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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.

Campylobacter jejuni is a major cause of pediatric diarrhea in developing countries-free-ranging chickens are presumed to be a common source. Campylobacter strains from monthly surveillance and diarrhea cases were compared by means of restriction-fragment length polymorphism (RFLP), rapid amplified polymorphic DNA, and Lior serotyping. RFLP analysis of 156 human and 682 avian strains demonstrated identical strains in chickens and humans in 29 (70.7%) of 41 families, and 35%-39% of human isolates from diarrhea and nondiarrhea cases were identical to a household chicken isolate. Isolation of the same RFLP type from a household chicken and a human within 1 month was highly protective against diarrhea (odds ratio, 0.07; P

A steady increase in the incidence of Guillain-Barré syndrome (GBS) with a seasonal preponderance, almost exclusively related to Campylobacter jejuni , and a rise in the incidence of laboratory-confirmed Campylobacter enteritis have been reported from Curaçao, Netherlands Antilles. We therefore investigated possible risk factors associated with diarrhea due to epidemic C. jejuni . Typing by pulsed-field gel electrophoresis identified four epidemic clones which accounted for almost 60% of the infections. One hundred six cases were included in a case-control study. Infections with epidemic clones were more frequently observed in specific districts in Willemstad, the capital of Curaçao. One of these clones caused infections during the rainy season only and was associated with the presence of a deep well around the house. Two out of three GBS-related C. jejuni isolates belonged to an epidemic clone. The observations presented point toward water as a possible source of Campylobacter infections.

BACKGROUND:Salmonellainfections cause gastrointestinal and systemic diseases worldwide and are the leading causes of food-borne illnesses in North America (1-4).Salmonellaserotype typhimurium (ST), in particular, is increasingly becoming a major public health concern because of its ability to acquire multiple resistant genes (5,6).OBJECTIVE: To describe demographic, temporal and geographical distributions, and reported risk factors of nonoutbreak cases of ST reported to a surveillance system in Ontario.METHODOLOGY: Descriptive analyses were performed on data on salmonellosiscases reported in Ontario between 1990 and 1998. Direct age- and sex-standardized rates were computed, and temporal trend analyses were performed using simple linear regression and a general additive model with alocally weighted regression (LOESS) smoother.RESULTS: The mean annual rates of infections with allSalmonellaserotypes and with ST were 27 cases per 100,000 persons and 3.7 cases per 100,000 persons, respectively. Males and children under five years of age had significantly higher rates of both ST and ST definitive type 104 (DT104) infections. There was also evidence of temporal clustering of all strains ofSalmonella,with significantly more cases being reported during the summer. Significantly higher rates of ST DT104 were observed in urban areas compared with rural areas, suggesting potential differences in the geographical distribution of risk factors.CONCLUSIONS: Information on demographic, temporal and geographical distributions, and risk factors is critical in planning disease control strategies. Further prospective analytical observation studies are needed to gain a better understanding of the epidemiology of ST and ST DT104 in Ontario, which will better guide disease control decisions.

Salmonella enterica subsp. enterica serotype Enteritidis is not readily subtyped beyond the level of phage type (PT). A recently developed method for ribotyping of this organism, which uses a mixture of Pst I and Sph I (PS) for restriction of DNA (PS ribotyping), has proved useful for further subtyping of a number of PTs of this organism, including PT 4. However, it has not been extensively tested with PT 8. In the present study the PS ribotyping method was used to investigate outbreaks of both S. enterica serotype Enteritidis PT 4 and PT 8 and provided subtyping data that were consistent with information obtained from epidemiologic investigations. The method proved to be more discriminatory than phage typing and pulsed-field gel electrophoresis (PFGE) combined and was useful for investigating a pseudo-outbreak involving isolates that had identical PTs and PFGE types but that could not be linked epidemiologically. Several PS ribotypes were found within the cluster of isolates indistinguishable by other subtyping methods, confirming the epidemiologic findings. Although the PS ribotyping method proved to have a superior discriminatory ability in resolving clusters, it did not have high enough throughput for use in outbreak investigations. This method has therefore been adapted for use in automated ribotyping with a RiboPrinter, and the results were compared with those obtained by manual ribotyping. Both methods produce equivalent results and are useful for obtaining epidemiologically relevant subtyping data for S. enterica serotype Enteritidis, including PT 8 strains not extensively tested previously.

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 PCR assay that uses primers whose sequences were obtained from the published sequence of the cdt-III gene was developed to determine the frequencies of the cdt-I , cdt-II , and cdt-III genes in Escherichia coli isolates from humans and animals. E. coli isolates producing cytolethal distending toxin (CDT) were infrequently detected. The cdt-I gene was preferentially detected in strains with the cnf1 gene, while the cdt-III gene was found in strains carrying the cnf2 gene. The cdt-III genotype was more prevalent in animal isolates, while the cdt-I and cdt-II genotypes were more evident in human isolates. The presence of further cdt gene variants was indicated by the presence of toxin activity in cell culture in the absence of PCR amplification of the cdt-I , cdt-II , or cdt-III gene.

OBJECTIVE: To determine the cause of an outbreak ofEscherichia coli0157:H7 related to animal exposures so that further transmission could be prevented.DESIGN: Description of laboratory investigations and a case control study.SETTING: Agricultural pavilion at an annual fair in Ontario.POPULATION: People with laboratory evidence ofE coli0157:H7 (seven people) and others with diarrhea (155 people) who called the health unit following a media release were interviewed. Animals that were accessed most frequently by the public in the agriculture pavilion were tested forE coli0157:H7. In the case control study, a case was defined as someone with laboratory-confirmedE coli0157:H7, or someone who developed severe or bloody diarrhea two to eight days after attending the agricultural pavilion at the fair (61 people). A convenience sample of people who attended the agricultural pavilion but did not develop diarrhea was selected as the control group (89 people).INTERVENTIONS: Human and animalE coli0157:H7 specimens were subtyped. Cases and controls were interviewed using a standardized questionnaire.RESULTS: Subtyping of the seven human isolates ofE coli0157:H7 revealed five that were of an extremely uncommon phage type. Three samples from goats and one from sheep at the petting zoo in the agricultural pavilion were of this same phage type. The case control study also implicated goats (odds ratio [OR] 3.65; 95% CI 1.63 to 8.52) and sheep (OR 2.94; 95% CI 1.33 to 6.57) from the petting zoo.CONCLUSIONS: Results of this investigation suggest strongly that the goats and sheep from the petting zoo were the source of this outbreak ofE coli0157:H7.

In the summer of 1999, the incidence of Salmonella enterica serotype Infantis infections in Alberta rose dramatically. Subsequent laboratory and epidemiological investigations established that an outbreak of human disease caused by this organism was occurring across Canada and was associated with pet treats for dogs produced from processed pig ears. Laboratory investigations using phage typing and pulsed-field gel electrophoresis (PFGE) established that isolates of Salmonella serotype Infantis from pig ear pet treats and humans exposed to pig ear pet treats comprised a well-defined subset of all isolates analyzed. Of the 53 subtypes of Salmonella serotype Infantis obtained around the time of the outbreak as defined by PFGE and phage typing, only 6 subtypes were associated with both human infection and isolation from pig ears. Together with information from epidemiological studies, these investigations established pig ear pet treats as the cause of the Salmonella serotype Infantis outbreak. The results are consistent with a model in which contaminated pig ear pet treats constitute a long-term, continuing vehicle for infection of the human population rather than causing temporally delimited point-source outbreaks. During the course of this outbreak, several other Salmonella serotypes were also isolated from pet treats, suggesting these products may be an important source of enteric infection in both humans and dogs. Though isolates of Salmonella serotypes other than Salmonella serotype Infantis from pet treats were also subjected to PFGE and phage typing, no link with human disease could be definitively established, and the contribution of pig ear pet treats to human disease remains unclear. Elimination of bacterial contamination from pet treats is required to reduce the risk of infection from these products.

To define relationships between Listeria monocytogenes genetic lineages, ribotypes, and serotypes, 235 L. monocytogenes isolates were characterized by serotyping and automated Eco RI ribotyping. Genetic lineage predicted the following serovar clusters: lineage I, comprising serotypes 1/2b, 3b, 3c, and 4b; lineage II, comprising serotypes 1/2a, 1/2c, and 3a; and lineage III, comprising serotypes 4a and 4c. Some Eco RI ribotypes contained multiple serotypes; a subset of these isolates was further differentiated with Pvu II ribotyping. Of the 12 resultant Eco RI- Pvu II combination types, only 4 contained multiple serotypes, demonstrating the potential of ribotyping for serotype prediction.

From 1997 to 1999 seven isolates of Campylobacter -like organisms from five patients that were exhibiting symptoms of gastroenteritis, including fever, stomach malaise, and diarrhea, were investigated. The organisms were isolated from stool samples and found to exhibit a diverse colony morphology; hence multiple isolates were submitted from one of the patients. All isolates were found to be identical. The organisms were catalase, urease, alkaline phosphatase, and nitrate negative but oxidase and indoxyl acetate positive. They grew at 37°C but not at 42°C, and three of the isolates from two different patients were sensitive to nalidixic acid and cephalothin. Full 16S rRNA sequence analysis not only grouped these organisms within the Helicobacter genus but also differentiated them from previously identified Helicobacter species. The closest relative by phylogenetic analysis was Helicobacter sp. flexispira taxon 1. Electron microscopy showed that these isolates had one or two bipolar flagella; however, the periplasmic fibers, a characteristic of the known Helicobacter sp. flexispira taxa, were not observed. The present isolates also lacked a flagellar sheath, a trait shared with four other Helicobacter spp., H. canadensis , H. mesocricetorum , H. pullorum , and H. rodentium . On the basis of the unique phenotypic properties of these isolates and 16S rRNA sequence analysis, we propose the classification of a new Helicobacter species, Helicobacter winghamensis sp. nov.

Canadian statistics show that children from birth to four years of age are more likely to be reported with an infection from Campylobacter, Giardia, Salmonella and Shigella species, and verotoxigenic Escherichia coli than any other age group. A review of the Canadian and international literature, and an analysis of case and outbreak data suggest that the risk factors for infection in young children (ages birth to four years) are different from the risk factors for older children and adults. In children from birth to four years of age, infant formula, fast foods, snacks and candies have caused major outbreaks of enteric and foodborne diseases; however, the contamination of a child's environment or the presence of ill individuals in a household may be highly significant to disease expression. Contact with animals (including family pets) and contaminated surfaces, together with experimental touching and testing behaviours, are important routes of infection for infants and preschool children. Risk factors for enteric infections in children appear to be related, occasionally, to specific foods that are particularly attractive to all children (all age groups from infants up to and including elementary school-aged childen), to an infected person or pet in the same household, or to the contamination of a child's environment. Nonfood-related risk factors may be of particular significance in infection in infants and very young children. Contact with animals, particularly exotic pets and farm animals, or their environments should be considered to be a potential source of infection in children in situations in which there is an absence of other risk factors. The evidence presented in the current paper emphasizes the importance of personal and home hygiene practices in limiting children's exposure to enteric pathogens. Strict hand washing practices and restrictions on touching birds, reptiles and other animals at petting zoos or in nursery and primary school facilities are recommended to avoid widespread infection. Public health authorities should consider the development of guidelines on the provision of hand washing facilities and instruction notices in settings where the public may come into contact with farm or other animals in jurisdictions where such guidelines do not already exist.

We recently analyzed 11 helicobacter isolates cultured from diarrhea patients in Canada. These isolates had been characterized biochemically by restriction fragment length polymorphism (RFLP; Alu I, Hha I) analysis and by fatty-acid analysis as Helicobacter pullorum . However, four of the isolates differed biochemically from H. pullorum by their inability to hydrolyze indoxyl acetate and their resistance to nalidixic acid. Using complete 16S rRNA analysis, we determined that these four strains clustered near H. pullorum but had a sequence difference of 2% and therefore represent a novel helicobacter, Helicobacter canadensis . This novel helicobacter could also be distinguished from H. pullorum by RFLP analysis using Apa LI. The number of novel Helicobacter spp. associated with gastrointestinal disease in humans and animals is rapidly increasing. There are now six Helicobacter spp. isolated from diarrheic humans, the other five being H. pullorum , H. canis , “ H. rappini ,” H. fennelliae , and H. cinaedi . This finding highlights the importance of careful molecular analysis in addition to standard biochemical tests in identifying the increasing number of Helicobacter spp. isolated from humans and animals.

A major Canada-wide outbreak of gastroenteritis due to Salmonella enterica serotype Enteritidis phage type (PT) 8 occurred in 1998, and this was traced to contaminated cheese in a commercial lunch pack product. Phage typing and pulsed-field gel electrophoresis linked the clinical and cheese isolates of serotype Enteritidis but failed to differentiate outbreak from nonoutbreak PT 8 strains. Further differentiation was made by biotyping based on melibiose fermentation.

The adhesion of listeriae to host cells employs mechanisms which are complex and not well understood. Listeria monocytogenes is a facultative intracellular pathogen responsible for meningoencephalitis, septicemia, and abortion in susceptible and immunocompromised individuals. Subsequent to colonization and penetration of the gut epithelium, the organism attaches to resident macrophages and replicates intracellularly, thus evading the humoral immune system of the infected host. The focus of these studies was to investigate the attachment of the organism to murine peritoneal macrophages in an opsonin-dependent and opsonin-independent fashion. Assessment of competitive binding experiments by immunofluorescence and enzyme-linked immunosorbent assays showed that adhesion of the organism to macrophages in the presence or absence of opsonins was inhibited (90%) by N -acetylneuraminic acid (NAcNeu). In addition, the lectin from Maackia amurensis , with affinity for NAcNeu-α(2,3)galactose, blocked binding of L. monocytogenes to host cells. Oxidation of the surface carbohydrates on the organism by using sodium metaperiodate resulted in a dose-dependent reduction (up to 98%) in adherence to macrophages. Monoclonal antibody to complement receptor 3 did not prevent listeriae from binding to mouse macrophages or from replicating within the infected cells whether or not normal mouse serum was present. Based on our results, we propose the involvement of NAcNeu, a member of the sialic acid group, in the attachment of L. monocytogenes to permissive murine macrophages.

Listeria monocytogenes adhered to and multiplied intracellularly in murine peritoneal macrophages in the absence of opsonins. The infective process in these cells was evaluated by viable bacterial cell colony counts of intracellular organisms and documented by transmission and scanning electron microscopy. Adherence of listeriae to macrophages involved surface interactions of the prokaryotic cell surface and eukaryotic cell membranes. Subsequent phagocytosis was seen to occur through a process in which host cell-derived pseudopodia surrounded and engulfed organisms leaving them within phagosomes in the cytoplasm of infected cells. This process of uptake of L. monocytogenes by macrophages occurred at 4 degrees C. Following invasion of the cell, escape of L. monocytogenes from the phagosome into the cytoplasm was initiated as early as 10 min into the infective process. Intracellular multiplication of bacteria continued for 8 h after inoculation at which point loss of adherent macrophages due to cell lysis was evident. The mean generation time of the organism in these cells was 58 min. The cellular and ultrastructural events of L. monocytogenes adherence to and phagocytosis by murine macrophages in the absence of antibody or complement have been defined.

Legionella pneumophila adhered to and multiplied intracellularly in the human histiocytic lymphoma U-937 cell line. The infectious process was evaluated by viable bacterial cell colony counts and documented by transmission and scanning electron microscopy. In the absence of opsonins, wash-resistant bacterial adherence to host cells occurred within 1 h and attachment of 1 or 2 organisms per U-937 host cell involved close surface interactions at the prokaryotic and eukaryotic membranes. Intracellular multiplication of bacteria was maximal by 24 h after inoculation of cell monolayers. Release of L. pneumophila from these cells appeared as a lytic process that resulted in an increase in the numbers of microorganisms in the extracellular fluids and a concomitant decline in the number of intracellular bacteria. The course of cellular infection was completed by 72 h. The cellular and ultrastructural events of L. pneumophila adherence and uptake by U-937 cells in the absence of antibody or complement have been defined. In addition, this work further establishes the U-937 cell as a suitable model for investigating Legionella – host cell interactions.Key words: Legionella, U-937 cells, intracellular replication, opsonin-independent uptake.

Analysis of 163 putative Shigella isolates from Canada and the USA showed biochemical reactions consistent with Shigella species, although none of the isolates reacted with antiserum raised against any of the well-established or provisional Shigella serotypes. All these isolates, provisionally designated serotype SH108, were positive for the ipaH gene and the invasion-associated locus. All fermented mannitol, were serologically indistinguishable from each other and showed no reaction in antisera prepared against Escherichia coli serotypes O1 to O181. PCR-RFLP analysis of the genes involved in O-antigen synthesis revealed a common pattern among these isolates that was distinct from recognized Shigella serotypes and E. coli. Between 1999 and 2003, isolates from across Canada were submitted to the National Laboratory for Enteric Pathogens for antibiotic susceptibility testing, phage typing and PFGE. These assays revealed heterogeneity among the members of this serotype. Antimicrobial susceptibility testing with seven antibiotics identified six profiles, with 90 % (45/50) of the isolates resistant to four or more antibiotics and 72 % (36/50) resistant to five or more. All isolates were typable using a panel of 16 phages, with 11 different phage types (PTs) represented. The most common PTs found were PT 3 (64 %), PT 6 (10 %) and PT 16 (6 %). Analysis of XbaI-restricted genomic DNA revealed 16 highly related patterns that were not readily distinguishable from those obtained for some other Shigella serotypes. The World Health Organization Collaborating Center for Shigella has added serotype SH108 to the Shigella scheme as S. boydii serotype 20 (serovar nov.). Strain SH108 (isolate 99-4528) is the reference strain for this serotype.

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.

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.

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.

For RT-PCR, removal of contaminating genomic DNA in RNA samples using manganese sulfate was more effective than magnesium. DNA contamination was removed in 3 microg of nucleic acid using 10 U of RNase-free DNase I in 10-microl reaction volumes. The digestion procedure was compatible with commercial RNA extraction kits and was suitable for RT-PCR assay.

Between 9 October and 12 November 1996, an outbreak of bloody diarrhea occurred in the neonatal nursery ward of the Policlínico Neuquén, in Neuquén, a city in the southwestern region of Argentina. Seven patients of the intermediate care unit were affected. Isolates of Escherichia coli O18ac:H31 that were non-lactose and -sorbitol fermenting were recovered from outbreak cases. Although the strains were negative for a number of virulence factors typically found in diarrheagenic groups of E. coli , all isolates from the present neonatal outbreak possessed the enterohemolysin gene, ehl1 . All isolates showed resistance to the antibiotics ampicillin and chloramphenicol. These isolates showed a low adherence property in HeLa cells without any recognizable pattern. In order to evaluate the outbreak dissemination in the neonatology ward, a prevalence study was conducted on 13 November. Stool specimens were obtained from 16 neonates hospitalized in the sector and from 33 medical staff members. E. coli isolates with identical biochemical characteristics of the outbreak strain were recovered from 11 of 16 inpatients and from 4 of 33 staff members during the prevalence study. A total of 15 E. coli strains recovered both from the outbreak and the prevalence study were processed by random amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE). By RAPD-PCR 14 of 15 strains showed patterns with 85 to 100% similarity, and by PFGE these strains were identical, each showing a unique pattern with 15 bands between 40 and 400 kb. One strain isolated from a nurse during the prevalence study presented a pattern not related to the others, and this was characterized as E. coli O81:HNM resistant to ampicillin only and negative for all the virulence factors studied. This outbreak occurred despite strict regulations in place to prevent cross-infection in the hospital. Postoutbreak prevalence studies were performed weekly thereafter without detecting new cases.

When a newly described infectious agent of human disease appears on the scene, there follows a major thrust to develop effective laboratory-based epidemiologic markers, including typing schemes to trace the source of the pathogen through to infection. Such was the case with Campylobacter jejuni and

BACKGROUND: VerotoxigenicEscherichia coli(VTEC) was first described in Canada during the 1980s as an emerging foodborne disease in association with morbidity and mortality in outbreaks of hemorrhagic colitis caused byE coliO157:H7.OBJECTIVE: To describe the surveillance activities and epidemiological laboratory markers of VTEC that are used at the National Laboratory for Enteric Pathogens (NLEP) to investigate sporadic cases and outbreaks ofE coliO157:H7 and non-O157 VTEC in Canada.METHODS: Passive surveillance was conducted by obtaining data on laboratory confirmed cases of VTEC from the Provincial Laboratories of Public Health across Canada. The laboratory epidemiological markers generated for isolates of VTEC included biotyping, serotyping, phage typing, toxin detection and characterization, and molecular typing using pulsed-field gel electrophoresis.RESULTS: Major outbreaks of VTEC O157:H7 disease have been associated with ground beef, unpasteurized apple juice, salami and untreated water. In 1999 and 2000, a total of 46 outbreaks ofE coliO157:H7 disease were investigated. Among those, one outbreak was associated with contact at a petting zoo and a second with the consumption of salami. An outbreak in 2000 in Ontario was associated with water and resulted in more than 1000 cases of human illness, with six deaths. The NLEP has also identified more than 100 non-O157 VTEC serotypes from cattle and meat products. At least 23 VTEC serotypes found in humans were also identical to those found in cattle and meat products.CONCLUSIONS: The laboratory-based information that is generated is used to define the incidence, sources of infection, risk factors, trends, distribution and transmission of VTEC to humans from food, water and animal sources. Prevention and control of outbreaks are high-priority health concerns.

The optimum method for isolating Campylobacter upsaliensis from stools has not been clearly defined. In a preliminary study, cefoperazone amphotericin teicoplanin (CAT) selective medium isolated six C. upsaliensis strains which were not detected using modified cefoperazone charcoal deoxycholate (mCCDA). In order to identify the factors that underlie the superiority of CAT over mCCDA for isolating C. upsaliensis , we examined the effect of incubation time and antibiotic content of culture media on the growth of C. upsaliensis isolates using semiquantitative methods. The recovery of a subgroup of C. upsaliensis isolates from seeded stool specimens was also evaluated. Differences in growth of C. upsaliensis on CAT and mCCDA were modest and were not explained by the antibiotic profiles of the two media. Recovery of C. upsaliensis from spiked human feces on CAT was superior to that on mCCDA at lower concentrations of organisms (10 3 CFU/ml). We conclude that although CAT is more suitable than mCCDA for the isolation of C. upsaliensis from stools, the superiority of CAT for detecting this organism is not accounted for by the antibiotic composition of the medium.

Background.Helicobacter pullorum, first detected in the liver and intestinal contents of poultry, was defined as a new species in 1994. This organism has since been isolated from humans with gastroenteritis. Phenotypic as well as genotypic methods have been used to identify H. pullorum associated with cases of human disease. Materials and Methods. Clinical isolates were submitted for identification to the National Laboratory for Enteric Pathogens by Provincial Public Health Laboratories within Canada. Phenotypic characterization was conducted using a variety of growth and biochemical tests including oxidase, catalase, indoxyl acetate, H2S production in triple sugar iron (TSI) agar, antimicrobial susceptibility testing, and fatty acid analysis. Genotypic identification was performed using a polymerase chain reaction–restriction fragment–length polymorphism (PCR-RFLP) analysis of a 1-kb fragment of the Helicobacter 16S rRNA gene. Results. During the last 7 years (1993–1999) a total of 11 isolates of H. pullorum were detected from patients with gastroenteritis for inclusion in this study. Typically, these isolates were oxidase and catalase positive, produced optimal growth at 42°C, and produced H2S in TSI. Of these 11 isolates, 1 showed DNase activity, while another did not produce H2S in TSI, and only 2 showed tolerance to 1% bile. Antimicrobial susceptibility assays indicated that 6 of the 11 strains were resistant to nalidixic acid. The fatty acid profiles of the isolates were similar to each other and provided a distinguishing profile from the other related species. Genetically identical and distinct species-specific restriction fragment–length polymorphism (RFLP) patterns were produced using the restriction enzymes Bsr I and Dde I. Conclusion. Phenotypic and genotypic procedures were used to identify H. pullorum. Interspecies phenotypic variability was apparent and supported the use of a polyphasic approach for identification. Similarities to the more prominent human pathogens Campylobacter coli and C. lari were also noted. The use of a combination of phenotypic and, in particular, genotypic markers for H. pullorum should prove valuable both for epidemiological investigations and for the diagnosis of disease related to this emerging human pathogen.

Campylobacter jejuni has been identified as the predominant cause of antecedent infection in Guillain-Barrésyndrome (GBS) and Miller Fisher syndrome (MFS). The risk of developing GBS or MFS may be higher after infection with specific C. jejuni types. To investigate the putative clonality, 18 GBS- or MFS-related C. jejuni strains from The Netherlands and Belgium and 17 control strains were analyzed by serotyping (Penner and Lior), restriction fragment length polymorphism analysis of PCR products of the flaA gene, amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis, and randomly amplified polymorphic DNA analysis. Serotyping revealed 10 different O serotypes and 7 different Lior serotypes, thereby indicating a lack of serotype clustering. Two new O serotypes, O:35 and O:13/65, not previously associated with GBS or MFS were found. Serotype O:19 was encountered in 2 of 18 strains, and none was of serotype O:41. The results of all genotypic methods also demonstrated substantial heterogeneity. No clustering of GBS- or MFS-related strains occurred and no molecular marker capable of separating pathogenic GBS or MFS from non-GBS- or non-MFS-related enteritis strains could be identified in this study. Sialic-acid-containing lipopolysaccharides (LPS) are thought to be involved in the triggering of GBS or MFS through molecular mimicry with gangliosides in human peripheral nerves. Therefore, further characterization of GBS- or MFS-related C. jejuni should target the genes involved in the synthesis of LPS and the incorporation of sialic acid.

A clinical isolate of Campylobacter jejuni, previously found to produce a toxin active in cell culture assays, was used for identification and characterisation of a cytotoxic porin-lipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by high-performance liquid chromatography of crude concentrated culture supernate and DEAE-anion exchange chromatography. The complex had a toxic activity of 20.1 tissue culture dose50 (TCD50)/microg of protein for HEp-2 cells, 7.49 TCD50/microg of protein for HeLa cells and 1.87 TCD50/microg of protein for Chinese hamster ovary cells. Analysis by SDS-PAGE revealed a single protein band of 45 kDa and a high mol. wt carbohydrate moiety. The complex gave a positive result in the Limulus amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins Galanthus nivalis agglutinin, Maackia amurensis agglutinin and Datura stramonium agglutinin. The cytotoxic activity associated with the complex was heat-labile at 70 degrees C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperiodate and the glycosidases neuraminidase and N-glycosidase F. Sequencing of the N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C. jejuni. The cytotoxic activity of the complex was neutralised by a polyclonal, homologous antiserum, which reacted on Western blot with the 45-kDa protein, but not by polyclonal antisera raised against a number of other bacterial toxins.

In the absence of serum, Legionella pneumophila demonstrated wash-resistant adherence to U-937 cells, primary guinea-pig alveolar macrophages, and MRC-5 cells. Neither complement nor antibody was required for binding. The dynamics of adherence following inoculation of L. pneumophila at increasing 10-fold multiplicities of infection to each of the three host cell types resulted in a first-order kinetic relationship of binding, indicative of one bacterial adhesin molecule recognized by one host cell receptor moiety. Host cell receptor saturation studies showed that depending on the cell type, 2–8% of the bacterial inoculum adhered to cells under these nonopsonic conditions. Preliminary adhesin and receptor characterization studies were preformed to define the chemical composition of the binding structures on both the organism and the three different host cell surfaces. The adherence phenomenon was investigated using competitive binding assays in the presence of putative adhesin analogs as well as following treatments modifying the microbial and host cell surface membranes. Attachment was evaluated both by viable bacterial cell colony counts and by indirect immunofluorescent assay. With the exception of aldehyde treatments, the various membrane-modifying regimes and the presence of the adhesin analogs were shown to have no effect on organism or host cell viability. Data suggested that the L. pneumophila adhesin responsible for opsonin-independent binding to these host cells was a protein structure with lectin-like properties. Furthermore, this protein would appear to be intimately associated with carbohydrate or lipid structures located on the bacterial outer membrane. The receptor moiety present on all host cells responsible for binding L. pneumophila had properties consistent with a carbohydrate or complex saccharide structure. To evaluate the role of complement receptors as the structures necessary for L. pneumophila infection of macrophages, a battery of monoclonal antibodies were used to block the complement receptor (CR) types 1 (CD35), CR3 (CD 18, CD11b), and CR4 (CD18, CD11c). Blocking studies with CR-specific monoclonal antibodies indicated that CR1 and the integrin receptors CR3 and CR4 were not involved in the opsonin-independent binding of L. pneumophila to macrophage-like cells.Key words: Legionella, opsonin-independent attachment, bacterial adherence, complement receptors, adhesion–receptor interactions.

SUMMARY: A genomic library derived from a virulent isolate of Legionella pneumophila was constructed in Escherichia coli JM 83 using the cloning vector pUC19. The clones were screened by filter immunoassay using L. pneumophila rabbit polyclonal antisera and in the absence of in situ bacterial lysis one such clone, LP 116, expressed L. pneumophila-specific antigens on the surface of E. coli. Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp. Southern hybridization confirmed that the fragment was L. pneumophila DNA. Sequencing data showed that the fragment was 810 bp in length with an open reading frame (ORF) of 678 bp. The outer-membrane profiles of the E. coli parent, the L. pneumophila DNA-contributing strain and clone LP 116 were compared by SDS-PAGE. A protein of 25 kDa was found in outer-membrane preparations of both the clone LP 116 and L. pneumophila but not in E. coli JM 83. This was in agreement with the molecular mass of the deduced peptide of the mature protein. Immunoblots using L. pneumophila-specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was a L. pneumophila polypeptide. Both direct immunofluorescence assay and immunoblots using the commercially produced monoclonal antibody specific for the common antigen of the major outer-membrane protein (MOMP) confirmed that the 25 kDa protein produced by LP 116 was involved with the MOMP complex. The gene encoding this protein has been designated ompM. Furthermore, using the fertile chicken egg virulence assay, clone LP 116 producing the 25 kDa MOMP of L. pneumophila showed an increase in virulence when compared to the E. coli parent strain.

The yeast Pityrosporum orbiculare is thought to cause the folliculitis associated with seborrheic eczema. However, a combination of mechanical and microbiological factors may be involved, with follicular occlusion leading to yeast overgrowth and folliculitis. Scanning electron microscopy was used to investigate this hypothesis. Skin biopsy specimens obtained from patients with Pityrosporum folliculitis were examined by scanning electron microscopy before and after oral ketoconazole therapy. Patients with active disease showed occlusion of noninflamed follicles, which resolved after ketoconazole treatment. Follicular occlusion was not present in biopsy specimens obtained from unaffected controls nor was it related to the presence of P orbiculare. These findings suggest that follicular occlusion may be a primary event in the development of this folliculitis, with yeast overgrowth a secondary occurrence. The beneficial effect of ketoconazole in this disease may be due to direct effects on the follicle.

A many-called magnetotactic prokaryote obtained from brackish water was observed to possess intercellular connections at points of contact between the outer membranes of constituent cells. Each aggregate organism consisted of 10 to 30 individual Gram-negative cells containing material with the appearance of poly-β-hydroxybutyrate and magnetosomes of unusual arrangement, structure and composition. The aggregate, which possessed prokaryotic-type flagella arranged at the outwards surfaces of each cell, showed motility indicative of co-ordination between individual component cells. These results suggest that this organism could be a multicellular prokaryote.

A 68,000-molecular-weight protein was isolated by polyacrylamide gel electrophoresis from the organism-free filtrate of a fully virulent clinical strain of Campylobacter jejuni. The eluted protein was heat labile, was inactivated at either pH 3.0 or 9.0, was sensitive to trypsin, and was lethal for fertile chicken eggs. It also had toxic effects on chicken embryo fibroblast, Chinese hamster ovary (CHO), and intestinal 407 (Int407) cells. A monoclonal antibody (CETPMAb4) raised to this eluted toxic protein (ETP) from C. jejuni abolished these toxic activities. Homology between C. jejuni ETP and Vibrio cholerae toxin was not observed in that specific antisera to each did not block their respective toxic activities. In enzyme-linked immunosorbent assays, ETP, unlike chlorea enterotoxin, did not bind to GM1 ganglioside. Furthermore, the C. jejuni toxin had cytotoxinlike properties and induced rounding of CHO cells. Binding of ETP to Int407 and primary chicken embryo fibroblast cells was maximal after 2 h as assessed by enzyme-linked immunosorbent assay, and this toxin adherence to host cell membranes was significantly reduced by prior treatment of the cells with proteolytic enzymes, neuraminidase, or glutaraldehyde but not by treatment with beta-galactosidase, lipase, Nonidet P-40, or sodium metaperiodate. In competitive binding assays, sugars, lectins, or GM1 ganglioside did not adversely influence uptake of ETP by these cells. These results suggest that the ETP produced by C. jejuni is a cytotoxin which binds to Int407 cells via a protein- or glycoproteinlike receptor on cell membranes and possesses properties dissimilar to those of V. cholerae toxin.

The ultrastructural pathology induced in organs of chick embryos previously infected with 100 times the yolk sac LD50 of Legionella pneumophila was examined by electron microscopy. Subtle pathological changes were observed three days post-inoculation while more extensive cellular and sub-cellular necrosis occurred at four days post-inoculation. Selected antimicrobial agents were administered three days after infection and evaluated for their ability to reduce or reverse ultrastructural lesions. In this study, ciprofloxacin was the only antibiotic that reversed tissue damage induced in response to infection with L. pneumophila while doxycycline, erythromycin, and rifampicin were also effective, but to a lesser degree. These results demonstrated the superior antimicrobial activity of ciprofloxacin and further illustrated the efficacy of the fertile hen's egg as a useful in vivo assay system for the evaluation at the ultrastructural level of putative chemotherapeutic agents for the treatment of Legionnaires' disease.

Summary The pathogenicity of Legionella pneumophila , serogroup 1, strain Nottingham N 7 , was assessed in terms of LD 50 data and the ability of the organism to induce pathological lesions in the fertile hen's egg. Histopathological examination of embryo organs after inoculation with 1, 10, 100 and 1000 times the yolk sac LD 50 revealed a disseminated infection. Systemic spread of the organism resulted in widespread necrosis and evidence of consolidation together with generation of copious amounts of oedema fluid. These were particularly severe in the liver, heart, spleen and kidney. The infection elicited a massive inflammatory response typified by infiltration with polymorphonuclear leucocytes and lymphocytes. Selected antimicrobial chemotherapeutic agents were investigated in protection studies for their capacity to ameliorate or control disease processes in this test system. Of those examined ciprofloxacin was most effective in reducing the incidence of lesions in these tissues and for prolonging embryo viability. Rifampicin, and to a lesser degree, erythromycin and doxycycline, also showed antimicrobial activity in these-in vivo trials. These results indicate that the fertile hen's egg may be a useful alternative to other animal systems for the in vivo testing of clinically putative antimicrobial agents in the treatment of Legionnaires' disease.

Summary: Ten strains representing six serogroups of Legionella pneumophila were examined by electron microscopy using freeze-etching, thin-sectioning and negative-staining techniques. In addition, selected strains were examined further as shadowed and freeze-dried preparations and by scanning electron microscopy. The cell envelope consisted of two membranes, evident in fractured specimens as four short ridges. The major fracture plane was through the inner membrane, and therefore the protoplasmic and extracellular fracture faces of this membrane were predominant. With the exception of one strain (Togus 1), the particle arrangement on these fracture faces was random. A peptidoglycan-like cell wall layer was revealed only in sections of partially plasmolysed cells. Membrane-bounded poly-β-hydroxybutyrate-like granules were evident in the cytoplasm and these frequently showed plastic deformation due to fracturing. Although appendages were present, the surfaces of organisms and of isolated cell membranes showed no regular arrays of particles.

SUMMARY: Legionella pneumophila attached to, penetrated and replicated within the three eukaryotic cell lines, MRC-5, HEp-2 and Vero. Multiplication occurred rapidly in these cells for an initial 48 h after inoculation and declined thereafter. Infected MRC-5 cell monolayers developed lytic-type cytopathic changes, with organisms being readily released. HEp-2 cells showed a more chronic infection, with slowly developing granular changes in the monolayers, and slow release of intracellular bacteria. In Vero cells, organisms were released rapidly along with a more progressively developing granular cytopathic effect in the monolayers. L. pneumophila was unable to grow in cell-free culture fluids. Uptake and intracellular development was similar for each cell type, and was initiated by ‘bacteriopexis’, a process in which the organisms bound via receptors and were surrounded by cellular microvilli which eventually fused, leading to bacterial engulfment. Replication of organisms in vacuoles within the cytoplasm of infected cells was confirmed by thorium labelling. These vacuoles were lined with ribosomes and, at the early stages of intracellular development, were found in close proximity to mitochondria, cytoplasmic filaments and banded enclosures. Ruthenium red staining showed that acid mucopolysaccharide capsular material was not present on these organisms during the attachment process or intracellular phase. Organism release was by lysis of the infected cells.

The pathogenicity of Campylobacter jejuni was examined in chicken embryos. In this system, mortality data and histopathological findings induced by organisms and by bacterium-free filtered broth were identical. The absence in chicken embryo tissues both of organisms and of an inflammatory infiltrate suggests a toxin etiology.