A Highly Unstable and Elusive Plasmid That Encodes the Type III Secretion System Is Necessary for Full Virulence in the Marine Fish Pathogen Photobacterium damselae subsp. piscicida.

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 two-component system RstAB regulates production of a polysaccharide capsule with a role in virulence in the marine pathogen Photobacterium damselae subsp. damselae.

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.

Highly Transferable pAQU-Related Plasmids Encoding Multidrug Resistance Are Widespread in the Human and Fish Pathogen Photobacterium damselae subsp. damselae in Aquaculture Areas in the Black Sea.

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, a generalist pathogen with unique virulence factors and high genetic diversity.

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.

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

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.

Photobacterium damselae subsp. damselae, an Emerging Fish Pathogen in the Black Sea: Evidence of a Multiclonal Origin.

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.

Persistence of Antibiotic Resistant Vibrio spp. in Shellfish Hatchery Environment.

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.

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.

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.

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

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.

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

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 ß-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.

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

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.

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

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.

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

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.

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

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.

Low salinity activates a virulence program in the generalist marine pathogen Photobacterium damselae subsp. damselae.

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.

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

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.

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

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.

Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse.

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.

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. piscicida Cleaves the Peptidoglycan of Potentially Competing Bacteria.

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 ( PhotobacteriumNlpC-like protein 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.

Exposure of the Opportunistic Marine Pathogen Photobacterium damselae subsp. damselae to Human Body Temperature Is a Stressful Condition That Shapes the Transcriptome, Viability, Cell Morphology, and Virulence.

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.