Genome-wide association studies of Shigella spp. and Enteroinvasive Escherichia coli isolates demonstrate an absence of genetic markers for prediction of disease severity

Abstract

Background: We investigated the association of symptoms and disease severity of shigellosis patients with genetic determinants of infecting Shigella and entero-invasive Escherichia coli (EIEC), because determinants that predict disease outcome per individual patient could be used to prioritize control measures. For this purpose, genome wide association studies (GWAS) were performed using presence or absence of single genes, combinations of genes, and k-mers. All genetic variants were derived from draft genome sequences of isolates from a multicenter cross-sectional study conducted in the Netherlands during 2016 and 2017. Clinical data of patients consisting of binary/dichotomous representation of symptoms and their calculated severity scores were also available from this study. To verify the suitability of the methods used, the genetic differences between the genera Shigella and Escherichia were used as control.

Results: The isolates obtained were representative of the population structure encountered in other Western European countries. No association was found between single genes or combinations of genes and separate symptoms or disease severity scores. Our benchmark characteristic, genus, resulted in eight associated genes and > 3,000,000 k-mers, indicating adequate performance of the algorithms used.

Conclusions: To conclude, using several microbial GWAS methods, genetic variants in Shigella spp. and EIEC that can predict specific symptoms or a more severe course of disease were not identified, suggesting that disease severity of shigellosis is dependent on other factors than the genetic variation of the infecting bacteria. Specific genes or gene fragments of isolates from patients are unsuitable to predict outcomes and cannot be used for development, prioritization and optimization of guidelines for control measures of shigellosis or infections with EIEC.

Keywords: Disease control guidelines; Disease severity; E. coli; EIEC; Escherichia coli; GWAS; Shigella; Shigellosis; Symptoms.

Fig. 1

Phylogenetic tree based on core genome SNPs with species indication, underlying diseases and severity scores. Within the salmon squares are the main lineages or phylogroups depicted. wzx6 = S. flexneri serotype 6. PGx = phylogenetic group of S. flexneri. STxxx = Warwick sequence type of EIEC. II and III = S. sonnei lineage II and III

Fig. 2

Results of Scoary: the expected versus the observed log transformed p-values. Lilac lines indicate the outcomes of the permutation dataset. a. Best comparison test for association of gene presence/absence with de Wit severity score. b. Best comparison test for association of gene presence/absence with Modified Vesikari score. c. Best comparison test for association of gene presence/absence with symptoms. d. Benjamini Hochberg’s test for association of gene presence/absence with genus

Fig. 3

Blast result of k-mers resulting consensus on used isolates. a. Blast results versus severity score. b. Histogram of the relative frequency of the severity scores in the dataset versus the severity score of de Wit, displayed for three bit-score categories