Does virulence assessment of Vibrio anguillarum using sea bass (Dicentrarchus labrax) larvae correspond with genotypic and phenotypic characterization?

Background: Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. Methodology/Principal Findings: We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog's Phenotype MicroArray (TM) technology and some virulence factor assays. Conclusions/Significance: Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum

Genomic Correlates of Virulence Attenuation in the Deadly Amphibian Chytrid Fungus, Batrachochytrium dendrobatidis.

Emerging infectious diseasespose a significant threat to global health, but predicting disease outcomes for particular species can be complicated when pathogen virulence varies across space, time, or hosts. The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused worldwide declines in frog populations. Not only do Bd isolates from wild populations vary in virulence, but virulence shifts can occur over short timescales when Bd is maintained in the laboratory. We leveraged changes in Bd virulence over multiple generations of passage to better understand mechanisms of pathogen virulence. We conducted whole-genome resequencing of two samples of the same Bd isolate, differing only in passage history, to identify genomic processes associated with virulence attenuation. The isolate with shorter passage history (and greater virulence) had greater chromosome copy numbers than the isolate maintained in culture for longer, suggesting that virulence attenuation may be associated with loss of chromosome copies. Our results suggest that genomic processes proposed as mechanisms for rapid evolution in Bd are correlated with virulence attenuation in laboratory culture within a single lineage of Bd. Moreover, these genomic processes can occur over extremely short timescales. On a practical level, our results underscore the importance of immediately cryo-archiving new Bd isolates and using fresh isolates, rather than samples cultured in the laboratory for long periods, for laboratory infection experiments. Finally, when attempting to predict disease outcomes for this ecologically important pathogen, it is critical to consider existing variation in virulence among isolates and the potential for shifts in virulence over short timescales

Evolution of virulence in opportunistic pathogens: generalism, plasticity, and control

Standard virulence evolution theory assumes that virulence factors are maintained because they aid parasitic exploitation, increasing growth within and/or transmission between hosts. An increasing number of studies now demonstrate that many opportunistic pathogens (OPs) do not conform to these assumptions, with virulence factors maintained instead because of advantages in non-parasitic contexts. Here we review virulence evolution theory in the context of OPs and highlight the importance of incorporating environments outside a focal virulence site. We illustrate that virulence selection is constrained by correlations between these external and focal settings and pinpoint drivers of key environmental correlations, with a focus on generalist strategies and phenotypic plasticity. We end with a summary of key theoretical and empirical challenges to be met for a fuller understanding of OPs

A Shift in Central Metabolism Accompanies Virulence Activation in Pseudomonas aeruginosa.

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach

The effect of treatment on pathogen virulence.

The optimal virulence of a pathogen is determined by a trade-off between maximizing the rate of transmission and maximizing the duration of infectivity. Treatment measures such as curative therapy and case isolation exert selective pressure by reducing the duration of infectivity, reducing the value of duration-increasing strategies to the pathogen and favoring pathogen strategies that maximize the rate of transmission. We extend the trade-off models of previous authors, and represents the reproduction number of the pathogen as a function of the transmissibility, host contact rate, disease-induced mortality, recovery rate, and treatment rate, each of which may be influenced by the virulence. We find that when virulence is subject to a transmissibility-mortality trade-off, treatment can lead to an increase in optimal virulence, but that in other scenarios (such as the activity-recovery trade-off) treatment decreases the optimal virulence. Paradoxically, when levels of treatment rise with pathogen virulence, increasing control efforts may raise predicted levels of optimal virulence. Thus we show that conflict can arise between the epidemiological benefits of treatment and the evolutionary risks of heightened virulence

Helicobacter pylori virulence factors in duodenal ulceration: A primary cause or a secondary infection causing chronicity

Reports from countries with a high prevalence of Helicobacter pylori. (H pylori) infection do not show a proportionately high prevalence of duodenal ulceration, suggesting the possibility that H pylori cannot be a primary cause of duodenal ulceration. It has been mooted that this discrepancy might be explained by variations in the prevalence of virulence factors in different populations. The aim of this paper is to determine whether the published literature gives support to this possibility. The relevant literature was reviewed and analyzed separately for countries with a high and low prevalence of H pylori infection and virulence factors. Although virulent strains of H pylori were significantly more often present in patients with duodenal ulcer than without the disease in countries with a low prevalence of H pylori infection in the population, there was no difference in the prevalence of virulence factors between duodenal ulcer, non - ulcer dyspepsia or normal subjects in many countries, where the prevalence of both H pylori infection and of virulence factors was high. In these countries, the presence of virulence factors was not predictive the clinical outcome. To explain the association between virulence factors and duodenal ulcer in countries where H pylori prevalence is low, only two papers were found that give little support to the usual model proposed, namely that organisms with the virulence factors are more likely than those without them to initiate a duodenal ulcer. We offer an alternative hypothesis that suggests virulence factors are more likely to interfere with the healing of a previously produced ulcer. The presence of virulence factors only correlates with the prevalence of duodenal ulcer in countries where the prevalence of H pylori is low. There is very little evidence that virulence factors initiate duodenal ulceration, but they may be related to failure of the ulcer to heal

Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence

Efflux pumps of the resistance-nodulation-cell-division (RND) family increase antibiotic resistance in many bacterial pathogens, representing candidate targets for the development of antibiotic adjuvants. RND pumps have also been proposed to contribute to bacterial infection, implying that efflux pump inhibitors (EPIs) could also act as anti-virulence drugs. Nevertheless, EPIs are usually investigated only for their properties as antibiotic adjuvants, while their potential anti-virulence activity is seldom taken into account. In this study it is shown that RND efflux pumps contribute to Pseudomonas aeruginosa PAO1 pathogenicity in an insect model of infection, and that the well-characterized EPI Phe-Arg-β-naphthylamide (PAβN) is able to reduce in vivo virulence of the P. aeruginosa PAO1 laboratory strain, as well as of clinical isolates. The production of quorum sensing (QS) molecules and of QS-dependent virulence phenotypes is differentially affected by PAβN, depending on the strain. Transcriptomic and phenotypic analyses showed that the protection exerted by PAβN from P. aeruginosa PAO1 infection in vivo correlates with the down-regulation of key virulence genes (e.g. genes involved in iron and phosphate starvation). Since PAβN impacts P. aeruginosa virulence, anti-virulence properties of EPIs are worthy to be explored, taking into account possible strain-specificity of their activit

Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein – a well known virulence determinant of NDV – but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex

Microfluidics-based liquid chromatography/mass spectrometry multiple reaction monitoring approach for the relative quantification of Burkholderia cenocepacia secreted virulence factors

Rationale: Burkholderia cenocepacia is an opportunistic pathogen that is commonly isolated from patients with cystic fibrosis (CF). Quorum sensing has been suggested to play a role in the activity of type II and type VI secretion systems and the release of virulence factors. Apart from the classical acyl homoserine lactone quorum sensing, B. cenocepacia also uses the diffusible signal factor system (DSF). Quantitative information on the true impact of DSF molecules on the release of ZmpA and other virulence factors is lacking. Methods: Based on results of a label-free proteomics analysis addressing changes in the secretome in response to DSFs, a panel of peptides was selected to develop a microfluidics liquid chromatography/mass spectrometry (LC/MS) method implementing single reaction monitoring (SRM) to quantify B. cenocepacia virulence factors. Results: Increase in secretion of virulence factors upon treatment with BDSF was observed for ZmpA and Aida, but not for ZmpB. Type VI secretion system dependent Hcp1 and TecA were decreased. However, non-physiological amounts of BDSF were needed to provoke the effect. DSFs from P. aeruginosa and S. maltophilia were also affecting virulence factor secretion, but the effect was smaller than for the endogenous BDSF. Conclusions: Microfluidics-based SRM is a useful tool to quantitatively assess the impact of quorum sensing on the release of virulence factors by (opportunistic) pathogens

Virulence behavior of uropathogenic Escherichia coli strains in the host model Caenorhabditis elegans

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Although a number of bacteria can cause UTIs, most cases are due to infection by uropathogenic Escherichia coli (UPEC). UPEC are a genetically heterogeneous group that exhibit several virulence factors associated with colonization and persistence of bacteria in the urinary tract. Caenorhabditis elegans is a tiny, free-living nematode found worldwide. Because many biological pathways are conserved in C. elegans and humans, the nematode has been increasingly used as a model organism to study virulence mechanisms of microbial infections and innate immunity. The virulence of UPEC strains, characterized for antimicrobial resistance, pathogenicity-related genes associated with virulence and phylogenetic group belonging was evaluated by measuring the survival of C. elegans exposed to pure cultures of these strains. Our results showed that urinary strains can kill the nematode and that the clinical isolate ECP110 was able to efficiently colonize the gut and to inhibit the host oxidative response to infection. Our data support that C. elegans, a free-living nematode found worldwide, could serve as an in vivo model to distinguish, among uropathogenic E. coli, different virulence behavior