PulseNet USA is the molecular surveillance network for foodborne infections in the United States. Since its inception in 1996, it has been instrumental in detection, investigation and control of numerous outbreaks caused by Shiga toxin-producing Escherichia coli O157:[H7] (STEC O157), Salmonella enterica, Listeria monocytogenes, Shigella spp., and Campylobacter. This paper describes the current status of the network, including the methodologies used and its future possibilities. The currently preferred subtyping method in the network is pulsed-field gel electrophoresis (PFGE), a proven highly discriminatory molecular subtyping method. New simpler sequencebased subtyping methods are under development and validation to complement and eventually replace PFGE. PulseNet is essentially a cluster detection network, but the data in the system will now also be used in attribution analyses of sporadic infections. The PulseNet platform will also be used as a primary tool in preparedness and response to acts of food bioterrorism.

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.

Infections by Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) are the predominant cause of bloody diarrhea and hemolytic uremic syndrome in the United States. In silico comparison of the two complete STEC O157 genomes (Sakai and EDL933) revealed a strikingly high level of sequence identity in orthologous protein-coding genes, limiting the use of nucleotide sequences to study the evolution and epidemiology of this bacterial pathogen. To systematically examine single nucleotide polymorphisms (SNPs) at a genome scale, we designed comparative genome sequencing microarrays and analyzed 1199 chromosomal genes (a total of 1,167,948 bp) and 92,721 bp of the large virulence plasmid (pO157) of eleven outbreak-associated STEC O157 strains. We discovered 906 SNPs in 523 chromosomal genes and observed a high level of DNA polymorphisms among the pO157 plasmids. Based on a uniform rate of synonymous substitution for Escherichia coli and Salmonella enterica (4.7 × 10 −9 per site per year), we estimate that the most recent common ancestor of the contemporary ␤-glucuronidase-negative, non-sorbitolfermenting STEC O157 strains existed ca. 40 thousand years ago. The phylogeny of the STEC O157 strains based on the informative synonymous SNPs was compared to the maximum parsimony trees inferred from pulsed-field gel electrophoresis and multilocus variable numbers of tandem repeats analysis. The topological discrepancies indicate that, in contrast to the synonymous mutations, parts of STEC O157 genomes have evolved through different mechanisms with highly variable divergence rates. The SNP loci reported here will provide useful genetic markers for developing high-throughput methods for fine-resolution genotyping of STEC O157. Functional characterization of nucleotide polymorphisms should shed new insights on the evolution, epidemiology, and pathogenesis of STEC O157 and related pathogens.

Contaminated diced celery caused a baffling, lengthy outbreak of hospital-acquired listeriosis. PCR testing often failed to detect the pathogen, suggesting its reliability should be further evaluated. Listeriosis risk should be considered in fresh produce selections for immunocompromised patients.

Infections caused by foodborne bacterial pathogens continue to be a major public health issue around the world. During the past decade, pulsed-field gel electrophoresis (PFGE) has become the gold standard for molecular subtyping and source tracking of most foodborne bacteria. Owing to problems inherent in PFGE technology, new methods have been developed focusing on DNA sequence-based subtyping. This review discusses the feasibility of using multilocus sequence typing, multiple-locus variable-number tandem repeat analysis, single nucleotide polymorphisms, microarrays, whole genome sequencing and mass spectrometry for subtyping foodborne bacterial pathogens.