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Enteroaggregative Escherichia coli (EAEC) is defined by aggregative adherence (AA) to HEp-2 cells, where bacteria display adherence to cell surfaces and also to the intervening substratum in a stacked-brick configuration. We previously showed that an AraC homologue designated AggR is required for the expression of plasmid-encoded genes that mediate AA of EAEC strain 042. In this study, we hypothesized that AggR also controls the expression of other virulence determinants in EAEC 042. Using proteomic and microarray analysis, we identified for the first time that AggR activates the expression of chromosomal genes, including 25 contiguous genes ( aaiA–Y), which are localized to a 117 kb pathogenicity island (PAI) inserted at pheU. Many of these genes have homologues in other Gram-negative bacteria and were recently proposed to constitute a type VI secretion system (T6SS). AaiC was identified as a secreted protein that has no apparent homologues within GenBank. EAEC strains carrying in-frame deletions of aaiB, aaiG, aaiO or aaiP still synthesized AaiC; however, AaiC secretion was abolished. Cloning of aai genes into E. coli HB101 suggested that aaiA–P are sufficient for AaiC secretion. A second T6SS was identified within the pheU PAI that secretes a protein unrelated by sequence identity to AaiC. Distribution studies indicated that aaiA and aaiC are commonly found in EAEC isolates worldwide, particularly in strains defined as typical EAEC. These data support the hypothesis that AggR is a global regulator of EAEC virulence determinants, and builds on the hypothesis that T6SS is an importance mediator of pathogenesis. [ABSTRACT FROM AUTHOR]

Enteroaggregative Escherichia coli (EAEC) is emerging as a significant diarrheal pathogen in multiple population groups. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in adults including HIV-positive patients and travelers and has been a cause of food-borne outbreaks in the industrialized world. Current data suggest that one set of virulence elements is not associated with all EAEC strains, but that combinations of multiple factors prevail. Pathogenesis is believed to be initiated with adherence to the terminal ileum and colon in an aggregative, stacked-brick-type pattern by means of one of several different hydrophobic aggregative adherence fimbriae. Some strains of EAEC may then elaborate cytotoxins including the plasmid-encoded toxin and the enterotoxins, EAST1 and ShET1. An AraC homolog termed AggR regulates several genes contributing to fimbrial biogenesis in ‘typical EAEC strains’. AggR has now also been shown to regulate genes on a chromosomal island. Sequencing of the EAEC type strain 042 completed at the Sanger Center has revealed two other chromosomal islands that are being explored for their pathogenetic potential. This article reviews these virulence elements and presents on-going areas of research in EAEC pathogenesis. [ABSTRACT FROM AUTHOR]

The Shiga toxin-producing Escherichia coli (STEC) strains, including those of 0157:H7 and the "big six" serogroups (i.e., sero-groups 026,045,0103, Olll, 0121, and 0145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for 026:H11,045:H2,0103:H2, OHl:H8,0121:H19,0145:H28, and 0157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 0157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each 0157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and Hll, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of 0157:H7 from its 055:H7 ancestor revealed that, over this ~ 7,000-year span, spacer deletion was the primary force generating CRISPR diversity. [ABSTRACT FROM AUTHOR]

Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks. [ABSTRACT FROM AUTHOR]