Genome-wide association study of hemolytic uremic syndrome causing Shiga toxin-producing Escherichia coli from Sweden, 1994–2018

Shiga toxin-producing Escherichia coli (STEC) represents a diverse group of E. coli producing one or two different types of Shiga toxin (Stx) [1]. STEC infection causes clinical manifestations ranging from mild, watery diarrhea to bloody diarrhea with severe abdominal pain (hemorrhagic colitis), and potentially fatal hemolytic uremic syndrome (HUS) characterized by the triad of non-immune hemolytic anemia, thrombocytopenia, and acute kidney injury. It has been reported that 5–15% of STEC cases progress to HUS [2, 3]. O157:H7 has been considered as the most common serotype associated with severe disease such as HUS. In recent years, the emerging clinical importance of non-O157 serotypes has been noted primarily due to the improvements in diagnostic tests [4‐6].

The key STEC virulence factor Stx encoded by stx located on bacteriophages can damage intestinal, vascular, and renal cells leading to gastrointestinal and renal diseases [7]. There are two immunologically distinct Stx types, i.e., Stx1 and Stx2, which can be further divided into various subtypes [8]. Different Stx subtypes display dramatic differences in potency [9]. The presence of stx2 especially stx2a subtype (with and without stx2c) correlates highly with the development of HUS, whereas, other Stx1/Stx2 subtypes are linked to mild symptoms [10]. Stx production is essential but not sufficient for STEC virulence. The majority of pathogenic STEC strains, particularly O157:H7 serotype, possess a pathogenicity island known as the locus of enterocyte effacement (LEE), which encodes genes involved in effacement of intestinal epithelial cell microvilli and in intimate adherence between bacteria and the epithelial cell membrane [11]. The major virulence factors encoded on the LEE are intimin (encoded by eae), translocated intimin receptor (tir), and a type III secretion system [11]. STEC strains harbor additional virulence genes that influence their pathogenic potential, such as astA (enteroaggregative E. coli heat-stable toxin 1), toxB (cytotoxin), ehxA (enterohemolysin), and non-LEE encoded adherence genes [12]. The molecular mechanism underlying the pathogenicity among diverse STEC strains remains to be further elucidated.

Previous epidemiological studies have evaluated the risk of development of STEC-associated HUS in correlation to serotypes, stx subtypes, and other virulence factors in STEC strains from Nordic countries such as Finland, Norway, and Denmark [13‐16], with various results. In Sweden, we have previously analyzed a collection of STEC strains from patients with HUS in correlation to clinical outcomes in HUS patients [17] and also strains from STEC-infected patients with/without bloody diarrhea [18], yet, a comparative study between HUS-STEC and non-HUS-STEC strains is lacking in Sweden. Herein, we performed a genome-wide association study on all clinical STEC strains isolated from patients with and without HUS in Sweden between 1994 and 2018, with the aim to identify genetic factors of STEC predicting the potential to cause HUS.

In this study, we performed a genome-wide association study on a large collection of clinical O157 and non-O157 STEC strains from patients with and without HUS in Sweden between 1994 and 2018. O157:H7 can be classified into nine phylogenetically distinct lineages, as determined by single nucleotide polymorphism genotyping; one lineage (clade 8) was found to be associated with more severe disease such as HUS [27, 28]. The majority of clinical human and bovine isolates belonged to the hypervirulent clade 8 in Argentina [29, 30]. Our study showed that strains of O157:H7 serotype, especially those from the clade 8, were most commonly found in patients with HUS in Sweden. An earlier study showed that clade 8 strains were overrepresented among isolates from cattle farms associated with human cases in Sweden [31]. Shiga toxin gene subtypes stx2a and stx2a + stx2c were found to be significantly associated with development of HUS, while stx1a was associated with a reduced risk of HUS, in line with studies from other Nordic countries [13, 14]. Other virulence markers associated with HUS mainly included genes encoding intimin (eae), adherence factors, toxins, and type III secretion system proteins. It should be noted that the association observed between bacterial factors and clinical outcomes does not indicate any causal link. Further studies are warranted to examine the functions of these identified genetic makers and their potential roles in HUS pathogenesis. It is notable that there is great geographical variation in genetic characteristics of pathogenic STEC strains that correlates with disease severity. For instance, a recent study from Finland indicated that eae was not statistically overrepresented in HUS-STEC strains from pediatric patients, while cytolethal distending toxin (CDT) encoding genes cdtA, cdtB, and cdtC were the most discriminative virulence genes overrepresented in the Finnish pediatric HUS-STEC strains [16]. In the present study, we did not find CDT genes overrepresented in Swedish HUS-STEC strains. Moreover, most HUS-associated O157:H7 strains in Finland were non-clade 8, while all HUS-associated O157:H7 with one exception belonged to clade 8 in this study. Another recent study in Argentina demonstrated no relationship between disease severity and serotypes and genotypes of STEC [32]. These data suggest genetic differences of pathogenic STEC strains in different geographical regions and populations (e.g., age and sex). It may also indicate that non-bacterial factors (e.g., human immunity) and/or bacteria-host interaction play a more important role in STEC-associated disease progression. Future large-scale studies with representative strains and clinical data from various geographical regions and populations are essential to gain further insights.

In the present pangenome-wide association study, we identified a large number of accessory genes differentially presented in HUS-STEC and non-HUS-STEC strains. Besides virulence genes mentioned above, other genes that were significantly overrepresented in HUS-STEC strains mainly encode outer membrane proteins, transcriptional regulators, and phage-related proteins. In addition, numbers of significant genes encode hypothetical proteins (HP) whose functions are poorly understood, further studies are needed to characterize these HP genes and to evaluate their potential role in STEC pathogenesis. Whole genome phylogeny and MCA of pangenomes could not separate HUS-STEC strains from non-HUS-STEC strains, which was in line with earlier studies from Finland and Norway [16, 33]. These results suggest that STEC strains from different phylogenetic lineages may independently acquire genes that determine their pathogenicity. It is noteworthy that O157:H7 strains from HUS patients grouped closely, separated from strains from non-HUS patients. Nevertheless, no significant difference in virulence genes was found between O157 strains from patients with and without HUS, and the majority of significant accessory genes identified at pangenome level were functionally uncharacterized. These data support that other factors, e.g., infection dose of pathogen, variations in host innate, and adaptive immunity, may play an important role in STEC pathogenesis and development of HUS. Further study is warranted to elucidate the host factors in correlation to HUS pathogenesis.

In conclusion, our study revealed that STEC strains of O157:H7 serotype especially clade 8 variants were most commonly found in patients with HUS in Sweden. Genetic factors identified as molecular predictor for development of HUS included stx subtype stx2a, stx2a + stx2c, and genes encoding intimin, toxins, secretion system proteins, and transcriptional regulators. Further studies are needed to evaluate the functions of these genes and their role in the development of HUS. Whole genome phylogeny and MCA of pangenomes could not differentiate HUS-STEC strains from non-HUS-STEC strains, suggesting that STEC strains of diverse genetic backgrounds may independently acquire genes that determine their pathogenicity, and that other non-bacterial factors may play a crucial role in the development of HUS, which warrants further investigation.