Population analysis of O26 Shiga Toxin-producing Escherichia coli causing Haemolytic Uraemic Syndrome in Italy, 1989-2020. undefined

Shiga toxin-producing Escherichia coli (STEC) belonging to O26 serogroup represent an important cause of Haemolitic Uraemic Syndrome (HUS) worldwide and the incidence of severe cases has been steadily increasing in the last decade in many countries and particularly in Europe. Cattle represent an important natural reservoir and the transmission to humans is mainly occurring by the ingestion of contaminated food or contact with wild animals. However, person to person transmission has also been reported. Novel O26 STEC clones have been described in the last decade, interestingly showing different combination of virulence genes. For example, the highly virulent ST29 stx2a-positive clone which emerged in Germany in mid-1990s and rapidly spread throughout Europe harboured ehxA and etpD plasmidborne virulence genes, while the ST29 stx2d-positive clone recently described as emerging in France instead completely lacked the virulence plasmid. In Italy O26 has been the most frequently detected STEC serogroup from HUS cases in the paediatric population since the late 1990s. In this work we describe O26 STEC strains isolated from cases of HUS and severe disease in Italy in a thirty years time span, for a total of 144 strains analysed. In detail, we report the genomics characterization of the strains performed through the Galaxy system developed at Sciensano Institute and further investigate on the population structure through Hierarchical Clustering on Principal Components (HCPC), performed by analysing the presence of genetic features including accessory virulence genes, antimicrobial resistance genes and plasmid replicon. All the strains isolated in the period 1989-1998 were typed as ST21 (n=89), while ST29 strains (n=59) started to be isolated from 1999. The HCPC approach allowed to classify the strains in six clusters each grouping 2, 13, 39, 63, 16 and 10 strains, with one outlier, highlighting specific assets of plasmidborne features significally associated with some of the clusters. The virulence genes ehxA, katP, espP, etpD and toxB were positively associated with the strains in the largest cluster, mostly belonging to ST21, while the antimicrobial resistance genes dfrA1, aadA1, sul1, sul2, tetA, tetB, strA and strB together with IncFIC, IncFII and other plasmid replicons were positively associated with the cluster composed of 10 ST21 strains. The second largest cluster containing 39 strains, all but one typed as ST29, was instead positively associated with etpD, cba and cma plasdmidborne virulence genes and IncFII replicon. The stx subtype was also found to be positively associated with certain clusters, with the most common profile identified consisting in the presence of stx2a only (n=118), followed by stx1a (n=20), stx1a stx2a (n=4) and stx2d (n=2). In detail, the stx1a genes were associated with clusters grouping ST21 strains. The analysis of the genomic backbone through Core Genome Multi Locus Sequence Typing highlighted that ST21 strains were dispersed in three main large groups showing high numbers of allelic differences, while ST29 strains formed three distinct groups, each corresponding to one HCPC cluster. In detail, one group included 38 ST29 strains differing for maximum 112 allelic differences. Our results showed high conservation of either the core or the accessory genomic fraction in populations of ST29 O26 STEC strains isolated from cases of severe infection in Italy, differently for what observed in ST21 strains, suggesting a different selective pressure could drive the evolution of different populations of these pathogens possibly involving different ecological niches. Future investigation of strains isolated from different sources through HCPC approach might be useful to unveil possible unknown transmission routes for human infection.