Serotyping of Non-O157 Shiga Toxin-producing Escherichia coli by Single Nucleotide Polymorphisms in gnd

Introduction: Shiga toxin-producing Escherichia coli (STEC) infections may result in serious disease such as hemolytic uremic syndrome, which may lead to kidney failure and death.  The most clinically significant STEC serotypes in the US include O157 and six non-O157 serotypes (i.e., O26, O45, O103, O111, O121, O145), which were recently estimated to cause >70% of non-O157 STEC infections in the US.  The lack of rapid methods to identify and confirm clinically important non-O157 STEC serotypes may contribute to underreporting of STEC related disease and hinder outbreak investigations.

Purpose: The purpose of this study was to develop a rapid and high-throughput molecular serotyping method to group STEC isolates into seven serotypes (i.e., O157 and the six clinically relevant non-O157 serotypes) by interrogating single nucleotide polymorphisms (SNPs) in gnd, which encodes 6-phosphogluconate dehydrogenase.

Methods: A collection of 191 STEC isolates, including isolates belonging to serotypes O103 (n=25), O111 (n=23), O121 (n=24), O145 (n=22), O26 (n=23), O45 (n=22), O157 (n=19), and nine other serotypes (n=33), was assembled and characterized by full sequencing of gnd to identify discriminatory SNPs for molecular serotyping.  A multiplex SNP genotyping assay was developed to interrogate 11 informative gnd SNPs by single base pair extension chemistry and used to characterize the STEC isolate collection assembled here.

Results: Allelic types were assigned to each isolate based on unique combinations of gnd SNPs, as determined by the assay, and polymorphisms were confirmed with DNA sequence data.  Serotype specific allelic types were identified for each of the seven clinically important STEC serotypes, which allowed the differentiation of clinically important STEC serotypes from non-clinically important STEC serotypes.  Three isolates were incorrectly grouped by the SNP assay.

Significance: Molecular serotyping of clinically important STEC isolates by interrogation of 11 SNPs in gnd represents an alternative to traditional serotyping for rapid and high-throughput identification and confirmation of STEC serotypes O26, O45, O103, O111, O121, O145, and O157 for surveillance and epidemiological investigations.