Functional analysis of non-resistance-factors of the pandemic Extended-spectrum beta -lactamase producing Escherichia coli sequence types ST131 and ST648

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Over the last decade the prevalence of Extended Spectrum Beta Lactamase (ESBL) producing E. coli in human and veterinary medicine has risen dramatically. Phylogenetic analyses via Multilocus sequence typing (MLST) gives evidence of an association of ESBL production to certain sequence types (ST) of E. coli. Within these ESBL sequence types pandemic lineages exist, which can be often found in clinical isolates as well as in wildlife and environmental samples, in the latter case independent from a selective antimicrobial pressure. Exemplary for these lineages are the ESBL sequence types ST131 and ST648, which both show an uncommon combination of antimicrobial resistance and virulence. This might be the underlying reason for their pandemic spread. In this grant proposal we therefore want to elucidate the molecular mechanisms of this phenomenon using the following hypothesis: strains of the lineages ST131 and ST648 posses (i) an increased plasmid uptake (ii) a core genome which interacts with the ESBL plasmids resulting in increased virulence and/or virulence independent adaption to habitats (iii) a core and accessory genome which independent from the ESBL plasmid uptake is responsible for extended habitat features of these strains.The very- and falsification of these hypothesis´ will be based on in silico analyses of a large set of whole genome sequences and plasmids of these pandemic ESBL sequence types. By using an in vivo screening in the avian gut , the natural habitat, wildtype ESBL strains will be chosen, which will be later used for the generation of deletion mutants of metabolic and virulence genes. The selection of these candidate genes will be based on results of the genome, transcriptom (RNA Sequencing) and phenotypic (Biolog Macroarray) analysis. The relevance of certain genes in vivo will be revealed using these deletion mutants in a similar in vivo screening with the chicken infection model.