Identification and functional analysis of virulence determinants of Verocytotoxin-producing Escherichia coli (VTEC)

Abstract

Verocytotoxin(VT)-producing Escherichia coli (VTEC) are important zoonotic pathogens whose natural reservoir is the gastrointestinal tract of ruminants. The transmission of the infections mainly occurs via the ingestion of contaminated food of animal origin. VTEC pathogenicity relies on the production of the VTs and on the action of accessory virulence factors constituting the virulome, which has not been completely identified yet. The main objective of this piece of research was the identification of the genomic structures forming the VTEC virulome. An additional goal was the analysis of their distribution in different VTEC sub-populations. Finally, a function for some of the factors identified and involved in the pathogenetic mechanism has been proposed. The work presented here has been largely based on the genomic comparison of VTEC strains isolated from human and animal sources and held in the collections of the EU RL VTEC and of the collaborating institutions Statens Serum Institut (Copenhagen, DK) and University of Extremadura (Caceres, ES). This approach led to the identification and characterisation of two pathogenicity islands (PAIs) proposed to be part of the virulome of VTEC strains commonly isolated from cases of human disease. The two PAIs harbour genes encoding factors involved in the colonization (adfO, OI-57, Chapter 4 and tia, SE-PAI, Chapter 3) or specifying an allelic variant of the Subtilase cytotoxin (subAB2, SE-PAI, Chapter 3). The identification and characterization of the open reading frames carried by the two PAIs allowed making inference on the function of the encoded proteins and on their role in the pathogenetic process. As a matter of fact, the tia and shiA genes (SE-PAI, Chapter 3) have been proposed to be part of the colonization machinery of VTEC strains lacking the ability to cause attaching-and-effacing (A/E) lesions on the intestinal mucosa, a typical feature of the VTEC associated with the most severe forms of the infection. The products of genes homologues to tia and shiA have been in fact described respectively to have a role in the process of cell invasion used by some enterotoxigenic E. coli strains or in the attenuation of host inflammatory response upon infection of another invasive enteric pathogen: Shigella flexneri. The genomic approach has also been used to investigate on the VT-producing Enteroaggregative E. coli O104:H4 that caused an outbreak in Germany in 2011. This peculiar VTEC is characterised by a rare combination of virulence traits from Enteroaggregative E. coli (EAEC) and VTEC, two different E. coli pathotypes responsible for enteric infections in low-income areas and common in industrialized countries respectively. Before the German outbreak, VTEC O104:H4 had been isolated in a few sporadic cases of infection, sometimes with an epidemiological connection with travel in North Africa. Similarly, the strain that caused the German outbreak was introduced into the EU with fenugreek seeds produced in Egypt. These observations, together with the genomic stability observed for some of the VTEC O104 strain (Chapter 5), led to the hypothesis that such chimeric strains may have emerged in developing countries. The high loads of enteric pathogens causing diarrhoea and lack of wastewater treatment in these geographic regions, cause the diffuse faecal contamination of the environment, particularly of the aquatic ecosystem often shared with ruminants, facilitating the exchange of mobile genetic elements between human and animal E. coli pathotypes and leading to the formation of new combinations of virulence features such as those characterising the VT-producing EAEC O104:H4.