Galleria mellonella as an infection model to investigate virulence of Vibrio parahaemolyticus

This is the final version. Available from Taylor & Francis via the DOI in this recordNon-toxigenic V. parahaemolyticus isolates (tdh-/trh-/T3SS2-) have recently been isolated from patients with gastroenteritis. In this study we report that the larvae of the wax moth (Galleria mellonella) are susceptible to infection by toxigenic or non-toxigenic clinical isolates of V. parahaemolyticus. In comparison larvae inoculated with environmental isolates of V. parahaemolyticus did not succumb to disease. Whole genome sequencing of clinical non-toxigenic isolates revealed the presence of a gene encoding a nudix hydrolase, identified as mutT. A V. parahaemolyticus mutT mutant was unable to kill G. mellonella at 24 h post inoculation, indicating a role of this gene in virulence. Our findings show that G. mellonella is a valuable model for investigating screening of possible virulence genes of V. parahaemolyticus and can provide new insights into mechanisms of virulence of atypical non-toxigenic V. parahaemolyticus. These findings will allow improved genetic tests for the identification of pathogenic V. parahaemolyticus to be developed and will have a significant impact for the scientific community.This work was partly supported by Wellcome Trust Institutional Strategic Support Fund (WT097835MF), Wellcome Trust Multi User Equipment Award (WT097835MF) Medical Research Council Clinical Infrastructure Funding (MR/M008924/1) and Biotechnology and Biological Sciences Research Council (BBSRC) funding (BB/N016513/1)

Burkholderia pseudomallei Is Spatially Distributed in Soil in Northeast Thailand

Melioidosis is a severe infection caused by the environmental bacterium Burkholderia pseudomallei. Soil sampling is important to identify geographic regions where humans and animals are at risk of exposure. The purpose of this study was to examine a factor that has a major bearing on the accuracy of soil sampling: the spatial distribution of B. pseudomallei in soil of a specified sampling site. Soil sampling was performed using a fixed-interval grid of 100 sampling points in each of two sites (disused land and rice field) in northeast Thailand, and the presence and amount of B. pseudomallei determined using culture. Mapping of the presence and B. pseudomallei count demonstrated that samples taken from areas adjacent to sampling points that were culture positive (negative) for B. pseudomallei were also likely to be culture positive (negative), and samples taken from areas adjacent to sampling points with a high (low) B. pseudomallei count were also likely to yield a high (low) count (spatial autocorrelation). These data were used as the basis for highlighting several pitfalls in current approaches to soil sampling, together with a discussion of the suitability of a range of sampling strategies in different geographical locations and for different study objectives

Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment

The soil dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis, a serious human infection that occurs in Southeast Asia and northern Australia. The purpose of this study was to evaluate the population genetic structure of B. pseudomallei in the environment. To achieve this, we undertook soil sampling and culture for the presence of B. pseudomallei in 100 equally spaced points within an area of disused land in northeast Thailand, and undertook detailed genotyping of primary plate colonies isolated from three independent sampling points. Our results demonstrated that multiple B. pseudomallei genotypes were present within a single soil sample, and that different genotypes were present at independent but nearby sampling points. The B. pseudomallei genetic population was unevenly distributed within a given sample, with a predominant genotype co-existing with several genotypes present as a minority population. We discuss the implications of this structuring of genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies

Inhibitory effects of human neutrophil granules and oxygen radicals on adherence of Candida albicans

The adherence of Candida albicans to dacron fibre microcolumns was significantly suppressed after interaction with human neutrophils. The adherence-inhibiting properties of neutrophils were shown to reside in their cytoplasmic granules and granular enzymes. Oxygen-derived free radicals produced by the respiratory burst may also be responsible, as shown by experiments in which oxygen radicals were generated by the cell-free hypoxanthine-xanthine oxidase system. Dose-response studies with HO and β-glucuronidase demonstrated that lower concentrations of these agents inhibited adherence without affecting viability of C. albicans. These results suggest that interference with adherence mechanisms may be an effective means of host defence by neutrophils against the colonisation of mucosal surfaces by C. albicans

A simple, rapid method for the microassay of adherence of Candida-albicans to nylon fiber

We describe a simple and reproducible assay for adherence of Candida albicans to synthetic surfaces. Suspensions of yeast are placed into nylon fibre microcolumns for 30 min, and the fluid extracted by vacuum pressure of 250 mbar for 1 min. The assay is best performed with a concentration of 5×10 yeasts/ml in phosphate-buffered saline and at a temperature of 25°C (room temperature). Alteration of the structural integrity of C. albicans by heat (75°C for 60 min) or formalin (2% for 24 h) resulted in significant impairment of adherence. Pre-treatment of C. albicans with amphotericin B for 1 h caused a dose-dependent inhibition of adherence. This assay may find application in studies on the mode of action and efficacy of antifungal agents, as well as in basic and applied research on adherence mechanisms and pathogenesis of C. albicans infections

Direct modulation of human neutrophil behaviour by Candida albicans

We examined the direct effect of unopsonized yeast particles of Candida albicans on two aspects of neutrophil behaviour, namely adherence and 3H-deoxyglucose uptake. The data show that brief exposure of C. albicans to human neutrophils resulted in decreased ability of the neutrophils to adhere to Dacron fibre and take up deoxyglucose. This inhibitory effect was further shown to be dependent on yeast concentration and on the integrity of the yeast cell wall. Additional experiments indicate that this effect was direct rather than indirect through soluble mediators in the supernatant. Interference experiments with glucan and mannan, the two major polysaccharide components of the yeast cell wall, suggest that the ligand in direct interaction between C. albicans and neutrophil contains glucan. Finally, it was shown that nonpathogenic species of Candida such as C. krusei, C. parapsilosis and C. guilliermondii did not display neutrophil-modulatory properties while the occasionally pathogenic C. tropicalis did. These results indicate that C. albicans has the ability to circumvent neutrophil defence mechanisms, and may in part explain the propensity of this fungus to cause infection

Effects of the newer antifungal agents (bifonazole, ICI 195,739 and amorolfin) on in vitro phagocytic, lymphocytic and natural-killer cell responses

Amphotericin B and some of the imidazole drugs have been shown to suppress certain neutrophil and lymphocyte functions both in vitro and in vivo. We present here the in vitro effects of: amorolfin, a morpholine derivative; the imidazoles clotrimazole and ketoconazole; the N-substituted imidazole bifonazole and a triazole (ICE 195, 739), on neutrophil and lymphocyte function. All of these drugs inhibited neutrophil random migration, chemotaxis and hexose monophosphate shunt activity. The effects of the drugs on neutrophil adherence, deoxyglucose transport and beta-glucuronidase release were variable while lysozyme release was unaffected. Natural Killer cell cytoxicity was depressed by all drugs tested except for amorolfin. Mitogen-induced lymphocyte blastogenesis was suppressed by all antifungal drugs tested. Similar results were obtained using the mitogens phytohaemagglutinin, concanavalin A and pokeweed mitogen. The mechanism of action of these drugs on these cell functions remains unknown, there may be a correlation between their effects on fungi and their effects on leukocytes. Clearance of systematic fungal infection is heavily dependent on integrity of the cellular immune system and it is clearly undesirable that antifungal drugs have immunosuppressive properties. Further studies are required to determine the in vivo and clinical relevance of our observations