Next Generation Typing Tools

Currently, surveillance for foodborne pathogens in PulseNet laboratories is based on the comparison of fingerprints (patterns) generated by restricting total genomic DNA with restriction endonucleases. The resulting DNA fragments are separated by pulsed-field gel electrophoresis (PFGE) to obtain a fingerprint pattern for each isolate tested. Existing standardized pulsed-field gel electrophoresis (PFGE) are highly discriminatory, allowing isolates to be subtyped according to differences in the banding pattern of genomic restriction fragments. While this technique is currently state-of-the-art, lab-to-lab comparisons are very difficult as strict adherence to standardized experimental conditions and labor-intensive manipulation of data are required in order to obtain comparable results.

To address these limitations, the PulseNet has begun to explore new subtyping methods based on genomic sequence analysis. The new methods are based on DNA sequence analysis, which has several advantages over PFGE:

there is minimal experimental variation in DNA sequence results, allowing direct inter-laboratory comparisons;
DNA sequence analysis has the potential for greater accuracy in determining the likelihood that two or more strains might or might not be related; and,
technologically, DNA sequencing approach is more automatable than PFGE, which would facilitate the acquisition of subtyping data and earlier identification of clusters of foodborne illness.

The first of these, multiple-locus variable number tandem repeat analysis (MLVA), leverages short repeat sequences throughout the bacterial genome. Multiplex PCR and high-resolution fragment analysis allow for subtype identification using differences in copy number across a series of loci. MLVA is currently used for E. coli O157:H7 in several PulseNet laboratories, and protocols for Salmonella, non-O157 STEC, and Listeria are under active development. A draft protocol for subtyping of Shigella sonnei has been developed by the PulseNet participant at Taiwan CDC.

The analysis of single nucleotide polymorphisms (SNPs) is also being evaluated at CDC as a subtyping platform, using patterns of SNPs to identify specific subtypes or characteristics of bacterial isolates. In collaboration with the USDA, PulseNet has also begun to evaluate multi-locus genome (MLGT) for high-resolution subtyping of Listeria spp. The application of these technologies, will compliment existing PFGE-based methods, leading to faster, more accurate data acquisition and an increased likelihood of identifying an outbreak earlier so that effective responses may be implemented.