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PubMed2004

Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA.

We describe a multiplex PCR assay to identify and discriminate between isolates of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis. The C. jejuni isolate F38011 lpxA gene, encoding a UDP-N-acetylglucosamine acyltransferase, was identified by sequence analysis of an expression plasmid that restored wild-type lipopolysaccharide levels in Escherichia coli strain SM105 [lpxA(Ts)]. With oligonucleotide primers developed to the C. jejuni lpxA gene, nearly full-length lpxA amplicons were amplified from an additional 11 isolates of C. jejuni, 20 isolates of C. coli, 16 isolates of C. lari, and five isolates of C. upsaliensis. The nucleotide sequence of each amplicon was determined, and sequence alignment revealed a high level of species discrimination. Oligonucleotide primers were constructed to exploit species differences, and a multiplex PCR assay was developed to positively identify isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis. We characterized an additional set of 41 thermotolerant isolates by partial nucleotide sequence analysis to further demonstrate the uniqueness of each species-specific region. The multiplex PCR assay was validated with 105 genetically defined isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis, 34 strains representing 12 additional Campylobacter species, and 24 strains representing 19 non-Campylobacter species. Application of the multiplex PCR method to whole-cell lysates obtained from 108 clinical and environmental thermotolerant Campylobacter isolates resulted in 100% correlation with biochemical typing methods.

Acyltransferases↗

PubMed1985

DNA homology studies of the catalase-negative campylobacters and "Campylobacter fecalis," an emended description of Campylobacter sputorum, and proposal of the neotype strain of Campylobacter sputorum.

Twenty-three strains of catalase-negative campylobacters and five strains of "Campylobacter fecalis," which is catalase-positive, were examined by DNA hybridization experiments. These organisms formed four distinct DNA homology groups corresponding to Campylobacter sputorum, Campylobacter mucosalis, Campylobacter concisus, and a currently unnamed group referred to as the "catalase-negative or weak" (CNW) strains. The strains were further characterized to determine which phenotypic characteristics provide the most reliable identification at the species level. Campylobacter sputorum ssp. sputorum, C. sputorum ssp. bubulus, and "C. fecalis" could not be distinguished by DNA homology; consequently, it is proposed that these three taxa be considered as biovars of C. sputorum. The description of C. sputorum is emended accordingly. ATCC strain 35980 is proposed as the neotype strain of C. sputorum.

Campylobacter↗

PubMed1999

Rapid identification of thermotolerant Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis from various geographic locations by a GTPase-based PCR-reverse hybridization assay.

Recently, a gene from Campylobacter jejuni encoding a putative GTPase was identified. Based on two semiconserved GTP-binding sites encoded within this gene, PCR primers were selected that allow amplification of a 153-bp fragment from C. jejuni, C. coli, C. lari, and C. upsaliensis. Sequence analysis of these PCR products revealed consistent interspecies variation, which allowed the definition of species-specific probes for each of the four thermotolerant Campylobacter species. Multiple probes were used to develop a line probe assay (LiPA) that permits analysis of PCR products by a single reverse hybridization step. A total of 320 reference strains and clinical isolates from various geographic origins were tested by the GTP-based PCR-LiPA. The PCR-LiPA is highly specific in comparison with conventional identification methods, including biochemical and whole-cell protein analyses. In conclusion, a simple method has been developed for rapid and highly specific identification of thermotolerant Campylobacter species.

Australia↗

PubMed1997

Molecular discrimination between Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis by polymerase chain reaction based on a novel putative GTPase gene.

Polymerase chain reaction (PCR) mediated DNA fingerprinting has resulted in the identification of a novel Campylobacter jejuni gene, encoding a GTPase protein. The gene, consisting of 383 amino acids contained semi-conserved GTP-binding sites (designated G-1 to G-4), that are characteristic for members of the GTPase protein superfamily. Remarkably, this gene from C. Jejuni appears to encode a member of a novel family of GTP-binding proteins, containing two separate putative GTP-binding domains, each comprising a series of semi-conserved GTP-binding motifs. Spacing between these motifs is highly conserved. Based on this novel gene, a general PCR strategy for the identification of C. jejuni, C. coli, C. lari and C. upsaliensis was developed. PCR primers were deduced from GTP-binding motifs G-1 and G-3 of the first GTP-binding domain. These GTP-binding sites flank a variable region of precisely 117 bp in the four Campylobacter spp. that allowed the development of species-specific probes. This PCR-hybridization assay offers a novel tool for rapid molecular detection and specific identification of the thermophilic Campylobacter spp.

Amino Acid Sequence↗

PubMed2002

Prevalence of Campylobacter jejuni, Campylobacter lari, and Campylobacter coli in different ecological guilds and taxa of migrating birds.

A total of 1,794 migrating birds trapped at a coastal site in southern Sweden were sampled for detection of Campylobacter spp. All isolates phenotypically identified as Campylobacter jejuni and a subset of those identified as non-C. jejuni were identified to the species level by PCR-based techniques. C. jejuni was found in 5.0% of the birds, Campylobacter lari was found in 5.6%, and Campylobacter coli was found in 0.9%. An additional 10.7% of the tested birds were infected with hippurate hydrolysis-negative Campylobacter spp. that were not identified to the species level. The prevalence of Campylobacter spp. differed significantly between ecological guilds of birds. Shoreline-foraging birds feeding on invertebrates and opportunistic feeders were most commonly infected (76.8 and 50.0%, respectively). High prevalence was also shown in other ground-foraging guilds, i.e., ground-foraging invertebrate feeders (11.0%), ground-foraging insectivores (20.3%), and plant-eating species (18.8%). Almost no Campylobacter spp. were found in ground-foraging granivores (2.3%), arboreal insectivores (0.6%), aerial insectivores (0%), or reed- and herbaceous plant-foraging insectivores (3.5%). During the autumn migration, a high proportion of samples from juveniles were positive (7.1% in passerines, 55.0% in shorebirds), indicating transmission on the breeding grounds or during the early part of migration. Prevalence of Campylobacter spp. was associated with increasing body mass among passerine bird species. Furthermore, prevalence was higher in short-distance migrants wintering in Europe than in long-distance migrants wintering in Africa, the Middle East, or Asia. Among ground-foraging birds of the Muscicapidae, those of the subfamily Turdinae (i.e., Turdus spp.) showed a high prevalence of Campylobacter spp., while the organism was not isolated in any member of the subfamily Muscicapinae (i.e., Erithacus and Luscinia). The prevalence of Campylobacter infection in wild birds thus seems to be linked to various ecological and phylogenetic factors, with great variations in carriership between different taxa and guilds.

Animals↗

PubMed2005

Advantages of peptide nucleic acid oligonucleotides for sensitive site directed 16S rRNA fluorescence in situ hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli and Campylobacter lari.

Traditionally fluorescence in situ hybridization (FISH) has been performed with labeled DNA oligonucleotide probes. Here we present for the first time a high affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting thermotolerant Campylobacter spp. using FISH. Thermotolerant Campylobacter spp, including the species Campylobacter coli, Campylobacter jejuni and Campylobacter lari, are important food and water borne pathogens. The designed PNA probe (CJE195) bound with higher affinity to a previously reported low affinity site on the 16S rRNA than the corresponding DNA probe. PNA also overcame the problem of the lack of affinity due to the location of the binding site and the variation of the target sequence within species. The PNA probe specificity was tested with several bacterial species, including other Campylobacter spp. and their close relatives. All tested C. coli, C. jejuni and C. lari strains were hybridized successfully. Aging of the Campylobacter cultures caused the formation of coccoid forms, which did not hybridize as well as bacteria in the active growth phase, indicating that the probe could be used to assess the physiological status of targeted cells. The PNA FISH methodology detected C. coli by membrane filtration method from C. coli spiked drinking water samples.

Animals↗

PubMed1985

Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men.

In a recent study in Seattle, we isolated Campylobacter-like organisms (CLOs) significantly more often from homosexual men with intestinal symptoms than from asymptomatic heterosexual and homosexual control subjects. Biochemical tests, morphology, and guanine-plus-cytosine content of whole-cell DNA showed that these organisms were similar to, but distinguishable from, other species in the genus Campylobacter. DNA homology tests showed that the Campylobacter-like organisms belong to four unique genetic groups, two of which were phenotypically identical. We developed a rapid screening test to genetically group these organisms and to assess the correlation of each genotype with enteric disease in homosexual men. Three of the four CLO groups were isolated only from men with proctitis, proctocolitis, and/or enteritis. The remaining group, isolated from both symptomatic and asymptomatic homosexual men, was significantly correlated with the presence of polymorphonuclear leukocytes in the rectal secretions of asymptomatic men, a finding that suggests subclinical disease. We have tentatively classified two of the CLO groups into species, Campylobacter cinaedi sp. nov. and Campylobacter fennelliae sp. nov.

Campylobacter↗

PubMed1993

Development of species-specific DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction fingerprinting.

The application of polymerase chain reaction (PCR) fingerprinting assays enables discrimination between species and strains of microorganisms. PCR primers aiming at arbitrary sequences in combination with primers directed against the repetitive extragenic palindrome (REP) or enterobacterial repetitive intergenic consensus (ERIC) motifs generate isolate-specific DNA banding patterns. Analysis of these PCR fingerprints obtained for 33 isolates of Campylobacter jejuni, 30 isolates of Campylobacter coli, and 8 isolates of Campylobacter lari revealed that besides generation of isolate-specific fragments, species-specific DNA fragments of identical size were synthesized. It appeared that these DNA fragments could be used as species-specific probes, since they are unique for the pattern which they are deriving from. The probes do not cross-react with amplified DNA originating from a large panel of nonrelated microorganisms. Moreover, these probes displayed species specificity, as they reacted with a single restriction fragment on Southern blots containing DNA from C. jejuni, C. coli, and C. lari and other Campylobacter species. This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information. The principle of the procedure holds great promise for the rapid isolation of DNA probes which, in combination with a general PCR assay, may lead to efficient typing and detection procedures for a multitude of medically important nonviral microorganisms.

Base Sequence↗

PubMed1984

New, extended biotyping scheme for Campylobacter jejuni, Campylobacter coli, and "Campylobacter laridis".

A biotyping scheme using improved media and methods for the detection of hippurate hydrolysis, rapid H2S production, and DNA hydrolysis was applied to 1,826 cultures of Campylobacter jejuni, Campylobacter coli and "Campylobacter laridis" isolates from human and nonhuman sources. Four biotypes were identified among C. jejuni: 57.3% of the isolates belonged to biotype I; 36.0%, to biotype II; 4.0%, to biotype III; and 2.7%, to biotype IV. C. coli organisms were differentiated into biotype I (67.0% of the isolates) and biotype II (33.0%). All "C. laridis" isolates belonged to biotype I. The combination of the biotyping scheme with the serotyping of campylobacters provided additional epidemiological markers by further differentiating the serogroups by species and biotypes.

Animals↗

PubMed1986

Cytotoxic and cytotonic factors produced by Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis.

Complete toxigenicity studies were performed on 341 strains of Campylobacter spp., including 23 nonhuman isolates. Toxin profiles based on both cytotonic and cytotoxic factors were determined after analyzing responses in Vero, HeLa, CHO and Y-1 cells. Suckling mouse assays were consistently negative for all culture filtrates tested. Toxin-producing strains were frequently encountered among both the human and nonhuman strains of Campylobacter jejuni, C. coli, and C. laridis investigated. Strains isolated from outbreaks demonstrated parallels in serotype, biotype, and toxigenicity profile, although no clear association could be demonstrated. Biphasic culture conditions conducive to the production of both toxic factors were delineated for the propagation of test Campylobacter strains. Cytotonic effects of Campylobacter culture filtrates were determined in Vero and CHO cells, and cyclic AMP accumulation in cells exposed to these culture filtrates was compared with that in cells exposed to reference toxigenic strains of Vibrio cholerae and Escherichia coli. Partial neutralization of C. jejuni enterotoxin was demonstrated by using antitoxins to cholera toxin and E. coli heat-labile enterotoxin. No neutralization of C. jejuni cytotoxin could be achieved by using antitoxins to either Clostridium difficile cytotoxin or E. coli Verotoxin (0157:H7).

Animals↗

PubMed1996

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis.

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg l-1), amphotericin (10 mg l-1) and teicoplanin (4 mg l-1) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0.65 micron filters and a 150-fold reduction using the 0.45 micron filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10(4) cfu mL-1). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0.65 microns) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.

Animals↗

PubMed1994

Identification of Campylobacter jejuni, Campylobacter coli and campylobacter lari by the nucleic acid amplification system NASBAR.

NASBAR, an isothermal amplification technique for nucleic acids, was evaluated for the specific identification of Campylobacter jejuni, Camp. coli and Camp. lari. A set of primers and a probe were chosen from the 16S rRNA sequence of Campylobacter. The probe was hybridized in solution with the amplified nucleic acids of 12 Campylobacter species and nine other Gram-negative bacteria. The probe was shown to hybridize specifically to the amplified single-stranded RNA of Camp. jejuni, Camp. coli and Camp. lari in an enzyme-linked gel assay (ELGA). In a Camp. jejuni model system the combination of NASBAR and ELGA was able to detect ca 1000 rRNA molecules. The presence of an excess of Gram-negative bacteria did not influence the sensitivity of detection. A number of 6 cfu of Camp. jejuni, present in a total count of 4 x 10(6) cfu of Gram-negative bacteria, resulted in a positive hybridization signal.

Base Sequence↗

PubMed1986

Campylobacter infection associated with raw milk. An outbreak of gastroenteritis due to Campylobacter jejuni and thermotolerant Campylobacter fetus subsp fetus.

Raw milk is identified with increasing numbers of outbreaks of gastroenteritis and is an important vehicle for transmission of Campylobacter infection. Unlike Campylobacter jejuni, Campylobacter fetus subsp fetus has not been associated with common-source outbreaks of gastroenteritis. This report describes an outbreak of gastroenteritis involving C jejuni and a thermotolerant strain of C fetus subsp fetus associated with raw milk. Fifteen (39%) of 38 persons who attended a banquet in Wisconsin in June 1982 developed acute gastroenteritis. Stool specimens were obtained from nine ill guests; four yielded C jejuni and three yielded C fetus subsp fetus. The C fetus subsp fetus isolates were identified fortuitously, in part because of unusual thermotolerance (growth at 42 degrees C), permitting isolation at temperature appropriate for C jejuni. Survey results implicated raw milk as the source of the outbreak. Findings provide evidence of a potentially emergent milkborne pathogen contributing to the risk of raw milk consumption and suggest that current diagnostic laboratory techniques may fail to identify significant foodborne agents.

Adolescent↗

PubMed2005

A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp.

AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.

Amino Acid Sequence↗

PubMed1986

The chicken embryo as a model for campylobacter invasion: comparative virulence of human isolates of Campylobacter jejuni and Campylobacter coli.

Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.

Animals↗

PubMed1983

Serogroups of Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus defined by direct immunofluorescence.

Rabbits were inoculated with whole, formalinized Campylobacter jejuni, C. coli, and C. fetus cells; the C. jejuni and C. coli immunogens were identified by their DNA relatedness at the species level to the type strains of C. jejuni or C. coli. The designation C. coli was not used among the other C. jejuni strains; they were classified as hippurate-positive or hippurate-negative C. jejuni. Immunoglobulin G was isolated from the antisera and labeled with fluorescein isothiocyanate. These conjugates defined 10 serogroups of C. jejuni, 2 serogroups of C. coli, and 2 serogroups of C. fetus. Of the 316 strains of C. jejuni tested, 258 (82%) were groupable; 173 were single-serogroup strains, and 85 were multiple-serogroup strains. Of the 226 strains in C. jejuni serogroups, 223 (98.7%) were hippurate positive; of the 27 strains in C. coli serogroups, 26 (96.3%) were hippurate negative. Five strains were equally reactive in immunofluorescent staining with a conjugate for a C. jejuni serogroup and a conjugate for a C. coli serogroup. A total of 58 strains of C. jejuni were ungroupable: 33 (13%) of the 259 hippurate-positive strains and 25 (44%) of the 57 hippurate-negative strains. All 121 strains of C. fetus tested were groupable as A, B, or A:B. The 14 conjugates used to define serogroups of C. jejuni, C. coli, and C. fetus reacted with the flagella but not the cells of other Campylobacter species and were negative on 256 other bacteria from 21 genera.

Animals↗

PubMed1985

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals.

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.

Animal Population Groups↗

PubMed1986

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.

Adolescent↗