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

在过去的十年中，产超广谱β-内酰胺酶（ESBL）的E.人类和兽医学中的大肠杆菌数量急剧上升。通过多位点序列分型（MLST）的系统发育分析提供了证据，ESBL的产生与某些序列类型（ST）的E。杆菌在这些ESBL序列类型中存在流行性谱系，其通常可以在临床分离株以及野生动物和环境样品中发现，在后一种情况下，独立于选择性抗微生物压力。这些谱系的示例是ESBL序列类型ST 131和ST 648，它们都显示出抗菌素耐药性和毒力的罕见组合。这可能是它们大流行蔓延的根本原因。因此，在这项资助提案中，我们希望使用以下假设来阐明这种现象的分子机制：谱系ST 131和ST 648的菌株具有（i）增加的质粒摄取（ii）与ESBL质粒相互作用的核心基因组，导致增加的毒力和/或毒力独立适应栖息地（iii）一个独立于ESBL质粒摄取的核心和辅助基因组负责这些菌株的扩展栖息地特征。这些假说的证伪将基于对这些流行性ESBL序列类型的大量全基因组序列和质粒的计算机分析。通过在禽类肠道（自然栖息地）中使用体内筛选，将选择野生型ESBL菌株，其随后将用于产生代谢和毒力基因的缺失突变体。这些候选基因的选择将基于基因组、转录组（RNA测序）和表型（Biolog Macroarray）分析的结果。将使用这些缺失突变体在与鸡感染模型类似的体内筛选中揭示某些基因在体内的相关性。

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.