Validation of three rapid screening methods for detection of verotoxin-producing Escherichia coli in foods: interlaboratory study.

An interlaboratory study was conducted for the validation of 3 methods for the detection of all verotoxin-producing Escherichia coli (VTEC) in foods. The methods were a multi-analyte 1-step lateral flow immunoassay (LFIA) for detection of E. coli O157 and verotoxin (VT); an enzyme-linked immunosorbent assay targeted against VT1, VT2, and VT2c (VT-ELISA); and a polymerase chain reaction (PCR) method for detection of VT genes (VT-PCR). Aliquots (25 g or 25 mL) of 4 food types (raw minced [ground] beef, unpasteurized milk, unpasteurized apple juice [cider], and salami) were individually inoculated with low numbers (<9 to 375 cells/25 g) of 6 test strains of E. coli (serogroups O26, O103, O111, O145, and O157) with differing VT-producing capabilities. Five replicates for each test strain and 5 uninoculated samples were prepared for each food type. Fourteen participating laboratories analyzed samples using the LFIA, 9 analyzed the samples by ELISA, and 9 by PCR. The LFIA for O157 and VT had a specificity (correct identification of negative samples) of 92 and 94%, respectively, and a sensitivity (correct identification of positive samples) of 94 and 55%, respectively. The VT-ELISA and VT-PCR had a specificity of 98 and 99%, respectively, and a sensitivity of 89 and 72%, respectively.

Phenotypic and genotypic relationship between Campylobacter spp isolated from humans and chickens in Northern Ireland--a comparison of three phenotyping and two genotyping schemes.

Human campylobacteriosis is currently the most common cause of acute bacterial gastroenteritis on the island of Ireland, accounting for over 3,000 laboratory reports per year, where circa 2,000 reports originate from the Republic of Ireland and circa 1,000 reports from Northern Ireland. Elsewhere, consumption of contaminated poultry has been associated with the zoonotic transmission of disease, therefore it was the aim of this study to examine the phenotypic and genotypic relatedness of campylobacters isolated from chickens and humans locally. Sixty isolates were subtyped using phenotyping techniques (biotyping, phage-typing), as well as genotyping techniques (multilocus enzyme electrophoresis (MEE), ribotyping) and the data compared. The frequency of shared phenotypes and genotypes between poultry and humans varied depending on the typing technique employed ranging from 98.2% of human isolates sharing a similar resistotyping (MAST) disc type with poultry strains to 20% similarity with MEE typing. Overall, this small study is the first report on phenotypic and genotypic relatedness between human and poultry campylobacters in Northern Ireland, isolated under controlled conditions. The study demonstrated an association between chicken and human sub-species types, taken from a relatively contained epidemiological environment. Further work is required with larger numbers of isolates coupled with typing schemes, which are able to reliably cluster strains from chicken and humans, which share high degrees of clonality, before local poultry can be conclusively proven to be a significant source of human campylobacteriosis.

A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture.

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.

Molecular characterization of Campylobacter jejuni clones: a basis for epidemiologic investigation.

A total of 814 isolates of the foodborne pathogen Campylobacter jejuni were characterized by multilocus sequence typing (MLST) and analysis of the variation of two cell-surface components: the heat-stable (HS) serotyping antigen and the flagella protein FlaA short variable region. We identified 379 combinations of the MLST loci (sequence types) and 215 combinations of the cell-surface components among these isolates, which had been obtained from human disease, animals, food, and the environment. Despite this diversity, 748 (92%) of the isolates belonged to one of 17 clonal complexes, 6 of which contained many (318, 63%) of the human disease isolates. Several clonal complexes exhibited associations with isolation source or particular cell-surface components; however, the latter were poorly predictive of clonal complex. These data demonstrate that the clonal complex, as defined by MLST, is an epidemiologically relevant unit for both long and short-term investigations of C. jejuni epidemiology.

Prevalence of thermophilic Campylobacter spp. in ready-to-eat foods and raw poultry in Northern Ireland.

Although there have been numerous studies investigating the prevalence of campylobacters in animals and raw meats, there are limited data on the persistence of these organisms in ready-to-eat (RTE) foodstuffs. Although poultry is now well established as a major reservoir of thermophilic campylobacters, it is widely assumed that hazard analysis critical control point (HACCP) controls in commercial and industrial settings are effective in eliminating this hazard through thorough cooking of RTE products. Therefore, it was the primary aim of this study to investigate the effectiveness of HACCP controls in eliminating campylobacters in such cooked RTE foods by attempting to isolate viable organisms from product. Concurrently, the results of this study demonstrate that local poultry is highly contaminated with campylobacters. Commercially available RTE foodstuffs (n = 2,030) consisting of 1,061 poultry-related cooked products and 969 other products were analyzed and were not found to contain thermophilic Campylobacter spp. In addition, 107 raw chickens (63 fresh birds and 44 frozen birds) were sampled, and 94% of the fresh birds and 77% of the frozen birds examined were demonstrated to be contaminated with campylobacters, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari accounting for 69, 30, and 1% of the contaminating organisms, respectively. In general, commercially available RTE foodstuffs, including cooked poultry, are not commonly contaminated with campylobacters and thus do not appear to represent a significant cause of clinical infection of Campylobacter spp. in Northern Ireland. However, raw poultry produce, including fresh and frozen chicken, frequently tested positive for campylobacters. Implementation of HACCP systems by food processors will help to minimize and/or eliminate the risk posed by this organism to the consumer.