Differentiation of Yersinia enterocolitica serotype O:5,27 strains by phenotypic and molecular techniques

Restriction endonuclease analyses of virulence plasmid DNA (REAP) and chromosomal DNA and other phenotypic characteristics were used to study the differentiation of Yersinia enterocolitica serotype O:5,27 strains. There was a close correlation between REAP patterns and the geographical distribution of serotype O:5,27. Human isolates produced only one REAP pattern, which was also found with isolates from pigs and dogs.     

Antimicrobial resistance of clinical strains of Campylobacter jejuni subsp. jejuni isolated from 1985 to 1997 in Quebec, Canada

The antimicrobial resistance of 158 Campylobacter jejuni strains isolated from humans in Quebec, Canada, from 1995 to 1997 was compared to the resistance of 47 and 86 strains of C. jejuni isolated in 1985 and 1986 and in 1992 and 1993, respectively. Of the 291 C. jejuni strains tested, no strain was resistant to erythromycin. Compared to the C. jejuni strains isolated in 1985 and 1986, the C. jejuni strains isolated in 1992 and 1993 were more resistant to tetracycline (40.7 versus 19.1%, respectively; P = 0. 01) but not to nalidixic acid or ciprofloxacin (P > 0.05). Compared to the C. jejuni strains isolated in 1992 and 1993 and in 1985 and 1986, the C. jejuni strains isolated from 1995 to 1997 were more resistant to tetracycline (55.7% versus 40.7 and 19.1%, respectively; P = 0.03 and P < 0.001, respectively) to nalidixic acid (13.9% versus 4.7 and 0%, respectively; P = 0.02 and P = 0.007, respectively), and to ciprofloxacin (12.7% versus 3.5 and 0%, respectively; P = 0.02 and P = 0.009, respectively).     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples

Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.     

Fisher syndrome associated with IgG anti-GQ1b antibody following infection by a specific serotype of Campylobacter jejuni

OBJECTIVE: The purpose of the study was to describe clinical and serologic features of Fisher syndrome associated with IgG anti-GQ1b ganglioside antibody following Campylobacter jejuni enteritis. DESIGN: A clinical trial. PARTICIPANTS: Four consecutive patients with Fisher syndrome were studied. INTERVENTION: Samples of sera from four patients were tested for reactivity to GQ1b ganglioside by enzyme-linked immunosorbent assay (ELISA). Campylobacter jejuni strains isolated from samples of stool from three patients were serotyped by the method of Penner and Hennessy and that of Lior. MAIN OUTCOME MEASURES: Serum IgG anti-GQ1b antibody titer and serotypes of C. jejuni. RESULTS: Diplopia occurred 8 to 14 days after the onset of diarrhea. Campylobacter jejuni was isolated from samples of stool from all of the patients. ELISA revealed a high serum IgG anti-GQ1b antibody titer for all four patients. Two patients had high serum titers of other antiganglioside antibodies frequently related to Guillain-Barré syndrome. These two patients developed limb weakness following the onset of ophthalmoplegia. The C. jejuni serotype was Penner's serotype 2 for all three of the patients tested. CONCLUSIONS: These findings suggest that C. jejuni, especially Penner's serotype 2, enteritis could trigger development of Fisher syndrome associated with IgG anti-GQ1b antibody.     

A putative adhesin gene cloned from Campylobacter jejuni

Thirteen Campylobacter jejuni strains of human origin showed differing behaviours when analysed for their ability to bind the Caco-2 cell line in vitro, suggesting variations in genetic complements and/or regulation. We designed an oligonucleotide probe corresponding to a highly conserved part of adhesins from various Gram-negative bacteria. Among our laboratory collection, Southern hybridization has demonstrated that only a discrete number of strains harbour this sequence. The corresponding gene has been cloned from our prototype strain and sequence analysis has confirmed homology with Gram-negative bacterial adhesins. The ORF corresponded to 869 amino acids; we named this protein P95. Protein sequence similarity assessment demonstrated that this gene product belongs to the family of proteins including the filamentous haemagglutinin of Bordetella pertussis and the high-molecular-weight surface-exposed adhesins of Haemophilus influenzae. Comparison of adhesion and hybridization results emphasized the involvement of this gene in an essential pathogenic process of Campylobacter.     

Evidence of genomic instability in Campylobacter jejuni isolated from poultry

Poultry isolates of Campylobacter jejuni derived from a survey of meat processing batches were genotyped by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA to establish the clonal relationships between single-colony isolates. In the majority of batches studied, one or two genotype patterns predominated. However, in one batch (batch A), 21 single-colony isolates gave 14 different PFGE genotypes. The banding patterns obtained with SmaI were sufficiently different to distinguish between genotypes, although the patterns also produced many common bands. The question of whether these isolates represented different clones or had a common clonal ancestry was addressed by additional genotypic and phenotypic methods. Restriction length polymorphism of PCR products obtained from the flagellin genes showed an identical flagellin genotype for all of these isolates. In contrast, unrelated control isolates resulted in different flagellin genotypes. Moreover, all 14 different PFGE genotypes of batch A had identical Penner serotypes and identical or similar biotypes and phage types. It was concluded that the isolates were of clonal origin and that the diversity in the PFGE banding patterns had most likely originated from genomic rearrangements. However, the PFGE genotypes were shown to be stable upon subculturing in vitro and after in vivo passage in chickens, and natural transformation between isogenic mutants carrying antibiotic markers did not occur in vivo in a chick colonization model. The possible mechanisms for the hypothesized genomic recombinations and the conditions that allow, induce, or select for such events are discussed.     

Isolation and molecular analysis of colonising and non-colonising strains of Campylobacter jejuni and Campylobacter coli following experimental infection of young chickens

Fourteen-day-old chickens were inoculated with selected Campylobacter coli and C. jejuni strains. C. jejuni strains were of two subgroups based on a polymorphism detected using a DNA probe and represented the profiles typical for the majority of strains of either chicken or human origin. All C. coli strains previously isolated from humans colonised chickens, whereas from 4/7 C. jejuni strains of human origin, failed to colonise. Of 12 Campylobacter strains of chicken origin, 10 established a persistent colonisation in the chickens, and 2 strains colonised poorly or not at all. Four strains that failed to colonise chickens were each inoculated into groups of five birds. Three strains again did not colonise any of the chickens and the fourth strain colonised four out of the five chickens, but was poorly excreted. When infected chickens were placed in the same enclosure to facilitate interchange of strains, C. jejuni strain 331 was found to be dominant and colonised all 12 chickens by 21 days, displacing all other strains. C. jejuni strain 331, was then inoculated into groups of five birds with previously established colonisation by C. jejuni and C. coli strains. Strain 331 was able to replace the C. jejuni strain in all five birds but established co-colonisation with C. coli strain. Naturally occurring co-colonisation by two C. jejuni strains was detected in one chicken out of 200 tested. There was no obvious correlation between the type of DNA polymorphism in strains of chicken origin and their ability to colonise chickens.     

Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant

A PCR method for the rapid identification and discrimination of thermophilic Campylobacter jejuni and Campylobacter coli was developed by using a gene encoding a protein involved in siderophore transport (ceuE). A nucleotide sequence divergence of approximately 13% in the ceuE genes of C. jejuni and C. coli facilitated the design of two species-specific PCR primer sets. The specificity of the PCR amplification reactions was confirmed by using two nonradioactively labelled species-specific internal oligonucleotide hybridization probes for each of these species.     

In vitro studies of Campylobacter jejuni/coli strains from hens and humans regarding adherence, invasiveness, and toxigenicity

Campylobacter jejuni/coli strains from hens and humans were compared for their ability to adhere to and invade HEp-2-cells and for toxigenicity to CHO-cells. In both hen and human strains, invasiveness was higher among non-toxigenic strains than among toxigenic ones. The frequency of adherence, invasiveness, and toxigenicity was the same in hen and human strains.     

Restriction fragment length polymorphism analysis and random amplified polymorphic DNA analysis of Campylobacter jejuni strains isolated from patients with Guillain-Barré syndrome

Campylobacter jejuni serotype O19 strains associated with the Guillain-Barré syndrome (GBS) and other strains were examined by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction products of the flaA genes and by random amplified polymorphic DNA (RAPD) analysis. RFLP analysis showed that regardless of LIO serotype, geographic origins, or association with GBS, the O19 isolates shared an identical digestion pattern by each of four restriction endonucleases, DdeI, MboI, MseI, and AluI. In contrast, among C. jejuni O1 or O2 strains, RFLP patterns were different even among strains of the same LIO serotype. The results of the RAPD analysis were consistent with the flaA RFLP data. These data indicate that all of the O19 strains that were tested were closely related to one another whether they were or were not associated with GBS.     

Typing of human Campylobacter jejuni isolates in Finland by pulsed-field gel electrophoresis

A total of 69 pulsed-field gel electrophoresis (PFGE) types were identified among 176 Campylobacter jejuni isolates from Finnish patients. In two geographic areas studied, five predominant PFGE types comprised over 40% of the isolates. One-third of the isolates had unique PFGE types. In small outbreaks, identical PFGE patterns were demonstrated, indicating a common source of infection.     

Antimicrobial susceptibilities of Campylobacter jejuni and coli by using E-test in Taiwan

Cytogenetic abnormalities in human malignancies frequently involve chromosome 7. The existence of several tumor suppressor genes on the long arm of chromosome 7 has been suggested in both epithelial and hematologic malignancies. From the Danish Cytogenetic Register, we identified 183 persons with constitutional abnormalities involving chromosome 7, including 16 patients with Williams syndrome. By linkage to the Danish Cancer Registry, we found five persons with cancer, including one thyroid carcinoma, three carcinomas of the digestive tract, and one malignant melanoma. There were no cases of leukemia. The overall risk of developing cancer was not increased.     

Characterization of the thermal stress response of Campylobacter jejuni

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage lambda to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46 degreesC was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.     

Toxic megacolon as a complication of Campylobacter jejuni enterocolitis

We report the case of a previously healthy 53-year-old white male who developed an extraordinary complication of acute Campylobacter jejuni colitis. Toxic megacolon occurred while the patient was treated with a fluoroquinolone antibiotic and glucocorticoids, which were given for endoscopically suspected Crohn's colitis. During the course of the disease no cause of colitis was found other than C. jejuni. Despite the extreme dilatation, the patient was treated conservatively with parenteral nutrition and repeated decompression colonoscopies and made a full, though slow, and uneventful recovery. Follow-up colonoscopies for up to 4 years showed persistent scarring of the transverse colon, probably due to the extreme dilatation, and mild unspecific inflammation of the terminal ileum without histological evidence of inflammatory bowel disease. A comparison with the 6 previously published cases leads to the following conclusions: in most cases the transverse colon is most severely affected. Treatment with either antimotility agents or systemic glucocorticoids does not seem to promote colonic dilatation. The complication has affected patients of both sexes (4 women, 3 men), in the age range of 21 to 83 years, most of them without an underlying disease. The interval between the start of diarrhea and development of the megacolon ranged widely from 3 to 33 days, as did recovery time (2 days to several months). Three of the 7 patients underwent colectomy for imminent or actual colonic perforation. The delayed recovery of our patient was partly attributed to colonic damage caused by extreme dilatation, leading to ischaemia and subsequent scarring of the mucosa, which persisted. Histologically no Crohn's disease or ulcerative colitis could be found at any stage. A rapid increase in resistance of C. species against fluoroquinolone antibodies has been observed in recent years, due to use of the antibiotics in farming. Our patient's severe illness may partly have resulted from delayed effective antibiotic treatment due to resistance. Antibiotic resistance to common enteropathogens should be considered in the case of unusually prolonged or severe enterocolitis. The level of suspicion for either infection or inflammatory bowel disease should remain high as it may be impossible to distinguish between them on the basis of clinical or endoscopic criteria alone.     

Plasmid profiles and antimicrobial susceptibility of Campylobacter jejuni isolated from Israeli children with diarrhea

Thirty Campylobacter jejuni (C. jejuni) strains isolated from stools of Israeli children with enteritis were tested for sensitivity to eight antimicrobial agents (MIC) and the presence of plasmids. It was found that all the isolates were sensitive to ciprofloxacin, ofloxacin, furazolidone and erythromycin. Of the 30 strains tested, 21 (70%) were found to be tetracycline-resistant, a relatively high resistance rate as compared with data from other countries and previous reports from Israel. Plasmids were detected in 17 out of 30 C. jejuni isolates (55.6%). A total of nine different plasmid profiles could be distinguished; six profiles were represented by one strain each. Of the 21 tetracycline-resistant strains, plasmids were found in 17 isolates (80%) carrying from 1-2 to 5 plasmids of various sizes. No plasmids were found in tetracycline-sensitive strains, with the exception of one isolate which contained a 24.4 MDa plasmid and was co-trimoxazole-resistant. Our studies indicate a relatively high percentage of tetracycline-resistant C. jejuni isolates in the Tel Aviv area. In 80% of these strains, various plasmid profiles were detected.     

Analysis of distribution of Campylobacter jejuni and Campylobacter coli in broilers by using restriction fragment length polymorphism of flagellin gene

The incidence of Campylobacter jejuni and Campylobacter coli in broiler farms was 33.9% (19/56). C. jejuni-positive flocks accounted for 20.0% (17/85) and C. coli-positive ones was 4.7% (4/85). There were 14 patterns (fla type) of restriction fragment length polymorphism (RFLP) of flagellin A gene among these 22 strains of C. jejuni and C. coli including the standard strain C. jejuni ATCC 33560. Different fla types of Campylobacter were isolated from broilers in different growing cycles on the same farms. Four strains of C. jejuni were isolated from four breeder farms and four fla types of C. jejuni were detected from their progenies reared on growing farms. Three fla types of C. jejuni detected from the progenies were different from those of each breeder. Also, the other three fla types of C. jejuni were detected from different progenies of each growing farm during the next growing cycle. These findings indicate that the RFLP analysis may contribute to epidemiological studies of C. jejuni and C. coli contamination of broilers and suggest the risk of contamination with different types of Campylobacter in every growing cycle of broilers on the farm even on the same farm. They also supported that there was little likeliness of the vertical transmission of C. jejuni and C. coli from breeders to broilers.