Epidemiology and comparative analysis of Yersinia in Ireland

Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently records only 3-7 notified cases of yersiniosis per year. At the same time pathogenic Yersinia enterocolitica is recovered from pigs (main source of pathogenic Y. enterocolitica) at the levels similar to that observed in Yersinia endemic countries. Introduction of Yersinia selective culture procedures may increase Yersinia isolation rates. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of appendix and throat swabs. Higher levels of anti-Yersinia seroprevalence than yersiniosis notification rates in endemic countries suggests that most yersiniosis cases are unrecognised by culture. Subsequently, in addition to a prospective culture study of clinical specimens, we carried out serological screening of Irish blood donors and environmental screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past. Y. enterocolitica is a heterogeneous group of microorganisms that comprises strains with different degree of pathogenicity. Although non-pathogenic Y. enterocolitica lack conventional virulence factors, these strains can be isolated from patients with diarrhoea. Insecticidal Toxin Complex (ITC) and Cytolethal Distending Toxins can potentially contribute to the virulence of non-pathogenic Y. enterocolitica in the absence of other virulence factors. We compared distribution of ITC and CDT loci among pathogenic and non-pathogenic Y. enterocolitica. Additionally, to demonstrate potential pathogenicity of non-pathogenic Y. enterocolitica we compared their virulence towards Galleria mellonella larvae (a non-mammalian model of human bacterial infections) with the virulence of highly and mildly pathogenic Y. enterocolitica strains. Surprisingly, virulence of pathogenic and non-pathogenic Y. enterocolitica in Galleria mellonella larvae observed at 37°C did not correlate with their pathogenic potential towards humans. Comparative phylogenomic analysis detects predicted coding sequences (CDSs) that define host-pathogen interactions and hence providing insights into molecular evolution of bacterial virulence. Comparative phylogenomic analysis of microarray data generated in Y. enterocolitica strains isolated in the Great Britain from humans with diarrhoea and domestic animals revealed high genetic heterogeneity of these species. Because of the extensive human, animal and food exchanges between the UK and Ireland the objective of this study was to gain further insight into genetic heterogeneity and relationships among clinical and non-clinical Y. enterocolitica strains of various pathogenic potential isolated in Ireland and Great Britain. No evidence of direct transfer of strains between the two countries was found

Cellular Bioreactivity of Micro- and Nano-Plastic Particles in Oysters

The global usage of plastics has increased dramatically over the last several decades. Polystyrene (PS) is the fourth most common plastic material produced annually due to its many versatile applications. Consequently, there has been a coinciding increase in PS wastes, much of which makes its way into waterways and oceanic habitats. While plastic debris has been shown to adversely affect many marine species as a result of ingestion and entanglement, less is known about the cellular uptake of small-scale plastic particles (nano and micro) by marine invertebrates. In this study, we investigated the potential for uptake of PS nano and micron-sized beads (50 nm and 3 μm) by the Eastern Oyster, Crassostrea virginica. This research was focused on two key issues: (1) how particle size would affect uptake by hepatopancreas (HP) cells in vitro and (2) the difference in uptake of micron and nano particles in vivo between gill and HP tissues. This research confirmed that oysters can accumulate PS beads in their tissues, especially HP tissues. Furthermore, using fluorescent deconvolution microscopy, it was observed that plastic nanoparticles exhibited a much greater propensity for intracellular accumulation in HP cells, primarily into lysosomes via endosomal pathways, indicating the potential for significant bioreactivity and sublethal impacts. While exposures of whole oysters or isolated HP cells to bare PS beads did not cause any significant toxicity (acute or sublethal), nanoplastics are more likely to accumulate intracellularly and to deliver adsorbed toxins directly into cells

Parallel independent evolution of pathogenicity within the genus Yersinia

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Pseudomonas aeruginosa uses multiple receptors for adherence to laminin during infection of the respiratory tract and skin wounds

Pseudomonas aeruginosa efficiently adheres to human tissues, including the lungs and skin, causing infections that are difficult to treat. Laminin is a main component of the extracellular matrix, and in this study we defined bacterial laminin receptors on P. aeruginosa. Persistent clinical P. aeruginosa isolates from patients with cystic fibrosis, wounds or catheter-related urinary tract infections bound more laminin compared to blood isolates. Laminin receptors in the outer membrane were revealed by 2D-immunblotting, and the specificities of interactions were confirmed with ELISA and biolayer interferometry. Four new high-affinity laminin receptors were identified in the outer membrane; EstA, OprD, OprG and PA3923. Mutated bacteria devoid of these receptors adhered poorly to immobilized laminin. All bacterial receptors bound to the heparin-binding domains on LG4 and LG5 of the laminin alpha chain as assessed with truncated laminin fragments, transmission electron microscopy and inhibition by heparin. In conclusion, P. aeruginosa binds laminin via multiple surface receptors, and isolates from lungs of cystic fibrosis patients bound significantly more laminin compared to bacteria isolated from the skin and urine. Since laminin is abundant in both the lungs and skin, we suggest that laminin binding is an important mechanism in P. aeruginosa pathogenesis

A novel vitronectin-binding protein of Pseudomonas aeruginosa for effective infection of the airways

Objectives Pseudomonas aeruginosa is a Gram-negative species that causes chronic and acute infections of the lung, skin, urinary tract and eyes. Most P. aeruginosa isolates are highly resistant to antibiotics and difficult to eradicate due to biofilm formation. The bacterium is known to utilize host proteins by diverse strategies in order to enhance its virulence. Vitronectin is a glycoprotein that is abundant in serum and the extracellular matrix, and is involved in cell adhesion, migration, tissue repair and regulation of the complement cascade. The concentration of vitronectin in the lung reflects the level of inflammation in patients with interstitial lung disease. Furthermore, the production is upregulated in patients with cystic fibrosis, which are often chronically colonised with P. aeruginosa. In this study, we analysed the vitronectin-binding capability of clinical strains and identified the P. aeruginosa surface proteins involved in vitronectin binding. Methods P. aeruginosa clinical isolates (n=64) from the airway (n=36), blood (n=15) and urine (n=13), in addition to the reference strain (PAO1) were analysed in a direct binding assay using [125I]-vitronectin. To identify the vitronectin-binding surface proteins of P. aeruginosa, the outer membrane proteins of PAO1 were separated by 2D-SDS-PAGE and western blotting. Vitronectin binding proteins of P. aeruginosa were recombinantly expressed in Escherichia coli and protein-protein interactions were evaluated by ELISA and flow cytometry. P. aeruginosa transposon mutants obtained from the “P. aeruginosa two-allele library” were analysed for vitronectin binding by [125I]-vitronectin or vitronectin coated to a glass surface. Results Our direct binding assay revealed that P. aeruginosa airway isolates bound significantly more vitronectin in comparison to blood (p=0.02) and urine isolates (p=0.04) (Fig. A). Using an approach consisting of 2D-SDS-PAGE and western blotting, we identified two outer membrane proteins that interacted with vitronectin (Fig. B). Expression of one of those (vitronectin binding protein 1; VnBp1) in an E. coli laboratory strain resulted in VnBp1 on the cell surface, and a vitronectin-binding phenotype. In addition, recombinantly expressed and purified VnBP1 showed a dose-dependent interaction with vitronectin in an ELISA (Fig. C). P. aeruginosa with a transposon insert in the vnBp1 gene bound significantly less vitronectin in comparison to the wild type (p=0.0009). Moreover, vnBp1 deficient mutants also showed significant reduced adherence to vitronectin coated glass slide (p≤0.001) in comparison to the wild type (Fig. D). Conclusions P. aeruginosa isolates cultured from the lung bind significantly more vitronectin in comparison to strains cultured from urine or blood. Vitronectin is recruited at the surface via VnBp1. This mechanism is likely to be of great importance for P. aeruginosa adhesion to the airway epithelial and basal lamina of disrupted airway epithelial cell layer and hence for the colonisation of the respiratory tract

All Yersinia enterocolitica are pathogenic: virulence of phylogroup 1 Y. enterocolitica in a Galleria mellonella infection model.

Yersinia enterocolitica is a zoonotic pathogen and a common cause of gastroenteritis in humans. The species is composed of six diverse phylogroups, of which strains of phylogroup 1 are considered non-pathogenic to mammals due to the lack of the major virulence plasmid pYV, and their lack of virulence in a mouse infection model. In the present report we present data examining the pathogenicity of strains of Y. enterocolitica across all six phylogroups in a Galleria mellonellla model. We have demonstrated that in this model strains of phylogroup 1 exhibit severe pathogenesis with a lethal dose of as low as 10 c.f.u., that this virulence is an active process and that flagella play a major role in the virulence phenotype. We have also demonstrated that the complete lack of virulence in Galleria of the mammalian pathogenic phylogroups is not due to carriage of the pYV virulence plasmid. Our data suggest that all Y. enterocolitica can be pathogenic, which may be a reflection of the true natural habitat of the species, and that we may need to reconsider the eco-evo perspective of this important bacterial species