Second Generation Subtyping: A Proposed PulseNet Protocol for Multiple-Locus Variable-Number Tandem Repeat Analysis of Shiga Toxin–ProducingEscherichia coliO157 (STEC O157)

Abstract

Most bacterial genomes contain tandem duplications of short DNA sequences, termed "variable-number tandem repeats" (VNTR). A subtyping method targeting these repeats, multiple-locus VNTR analysis (MLVA), has emerged as a powerful tool for characterization of clonal organisms such as Shiga toxin-producing Escherichia coli O157 (STEC O157). We modified and optimized a recently published MLVA scheme targeting 29 polymorphic VNTR regions of STEC O157 to render it suitable for routine use by public health laboratories that participate in PulseNet, the national and international molecular subtyping network for foodborne disease surveillance. Nine VNTR loci were included in the final protocol. They were amplified in three PCR reactions, after which the PCR products were sized using capillary electrophoresis. Two hundred geographically diverse, sporadic and outbreak- related STEC O157 isolates were characterized by MLVA and the results were compared with data obtained by pulsed-field gel electrophoresis (PFGE) using XbaI macrorestriction of genomic DNA. A total of 139 unique XbaI PFGE patterns and 162 MLVA types were identified. A subset of 100 isolates characterized by both XbaI and BlnI macrorestriction had 62 unique PFGE and MLVA types. Although the clustering of isolates by the two subtyping systems was generally in agreement, some discrepancies were observed. Importantly, MLVA was able to discriminate among some epidemiologically unrelated isolates which were indistinguishable by PFGE. However, among strains from three of the eight outbreaks included in the study, two single locus MLVA variants and one double locus variant were detected among epidemiologically implicated isolates that were indistinguishable by PFGE. Conversely, in three other outbreaks, isolates that were indistinguishable by MLVA displayed multiple PFGE types. An additional more extensive multi-laboratory validation of the MLVA protocol is in progress in order to address critical issues such as establishing epidemiologically relevant interpretation guidelines for the MLVA data.

Investigation of a Diffused Outbreak in Yamaguchi Prefecture in 2012 Using Multiple Molecular Typing Methods

“…As reported previously, the discrimination power of MLVA is equal to, or even higher than, that of PFGE (3,4). In the present study, five MLVA profiles were found among seven strains with an identical PFGE pattern, demonstrating that MLVA can discriminate in great detail among epidemiologically unrelated strains with an identical PFGE type.…”

Molecular characterization of Verocytotoxigenic Escherichia coli O157:H7 isolates from Argentina by Multiple-Loci VNTR Analysis (MLVA)

“…In spite of the universal implementation of PFGE as a subtyping method and as a foundation of the PulseNet, this technology suffers from some limitations (3,6,7,22).…”

Detection of microbial food contaminants and their products by capillary electromigration techniques

“…Using labeled primers with different dyes, it is possible to perform multiplex PCR followed by CGE-LIF to provide microbiological identification with less uncertainty than with AGE analysis. This methodology has proven to be a powerful tool for differentiating serovars of S. enterica and Shiga toxin-producing E. coli O157, and therefore, it may be a helpful tool in foodborne disease surveillance programs [74,75].…”