Publications by authors named "Manuel L Lemos"

57 Publications

Draft Genome Sequences of Five Vibrio neptunius Strains Isolated from Hatcheries of Bivalve Mollusks.

Microbiol Resour Announc 2021 Apr 29;10(17). Epub 2021 Apr 29.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura y CIBUS-Facultad de Biología, Universidade de Santiago de Compostela, A Coruña, Spain

is a Gram-negative bacterium that has been shown to cause disease in marine bivalve mollusk larvae. Here, we report the draft genome sequences and annotations of five strains isolated from larvae of European oyster () and Manila clam () at hatcheries in Galicia, northwest Spain.
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http://dx.doi.org/10.1128/MRA.00237-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086213PMC
April 2021

The Vibriolysin-Like Protease VnpA and the Collagenase ColA Are Required for Full Virulence of the Bivalve Mollusks Pathogen .

Antibiotics (Basel) 2021 Apr 6;10(4). Epub 2021 Apr 6.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura y CIBUS-Facultad de Biología, Universidade de Santiago de Compostela, 15705 Santiago de Compostela, A Coruña, Spain.

is an important pathogen of bivalve mollusks worldwide. Several metalloproteases have been described as virulence factors in species of that are pathogenic to bivalves, but little is known about the contribution of these potential virulence factors to pathogenesis. In silico analysis of the genome of strain PP-145.98 led to the identification of two hitherto uncharacterized chromosomal loci encoding a probable vibriolysin-like metalloprotease and a putative collagenase, which were designated VnpA and ColA, respectively. Single defective mutants of each gene were obtained in PP-145.98, and the phospholipase, esterase and collagenase activities were studied and compared with those of the wild-type strain. The results showed that the single inactivation of resulted in a 3-fold reduction in phospholipase/esterase activity. Inactivation of reduced the collagenase activity by 50%. Finally, infection challenges performed in oyster larvae showed that Δ and Δ-single mutant strains of -are between 2-3-fold less virulent than the wild-type strain. Thus, the present work demonstrates that the production of both VnpA and ColA is required for the full virulence of the bivalve pathogen .
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http://dx.doi.org/10.3390/antibiotics10040391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067407PMC
April 2021

The marine bivalve molluscs pathogen Vibrio neptunius produces the siderophore amphibactin, which is widespread in molluscs microbiota.

Environ Microbiol 2020 12 1;22(12):5467-5482. Epub 2020 Dec 1.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura y Facultad de Biología-CIBUS, Universidade de Santiago de Compostela, Campus Sur, Santiago de Compostela, Spain.

Amphiphilic siderophores, including amphibactins, are the most abundant siderophores in oceans. Genes putatively encoding the amphibactin system were proposed in some bacteria and homologues of these genes are particularly abundant in multiple bacterial lineages inhabitant of low-iron seawater. However, since no defective mutant strains in any of these genes were studied to date, their role in amphibactin synthesis or uptake was not demonstrated. In this work, an in silico analysis of the genome of the mollusc pathogen Vibrio neptunius leads us to identify a gene cluster (denoted absABDEF) that is predicted to encode an amphibactin-like siderophore and several mutant strains unable to synthesize or use siderophores were constructed. The results showed that genes absABDEF are required for amphibactin synthesis. A comparative chemical analysis of V. neptunius wild type and biosynthesis mutants allowed us to identify a mixture of nine amphibactin forms produced by this bacterium. In addition, the gene abtA is predicted to encode the ferri-amphibactin outer membrane transporter. The prevalence of the amphibactin system in bivalve hemolymph microbiota was also studied. We found that the amphibactin system is widespread in hemolymph microbiota including both commensal and pathogenic bacterial species. Thus, its contribution to bacterial fitness must be more related to environmental persistence than to pathogenicity.
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http://dx.doi.org/10.1111/1462-2920.15312DOI Listing
December 2020

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection.

J Vis Exp 2020 06 16(160). Epub 2020 Jun 16.

Centro de Investigacións Científicas Avanzadas (CICA), Departamento de Química, Facultade de Ciencias, Universidade da Coruña;

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described. Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 ([email protected]) using the Stöber process followed by functionalization with APTES ([email protected]@NH2). Then, feroxamine was conjugated with the [email protected]@NH2 by carbodiimide coupling to give [email protected]@[email protected] The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.
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http://dx.doi.org/10.3791/60842DOI Listing
June 2020

The Expression of Virulence Factors in Is Dually Regulated by Iron Levels and Temperature.

Front Microbiol 2019 15;10:2335. Epub 2019 Oct 15.

Department of Microbiology and Parasitology, Institute of Aquaculture, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

causes a hemorrhagic septicemia that affects cold- and warm-water adapted fish species. The main goal of this work was to determine the temperature-dependent changes in the virulence factors that could explain the virulence properties of for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25°C, the degree of virulence of RV22 is higher at 15°C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of RV22 cultured under low-iron availability at either 25 or 15°C, which would mimic the conditions that finds during colonization of fish cultivated at warm- or cold-water temperatures. The comparative analysis of transcriptomes at high- and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25°C than at 15°C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15°C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25°C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.
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http://dx.doi.org/10.3389/fmicb.2019.02335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803810PMC
October 2019

Outer membrane protein FrpA, the siderophore piscibactin receptor of Photobacterium damselae subsp. piscicida, as a subunit vaccine against photobacteriosis in sole (Solea senegalensis).

Fish Shellfish Immunol 2019 Nov 30;94:723-729. Epub 2019 Sep 30.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, 15782, Spain. Electronic address:

Photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) remains one of the main infectious diseases affecting cultured fish in Mediterranean countries. Diverse vaccine formulations based in the use of inactivated bacterial cells have been used with unsatisfactory results, especially in newly cultured species like sole (Solea senegalensis). In this work, we describe the use of the outer membrane receptor (FrpA) of the siderophore piscibactin produced by Pdp as a novel subunit vaccine against photobacteriosis. FrpA has been cloned and expressed in Escherichia coli under an arabinose-inducible promoter. A recombinant protein (rFrpA) containing the pelB localization signal and a His tag was constructed to obtain a pure native form of the protein from E. coli outer membranes. The immunogenicity of rFrpA, and its protective effect against photobacteriosis, was tested by i.p. injection of 30  μg of the protein, mixed with Freund's adjuvant, in sole fingerlings with two immunizations separated by 30 days. Results showed that using either pure rFrpA or whole cells as immobilized antigens in ELISA assays, rFrpA induces the production of specific antibodies in sole. An experimental infection using fish vaccinated with rFrpA or formalin-killed whole cells of Pdp showed that both groups were protected against Pdp infection at similar levels, with no significant differences, reaching RPS values of 73% and 79%, respectively. Thus, FrpA constitutes a promising antigen candidate for the development of novel more effective vaccines against fish photobacteriosis.
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http://dx.doi.org/10.1016/j.fsi.2019.09.066DOI Listing
November 2019

The Outer Membrane Protein FstC of subsp. Acts as Receptor for Amonabactin Siderophores and Displays a Wide Ligand Plasticity. Structure-Activity Relationships of Synthetic Amonabactin Analogues.

ACS Infect Dis 2019 11 9;5(11):1936-1951. Epub 2019 Oct 9.

Centro de Investigacións Científicas Avanzadas (CICA), Departamento de Química, Facultade de Ciencias , Universidade da Coruña , 15071 A Coruña , Spain.

Amonabactins are a group of four related catecholate siderophores produced by several species of the genus , including and the fish pathogen subsp. Although the gene cluster encoding amonabactin biosynthesis also contains a gene that could encode the ferri-siderophore receptor (), to date there is no experimental evidence to explain its role. In this work, we report the identification of the amonabactins' outer membrane receptor and the determination of the minimal structural parts of these siderophores involved in the molecular recognition by their cognate receptor. The four natural amonabactin forms (P750, T789, P693, and T732) and some mono and biscatecholate amonabactin analogues were chemically synthesized, and their siderophore activity on FstC(+) and FstC(-) strains was evaluated. The results showed that each amonabactin form has quite different growth promotion activity, with P750 and T789 the most active. The outer membrane receptor FstC recognizes more efficiently biscatecholate siderophores in which the length of the linker between the two iron-binding catecholamide units is 15 atoms (P750 and T789) instead of 12 atoms (P693 and T732). Analysis of the siderophore activity of synthetic analogues indicated that the presence of Phe or Trp residues is not required for siderophore recognition. The results together point toward evidence that the amonabactin receptor FstC admits a high degree of ligand plasticity. We also showed that FstC is present in most species, including relevant human and animal pathogens as . From the results obtained, we concluded that the ferri-amonabactin uptake pathway involving the outer membrane transporter FstC possesses a considerable functional plasticity that could be exploited for delivery of antimicrobial compounds into the cell. This would allow the use of the siderophore-based iron uptake mechanisms to combat infections caused by species of the genus .
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http://dx.doi.org/10.1021/acsinfecdis.9b00274DOI Listing
November 2019

The Fish Pathogen Under Iron Deprivation Produces the Siderophore Piscibactin.

Microorganisms 2019 Sep 3;7(9). Epub 2019 Sep 3.

Departamento de Microbiología y Parasitología, CIBUS-Facultad de Biología and Instituto de Acuicultura, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by . Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in , which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in . Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like subsp. and , which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.
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http://dx.doi.org/10.3390/microorganisms7090313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780188PMC
September 2019

The Siderophore Piscibactin Is a Relevant Virulence Factor for Favored at Low Temperatures.

Front Microbiol 2018 2;9:1766. Epub 2018 Aug 2.

Department of Microbiology and Parasitology, Institute of Aquaculture, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

causes vibriosis, a hemorrhagic septicaemia that affects many cultured marine fish species worldwide. Two catechol siderophores, vanchrobactin and anguibactin, were previously identified in this bacterium. While vanchrobactin is a chromosomally encoded system widespread in all pathogenic and environmental strains, anguibactin is a plasmid-encoded system restricted to serotype O1 strains. In this work, we have characterized, from a serotype O2 strain producing vanchrobactin, a novel genomic island containing a cluster of genes that would encode the synthesis of piscibactin, a siderophore firstly described in the fish pathogen subsp. . The chemical characterization of this siderophore confirmed that some strains of produce piscibactin. An analysis of the available genomes showed that this genomic island is present in many of the highly pathogenic strains lacking the anguibactin system. The construction of single and double biosynthetic mutants for vanchrobactin and piscibactin allowed us to study the contribution of each siderophore to iron uptake, cell fitness, and virulence. Although both siderophores are simultaneously produced, piscibactin constitute a key virulence factor to infect fish, while vanchrobactin seems to have a secondary role in virulence. In addition, a transcriptional analysis of the gene cluster encoding piscibactin in showed that synthesis of this siderophore is favored at low temperatures, being the transcriptional activity of the biosynthetic genes three-times higher at 18°C than at 25°C. We also show that iron levels and temperature contribute to balance the synthesis of both siderophores.
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http://dx.doi.org/10.3389/fmicb.2018.01766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083037PMC
August 2018

Secreted Citrate Serves as Iron Carrier for the Marine Pathogen subsp .

Front Cell Infect Microbiol 2017 8;7:361. Epub 2017 Aug 8.

Department of Microbiology and Parasitology, Institute of Aquaculture, University of Santiago de CompostelaSantiago de Compostela, Spain.

subsp () is a 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 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 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 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 secretes endogenous citrate and use it for iron scavenging from the cell environment.
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http://dx.doi.org/10.3389/fcimb.2017.00361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550697PMC
April 2018

Genomic analysis of the marine fish pathogen Photobacterium damselae subsp. piscicida: Insertion sequences proliferation is associated with chromosomal reorganisations and rampant gene decay.

Infect Genet Evol 2017 10 5;54:221-229. Epub 2017 Jul 5.

Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.meegid.2017.07.007DOI Listing
October 2017

Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis.

Int Microbiol 2017 Mar;20(1):1-10

Universidad de Málaga, Andalucía Tech, Departamento de Microbiología, Campus de Teatinos, Málaga, Spain.

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus. [Int Microbiol 20(1): 1-10 (2017)].
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http://dx.doi.org/10.2436/20.1501.01.280DOI Listing
March 2017

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish.

Appl Environ Microbiol 2017 06 17;83(11). Epub 2017 May 17.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Galicia, Spain

subsp. 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 and 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. This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, subsp. 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.
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http://dx.doi.org/10.1128/AEM.00401-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440696PMC
June 2017

A proteomic analysis of the iron response of Photobacterium damselae subsp. damselae reveals metabolic adaptations to iron levels changes and novel potential virulence factors.

Vet Microbiol 2017 Mar 6;201:257-264. Epub 2017 Feb 6.

Department of Microbiology and Parasitology, Institute of Aquaculture, Universidade de Santiago de Compostela, Campus Sur, Santiago de Compostela 15782, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.vetmic.2017.01.040DOI Listing
March 2017

Identification of the Ferric-Acinetobactin Outer Membrane Receptor in Aeromonas salmonicida subsp. salmonicida and Structure-Activity Relationships of Synthetic Acinetobactin Analogues.

ACS Chem Biol 2017 02 28;12(2):479-493. Epub 2016 Dec 28.

Centro de Investigacións Científicas Avanzadas (CICA), Departamento de Química Fundamental, Facultade de Ciencias, Universidade da Coruña , 15071 A Coruña, Spain.

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.
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http://dx.doi.org/10.1021/acschembio.6b00805DOI Listing
February 2017

Unveiling the pan-genome of the SXT/R391 family of ICEs: molecular characterisation of new variable regions of SXT/R391-like ICEs detected in Pseudoalteromonas sp. and Vibrio scophthalmi.

Antonie Van Leeuwenhoek 2016 Aug 26;109(8):1141-52. Epub 2016 May 26.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, 15782, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1007/s10482-016-0716-3DOI Listing
August 2016

Iron assimilation and siderophore production by Vibrio ordalii strains isolated from diseased Atlantic salmon Salmo salar in Chile.

Dis Aquat Organ 2016 Mar;118(3):217-26

Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile.

Vibrio ordalii is the causative agent of vibriosis in several cultured salmonid species worldwide. Despite its impact on aquaculture, relatively little information is available about its virulence factors. The present study demonstrates for the first time that V. ordalii possesses different systems of iron acquisition, one involving siderophore synthesis and another one that uses direct binding of heme to use iron. Using 6 strains of V. ordalii from Atlantic salmon Salmo salar and the V. ordalii type strain, we could demonstrate that all strains could grow in presence of the chelating agent 2,2'-dipyridyl and produced siderophores in solid and liquid media. Cross-feeding assays among V. ordalii strains evidenced variability in the siderophores produced. Bioassays and PCR data suggest that V. ordalii could produce a siderophore with a structure similar to piscibactin, although the production of a second siderophore in certain strains cannot be discarded. Furthermore, all strains were able to use hemin and hemoglobin as the only iron sources, although the cell yield was higher when using hemoglobin. A hemin-binding assay indicated the presence of constitutive heme-binding molecules at the cell surface of V. ordalii. Virulence tests using rainbow trout as a model of infection revealed a clear relationship between iron-uptake ability and pathogenicity in V. ordalii.
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http://dx.doi.org/10.3354/dao02976DOI Listing
March 2016

Two Catechol Siderophores, Acinetobactin and Amonabactin, Are Simultaneously Produced by Aeromonas salmonicida subsp. salmonicida Sharing Part of the Biosynthetic Pathway.

ACS Chem Biol 2015 Dec 22;10(12):2850-60. Epub 2015 Oct 22.

Department of Microbiology and Parasitology, Institute of Aquaculture, Universidade de Santiago de Compostela , Campus Sur, Santiago de Compostela 15782, Spain.

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.
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http://dx.doi.org/10.1021/acschembio.5b00624DOI Listing
December 2015

Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae.

Infect Immun 2015 Nov 24;83(11):4335-48. Epub 2015 Aug 24.

Institut für Medizinische Mikrobiologie und Hygiene, Universitätsmedizin der Johannes Gutenberg-Universität, Mainz, Germany

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.
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http://dx.doi.org/10.1128/IAI.00277-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598394PMC
November 2015

A Transmissible Plasmid-Borne Pathogenicity Island Confers Piscibactin Biosynthesis in the Fish Pathogen Photobacterium damselae subsp. piscicida.

Appl Environ Microbiol 2015 Sep 19;81(17):5867-79. Epub 2015 Jun 19.

Division of Infectious Diseases, Brigham and Women's Hospital and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.

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.
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http://dx.doi.org/10.1128/AEM.01580-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551267PMC
September 2015

Photobacterium damselae subsp. damselae major virulence factors Dly, plasmid-encoded HlyA, and chromosome-encoded HlyA are secreted via the type II secretion system.

Infect Immun 2015 Apr 12;83(4):1246-56. Epub 2015 Jan 12.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Galicia, Spain

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.
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http://dx.doi.org/10.1128/IAI.02608-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363413PMC
April 2015

Evidence for horizontal gene transfer, gene duplication and genetic variation as driving forces of the diversity of haemolytic phenotypes in Photobacterium damselae subsp. damselae.

FEMS Microbiol Lett 2014 Jun 4;355(2):152-62. Epub 2014 Jun 4.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1111/1574-6968.12464DOI Listing
June 2014

Host-nonspecific iron acquisition systems and virulence in the zoonotic serovar of Vibrio vulnificus.

Infect Immun 2014 Feb 2;82(2):731-44. Epub 2013 Dec 2.

Department of Microbiology and Ecology, University of Valencia, Valencia, Spain.

The zoonotic serovar of Vibrio vulnificus (known as biotype 2 serovar E) is the etiological agent of human and fish vibriosis. The aim of the present work was to discover the role of the vulnibactin- and hemin-dependent iron acquisition systems in the pathogenicity of this zoonotic serovar under the hypothesis that both are host-nonspecific virulence factors. To this end, we selected three genes for three outer membrane receptors (vuuA, a receptor for ferric vulnibactin, and hupA and hutR, two hemin receptors), obtained single and multiple mutants as well as complemented strains, and tested them in a series of in vitro and in vivo assays, using eels and mice as animal models. The overall results confirm that hupA and vuuA, but not hutR, are host-nonspecific virulence genes and suggest that a third undescribed host-specific plasmid-encoded system could also be used by the zoonotic serovar in fish. hupA and vuuA were expressed in the internal organs of the animals in the first 24 h of infection, suggesting that they may be needed to achieve the population size required to trigger fatal septicemia. vuuA and hupA were sequenced in strains representative of the genetic diversity of this species, and their phylogenies were reconstructed by multilocus sequence analysis of selected housekeeping and virulence genes as a reference. Given the overall results, we suggest that both genes might form part of the core genes essential not only for disease development but also for the survival of this species in its natural reservoir, the aquatic environment.
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http://dx.doi.org/10.1128/IAI.01117-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911369PMC
February 2014

Photobacterium damselae subsp. damselae, a bacterium pathogenic for marine animals and humans.

Front Microbiol 2013 Sep 25;4:283. Epub 2013 Sep 25.

Institute of Aquaculture, University of Santiago de Compostela Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.3389/fmicb.2013.00283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782699PMC
September 2013

Synergistic and additive effects of chromosomal and plasmid-encoded hemolysins contribute to hemolysis and virulence in Photobacterium damselae subsp. damselae.

Infect Immun 2013 Sep 24;81(9):3287-99. Epub 2013 Jun 24.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Galicia, Spain.

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.
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http://dx.doi.org/10.1128/IAI.00155-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754223PMC
September 2013

Genetic characterization of pPHDP60, a novel conjugative plasmid from the marine fish pathogen Photobacterium damselae subsp. piscicida.

Plasmid 2013 Jul 7;70(1):154-9. Epub 2013 Mar 7.

Department of Microbiology and Parasitology, Institute of Aquaculture and Faculty of Biology, University of Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain.

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.
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http://dx.doi.org/10.1016/j.plasmid.2013.02.004DOI Listing
July 2013

Synthesis and antibacterial activity of conjugates between norfloxacin and analogues of the siderophore vanchrobactin.

Bioorg Med Chem 2013 Jan 29;21(1):295-302. Epub 2012 Oct 29.

Departamento de Química Fundamental, Facultade de Ciencias, Universidade de A Coruña, Campus da Zapateira, A Coruña 15071, Spain.

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.
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http://dx.doi.org/10.1016/j.bmc.2012.10.028DOI Listing
January 2013

Integrating conjugative elements of the SXT/R391 family from fish-isolated Vibrios encode restriction-modification systems that confer resistance to bacteriophages.

FEMS Microbiol Ecol 2013 Feb 10;83(2):457-67. Epub 2012 Oct 10.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Galicia, Spain.

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.
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http://dx.doi.org/10.1111/1574-6941.12007DOI Listing
February 2013

Integrating conjugative elements as vectors of antibiotic, mercury, and quaternary ammonium compound resistance in marine aquaculture environments.

Antimicrob Agents Chemother 2012 May 6;56(5):2619-26. Epub 2012 Feb 6.

Department of Microbiology and Parasitology, Institute of Aquaculture, University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain.

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.
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http://dx.doi.org/10.1128/AAC.05997-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346659PMC
May 2012

The Photobacterium damselae subsp. damselae hemolysins damselysin and HlyA are encoded within a new virulence plasmid.

Infect Immun 2011 Nov 29;79(11):4617-27. Epub 2011 Aug 29.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela 15782, Galicia, Spain.

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.
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http://dx.doi.org/10.1128/IAI.05436-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257948PMC
November 2011