Evaluation of 11 PCR assays for species-level identification of Campylobacter jejuni and Campylobacter coli

We examined the sensitivity and specificity of 11 PCR assays described for the species identification of Campylobacter jejuni and Campylobacter coli by using 111 type, reference, and field strains of C. jejuni, C. coli, and Campylobacter lari. For six assays, an additional 21 type strains representing related Campylobacter, Arcobacter, and Helicobacter species were also included. PCR tests were initially established in the laboratory by optimizing conditions with respect to five type and reference strains of C. jejuni, C. coli, and C. lari. One PCR test for C. coli failed to give appropriate results during this initial setup phase and was not evaluated further. The remaining 10 assays were used to examine heated lysate and purified DNA templates as appropriate of well-characterized type, reference, and field strains of C. jejuni (n = 62), C. coli (n = 34), and C. lari (n = 15). The tests varied considerably in their sensitivity and specificity for their respective target species. No assay was found to be 100% sensitive and/or specific for all C. jejuni strains tested, but four assays for C. coli gave appropriate responses for all strains examined. Between one and six strains of C. jejuni gave amplicons in four of seven C. jejuni PCR tests only where purified DNA was used as the template; corresponding results were seen with one strain of C. coli in each of three assays for the latter species. Our findings indicate that a polyphasic strategy for PCR-based identification should be used to identify C. jejuni and C. coli strains. The data may assist laboratories in selecting assays suited for their needs and in designing evaluations of future PCR tests aimed to identify these species.     

Application of Lior biotyping by use of genetically identified Campylobacter strains.

We used the scheme of Lior to biotype 140 genetically identified Campylobacter strains. Our results confirmed previous studies and extended Lior biotyping to show that nine C. jejuni subsp. doylei strains (100%) were one biotype and nine C. jejuni subsp. jejuni nalidixic acid-resistant strains (100%) were C. jejuni biotype I or II. All C. jejuni subsp. jejuni hippurate-negative strains studied and 6 of 35 C. lari strains (17%) were grouped with C. coli biotypes. These findings may be useful in epidemiologic investigations.     

Identification of Campylobacter jejuni by using a biotinylated probe

A DNA biotinylated probe for the rapid identification of Campylobacter jejuni in culture (Enzo Biochem, New York) has been evaluated. The hybridized biotinylated DNA probe is detected by its interaction with streptavidin linked to horseradish peroxidase. Sixteen strains of C. jejuni, including type strain. 24 strains of other Campylobacter and Helicobacter species, and 59 strains of other general have been studied. The probe was highly sensitive (100%) and specific (100% inside the genera Campylobacter and Helicobacter). All Campylobacter strains gave strong signals, and only three weak signals have been observed with non-Campylobacter strains. Our results indicate that specific recombinant DNA probe should offer a reliable and rapid method for routine diagnosis of Campylobacter jejuni enteritis.     

Prevalence and characterization of hippurate-negative Campylobacter jejuni in King County, Washington

A total of 593 strains of thermophilic Campylobacter species were isolated either from humans with diarrhea or from poultry in King County, Washington. Of these strains, 98 (52 hippurate-positive strains and all 46 of the hippurate-negative strains) were selected for further phenotypic characterization and genetic classification. Hippurate hydrolysis, the test typically used to differentiate Campylobacter jejuni and C. coli, did not always correlate with the genetic classification. All hippurate-positive strains were classified as C. jejuni. Of the hippurate-negative strains, 20% were C. jejuni, 78% were C. coli, and 2% were C. laridis. Assuming that the remaining hippurate-positive strains were all C. jejuni, then hippurate-negative C. jejuni represented a small percentage (9 of 556 or 1.6%) of C. jejuni strains but a significant percentage (9 of 46 or 20%) of hippurate-negative strains. This finding suggests that hippurate hydrolysis should not be used as the sole criterion for differentiating thermophilic Campylobacter species, particularly when describing the disease states associated with these organisms.     

Culture confirmation of Campylobacter spp. by latex agglutination.

A commercial latex agglutination test [Meritec-Campy (jcl), Meridian Diagnostics, Cincinnati, Ohio] was evaluated for identification of Campylobacter jejuni, C. coli, C. laridis, and other Campylobacter isolates. The test had 100% sensitivity in detecting C. jejuni and C. coli but low sensitivity with C. laridis isolates. C. upsaliensis strains reacted with the test. The test had 100% specificity for 101 non-Campylobacter organisms.     

Longitudinal study of the excretion patterns of thermophilic Campylobacter spp. in young pet dogs in Denmark

The Campylobacter excretion patterns of 26 domestic pet dogs were described in a longitudinal study. The dogs entered the study between 3 and 8 months of age and were monitored until 2 years of age. They were tested monthly for Campylobacter carriage in stool samples that were cultured on the Campylobacter-selective media CAT and modified CCDA agar at 37 and 42 degrees C. This study comprised 366 fecal swab samples, of which 278 (76.2%) were found to be Campylobacter positive, with the following distribution of species: 75.0% Campylobacter upsaliensis, 19.4% Campylobacter jejuni, 2.1% Campylobacter lari, 0.7% Campylobacter coli, and 2.8% Campylobacter spp. Isolates were typed by pulsed-field gel electrophoresis (PFGE) to elucidate the strain excretion pattern. All study dogs excreted Campylobacter spp. during the study period. At 3 months of age, 60% of the dogs carried Campylobacter, increasing to nearly 100% carriers at 1 year of age, whereafter the carriage rate decreased to 67% at 24 months of age. The PFGE types showed that individual dogs were often colonized by unique strains of C. upsaliensis for several months, up to 21 months or longer. These C. upsaliensis strains were either clonal (or underwent concurrent minor mutative changes) or independent strains. In contrast, the excreted C. jejuni isolates were much more diverse and, in most cases, only seen in one sample from each dog. A high degree of diversity among different dogs was seen. We conclude that young domestic pet dogs excreted Campylobacter spp. during the majority of their puppyhood and adolescent period. In general C. upsaliensis strains were excreted for months, with short-term interruptions by or cocolonization with other transitory Campylobacter spp., predominantly C. jejuni. C. jejuni was more prevalent in dogs between 3 months and 1 year of age than in dogs between 1 and 2 years of age.     

Identification of thermotolerant campylobacter species by fluorescence in situ hybridization

Thermotolerant Campylobacter spp. (Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis) are leading causes of food-borne diarrhea in humans. In this study, the usefulness of fluorescence in situ hybridization (FISH) for the identification of Campylobacter isolates was investigated. A hierarchical FISH probe set that included six group-, genus-, and species-specific probes was developed and evaluated with 12 reference strains and 94 clinical isolates of Campylobacter, Arcobacter, and Helicobacter. FISH correctly identified all isolates to the genus level and detected all thermotolerant Campylobacter isolates. The assay showed high degrees of sensitivity for the identification of C. jejuni (90%), C. coli (97%), C. lari (81%), and C. upsaliensis (100%) to the species level.     

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.     

Comparative genomic analysis of Campylobacter jejuni strains reveals diversity due to genomic elements similar to those present in C. jejuni strain RM1221

Analysis of the complete genomic sequence of Campylobacter jejuni strain RM1221 identified four large genomic elements, Campylobacter jejuni-integrated elements (CJIEs), that were absent from C. jejuni strain NCTC 11168. To further investigate the genomic diversity of Campylobacter, we conducted a comparative genomic analysis from a collection of 67 C. jejuni and 12 Campylobacter coli strains isolated from various geographical locations and clinical and veterinary sources. Utilizing PCR, we demonstrated that 55% of the C. jejuni strains examined were positive for at least one RM1221-like genomic element and 27% were positive for two or more of these CJIEs. Furthermore, many C. coli strains were positive for either genomic element CJIE1 or CJIE3. To simultaneously assess for the presence or absence of several genes that comprise the various CJIEs, we developed a multistrain C. jejuni DNA microarray that contained most of the putative coding sequences for strains NCTC 11168 and RM1221. A comparative genomic hybridization (CGH) analysis of 35 of the 67 C. jejuni strains confirmed the presence of genomic elements similar to those in strain RM1221. Interestingly, the DNA microarray analysis demonstrated that these genomic elements in the other C. jejuni strains often exhibited modular patterns with some regions of the CJIEs present and other regions either absent or highly divergent compared to strain RM1221. Our CGH method also identified 18 other intraspecies hypervariable regions, such as the capsule and lipooligosaccharide biosynthesis regions. Thus, the inclusion of genes from these integrated genomic elements and the genes from the other intraspecies hypervariable regions contributes to a better assessment of the diversity in C. jejuni and may increase the usefulness of DNA microarrays as an epidemiological genotyping tool. Finally, we also showed that in CJIE1, a Campylobacter Mu-like phage, is located differentially in other strains of C. jejuni, suggesting that it may integrate essentially randomly.     

Biotype and Lior serogroup distribution of enteric Campylobacter isolated from children in Bangui (Central African Republic), and comparison with Penner serotypes

The new extended biotyping scheme of Lior as well as the slide agglutination technique were applied to 209 strains of enteric Campylobacter isolated from children in Bangui (Central African Republic). Three biotypes of C. jejuni and 2 biotypes of C. coli were identified among the strains; 31.1% were C. jejuni I, 11% C. jejuni II, 2.4% C. jejuni III, 44% C. coli I and 11.5% C. coli II. We were able to serotype 71.3% of the strains with 20 immune sera prepared against strains of Campylobacter isolated previously; 63% of the strains were distributed among the ten most common serogroups. No significant difference was observed in the distribution of biotypes or serogroups between strains from healthy and diarrhoeic children. Comparison of Lior serogroups with Penner serotypes showed that different Penner serotypes may correspond to a Lior serogroup and vice versa.     

Development and evaluation of immunochromatographic assay for simple and rapid detection of Campylobacter jejuni and Campylobacter coli in human stool specimens

An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA, they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 x 10(4) to 8.2 x 10(5) CFU/ml of cell suspension, and those for the C. coli strains ranged from 1.4 x 10(5) to 4.6 x 10(6) CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens, suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent, and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens.     

Detection of Campylobacter species by using polymerase chain reaction and nonradioactive labeled DNA probe]

We have detected Campylobacter species which are now recognized as major pathogens of acute diarrheal disease in humans using polymerase chain reaction (PCR) and a nonradioactive labeled DNA probe. Diagnosis of Campylobacter enteritis without doing culture from stool samples is of great benefit in the laboratory. Two oligonucleotide primers (20 mer) complementary to a unique sequence of the DNA encoding ribosomal RNA (rRNA) of Campylobacter jejuni for PCR were synthesized by solid-phase phosphoamidite method. Amplified target DNA of 275 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with an oligodeoxynucleotide probe (28 mer) conjugated to alkaline phosphatase. In identification experiments, it was shown that the nonradioactive probe was hybridized to clinical strains of C. jejuni (104), C. coli (5), C. laridis (5), C. hyointestinalis (1) and C. fetus subsp. fetus (1) with an accuracy of 99-100%, while it was not for Helicobacter pylori. Further, there was no evidence of amplification in strains of K. pneumoniae, S. marcescens and E. coli. Using direct detection to stool specimens, this method could be performed in C. jejuni in 39 of 43 culture-positive specimens (91%), and in 19 of 141 culture-negative specimens (13.5%), respectively. The results of this comparative study suggested that the DNA probe assay became a rapid and reliable technique to confirm culture of Campylobacter species.     

Development and application of a new scheme for typing Campylobacter jejuni and Campylobacter coli by PCR-based restriction fragment length polymorphism analysis

A molecular typing approach for Campylobacter jejuni and Campylobacter coli was developed with restriction fragment length polymorphism analysis of a 9.6-kb PCR-amplified portion of the lipopolysaccharide gene cluster. Sixty-one Penner serotype reference strains were analyzed with this new genotyping scheme, and 32 genogroups were found. Eleven additional genogroups were obtained from 87 clinical C. jejuni strains tested. This molecular typing method shows a correlation with the Penner heat-stable serotyping method, a phenotypic typing method based on lipopolysaccharide structures that is often used as a "gold standard" for subtyping Campylobacter spp. This strong correlation suggests that the data obtained can be directly compared with epidemiological data collected in the past by classical serotyping of C. jejuni and C. coli. In contrast to the high percentage of nontypeability by phenotyping, this molecular typing method results in 100% typeability and provides a superior alternative to serotyping.     

"Campylobacter upsaliensis" isolated from cats as identified by DNA relatedness and biochemical features

Campylobacter spp. were isolated from the feces of 20 (58%) of 53 asymptomatic cats during routine physical examination while the cats were maintained in an accredited quarantine facility. Fifteen of these Campylobacter spp. were identified phenotypically as Campylobacter jejuni, and two were identified as C. coli. DNA-DNA hybridization (hydroxyapatite method) was used to confirm the identification of three thermotolerant catalase-negative isolates. They were 80 to 100% related to each other and to the type strain of "C. upsaliensis" in reassociation reactions under optimal conditions and a stringent hybridization criterion. These strains were 75 to 100% interrelated and less than 12% related to type strains of other Campylobacter species. These strains represent the first reported feline isolate of "C. upsaliensis" and show that cats used in biomedical research can harbor this and other Campylobacter species.     

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.     

Comparative performance of different PCR assays for the identification of Campylobacter jejuni and Campylobacter coli

The sensitivity and specificity of 7 PCR assays described for the identification of Campylobacter jejuni and Campylobacter coli were examined using alkaline cell lysates from a collection of 100 well characterized reference strains of C. jejuni, C. coli, Campylobacter lari and related Campylobacter, Helicobacter and Arcobacter species. Based on a preliminary evaluation, one multiplex test was excluded from further evaluation. The various assays differed considerably in sensitivity and specificity towards their target species. For C. coli, 4 of the 5 assays were 100% specific and sensitive, but for C. jejuni, none of the 5 assays were found to be 100% specific or sensitive. Subsequently, a statistically valid sample (n=263) was taken from a Belgian collection of 1906 human Campylobacter field isolates. This second collection was used to further evaluate two selected multiplex PCR assays. The present study indicates that PCR-based identification using each of the two selected multiplex PCR assays was highly reliable. The R-mPCR assay, followed by species-specific PCR assays or the ceu-oxr mPCR assay if necessary, is our current strategy of choice for the molecular identification of C. jejuni and C. coli. Results presented here should aid researchers in selecting a PCR assay suitable for their specific needs.     

DNA relatedness among strains of Campylobacter jejuni and Campylobacter coli with divergent serogroup and hippurate reactions.

Eleven strains of Campylobacter from earlier fluorescent-antibody studies were examined by DNA hybridization to determine their species. Three of the strains hydrolyzed sodium hippurate, and eight did not. Four of the hippurate-negative strains were in Campylobacter jejuni serogroups, and the remaining strains were in both C. jejuni and Campylobacter coli serogroups. DNA relatedness to type strains of C. jejuni and C. coli indicated that all three of the hippurate-positive strains and two of the hippurate-negative strains were C. jejuni. The six remaining hippurate-negative strains were C. coli. Two of the hippurate-negative strains in C. jejuni serogroups were C. jejuni, and two were C. coli. Three of the strains in serogroups of both species were C. jejuni, and four were C. coli. These studies confirm that a few strains of C. jejuni are hippurate negative and show that identical or highly related antigens are found in C. coli and C. jejuni.     

Incidence of toxigenic Campylobacter strains in South Africa.

Campylobacter strains can produce a heat-labile cytotonic toxin (CTON) and various cytotoxins (CTOX). Of 22 South African Campylobacter strains tested, 86% were toxigenic (77% produced CTON, 41% produced CTOX, and 32% produced both types) and 14% were toxin negative. Campylobacter jejuni strains were 67% CTON positive and 47% CTOX positive, whereas Campylobacter coli strains were 100 and 29% positive, respectively.     

30 years of campylobacters: biochemical characteristics and a biotyping proposal for Campylobacter jejuni.

Several biochemical test systems were studied for their potential usefulness for the examination of strains of Campylobacter species. Most (81%) of the C. jejuni strains hydrolyzed sodium hippurate, but strains of C. fetus, C. sputorum, and C. fecalis did not. Some (46%) of the C. jejuni strains and all of the C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, and C. fecalis strains hydrolyzed DNA, but the C. fetus and C. sputorum subsp. mucosalis strains did not. Strains of all species of Campylobacter grew on charcoal-yeast extract agar, but 47% of the C. jejuni strains did not. Alkaline phosphatase activity was recorded for some strains of C. jejuni, but all other species were negative for this activity. Aryl sulfatase activity was detected in 7% of the C. jejuni, 15% of the C. fetus subsp. fetus, and all of the C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, and C. fecalis strains, but it was not detected in the C. fetus subsp. venerealis and C. sputorum subsp. mucosalis strains. Most (93%) of the C. jejuni but none of the other Campylobacter strains contained lactobacillic acid when examined for cellular fatty acids. On the basis of results from three of these tests (hippurate hydrolysis, DNA hydrolysis, and growth on charcoal-yeast extract agar), clinical strains of C. jejuni were placed in eight biotypes.     

Phylogenetic analysis and PCR-restriction fragment length polymorphism identification of Campylobacter species based on partial groEL gene sequences

The phylogeny of 12 Campylobacter species and reference strains of Arcobacter butzleri and Helicobacter pylori was studied based on partial 593-bp groEL gene sequences. The topology of the phylogenetic neighbor-joining tree based on the groEL gene was similar to that of the tree based on the 16S rRNA gene. However, groEL was found to provide a better resolution for Campylobacter species, with lower interspecies sequence similarities (range, 65 to 94%) compared with those for the 16S rRNA gene (range, 90 to 99%) and high intraspecies sequence similarities (range, 95 to 100%; average, 99%). A new universal reverse primer that amplifies a 517-bp fragment of the groEL gene was developed and used for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of 68 strains representing 11 Campylobacter species as well as reference strains of A. butzlerii and H. pylori. Digestion with the AluI enzyme discriminated all Campylobacter species included in the study but showed more intraspecies diversity than digestion with the ApoI enzyme. A hippurate-negative variant of Campylobacter jejuni with a high level of groEL sequence similarity to both C. jejuni (96%) and C. coli (94%) gave a unique AluI profile and an ApoI profile identical to those of other C. jejuni strains. In conclusion, groEL gene sequencing and PCR-RFLP analysis are recommended as valuable tools for the identification of Campylobacter species.