous to proteins of type IV conjugation systems described in IncP-type plasmids. Two modules could be distinguished within pPHDP60 sequence. One module included 10 genes of a putative type II secretion system with homologues in other Photobacterium and Vibrio plasmids. A second module exhibiting a transposon structure included a functional haloalkane dehalogenase gene linB as well as a toxin/antitoxin system. Additional interesting features of pPHDP60 include its ability to be conjugally transferred to several Gram negative bacteria., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Integrating conjugative elements (ICEs) of the SXT/R391 family have been described in Vibrios, mainly Vibrio cholerae, and other bacteria as carriers of variable gene content conferring adaptive advantages upon their hosts, including antimicrobial resistance and motility regulation. However, our knowledge on their host range and ecological significance is still limited. Here, we report the identification and characterization of ICEVspPor3 and ICEValSpa1, two novel ICEs of the SXT/R391 family from fish-isolated Vibrio splendidus and Vibrio alginolyticus, respectively. We found that ICEVspPor3 carries tetracycline and HgCl(2) resistance determinants and can be transferred by conjugation to Escherichia coli and to several species of marine bacteria including some of the major bacterial fish pathogens in marine aquaculture, whereas ICEValSpa1 lacks resistance genes. Interestingly, both ICEs harbor genes encoding distinct restriction-modification (RM) systems. We demonstrate here that these RM systems, when expressed in E. coli, confer protection to infection by T1 bacteriophage and by environmental water bacteriophages. Our results provide evidences that the variable gene content of ICEs of the SXT/R391 family encodes fitness functions beyond those related to antimicrobial resistance and motility regulation and suggest that the host range of these elements in the marine environment might be broader than expected., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

From synthetic functionalized analogues of vanchrobactin, a siderophore produced by the fish pathogenic bacteria Vibrio anguillarum serotype O2, several vanchrobactin analogues-norfloxacin conjugates were obtained and their antimicrobial activities against the wild-type and mutant strains of Vibrio anguillarum serotype O2 have been determined., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The presence of SXT/R391-related integrating conjugative elements (ICEs) in bacterial strains isolated from fish obtained from marine aquaculture environments in 2001 to 2010 in the northwestern Iberian Peninsula was studied. ICEs were detected in 12 strains taxonomically related to Vibrio scophthalmi (3 strains), Vibrio splendidus (5 strains), Vibrio alginolyticus (1 strain), Shewanella haliotis (1 strain), and Enterovibrio nigricans (2 strains), broadening the known host range able to harbor SXT/R391-like ICEs. Variable DNA regions, which confer element-specific properties to ICEs of this family, were characterized. One of the ICEs encoded antibiotic resistance functions in variable region III, consisting of a tetracycline resistance locus. Interestingly, hot spot 4 included genes providing resistance to rifampin (ICEVspPor2 and ICEValPor1) and quaternary ammonium compounds (QACs) (ICEEniSpa1), and variable region IV included a mercury resistance operon (ICEVspSpa1 and ICEEniSpa1). The S exclusion group was more represented than the R exclusion group, accounting for two-thirds of the total ICEs. Mating experiments allowed ICE mobilization to Escherichia coli strains, showing the corresponding transconjugants' rifampin, mercury, and QAC resistance. These results show the first evidence of ICEs providing rifampin and QAC resistances, suggesting that these mobile genetic elements contribute to the dissemination of antimicrobial, heavy metal, and QAC resistance determinants in aquaculture environments.

Photobacterium damselae subsp. damselae (formerly Vibrio damsela) is a marine bacterium that causes infections and fatal disease in a wide range of marine animals and in humans. Highly hemolytic strains produce damselysin (Dly), a cytolysin encoded by the dly gene that is lethal for mice and has hemolytic activity. We found that Dly is encoded in the highly hemolytic strain RM-71 within a 153,429-bp conjugative plasmid that we dubbed pPHDD1. In addition to Dly, pPHDD1 also encodes a homologue of the pore-forming toxin HlyA. We found a direct correlation between presence of pPHDD1 and a strong hemolytic phenotype in a collection of P. damselae subsp. damselae isolates. Hemolysis was strongly reduced in a double dly hlyA mutant, demonstrating the role of the two pPHDD1-encoded genes in hemolysis. Interestingly, although single hlyA and dly mutants showed different levels of hemolysis reduction depending on the erythrocyte source, hemolysis was not abolished in any of the single mutants, suggesting that the hemolytic phenotype is the result of the additive effect of Dly and HlyA. We found that pPHDD1-encoded dly and hlyA genes are necessary for full virulence for mice and fish. Our results suggest that pPHDD1 can be considered as a driving force for the emergence of a highly hemolytic lineage of P. damselae subsp. damselae.

Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD(50) value in rainbow trout obtained for strain 074 was 2.1 × 10(2) ± 84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.

The marine fish pathogen Photobacterium damselae subsp. piscicida utilizes haem compounds as the sole iron source. In a previous work, we characterized a gene cluster with ten potential haem uptake and utilization genes. Two of these genes, hutC and hutD, which are iron-regulated, conform a putative inner membrane haem ABC transporter. In this study, we constructed an insertional mutant, leading to the inactivation of hutCD genes. Reverse transcriptase-PCR analyses demonstrated that an insertion between the hutB and hutC genes abolished transcription of the downstream hutC and hutD genes. The hutCD mutant was unable to utilize haem as the sole iron source, demonstrating that the putative ABC-transporter proteins HutC and HutD are essential for haem utilization as an iron source in P. damselae subsp. piscicida. In addition, reverse transcriptase-PCR assays conducted with RNA samples isolated from experimentally infected fish revealed the presence of hutCD transcripts. The results demonstrate for the first time that haem uptake genes of a fish pathogen are expressed during the infective process in fish.

Vibrio anguillarum is a marine bacterium that is present in many marine aquatic environments and that is the main cause of vibriosis in diverse wild and cultured fish species. Two siderophore-mediated iron uptake systems have been described in V. anguillarum. One, mediated by the siderophore anguibactin, is encoded by the pJM1-type plasmids and is restricted to serotype O1 strains. The second one is mediated by the vanchrobactin siderophore and is widespread in many strains belonging to different serotypes. Both siderophores belong to the catecholate group of siderophores, sharing a 2,3-dihydroxybenzoic acid moiety. Vanchrobactin biosynthesis and transport genes are present in all strains examined although the siderophore is not produced in serotype O1 strains harbouring a pJM1-type plasmid. In these strains the insertion of an IS element in the main vanchrobactin biosynthetic gene vabF leads to the fact that only anguibactin is produced. From our current knowledge we can presume that vanchrobactin is the ancestral siderophore in this species and that the anguibactin-mediated system was later acquired during evolution, likely by horizontal transfer. The role of these two different iron uptake mechanisms in the biology, evolution and ecology of V. anguillarum is discussed although they are still far from being completely understood., (© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Some strains of Vibrio anguillarum, the causative agent of vibriosis in a variety of marine animals, produce a catechol-type siderophore named vanchrobactin. The biosynthetic pathway and regulation of vanchrobactin are quite well understood. However, aspects concerning its entry into the cell have remained uncharacterized. In the present study we characterized two genes, fvtA and orf13, encoding potential TonB-dependent ferric-vanchrobactin receptors in serotype O2 V. anguillarum strain RV22. We found that an fvtA mutant was defective for growth under iron limitation conditions and for utilization of vanchrobactin, suggesting that fvtA encodes the vanchrobactin receptor of V. anguillarum. Interestingly, an orf13 mutant was not significantly affected, and results of reverse transcriptase PCR, as well as analysis of outer membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggested that this gene is not expressed. Furthermore, fatA, a plasmid gene coding for the anguibactin receptor in plasmid pJM1-harboring strains, is also present in the chromosome of RV22, although it is inactivated by insertion of transposases. In addition, we found that FvtA is the route of entry for vanchrobactin analogues, and there is evidence that it recognizes primarily the catechol-iron center. These analogues are potential candidate vectors for a Trojan horse strategy aimed at generating antimicrobial compounds exploiting the same route of entry for native siderophores. We found that fvtA and vanchrobactin biosynthesis genes are ubiquitous in both vanchrobactin- and anguibactin-producing V. anguillarum strains, which reinforces the utility of the vanchrobactin route of entry for the design of future strategies for the control of vibriosis.

A new plasmid designated pAsa6 from an Aeromonas salmonicida subsp. salmonicida strain isolated from diseased turbot has been characterized. pAsa6 consists of 18536bp, has a G+C content of 53.8% and encodes 20 predicted open-reading frames (ORFs). Eight ORFs showed homology to transposases, of which six are complete and two are partial IS sequences. Two ORFs showed homology to replication proteins, and six ORFs showed homology to hypothetical proteins. Two ORFs are truncated homologs of putative A. salmonicida sulfatases. Two genes, aopH and sycH encode homologs of an effector protein for which a role in fish colonization by A. salmonicida has been previously reported, and its chaperone, respectively. The results of filter conjugation experiments suggested that pAsa6 is not mobilizable, as it failed to be conjugally-transferred to several species of marine bacteria tested. All the ORFs of pAsa6 with the exception of four copies of a IS1 transposase gene, have a counterpart in the recently sequenced 155-kb A. salmonicida plasmid pAsa5, suggesting either that pAsa6 is a derivative of pAsa5, or that pAsa5 is the result of the fusion of a pAsa6-like plasmid and a larger plasmid of ca. 135-kb. The pAsa6-encoded repA and aopH genes could be PCR-amplified from strains lacking pAsa6, suggesting presence of a large, possibly pAsa5-like plasmid that was not detected on agarose gels, or the existence of chromosome-integrated plasmid sequences. This study demonstrates that genomic locations for the aopH gene different to pAsa5 or pAsa5-like plasmids exist in A. salmonicida.

In this study we applied the Fur titration assay (FURTA) to isolate iron regulated genes in Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in fish. We have identified genes for siderophore biosynthesis and for ferri-siderophore transport, some of which have been previously described in this species. A gene for a hitherto uncharacterized functional hemin receptor HutE was identified, but its inactivation did not affect significantly the use of hemin as sole iron source, suggesting that redundant genes encoding hemin receptors exist in the A. salmonicida genome. Additional FURTA positive clones contained genes encoding a Flavoprotein, a transcriptional regulator protein, a DNA binding protein Fis, as well as genes encoding putative DapD and Flp/Fap-pilin proteins. A screening of gene distribution demonstrated that all the analyzed strains shared genes for siderophore biosynthesis and transport and for heme utilization, indicating that these two systems of iron acquisition are a conserved trait in this subspecies, whereas other genes are not common to all the isolates. Thus, the Fur regulon of A. salmonicida subsp. salmonicida includes both conserved and differentially occurring genes, suggesting that the genetic diversity within this fish pathogen might be higher than expected.