A robust 5 nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10 3 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10 4 CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.

An outbreak that comprised 3,842 cases of human infections with enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 occurred in Germany in May 2011. The high proportion of adults affected in this outbreak and the unusually high number of patients that developed hemolytic uremic syndrome makes this outbreak the most dramatic since enterohemorrhagic E. coli (EHEC) strains were first identified as agents of human disease. The characteristics of the outbreak strain, the way it spread among humans, and the clinical signs resulting from EAHEC infections have changed the way Shiga toxin–producing E. coli strains are regarded as human pathogens in general. EAHEC O104:H4 is an emerging E. coli pathotype that is endemic in Central Africa and has spread to Europe and Asia. EAHEC strains have evolved from enteroaggregative E. coli by uptake of a Shiga toxin 2a (Stx2a)–encoding bacteriophage. Except for Stx2a, no other EHEC-specific virulence markers including the locus of enterocyte effacement are present in EAHEC strains. EAHEC O104:H4 colonizes humans through aggregative adherence fimbrial pili encoded by the enteroaggregative E. coli plasmid. The aggregative adherence fimbrial colonization mechanism substitutes for the locus of enterocyte effacement functions for bacterial adherence and delivery of Stx2a into the human intestine, resulting clinically in hemolytic uremic syndrome. Humans are the only known natural reservoir known for EAHEC. In contrast, Shiga toxin–producing E. coli and EHEC are associated with animals as natural hosts. Contaminated sprouted fenugreek seeds were suspected as the primary vehicle of transmission of the EAHEC O104:H4 outbreak strain in Germany. During the outbreak, secondary transmission (human to human and human to food) was important. Epidemiological investigations revealed fenugreek seeds as the source of entry of EAHEC O104:H4 into the food chain; however, microbiological analysis of seeds for this pathogen produced negative results. The survival of EAHEC in seeds and the frequency of human carriers of EAHEC should be investigated for a better understanding of EAHEC transmission routes.

BackgroundEnterohaemorrhagic E. coli (EHEC) can cause severe disease such as bloody diarrhoea and haemolytic uraemic syndrome in humans. Besides production of Shiga toxins, the presence of LEE (eae-gene) and non-LEE (nle) encoded effector genes harboured on O-islands OI-122, OI-71 and OI-57 is associated with EHEC virulence and their frequency in outbreaks. Genes encoded by the EHEC-plasmid are putative virulence markers of EHEC. EHEC-plasmids, LEE and non-LEE effector genes have also been detected in some strains of enteropathogenic E. coli (EPEC). The objective of this study was to analyze the relationship between EHEC and EPEC for virulence genes encoded by genomic O-islands and by the EHEC-plasmids.ResultsNle genes ent/espL2, nleB and nleE (OI-122), nleA, nleF and nleH1-2 (OI-71), nleG5-2 and nleG6-2 (OI-57), espK (CP-933N) and the EHEC-plasmid encoded genes ehxA, espP, etpD and katP were searched in 73 typical and in 235 atypical enteropathogenic E. coli (EPEC) strains. Typical and atypical EPEC each fall into two clusters. Cluster 1 typical (n = 46) and atypical (n = 129) EPEC strains were characterized by the presence of OI-122 encoded genes and grouped together with 64 investigated EHEC strains. Cluster 2 typical (n = 27) and atypical (n = 106) strains grouped together with 52 LEE-negative, Shiga toxin-producing E. coli (STEC) and with 21 apathogenic E. coli strains. Typical EPEC Cluster 1 strains belonged to serotypes frequently involved in severe illness and outbreaks in children (O111:H2, O114:H2, O55:H6, O127:H6 and O142:H6). Atypical EPEC Cluster 1 strains were characterized by serotypes related to EHEC (O26:H11, O55:H7, O145:H28, O103:H2 and O103:H25).ConclusionThe OI-122 encoded nleB gene was found to be most closely associated with Cluster 1 strains and may serve as a diagnostic tool for the identification of virulent EHEC and EPEC seropathotypes. OI-71 encoded genes nleA, nleF and nleH1-2 are less associated with Cluster 1 strains. EHEC-plasmid, OI-57 and CP-933 associated genes showed only weak similarities with virulent Cluster 1 EHEC and EPEC strains.

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Shiga toxin 2e (Stx2e)-producing strains from food (n ‫؍‬ 36), slaughtered pigs (n ‫؍‬ 25), the environment (n ‫؍‬ 21), diseased pigs (n ‫؍‬ 19), and humans (n ‫؍‬ 9) were investigated for production of Stx2e by enzymelinked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of stx 2e genes in high-and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of stx 2e genes. DNA sequences specific for the int, Q, dam, and S genes of the stx 2e bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (eae), enterohemorrhagic Escherichia coli hemolysin (ehx), or other stx 1 or stx 2 variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.

Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H20, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was