Serogrouping of clinically significant Shiga toxin-producing Escherichia coli by single nucleotide polymorphisms in the GND gene



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Shiga toxin-producing Escherichia coli belonging to serogroups O26, O45, O103, O111, O121, and O145 were recently estimated to cause >70% of non-O157 infections in the US. Conventional serotyping of E. coli relies on agglutination of antisera raised against the O-antigen of the approximately 179 reference serogroups followed by typing of the H antigen either with antisera or by a molecular approach. The lack of a standardized methodology to identify non-O157 STEC serogroups as well as less emphasis on the surveillance of non-O157 STEC may contribute to underreporting of STEC related disease and hinder outbreak investigations. The purpose of this study was to develop a rapid and high-throughput molecular serogrouping method to group STEC isolates into the seven serogroups (i.e., O157 and the six clinically relevant non-O157 serogroups) by interrogating single nucleotide polymorphisms (SNPs) in gnd, which encodes 6-phosphogluconate dehydrogenase. A collection of 195 STEC isolates, including isolates belonging to serogroups O26 (n=23), O45 (n=22), O103 (n=25), O111 (n=24), O121 (n=24), O145 (n=24), O157 (n=19), and ten other serogroups (n=34), was assembled and characterized by full sequencing of gnd to identify discriminatory SNPs for molecular serogrouping. A multiplex SNP typing assay was developed to interrogate twelve informative gnd SNPs by single base pair extension chemistry and used to characterize the STEC isolate collection assembled here. SNP 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. O-serogroup-specific SNP types were identified for each of the seven clinically important STEC serogroups, which allowed the differentiation of clinically important STEC serogroups from non-clinically important STEC serogroups. Although serogroups of the big six and O157 had multiple SNP types per O-serogroup, there were no overlapping SNP types between different serogroups. One isolate produced conflicting results for traditional serogrouping and sequence analysis (classical serogrouping identified the strain as O20 while sequencing and SNP typing found the strain carried the predominant O111 allele and SNP type). Molecular serogrouping of clinically important STEC isolates by interrogation of twelve SNPs in gnd represents an alternative to traditional serogrouping for rapid and high-throughput identification of STEC serogroups O26, O45, O103, O111, O121, O145, and O157 for surveillance and epidemiological investigations.



Shiga toxin-producing Escherichia coli (STEC), Serogrouping, Single nucleotide polymorphisms (SNPs)