Multiple-Locus Variable-Number Tandem Repeat Analysis for Strain Discrimination of Non-O157 Shiga Toxin-Producing Escherichia coli

Introduction: Non-O157 Shiga toxin-producing Escherichia coli (STEC) are emerging pathogens of growing concern worldwide.  Rapid and sensitive strain discrimination methods are critical for quick identification of outbreaks and fast traceback to the contamination source.  

Purpose: The objective of this study was to develop a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) assay for intra- and inter-serogroup discrimination of 6 major non-O157 STEC serogroups: O26, O111, O103, O121, O45, and O145.

Methods: Twelve VNTR loci were selected, amplified in 3 multiplex PCR reactions, and the amplicons were sized using multicolor capillary electrophoresis.  Validation of the developed MLVA method was conducted using 84 clinical isolates of non-O157 STEC.

Results: Sixty-five unique MLVA types were obtained among the 84 clinical non-O157 STEC isolates comprised of geographically diverse sporadic and outbreak related strains.  Four of the 6 serogroups clustered separately in a minimum spanning tree.  The developed MLVA method allowed a higher level of discrimination among serogroups O26, O111, O103, and O121, with discriminatory powers of 1.0, 0.96. 0.97, and 0.97, respectively, than it did for serogroups O45 and O145, with discriminatory powers of 0.90 and 0.82, respectively.  The overall discriminatory power of the MLVA method for 84 non-O157 STEC isolates was 0.99. Compared to pulsed-field gel electrophoresis (PFGE), a higher level of discrimination was observed for serogroup O26.

Significance: The developed non-O157 STEC MLVA method allows inter-and intra-serogroup strain discrimination among 6 major serogroups of non-O157 STEC and provides a discriminatory power similar to slightly better than PFGE.