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Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by Campylobacter jejuni in up to 40% of cases. Nucleotide sequence-based typing of 25 C. jejuni isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800 C. jejuni isolates not associated with neuropathy. A total of 13 genetic lineages and 20 flaA short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the flaA short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.

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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 degrees C but not at 42 degrees 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.

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To define relationships between Listeria monocytogenes genetic lineages, ribotypes, and serotypes, 235 L. monocytogenes isolates were characterized by serotyping and automated EcoRI 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 EcoRI ribotypes contained multiple serotypes; a subset of these isolates was further differentiated with PvuII ribotyping. Of the 12 resultant EcoRI-PvuII combination types, only 4 contained multiple serotypes, demonstrating the potential of ribotyping for serotype prediction.

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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.

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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 degrees 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.

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We recently analyzed 11 helicobacter isolates cultured from diarrhea patients in Canada. These isolates had been characterized biochemically by restriction fragment length polymorphism (RFLP; AluI, HhaI) 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 ApaLI. 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.

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Campylobacter jejuni has been identified as the predominant cause of antecedent infection in Guillain-Barré syndrome (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.

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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.

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A rapid two-step identification scheme based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene was developed in order to differentiate isolates belonging to the Campylobacter, Arcobacter, and Helicobacter genera. For 158 isolates (26 reference cultures and 132 clinical isolates), specific RFLP patterns were obtained and species were successfully identified by this assay.

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The gene encoding the 25 kDa major outer membrane protein (MOMP) of Legionella pneumophila was transformed into Escherichia coli JM 83 and the resultant E. coli LP 116 clone expressed the Legionella-MOMP. Compared with the parent E. coli strain, the clone showed a fivefold increase in opsonin-independent binding to U-937 cells. Furthermore, this gene was incorporated by electroporation into a low virulence derivative of Leg. pneumophila which showed reduced expression of the MOMP but enhanced expression of a 31 kDa protein in the OMP profile. After electroporation, the attenuated strain showed an increased expression of the MOMP while the 31 kDa protein was eliminated and virulence for the chick embryo was re-established. The use of a monoclonal antibody specific for the MOMP abolished virulence and adherence. These studies suggest that the 25 kDa MOMP of Leg. pneumophila serves as an adhesive molecule for host cells and that this protein plays a major role in the virulence of the organism for the chick embryo.

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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-alpha(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.

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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.

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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 performed 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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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.(ABSTRACT TRUNCATED AT 250 WORDS)

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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.

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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.

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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.

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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.

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The response of Legionella pneumophila to antibiotics that inhibit cell-wall, protein and DNA synthesis was examined by electronmicroscopy, MIC estimations and viable counts. Ampicillin, cefotaxime, methicillin, erythromycin, rifampicin and ciprofloxacin, each used separately at 20 times their respective MIC values, showed activity against L. pneumophila in these studies. The inhibitors of cell-wall synthesis--ampicillin, cefotaxime and methicillin--effected the greatest bactericidal activity and induced the most extensive morphological changes, which included the formation of membranous lesions through which cytoplasmic contents were lost. In terms of ultrastructural damage and loss of viability, the inhibitors of protein and DNA synthesis were less effective than the antibiotics that acted on the microbial cell wall. Erythromycin- and rifampicin-treated cells possessed irregular membranes and were partially or fully lysed, whereas ciprofloxacin induced abnormally elongated organisms with intermittently lysed and detached inner membranes. These results illustrated the ability of antibiotics of putative clinical value, with diverse modes of action, to affect the ultrastructural cytology as well as the viability of L. pneumophila in vitro.

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