The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing

A multiplex PCR assay was used to simultaneously detect genes from the five major clinically relevant Campylobacter spp. Those genes selected were hipO and 23S rRNA from Campylobacter jejuni; glyA from each of C. coli, C. lari, and C. upsaliensis; and sapB2 from C. fetus subsp. fetus. The assay was evaluated with 137 clinical and environmental isolates and was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing isolates, even in mixed cultures.

Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include enterohemorrhagic E. coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E. coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control. Those genes detected were stx 1 , stx 2 , stx 2c , stx 2d , stx 2e , stx 2f , EHEC hlyA, and eaeA, as well as rfbE, which encodes the E. coli O157 serotype; fliC, which encodes the E. coli flagellum H7 serotype; and the E. coli 16S rRNA, which was included as an internal control. A total of 129 E. coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated. Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx. Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive. All STEC strains were identified by this procedure. In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method. A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion. The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E. coli while differentiating O157:H7 from non-O157 isolates.

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.

No abstract

The Walkerton, Canada, waterborne outbreak of 2000 resulted from entry of Escherichia coli O157:H7 and Campylobacter spp. from neighboring farms into the town water supply. Isolates of Campylobacter jejuni and Campylobacter coli obtained from outbreak investigations were characterized by phenotypic and genotypic methods, including heat-stable and heat-labile serotyping, phage typing, biotyping, fla–restriction fragment length polymorphism (RFLP) typing, and pulsed-field gel electrophoresis. Two main outbreak strains were identified on the basis of heat-stable serotyping and fla-RFLP typing. These strains produced a limited number of types when tested by other methods. Isolates with types indistinguishable from, or similar to, the outbreak types were found only on one farm near the town of Walkerton, whereas cattle from other farms carried a variety of Campylobacter strains with different type characteristics. Results of these analyses confirmed results from epidemiologic studies and the utility of using several different typing and subtyping methods for completely characterizing bacterial populations.

During the winter of 2000 to 2001, an outbreak due to Salmonella Enteritidis (SE) phage type 30 (PT30), a rare strain, was detected in Canada. The ensuing investigation involved Canadian and American public health and food regulatory agencies and an academic research laboratory. Enhanced laboratory surveillance, including phage typing and pulsed-field gel electrophoresis, was used to identify cases. Case questionnaires were administered to collect information about food and environmental exposures. A case-control study with 16 matched case-control pairs was conducted to test the hypothesis of an association between raw whole almond consumption and infection. Almond samples were collected from case homes, retail outlets, and the implicated processor, and environmental samples were collected from processing equipment and associated farms for microbiological testing. One hundred sixty-eight laboratory-confirmed cases of SE PT30 infection (157 in Canada, 11 in the United States) were identified between October 2000 and July 2001. The case-control study identified raw whole almonds as the source of infection (odds ration, 21.1; 95% confidence interval, 3.6 to infinity). SE PT30 was detected in raw whole natural almonds collected from home, retail, distribution, and warehouse sources and from environmental swabs of processing equipment and associated farmers' orchards. The frequent and prolonged recovery of this specific organism from a large agricultural area was an unexpected finding and may indicate significant diffuse contamination on these farms. Identification of almonds as the source of a foodborne outbreak is a previously undocumented finding, leading to a North American recall of this product and a review of current industry practices.

e Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide. The need for molecular subtyping methods with enhanced discrimination in the context of surveillance-and outbreak-based epidemiologic investigations of Campylobacter spp. is critical to our understanding of sources and routes of transmission and the development of mitigation strategies to reduce the incidence of campylobacteriosis. We describe the development and validation of a rapid and high-resolution comparative genomic fingerprinting (CGF) method for C. jejuni. A total of 412 isolates from agricultural, environmental, retail, and human clinical sources obtained from the Canadian national integrated enteric pathogen surveillance program (C-EnterNet) were analyzed using a 40-gene assay (CGF40) and multilocus sequence typing (MLST). The significantly higher Simpson's index of diversity (ID) obtained with CGF40 (ID ‫؍‬ 0.994) suggests that it has a higher discriminatory power than MLST at both the level of clonal complex (ID ‫؍‬ 0.873) and sequence type (ID ‫؍‬ 0.935). High Wallace coefficients obtained when CGF40 was used as the primary typing method suggest that CGF and MLST are highly concordant, and we show that isolates with identical MLST profiles are comprised of isolates with distinct but highly similar CGF profiles. The high concordance with MLST coupled with the ability to discriminate between closely related isolates suggests that CFG40 is useful in differentiating highly prevalent sequence types, such as ST21 and ST45. CGF40 is a high-resolution comparative genomics-based method for C. jejuni subtyping with high discriminatory power that is also rapid, low cost, and easily deployable for routine epidemiologic surveillance and outbreak investigations. Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide (20), and most cases are thought to be the direct result of infection by C. jejuni or C. coli (20,43). Risk factors for campylobacteriosis include exposure to contaminated water, milk, and various food products, such as poultry (1,3,6,21). The development and implementation of effective control measures for these pathogens hinge on the identification of sources of infection. Although the ingestion of contaminated food or water and animal contact play a significant role in the epidemiology of campylobacteriosis, efforts to track sources of Campylobacter infection are hampered by the sporadic nature of campylobacteriosis (25), the infrequent association with outbreaks of disease, and widespread reservoirs that include water, livestock, domestic animals, and wildlife (8,17,46,65,67).A number of different molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism analysis of the flagellin gene (flaA RFLP), and the DNA sequencing of the flagellin gene short variable region (flaA SVR), have been used to identify genotypic clusters of Campylobacter in the context of molecular epidemiology (18, 33). More recently, a multilocus sequence typing (ML...