Evidence that fish death after Vibrio vulnificus infection is due to an acute inflammatory response triggered by a toxin of the MARTX family.

Vibrio vulnificus is an emerging zoonotic pathogen associated with fish farms that is capable of causing a hemorrhagic septicemia known as warm-water vibriosis. According to a recent transcriptomic and functional study, the death of fish due to vibriosis is more related to the inflammatory response of the host than to the tissue lesions caused by the pathogen. In this work, we hypothesize that the RtxA1 toxin (a V. vulnificus toxin of the MARTX (Multifunctional Autoprocessing Repeats in Toxin) family) is the key virulence factor that would directly or indirectly trigger this fatal inflammatory response. Our hypothesis was based on previous studies that showed that rtxA1-deficient mutants maintained their ability to colonize and invade, but were unable to kill fish. To demonstrate this hypothesis, we infected eels (model of fish vibriosis) by immersion with a mutant deficient in RtxA1 production and analyzed their transcriptome in blood, red blood cells and white blood cells during early vibriosis (0, 3 and 12 h post-infection). The transcriptomic results were compared with those obtained in the previous study in which eels were infected with the V. vulnificus parental strain, and were functionally validated. Overall, our results confirm that fish death after V. vulnificus infection is due to an acute, early and atypical inflammatory response triggered by RtxA1 in which red blood cells seem to play a central role. These results could be relevant to other vibriosis as the toxins of this family are widespread in the Vibrio genus.

Corrigendum: A Transcriptomic Study Reveals That Fish Vibriosis Due to the Zoonotic Pathogen Vibrio vulnificus Is an Acute Inflammatory Disease in Which Erythrocytes May Play an Important Role.

[This corrects the article DOI: 10.3389/fmicb.2022.852677.].

A multiplex PCR for the detection of Vibrio vulnificus hazardous to human and/or animal health from seafood.

Vibrio vulnificus is a zoonotic pathogen linked to aquaculture that is spreading due to climate change. The pathogen can be transmitted to humans and animals by ingestion of raw shellfish or seafood feed, respectively. The aim of this work was to design and test a new procedure to detect V. vulnificus hazardous to human and/or animal health in food/feed samples. For this purpose, we combined a pre-enrichment step with multiplex PCR using primers for the species and for human and animal virulence markers. In vitro assays with mixed DNA from different Vibrio species and Vibrio cultures showed that the new protocol was 100 % specific with a detection limit of 10 cfu/mL. The protocol was successfully validated in seafood using artificially contaminated live shrimp and proved useful also in pathogen isolation from animals and their ecosystem. In conclusion, this novel protocol could be applied in health risk studies associated with food/feed consumption, as well as in the routine identification and subtyping of V. vulnificus from environmental or clinical samples.

A Transcriptomic Study Reveals That Fish Vibriosis Due to the Zoonotic Pathogen Vibrio vulnificus Is an Acute Inflammatory Disease in Which Erythrocytes May Play an Important Role.

Vibrio vulnificus is a marine zoonotic pathogen associated with fish farms that is considered a biomarker of climate change. Zoonotic strains trigger a rapid death of their susceptible hosts (fish or humans) by septicemia that has been linked to a cytokine storm in mice. Therefore, we hypothesize that V. vulnificus also causes fish death by triggering a cytokine storm in which red blood cells (RBCs), as nucleated cells in fish, could play an active role. To do it, we used the eel immersion infection model and then analyzed the transcriptome in RBCs, white BCs, and whole blood using an eel-specific microarray platform. Our results demonstrate that V. vulnificus triggers an acute but atypical inflammatory response that occurs in two main phases. The early phase (3 h post-infection [hpi]) is characterized by the upregulation of several genes for proinflammatory cytokines related to the mucosal immune response (il17a/f1 and il20) along with genes for antiviral cytokines (il12ß) and antiviral factors (ifna and ifnc). In contrast, the late phase (12 hpi) is based on the upregulation of genes for typical inflammatory cytokines (il1ß), endothelial destruction (mmp9 and hyal2), and, interestingly, genes related to an RNA-based immune response (sidt1). Functional assays revealed significant proteolytic and hemolytic activity in serum at 12 hpi that would explain the hemorrhages characteristic of this septicemia in fish. As expected, we found evidence that RBCs are transcriptionally active and contribute to this atypical immune response, especially in the short term. Based on a selected set of marker genes, we propose here an in vivo RT-qPCR assay that allows detection of early sepsis caused by V. vulnificus. Finally, we develop a model of sepsis that could serve as a basis for understanding sepsis caused by V. vulnificus not only in fish but also in humans.

Baseline Methods for the Parameter Estimation of the Generalized Pareto Distribution.

In the parameter estimation of limit extreme value distributions, most employed methods only use some of the available data. Using the peaks-over-threshold method for Generalized Pareto Distribution (GPD), only the observations above a certain threshold are considered; therefore, a big amount of information is wasted. The aim of this work is to make the most of the information provided by the observations in order to improve the accuracy of Bayesian parameter estimation. We present two new Bayesian methods to estimate the parameters of the GPD, taking into account the whole data set from the baseline distribution and the existing relations between the baseline and the limit GPD parameters in order to define highly informative priors. We make a comparison between the Bayesian Metropolis-Hastings algorithm with data over the threshold and the new methods when the baseline distribution is a stable distribution, whose properties assure we can reduce the problem to study standard distributions and also allow us to propose new estimators for the parameters of the tail distribution. Specifically, three cases of stable distributions were considered: Normal, Lévy and Cauchy distributions, as main examples of the different behaviors of the tails of a distribution. Nevertheless, the methods would be applicable to many other baseline distributions through finding relations between baseline and GPD parameters via studies of simulations. To illustrate this situation, we study the application of the methods with real data of air pollution in Badajoz (Spain), whose baseline distribution fits a Gamma, and show that the baseline methods improve estimates compared to the Bayesian Metropolis-Hastings algorithm.

The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms.

Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, genomic and functional studies to show that other zoonotic clades are in fact present in 4 of the 5 lineages of the species. Further, we associate these clades, most of them previously but incompletely described, with the acquisition of a family of fish virulence plasmids containing genes essential for resistance to the immune system of certain teleosts of interest in aquaculture. Consequently, our results provide several pieces of evidence about the importance of this species as a zoonotic agent linked to fish farms, as well as on the relevance of these artificial environments acting as drivers that accelerate the evolution of the species.

Draft Genome Sequences of Vibrio vulnificus Strains Recovered from Moribund Tilapia.

Potentially zoonotic Vibrio vulnificus strains were isolated from vibriosis outbreaks occurring on eastern Mediterranean tilapia farms between 2016 and 2019. In this work, the draft genome sequences of three representative isolates are presented.

Baseline Methods for Bayesian Inference in Gumbel Distribution.

Usual estimation methods for the parameters of extreme value distributions only employ a small part of the observation values. When block maxima values are considered, many data are discarded, and therefore a lot of information is wasted. We develop a model to seize the whole data available in an extreme value framework. The key is to take advantage of the existing relation between the baseline parameters and the parameters of the block maxima distribution. We propose two methods to perform Bayesian estimation. Baseline distribution method (BDM) consists in computing estimations for the baseline parameters with all the data, and then making a transformation to compute estimations for the block maxima parameters. Improved baseline method (IBDM) is a refinement of the initial idea, with the aim of assigning more importance to the block maxima data than to the baseline values, performed by applying BDM to develop an improved prior distribution. We compare empirically these new methods with the Standard Bayesian analysis with non-informative prior, considering three baseline distributions that lead to a Gumbel extreme distribution, namely Gumbel, Exponential and Normal, by a broad simulation study.

The Effect of the Environmental Temperature on the Adaptation to Host in the Zoonotic Pathogen Vibrio vulnificus.

Vibrio vulnificus is a zoonotic pathogen that lives in temperate, tropical and subtropical aquatic ecosystems whose geographical distribution is expanding due to global warming. The species is genetically variable and only the strains that belong to the zoonotic clonal-complex can cause vibriosis in both humans and fish (being its main host the eel). Interestingly, the severity of the vibriosis in the eel and the human depends largely on the water temperature (highly virulent at 28°C, avirulent at 20°C or below) and on the iron content in the blood, respectively. The objective of this work was to unravel the role of temperature in the adaptation to the host through a transcriptomic and phenotypic approach. To this end, we obtained the transcriptome of a zoonotic strain grown in a minimum medium (CM9) at 20, 25, 28, and 37°C, and confirmed the transcriptomic results by RT-qPCR and phenotypic tests. In addition, we compared the temperature stimulon with those previously obtained for iron and serum (from eel and human, respectively). Our results suggest that warm temperatures activate adaptive traits that would prepare the bacteria for host colonization (metabolism, motility, chemotaxis, and the protease activity) and fish septicemia (iron-uptake from transferrin and production of O-antigen of high molecular weight) in a generalized manner, while environmental iron controls the expression of a host-adapted virulent phenotype (toxins and the production of a protective envelope). Finally, our results confirm that beyond the effect of temperature on the V. vulnificus distribution in the environment, it also has an effect on the infectious capability of this pathogen that must be taken into account to predict the real risk of V. vulnificus infection caused by global warming.

Corrigendum: Phylogeny of Vibrio vulnificus From the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy.

[This corrects the article DOI: 10.3389/fmicb.2017.02613.].

Adaptation to host in Vibrio vulnificus, a zoonotic pathogen that causes septicemia in fish and humans.

Vibrio vulnificus is a siderophilic pathogen spreading due to global warming. The zoonotic strains constitute a clonal-complex related to fish farms that are distributed worldwide. In this study, we applied a transcriptomic and single gene approach and discover that the zoonotic strains bypassed the iron requirement of the species thanks to the acquisition of two iron-regulated outer membrane proteins (IROMPs) involved in resistance to fish innate immunity. Both proteins have been acquired by horizontal gene transfer and are contributing to the successful spreading of this clonal-complex. We have also discovered that the zoonotic strains express a virulent phenotype in the blood of its main susceptible hosts (iron-overloaded humans and healthy eels) by combining a host-specific protective envelope with the common expression of two toxins (VvhA and RtxA1), one of which (RtxA1) is directly involved in sepsis. Finally, we found that both IROMPs are also present in other fish pathogenic species and have recently been transmitted to the phylogenetic lineage involved in human primary sepsis after raw seafood ingestion. Together our results highlight the potential hazard that the aquaculture industry poses to public health, which is of particular relevance in the context of a warming world.

In vitro study of antimicrobial activity on Klebsiella Pneumoniae biofilms in endotracheal tubes.

Effective treatment approaches for biofilms in endotracheal tubes (ETTs) are lacking. In this study, we evaluated the in vitro effects of five antimicrobials against biofilms formed by Klebsiella pneumoniae in ETTs. K. pneumoniae was added to minimal mucin medium prior to inoculation in microtiter plates containing ETT fragments. Biofilm susceptibility was assessed by crystal violet staining. At 24 h, the antimicrobials significantly reduced biofilm formation. At 48 h, all of the antimicrobial agents exhibited significant reductions in biofilm formation, even at concentrations above the minimum inhibitory concentration (MIC). Tigecycline and fosfomycin showed the greatest inhibition capacity, with good activity at concentrations twofold greater than the MIC. K. pneumoniae exhibited excellent biofilm formation ability, with formation in the first 24 h and significantly reduced antimicrobial activity. These results contribute to the establishment of new antibiotic breakpoints for the adequate management of infections associated with biofilm formation. Abbreviations ETT Endotracheal tube MIC Minimum inhibitory concentration MBIC Minimum biofilm inhibitory concentration OD Optical density PBS Phosphate-buffered saline VAP Ventilator-associated pneumonia.

Phylogeny of Vibrio vulnificus from the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy.

Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). L1 comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. L2 is formed by a mixture of Bt1 and Bt2 strains from various sources, including diseased fish, and is related to the aquaculture industry. L3 is also linked to the aquaculture industry and includes Bt3 strains exclusively, mostly related to wound infections or secondary septicemia after farmed-fish handling. Lastly, L4 and L5 include a few strains of Bt1 associated with specific geographical areas. The phylogenetic trees for ChrI and II are not congruent to one another, which suggests that inter- and/or intra-chromosomal rearrangements have been produced along Vv evolution. Further, the phylogenetic trees for each chromosome and the virulence plasmid were also not congruent, which also suggests that pVvbt2 has been acquired independently by different clones, probably in fish farms. From all these clones, the one with zoonotic capabilities (Bt2-Serovar E) has successfully spread worldwide. Based on these results, we propose a new updated classification of the species based on phylogenetic lineages rather than on Bts, as well as the inclusion of all Bt2 strains in a pathovar with the particular ability to cause fish vibriosis, for which we suggest the name "piscis."

Iron and Fur in the life cycle of the zoonotic pathogen Vibrio vulnificus.

In this study, we aimed to analyze the global response to iron in the broad-range host pathogen Vibrio vulnificus under the hypothesis that iron is one of the main signals triggering survival mechanisms both inside and outside its hosts. To this end, we selected a strain from the main zoonotic clonal-complex, obtained a mutant in the ferric-uptake-regulator (Fur), and analyzed their transcriptomic profiles in both iron-excess and iron-poor conditions by using a strain-specific microarray platform. Among the genes differentially expressed, we identified around 250 as putatively involved in virulence and survival-related mechanisms. Then, we designed and performed a series of in vivo and in vitro tests to find out if the processes highlighted by the microarray experiments were in fact under iron and/or Fur control. Our results support the hypothesis that iron acts as a niche marker, not always through Fur, for V. vulnificus controlling its entire life cycle. This ranges from survival in the marine environment, including motility and chemotaxis, to survival in the blood of their hosts, including host-specific mechanisms of resistance to innate immunity. These mechanisms allow the bacterium to multiply and persist inside and between their hosts.

The Fish Pathogen Vibrio vulnificus Biotype 2: Epidemiology, Phylogeny, and Virulence Factors Involved in Warm-Water Vibriosis.

Vibrio vulnificus biotype 2 is the etiological agent of warm-water vibriosis, a disease that affects eels and other teleosts, especially in fish farms. Biotype 2 is polyphyletic and probably emerged from aquatic bacteria by acquisition of a transferable virulence plasmid that encodes resistance to innate immunity of eels and other teleosts. Interestingly, biotype 2 comprises a zoonotic clonal complex designated as serovar E that has extended worldwide. One of the most interesting virulence factors produced by serovar E is RtxA13, a multifunctional protein that acts as a lethal factor for fish, an invasion factor for mice, and a survival factor outside the host. Two practically identical copies of rtxA13 are present in all biotype 2 strains regardless of the serovar, one in the virulence plasmid and the other in chromosome II. The plasmid also contains other genes involved in survival and growth in eel blood: vep07, a gene for an outer membrane (OM) lipoprotein involved in resistance to eel serum and vep20, a gene for an OM receptor specific for eel-transferrin and, probably, other related fish transferrins. All the three genes are highly conserved within biotype 2, which suggests that they are under a strong selective pressure. Interestingly, the three genes are related with transferable plasmids, which emphasizes the role of horizontal gene transfer in the evolution of V. vulnificus in nutrient-enriched aquatic environments, such as fish farms.

Genome-wide SNP-genotyping array to study the evolution of the human pathogen Vibrio vulnificus biotype 3.

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae, horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen.

Polyphyletic origin of Vibrio vulnificus biotype 2 as revealed by sequence-based analysis.

A sequence-based analysis of seven housekeeping and virulence-related genes shows that the species Vibrio vulnificus is subdivided into three phylogenetic lineages that do not correspond with the biotypes and that biotype 2 is polyphyletic. These results support the reclassification of biotype 2 as a pathovar that would group the strains with pathogenic potential to develop vibriosis in fish.

CsrA modulates levels of lipoproteins and key regulators of gene expression critical for pathogenic mechanisms of Borrelia burgdorferi.

Carbon storage regulator A (CsrA) is an RNA binding protein that has been characterized in many bacterial species to play a central regulatory role by modulating several metabolic processes. We recently showed that a homolog of CsrA in Borrelia burgdorferi (CsrA(Bb), BB0184) was upregulated in response to propagation of B. burgdorferi under mammalian host-specific conditions. In order to further delineate the role of CsrA(Bb), we generated a deletion mutant designated ES10 in a linear plasmid 25-negative isolate of B. burgdorferi strain B31 (ML23). The deletion mutant was screened by PCR and Southern blot hybridization, and a lack of synthesis of CsrA(Bb) in ES10 was confirmed by immunoblot analysis. Analysis of ES10 propagated at pH 6.8/37°C revealed a significant reduction in the levels of OspC, DbpA, BBK32, and BBA64 compared to those for the parental wild-type strain propagated under these conditions, while there were no significant changes in the levels of either OspA or P66. Moreover, the levels of two regulatory proteins, RpoS and BosR, were also found to be lower in ES10 than in the control strain. Quantitative real-time reverse transcription-PCR analysis of total RNA extracted from the parental strain and csrA(Bb) mutant revealed significant differences in gene expression consistent with the changes at the protein level. Neither the csrA(Bb) mutant nor the trans-complemented strain was capable of infection following intradermal needle inoculation in C3H/HeN mice at either 10³ or 105 spirochetes per mouse. The further characterization of molecular basis of regulation mediated by CsrA(Bb) will provide significant insights into the pathophysiology of B. burgdorferi.

pilF Polymorphism-based PCR to distinguish vibrio vulnificus strains potentially dangerous to public health.

Vibrio vulnificus is a heterogeneous species that comprises strains virulent and avirulent for humans and fish, and it is grouped into three biotypes. In this report, we describe a PCR-based methodology that allows both the species identification and discrimination of those isolates that could be considered dangerous to public health. Discrimination is based on the amplification of a variable region located within the gene pilF, which seems to be associated with potential human pathogenicity, regardless of the biotype of the strain.