Occurrence of plasmid DNA in serologically defined strains of Campylobacter jejuni and Campylobacter coli

Forty Campylobacter jejuni and 17 Campylobacter coli strains that constitute the set of reference strains for our serotyping scheme were each examined for the presence of plasmid DNA. Agarose gel electrophoresis of alkaline-extracted DNA showed the occurrence of 29 bands in 11 C. jejuni strains and 40 bands in C. coli strains. Plasmids ranged in size from 1.6 to 70 megadaltons. Most strains that carried plasmids had between 2 and 6 of them; however, one strain had 14 plasmids, and two strains contained only 1 plasmid each. Repeated electrophoresis demonstrated that all plasmid profiles were stable. A different plasmid profile was seen for each of the 19 plasmid-carrying strains, but it was clear that plasmids of the same or similar molecular weight could be found in different strains. On the basis of these findings, we are persuaded that plasmid profiles determined by a rapid procedure for DNA extraction will play a significant role in resolving complexities among strains that are difficult to serotype and could be useful in epidemiological studies in which the implicated isolates are plasmid bearers.     

Differentiation of Campylobacter jejuni and Campylobacter coli strains by using restriction endonuclease DNA profiles and DNA fragment polymorphisms.

The chromosomal DNA fragment patterns from a total of 169 Campylobacter jejuni and Campylobacter coli isolates from poultry and humans were analyzed by using DNA restriction endonucleases ClaI and EcoRV. The DNA restriction patterns produced by ClaI and EcoRV consisted of unique DNA fragments of 9 to 9.5 kb and 3.5 kb generated with ClaI and a single unique fragment of 3.0 kb produced by EcoRV. These patterns were obtained with all strains of C. jejuni tested. The DNA restriction patterns were further examined by Southern blot analysis with a previously constructed DNA probe, pMO2005, which is also able to distinguish between C. jejuni and C. coli spp. (5). Two types of patterns were produced by hybridization with the ClaI-cleaved DNA of C. jejuni strains, one of a single 18.5-kb genomic fragment and the other of 14.5- and 4.0-kb fragments. This indicated the presence of an extra ClaI site in this genomic fragment in the strains with the duplex pattern. The Southern blot analysis of 169 C. jejuni and C. coli isolates from poultry and from humans with DNA probe pMO2005 demonstrated that 78% of C. jejuni strains isolated from chickens hybridized with DNA probe pMO2005 with a characteristic 14.5- and 4.0-kb banding pattern and 22% hybridized with a single 18.5-kb fragment, whereas 71% of human isolates hybridized with the single 18.5-kb fragment and only 29% hybridized with 14.5- and 4.0-kb fragments. These findings suggest that only a small proportion of C. jejuni strains that colonize chickens may cause disease in humans.     

Rapid and improved gas-liquid chromatography technique for detection of hippurate hydrolysis by Campylobacter jejuni and Campylobacter coli.

A gas-liquid chromatographic method which requires no chloroform extraction of the split products has been investigated for the detection of hippurate hydrolysis by Campylobacter spp. This technique gave better reproducibility than other tests also used in this study and allows the routine use of the gas-liquid chromatographic method for identification of Campylobacter isolates.     

Polynucleotide sequence relationships among flagellin genes of Campylobacter jejuni and Campylobacter coli.

DNA probes that encode a complete flagellin gene and various internal regions of the Campylobacter coli VC167 flagellin genes were hybridized to 30 strains of C. coli or C. jejuni from 20 different Lior serogroups. The results indicated a high overall degree of homology among all of the strains examined. Although the most variable regions occurred within the middle of the gene, significant DNA homology was observed among many serogroups in this region of the molecule.     

Monoclonal antibodies specific for hippurate hydrolase of Campylobacter jejuni

Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.     

Naturally occurring auxotrophs of Campylobacter jejuni and Campylobacter coli.

The nutritional requirements for 439 Campylobacter jejuni isolates and 46 Campylobacter coli isolates were determined by using a previously described chemically defined medium, campylobacter defined medium. With this medium, 45% of both human and nonhuman C. jejuni isolates demonstrated auxotrophic requirements. None of the 46 C. coli isolates studied demonstrated requirements for amino acids on campylobacter defined medium. The most common auxotrophic requirement among C. jejuni isolates was for methionine, which was present as a single requirement or in combination with other markers in 21% of human and 28% of nonhuman isolates. There was no correlation between plasmid carriage and auxotype, and a comparison of the Lior serotypes of 472 of the strains showed a correlation only between proline auxotrophs and Lior serotype 11 for strains isolated in the Seattle-King County region.     

Genetic relatedness and quinolone resistance of Campylobacter jejuni strains isolated in 2002 in Hong Kong

Pulsed-field gel electrophoresis fingerprints of 98 Campylobacter jejuni isolates from patients (85) and chicken carcasses (13) in Hong Kong in 2002 demonstrated high genetic diversity. The prevalence of quinolone resistance among the isolates was 85.9%, and replacement of the threonine-86 residue in the gyrase subunit A was the major resistance mechanism.     

Improved biotyping schemes for Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni (20 strains) and Campylobacter coli (12 strains) were assigned to four biovars for each species based on phenotypic tests that were easy to perform and interpret. The resulting biotyping schemes offer a greater degree of distinction among C. jejuni and C. coli strains than any of the other biotyping schemes previously described for these organisms.     

Evidence of reinfection with multiple strains of Campylobacter jejuni and Campylobacter coli in Macaca nemestrina housed under hyperendemic conditions.

A prospective bacteriologic study of 18 infant pig-tailed macaques (Macaca nemestrina) housed in a nursery facility in which Campylobacter spp. are endemic was undertaken to determine the epidemiology of infection and reinfection. The isolates of Campylobacter jejuni and C. coli cultured from 8 of the 18 infants were characterized by serotyping, DNA hybridization, and polyacrylamide gel electrophoresis protein profiles. The chronology of infection was indicative of multiple reinfections with different strains of C. jejuni and C. coli during the 12-month study of each infant. The duration of infection with a particular strain was 3 to 4 weeks. Infants were also infected with nalidixic acid-resistant campylobacters. These observations indicated that long-term infections under endemic conditions are caused by continual reinfection. C. jejuni or C. coli infection correlated with diarrhea in 5 of the 18 infants at 1 to 4 months of age.     

Serotyping of Campylobacter jejuni/coli.

Antisera were prepared from strains of Campylobacter jejuni/coli isolated from patients in six outbreaks of enteritis. Bactericidal antibodies, and agglutinating antibodies to heat-labile and heat-stable antigens, were demonstrated. These reactions were used to type a number of strains isolated from patients in each outbreak, and to distinguish 'epidemic' from 'non-epidemic' strains.     

Interaction of Campylobacter jejuni and Campylobacter coli with lectins and blood group antibodies.

Lectins and blood group antibodies were used to probe the surface structures of Campylobacter jejuni and Campylobacter coli. Of the 29 strains tested, there were distinct reaction patterns. The lectin-reactive and blood group antibody-reactive sites on the bacterial surface were distinguishable from the heat-stable (lipopolysaccharide) antigenic determinants. The interactions were strain specific. The reactive sites were stable with respect to culture media and passage and may be useful as additional markers for strain characterization.     

Identification of Campylobacter jejuni and Campylobacter coli antigens with mucosal and systemic antibodies.

The development of a rapid and specific diagnostic assay for Campylobacter infections is important in determining the etiology of acute diarrhea in humans. Studies have shown that sonicated whole bacteria or partially purified antigens cross-reacted with antibodies against other closely related bacteria. To solve the problems of specificity, we identified specific antigens of Campylobacter jejuni and Campylobacter coli for use in diagnostic assays. We investigated the responses of serum, urine, and intestinal lavage antibodies in infected (fed live bacteria) and parenterally immunized (intraperitoneal injection of sonicated whole bacteria with adjuvant) mice directed against C. jejuni or C. coli by Western blot (immunoblot) analysis. Antibody responses were examined weekly for up to 28 days. Fewer antigens were detected by urinary and intestinal lavage fluid immunoglobulin A (IgA) than serum IgG and IgM for both parenterally immunized and infected mice. Serum from parenterally immunized mice detected more antigens than that from infected mice. Two high-molecular-weight antigens (62,000 and 43,000) were predominantly detected by serum, urine, and intestinal lavage fluids of both parenterally immunized and infected mice. Serum antibodies from 28-day parenterally immunized mice detected one antigen specific to C. coli with a molecular weight of 38,000 and one antigen specific to C. jejuni with a molecular weight of 27,000. An immunodominant protein with a molecular weight of 31,000 common to both C. jejuni and C. coli was also recognized by serum antibodies from parenterally immunized mice.     

Correct identification and discrimination between Campylobacter jejuni and C. coli by a standardized hippurate test and species-specific polymerase chain reaction

Hippurate hydrolysis test results of 240 Campylobacter strains were compared with those of two multiplex polymerase chain reaction (PCR) assays. Of the 152 strains identified in Finnish clinical microbiology routine laboratories as C. coli (hippurate-negative), 11% were C. jejuni (hippurate-positive) by standardized hippurate test and 39% by PCR in the reference laboratory. Two of the 81 hippurate-positive strains were identified as C. coli. Standardizing the hippurate test by determining minimum and maximum turbidity limits (McFarland 6 and McFarland 10, OD(450) values 0.8 and 1.4, respectively) for the bacterial cell suspension eliminated the false-positive results, but 32% of the 145 hippurate-negative strains were still identified as C. jejuni by PCR. The species identification of Campylobacter isolates in Finland could be improved by using a standardized hippurate hydrolysis test to identify hippurate-positive C. jejuni and testing hippurate-negative strains by molecular methods. This would also improve the epidemiological data on this important zoonotic pathogen.     

Fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni

The aim of this study was to investigate the fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni. The in vitro growth, the survival on food matrix, and the in vivo colonization of C. jejuni and C. coli susceptible isolates and their isogenic resistant mutants were studied. In vitro experiments demonstrated that macrolide resistance imposed a fitness cost when the susceptible strains and their isogenic resistant mutants were cultured in competition. When inoculated in food matrix, the resistant C. jejuni mutant was no longer detectable after 3 to 5 days but the susceptible strain remained detectable for over 18 days. No difference in survival in food matrix was observed between susceptible and resistant C. coli. When inoculated in vivo in chickens, the macrolide susceptible and resistant C. coli displayed similar levels of colonization, both in separated inoculations and during competitive assays. Strikingly, when mono-inoculated or co-inoculated into chickens, macrolide susceptible C. jejuni outcompeted the macrolide resistant population. However, a spontaneous mutant that evolved in vivo showed a colonization capacity similar to the susceptible strain. Our findings demonstrate the effect of macrolide resistance on the fitness of Campylobacter but suggest that evolved mutants may be as fit as susceptible strains.     

Distribution and serotypes of Campylobacter jejuni and Campylobacter coli in enteric Campylobacter strains isolated from children in the Central African Republic.

One hundred eighty-five enteric Campylobacter strains isolated from diarrheic or healthy children in Bangui (Central African Republic) were studied to determine their species and serotypes. C. coli was identified in 38.9% of all strains and in 43.9% of strains from diarrheic children. By the hemagglutination technique for heat-stable antigens, 73.5% of the strains could be serotyped. Of the typeable strains, 75% were distributed among 13 more frequent serotypes. C. coli serotype Pen 37,56 was the most common serotype from diarrheic children.     

Strain characterization and grouping of Campylobacter jejuni and Campylobacter coli by interaction with lectins.

Strains of Campylobacter jejuni and Campylobacter coli were characterized and grouped by their distinct reaction patterns with lectins. Heating of the Campylobacter cultures to 100 degrees C and holding for 30 to 60 min greatly enhanced their reactivity with lectins and permitted the grouping of all but 3 of 155 cultures tested in this study without interference of autoagglutination and other nonspecific activities. The lectin reaction patterns of the heated cultures were stable and reproducible. They were strain specific and independent of the heat-stable antigenic types. The lectin-reactive sites of C. jejuni and C. coli may be useful as additional markers for strain characterization. Based on these observations, a simple slide agglutination procedure is described for differentiating strains of C. jejuni and C. coli by their interaction with a selected group of commercially available lectins.     

Comparison of basal media for culturing Campylobacter jejuni and Campylobacter coli.

Four strains of Campylobacter jejuni and four strains of Campylobacter coli were used to compare the quantitative growth of Campylobacter cells on blood agar base no. 2 (Oxoid), brucella agar (BBL Microbiology Systems and Difco Laboratories), campylobacter agar base (Difco), Columbia blood agar base (Difco and Oxoid), and Mueller-Hinton agar (Difco and Oxoid). Columbia blood agar base and blood agar base no. 2 were inhibitory to most of the strains tested, as evidenced by reduced (10- to 1,000-fold) colony counts compared with other basal media. One of the brucella agars was inhibitory to two of the C. coli strains. The inhibitory effect of these media could be eliminated by addition of FBP (0.05% each ferrous sulfate hydrate, sodium metabisulfite, and sodium pyruvate) or 7% defibrinated sheep blood. However, addition of FBP or blood to brucella agar, campylobacter agar base, or Mueller-Hinton agar did not significantly affect the count, indicating that supplements are not required in these media for growth of Campylobacter in pure culture.     

Prevalence of cytolethal distending toxin production in Campylobacter jejuni and relatedness of Campylobacter sp. cdtB gene.

Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). The genes encoding this toxin in C. jejuni 81-176 were cloned and sequenced. The nucleotide sequence of the genes revealed that there are three genes, cdtA, cdtB, and cdtC, encoding proteins with predicted sizes of 30,11-6, 28,989, and 21,157 Da, respectively. All three proteins were found to be related to the Escherichia coli CDT proteins, yet the amino acid sequences have diverged significantly. All three genes were required for toxic activity in a HeLa cell assay. HeLa cell assays of a variety of C. jejuni and C. coli strains suggested that most C. jejuni strains produce significantly higher CDT titers than do C. coli strains. Southern hybridization experiments demonstrated that the cdtB gene is present on a 6.0-kb ClaI fragment in all but one of the C. jejuni strains tested; the cdtB gene was on a 6.9-kb ClaI fragment in one strain. The C. jejuni 81-176 cdtB probe hybridized weakly to DNAs from C. coli strains. The C. jejuni 81-176 cdtB probe did not hybridize to DNAs from representative C. fetus, C. lari, C. "upsaliensis," and C. hyointestinalis strains, although the HeLa cell assay indicated that these strains make CDT. PCR experiments indicated the probable presence of cdtB sequences in all of these Campylobacter species.     

Occurrence of plasmids and antibiotic resistance among Campylobacter jejuni and Campylobacter coli isolated from healthy and diarrheic animals.

Serologically defined strains of Campylobacter jejuni and Campylobacter coli from healthy and diarrheic animals were examined for the occurrence of plasmid DNA in association with the antibiotic susceptibility of the bacterial host and the health status of the animal host. Of all campylobacter organisms surveyed, 53% (116 of 200) contained plasmid DNA. A plasmid occurrence rate of 73.8% was obtained for C. coli from healthy pigs, contrasted by lower plasmid occurrence rates for C. coli from diarrheic pigs (30%) and from all diarrheic animals (21.4%). For C. jejuni, in contrast, only 13.6% of healthy cattle contained plasmid DNA, contrasted by a higher plasmid occurrence rate of 31.2% from diarrheic cattle. A high plasmid occurrence rate of 75.8% was observed for C. jejuni from healthy chickens. Campylobacter plasmids ranged in size from less than or equal to 1 to 86 megadaltons. Antibiotic susceptibility for 52 animal isolates (excluding chickens) indicated that most isolates were susceptible to kanamycin, erythromycin, gentamicin, tetracycline, and compound sulfonamide, whereas few were susceptible to bacitracin (19.2%); approximately half were susceptible to ampicillin (55.8%) and streptomycin (51.9%), and no isolates were susceptible to penicillin G. More isolates containing plasmids were resistant to ampicillin, tetracycline, and gentamicin than were isolates not carrying plasmids, there being a statistically significant difference for tetracycline and gentamicin, which suggested that these two antibiotics were probably plasmid mediated. The antibiotic susceptibility patterns of 21 chicken isolates of C. jejuni, by contrast, were different in that most were susceptible to ampicillin in addition to kanamycin, erythromycin, and gentamicin, whereas few wer susceptible to compound sulfonamide, streptomycin, and tetracycline in addition to penicillin G and bacitracin. A 30- or 39-megadalton plasmid, or both, common to many of the chicken isolates was usually associated with tetracycline resistance.