The wax moth Galleria mellonella as a novel model system to study Enteroaggregative Escherichia coli pathogenesis

Enteroaggregative Escherichia coli (EAEC) comprise of a large, diverse group of diarrheagenic E. coli defined by their characteristically ‘stacked brick’ pattern on HEp-2 cells. EAEC was first associated with children’s diarrhea in developing countries but studies have since shown that EAEC is the cause of both acute and persistent diarrhea in all ages worldwide. Moreover, EAEC has been implicated in numerous outbreaks, most notably the large outbreak in Germany in 2011 with an EAEC strain lysogenized with a prophage harboring a stx2a-converting phage, resulting in 855 cases of hemolytic-uremic syndrome and 54 deaths. The pathogenesis of EAEC is not yet fully understood due to the heterogeneity among strains. EAEC are recognized as a diarrheal pathogen but are also isolated from healthy individuals stressing the need to be able to distinguish pathogenic from nonpathogenic EAEC strains. A variety of putative virulence factors have been identified, although none of these have been present only in the strains isolated from symptomatic patients. Even though the pathogenesis of EAEC is unclear, the 3 following stages have been suggested to occur upon infection; 1) initial adherence to the intestinal mucosa, possible by the aggregative adherence fimbriae (AAFs), 2) biofilm formation and 3) induction of an inflammatory response and the release of toxins. Understanding the complex relationship between the host and the bacterium is a crucial step for revealing the pathogenicity of a certain strain. Although many different animal models have been proposed for this pathogen, none have been able to show all of the clinical manifestation of disease. Thus, there is still an urgent need for a reproducible animal model that is able to show all aspects of EAEC pathogenesis. Recently, larvae of the greater wax moth Galleria mellonella were established as an acceptable model to study bacterial infections caused by several pathogens including Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa and extra-intestinal E. coli. The model has shown many advantages such as decreasing rearing costs, convenient feasibility, ability to carry out experiments at 37 C and most importantly, correlation was observed between the G. mellonella model and well established vertebrate models. In this study, we sought to determine whether G. mellonella would be a suitable model for studying the virulence of EAEC infections. The G. mellonella assays were performed as previously described by Morgan et al. Briefly, bacterial overnight cultures were pelleted by centrifugation (4000 £ g) and washed twice in PBS and finally re-suspended in PBS containing 10 % glucose. Groups of 10 larvae (200– 250 mg) were infected with 10 ml aliquots of serially diluted bacterial suspensions (from 10 to 10 bacterial cells per larvae) by injection with a Hamilton syringe (26 gauge) via the last right proleg. Larvae were incubated at 37 C after infection and survival was monitored for 96 hours. Firstly, we investigated the virulence of 6 well characterized EAEC strains in the larvae model. One of the major challenges with EAEC is that the strains are highly heterogeneous with respect to genomic background, phylogroup, serotype, as well as their virulence genes. All strains tested harbored the 4 classical EAEC virulence factors: the transcriptional factor AggR, the aii island encoding a type VI secretion system, as well as dispersin and the dispersin transporter. Infection of G. mellonella with the EAEC strains resulted in rapid killing of the