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A variety of non-O157 EHEC have emerged as serious causes of hemolytic uremic syndrome (HUS) and diarrhea worldwide. The most important non-O157 O serogroups causing one third of the EHEC infections in Germany are O26, O103, O111 and O145. In a previous study we observed cluster formation of O26 and O111 EHEC in one single sequence type complex, STC29. STC29 also harbors aEPEC of the same O serogroups, which differ from EHEC merely in absence of stx-converting bacteriophages. This suggests an ongoing microevolutionary scenario of bidirectionalconversion, in which the phage-encoded stx-gene is transferred between aEPEC and EHEC . In this study we aim to develop a microevolutionary model of aEPEC and EHEC strain conversion of STC29 strains by whole genome sequencing (WGS), concentrating on epidemiologically highly important EHEC lineages of O serogroups O26 and O111. WGS of 99 selected strains (aEPEC (n=20), EHEC (n=79) of human (n=47) and bovine (n=58) origin) was performed, analyzing the maximum common genome (MCG) for single nucleotide polymorphisms (SNPs) as well as the presence of virulence associated genes (VAGs) and the occupation of insertion sites for mobile genetic elements in those strains. The resulting minimum spanning tree of the MCG-based SNPanalysis revealed three distinct clusters. Cluster 1 harbored O111
strains also designated as ST16 with MLST. Interestingly, the distinct Cluster 2 included only O26 aEPEC strains of ST29, while the more heterogeneous Cluster 3 combined EHEC as well as aEPEC strains of O serogroup O26 that were only roughly separated into strains of ST29 and ST21. The analysis of the presences or absence of accessory VAGs confirmed the results of the SNP-analysis, and suggests a parallel evolution of the MCG of those strains and the acquisition of virulence genes. Furthermore, the analysis of insertion sites for mobile genetic elements resulted in a similar relation of the analyzed strains. Our cumulative results of MLST, SNP-analysis of the MCG, the presence of VAGs and occupation of insertion sites led to the development of a microevolutionary model of EHEC of O serogroups O26 and O111, which developed as two distinct lineages from a common aEPEC ancestor of ST29 by lysogenic conversion with stx-converting bacteriophages. None of the analyses revealed a separated grouping of strains based on the host species they had been isolated from. Hence, these strains do not appear to harbor host-specific genomic alterations, neither within the MCG nor in the acquired VAGs, and therefore do not appear to emerge by adaptation to a specific niche, supporting the zoonotic nature of aEPEC and EHEC of these STC29 strains. In conclusion, EHEC belonging to STC29 of O serogroups O26 and O111 originate from a common ancestor; further aEPEC and EHEC of those serogroups share a common phylogeny and are bona fide zoonotic agents.