Comprehensive Characterization of Escherichia coli O104:H4 Isolated from Patients in the Netherlands

Abstract

In 2011, a Shiga toxin-producing Enteroaggregative Escherichia coli (EAEC Stx2a+) O104:H4 strain caused a serious outbreak of acute gastroenteritis and hemolytic-uremic syndrome (HUS) in Germany. In 2013, E. coli O104:H4 isolates were obtained from a patient with HUS and her friend showing only gastrointestinal complaints. The antimicrobial resistance and virulence profiles of these isolates together with three EAEC Stx2a+ O104:H4 isolates from 2011 were determined and compared. Whole-genome sequencing (WGS) was performed for detailed characterization and to determine genetic relationship of the isolates. Four additional genomes of EAEC Stx2a+ O104:H4 isolates of 2009 and 2011 available on NCBI were included in the virulence and phylogenetic analysis. All E. coli O104:H4 isolates tested were positive for stx2a, aatA, and terD but were negative for escV. All, except one 2011 isolate, were positive for aggR and were therefore considered EAEC. The EAEC Stx2a+ O104:H4 isolates of 2013 belonged to sequence type (ST) ST678 as the 2011 isolates and showed slightly different resistance and virulence patterns compared to the 2011 isolates. Core-genome phylogenetic analysis showed that the isolates of 2013 formed a separate cluster from the isolates of 2011 and 2009 by 27 and 20 different alleles, respectively. In addition, only a one-allele difference was found between the isolate of the HUS-patient and that of her friend. Our study shows that EAEC Stx2a+ O104:H4 strains highly similar to the 2011 outbreak clone in their core genome are still circulating necessitating proper surveillance to prevent further outbreaks with these potentially pathogenic strains. In addition, WGS not only provided a detailed characterization of the isolates but its high discriminatory power also enabled us to discriminate the 2013 isolates from the isolates of 2009 and 2011 expediting the use of WGS in public health services to rapidly apply proper infection control strategies.

Keywords: Shiga toxin-producing E. coli –STEC; antimicrobial resistance; enterohemorrhagic E. coli –EHEC; hemolytic uremic syndrome; outbreak; phylogenetic analysis; whole genome sequencing.

FIGURE 1

Dendrogram of Escherichia coli isolates based on rep-PCR results. Stx2a+ O104:H4 isolates of 2011 and 2013 are separated into two clusters with a similarity of less than 95% whereas the non O104:H4 E. coli isolates are clearly separated with a similarity of less than 80%.

FIGURE 2

Core-genome phylogenetic analysis of E. coli strains. The minimum spanning tree was generated by SeqSphere and based on allelic profiles comparing 3764 alleles present in all analyzed strains and defined as their core genome. Numbers in the lines indicate the number of allele differences between isolates. Black lines indicate minimum distances and red lines connect isolates with more than minimum distances. Different colors represent isolates of different time periods except isolate 381-3, which was isolated in 2013 but marked in a different color as it is stx negative. EAEC 55989 was used as the hypothetical EAEC Stx2a+ O104:H4 progenitor.