Whole-Genome Single-Nucleotide Polymorphism (SNP) Analysis Applied Directly to Stool for Genotyping Shiga Toxin-Producing Escherichia coli: an Advanced Molecular Detection Method for Foodborne Disease Surveillance and Outbreak Tracking.

Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and comprehensive results at a cost that is advantageous compared with traditional diagnostic methods. Sequencing pathogens directly from a primary clinical specimen would help circumvent the need for culture and, in the process, substantially shorten the time to diagnosis and public health reporting. Unfortunately, this approach poses significant challenges because of the mixture of multiple sequences from a complex fecal biomass. The aim of this project was to develop a proof of concept protocol for the sequencing and genotyping of Shiga toxin-producing Escherichia coli (STEC) directly from stool specimens. We have developed an enrichment protocol that reliably achieves a substantially higher DNA yield belonging to E. coli, which provides adequate next-generation sequencing (NGS) data for downstream bioinformatics analysis. A custom bioinformatics pipeline was created to optimize and remove non-E. coli reads, assess the STEC versus commensal E. coli population in the samples, and build consensus sequences based on population allele frequency distributions. Side-by-side analysis of WGS from paired STEC isolates and matched primary stool specimens reveal that this method can reliably be implemented for many clinical specimens to directly genotype STEC and accurately identify clusters of disease outbreak when no STEC isolate is available for testing.

Enhanced identification and characterization of non-O157 Shiga toxin-producing Escherichia coli: a six-year study.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are emerging pathogens with the potential to cause serious illness and impact public health due to diagnostic challenges. Between 2005 and 2010, the Wadsworth Center (WC), the public health laboratory of the New York State (NYS) Department of Health, requested that Shiga toxin enzyme immunoassay (EIA)-positive stool enrichment broths and/or stool specimens be submitted by clinical and commercial reference laboratories testing NYS patient specimens. A total of 798 EIA-positive specimens were received for confirmation and serotyping, and additionally a subset of STEC was assessed for the presence of six virulence genes (stx1, stx2, eaeA, hlyA, nleA, and nleB) by real-time polymerase chain reaction. We confirmed 591 specimens as STEC, 164 (28%) as O157 STEC, and 427 (72%) as non-O157 STEC. Of the non-O157 STEC serogroups identified, over 70% were O103, O26, O111, O45, O121, or O145. During this time period, WC identified and characterized a total of 1282 STEC received as E. coli isolates, stool specimens, or EIA broths. Overall, the STEC testing identified 59% as O157 STEC and 41% as non-O157 STEC; however, out of 600 isolates submitted to the WC as E. coli cultures, 543 (90%) were identified as O157 STEC. This report summarizes a 6-year study utilizing enhanced STEC testing that resulted in increased identification and characterization of non-O157 STEC in NYS. Continued utilization of enhanced STEC testing may lead to effective and timely outbreak response and improve monitoring of trends in STEC disease epidemiology.

Multidrug-resistant nontyphoidal Salmonella in New York state's foodborne diseases active surveillance network counties.

With the emergence of multidrug-resistant nontyphoidal (NT) Salmonella, knowledge of resistance patterns is critical for appropriate presumptive treatment. This report describes the prevalence and trends of NT Salmonella antimicrobial susceptibility within the New York State (NYS) Foodborne Diseases Active Surveillance Network (FoodNet). The NYS Department of Health, Wadsworth Center Public Health Laboratory tested all Salmonella isolates from the NYS FoodNet catchment area between May 2003 and December 2007 for antimicrobial susceptibility to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline, nalidixic acid, and ciprofloxacin. Isolate susceptibility results were linked to their corresponding demographic and clinical data and analyzed. Multidrug-resistant isolates were defined as resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline (R-type ACSSuT). Antimicrobial susceptibility for 2189 FoodNet cases (98.5% of total cases) showed 79.6% pansusceptible, 6.9% R-type ACSSuT, and 13.5% resistant to at least one antimicrobial agent but not R-type ACSSuT. Four (0.2%) isolates were resistant to ciprofloxacin. From 2004 to 2007, cases with R-type ACSSuT significantly decreased from 8.7% (37/424) to 4.8% (24/499) (p < 0.01). Serotypes with the highest proportion of R-type ACSSuT included Salmonella Typhimurium 17.9% (79/444), and Salmonella Newport 29.1% (51/175). Among Salmonella Typhimurium isolates, over 40% of the African-American cases (19/46) had R-type ACSSuT isolates, compared with 15.7% of the Caucasian cases (58/369) (p < 0.01). R-type ACSSuT Salmonella Typhimurium cases were hospitalized (41.8%) more frequently than pansusceptible Salmonella Typhimurium cases (24.9%), after controlling for age (p < 0.05). Length of hospitalization was not significantly different. Although R-type ACSSuT NT Salmonella has decreased since 2003 within the NYS FoodNet catchment area, monitoring resistance patterns remains important in identifying emerging resistant strains, vulnerable populations, and determining appropriate presumptive treatment regimens. The higher rate of R-type ACSSuT among the African-American cases requires further study.