Escherichia coli O157:H7 is a foodborne pathogen distinguished from typical E. coli by the production of Shiga toxins (Stx) and the inability to ferment sorbitol (SOR) and to express beta-glucuronidase (GUD) activity. An allele-specific probe for the GUD gene (uidA) and multilocus enzyme electrophoresis were used to elucidate stages in the evolutionary emergence of E. coli O157: H7. A point mutation at +92 in uidA was found only in O157:H7 and its nonmotile relatives, including a SOR+ O157:H clone implicated in outbreaks of hemolytic-uremic syndrome in Germany. The results support a model in which O157:H7 evolved sequentially from an O55:H7 ancestor, first by acquiring the Stx2 gene and then by diverging into two branches; one became GUD- SOR- , resulting in the O157:H7 clone that spread worldwide, and the other lost motility, leading to the O157:H clone that is an increasing public health problem in Europe.

Our recent studies have shown that the dendritic cell-specific ICAM nonintegrin CD209 (DC-SIGN) specifically binds to the core LPS of Escherichia coli K12 (E. coli), promoting bacterial adherence and phagocytosis. In this current study, we attempted to map the sites within the core LPS that are directly involved in LPS-DC-SIGN interaction. We took advantage of four sets of well-defined core LPS mutants, which are derived from E. coli, Salmonella enterica serovar Typhimurium, Neisseria gonorrhoeae, and Haemophilus ducreyi and determined interaction of each of these four sets with DC-SIGN. Our results demonstrated that N-acetylglucosamine (GlcNAc) sugar residues within the core LPS in these bacteria play an essential role in targeting the DC-SIGN receptor. Our results also imply that DC-SIGN is an innate immune receptor and the interaction of bacterial core LPS and DC-SIGN may represent a primeval interaction between Gram-negative bacteria and host phagocytic cells.

Recent advancements in biotechnology are rapidly altering the diagnostic procedures used in microbiologic analysis of foods. Biochemical identification tests have been miniaturized and automated, making them faster and more economical. Pathogenic bacteria that were previously isolated and identified after labor- and time-intensive enrichment and plating procedures can now be detected by measuring specific physicochemical changes resulting from their growth or metabolic activity. Nucleic acid and antibody-based assays are now used to rapidly and reliably detect pathogenic bacteria in foods. Nevertheless, foods offer unique challenges to the application of these techniques because of their complexity and variety, their interference with the rapid detection methods, and the need to detect pathogenic bacteria when they are present in foods at very low levels. Methods to sequester target pathogenic bacteria from interfering food components and to concentrate them in small volumes are needed to enable the efficient application of rapid detection and identification methods.

Mismatch amplification mutation assay primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, was coupled with primers for the Shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. Analysis of 108 bacteria showed that all Escherichia coli serotype O157:H7 strains were identified simultaneously with the SLT types encoded by these strains.

Rapid assays for Escherichia coli were developed by using the compound 4methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 ,ug/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35°C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45°C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-mostprobable-number assay was superior to violet red bile agar for the detection of heatand chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).

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Molecular characterization and subtyping show genetic diversities within clonal complexes.

Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtyping and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsed-field gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. A variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.