INTRODUCTION

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Campylobacter jejuni is a gram-negative microaerophilic bacterial pathogen of humans and animals. In humans, C. jejuni infection is characterized by acute diarrheal disease (5) and recently has been associated with Guillain-Barré syndrome, a peripheral neuropathy characterized by limb weakness and other neurological and systemic sequelae (5, 17).

Erythromycin, fluoroquinolones, and tetracyclines are the antimicrobial agents commonly used to treat severe C. jejuni infection (1, 5). Resistance to these antibiotics in human and animal Campylobacter isolates has been established (10, 11, 21, 22, 31, 32). The predominant mechanism for high-level ciprofloxacin (a fluoroquinolone) resistance (MIC 16 µg/ml) in C. jejuni appears to be a CT transition in codon 86 in the quinolone resistance-determining region (QRDR) of gyrA. This gene encodes one subunit of DNA gyrase, which is a target for fluoroquinolone antibiotics. The codon 86 mutation results in a threonine-to-isoleucine substitution in the functional protein (6, 12, 18, 29, 36). Ciprofloxacin susceptibility testing of Campylobacter is commonly performed using standard methods such as broth or agar dilution (6, 10, 11, 29). To more efficiently monitor C. jejuni resistance to quinolones, our laboratory developed a rapid PCR-based TaqMan method (15) for the detection of C. jejuni isolates that carry the CT transition in codon 86 of gyrA.

In addition to two standard DNA primers, TaqMan PCR uses a dual-labeled fluorescent probe that binds to target DNA between the flanking primers. Taq polymerase digests the bound probe during amplification releasing the 5' reporter fluor, 6-carboxy-fluorescein (FAM), from the blocking effects of the 3'-quenching fluor, 6-carboxy-tetramethyl-rhodamine (TAMRA). Fluorescence emission is monitored during the reaction and is directly proportional to the amount of amplification product produced. The TaqMan assay allows real-time detection of specific DNA and provides a powerful tool for pathogen identification (3, 16, 25).

To discriminate between wild-type and ciprofloxacin-resistant strains of C. jejuni, a variation of TaqMan, allelic discrimination (AD), was employed. AD is dependent on competition between two probes labeled with the same quenching fluor but different reporter fluors, FAM or tetrachloro-6-carboxy-fluoroscein (TET). One probe is specific for wild-type QRDR DNA (codon 86, ACA), and the other probe is specific for the mutant QRDR (codon 86, ATA). The genotype of the template is indicated by the relative fluorescence emissions of the two reporter tags.

Nucleotide sequence analysis of the QRDRs of several dozen Campylobacter isolates enabled the design of C. jejuni-specific PCR primers and TaqMan probes. A TaqMan assay which detected femtogram levels of C. jejuni chromosomal DNA was developed. The AD variation of this assay could effectively distinguish between wild-type strains and strains that carry the CT transition in codon 86 of gyrA. Sequence analysis of QRDRs of gyrA was also shown to be useful for tentative identification of Campylobacter isolates to the species level.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. Campylobacter strains used for assay development are listed in Table 1. Strains CS34, CS42, CS50, CS143, CS161, and CS165 are human clinical isolates that contain the CT transition in codon 86 of gyrA and that are resistant to high levels of ciprofloxacin (37). The other codon 86 mutant C. jejuni strains used in this study were derived in our laboratory. Strain identification was confirmed either with the API CAMPY system (Biomerieux, Marcy l'Etoile, France) or in collaboration with the diagnostic laboratory at the Michigan Department of Community Health (Lansing, Mich.) using biochemical analyses (24) in combination with fatty acid profiling.

DNA isolation and sequencing. Bacterial cultures were pelleted and DNA was extracted by standard methods (2). Briefly, cells were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) and lysed with 0.5% sodium dodecyl sulfate in the presence of 100 µg of proteinase K/ml. Cellular debris was removed by complexing with hexadecyltrimethyl ammonium bromide followed by phenol-chloroform extraction and RNase A digestion. DNA was precipitated with 0.6 volume of isopropanol, redissolved in TE, and quantitated using a DU 530 spectrophotometer (Beckman Instruments, Schaumburg, Ill.).

DNA sequence analysis. Multiple sequence alignment (Fig. 1) and phylogram analysis (Fig. 2) of the Campylobacter QRDRs was performed using the Pileup and GrowTree programs of Genetics Computer Group (Madison, Wis.) SeqWeb software. Sequence alignment allowed the design of primers JL238 (5' TGG GTG CTG TTA TAG GTC GT 3') and JL239 (5' GCT CAT GAG AAA GTT TAC TC 3') and TaqMan probes TAQ1 (5' FAM-TTT GCT TCA GTA TAA CGC ATC GCA GC-TAMRA 3'), TAQ2 (5' FAM-CCA CAT GGA GAT ACA GCA GTT TAT GAT G-TAMRA 3'), and TAQ3 (5' TET-CCA CAT GGA GAT ATA GCA GTT TAT GAT GC-TAMRA 3'). Primers were synthesized at the MSU Macromolecular Structure Facility. TaqMan probes were produced at Integrated DNA Technologies (Coralville, Iowa). The FAM or TET reporter dye of each TaqMan probe was attached to the 5' nucleotide, and the TAMRA quencher was positioned at the 3' nucleotide. Probes were phosphorylated at the 3' end to prevent extension during PCR amplification.

Mutant isolation. Ciprofloxacin-resistant C. jejuni mutants (Table 1) were acquired using the following method. Bacterial cultures were grown as indicated above and resuspended in brucella broth to a concentration of ~10¹⁰ CFU/ml. Cultures were plated onto BASB supplemented with ciprofloxacin (Bayer, Kankakee, Ill.) at concentrations of 2 or 16 µg/ml. Colonies from these plates were transferred to BASB containing ciprofloxacin at a concentration of 16 µg/ml to confirm the resistant phenotype. Chromosomal DNA samples of these mutants were sequenced as described above, and the presence of a CT transition in codon 86 of gyrA was confirmed for each mutant.

TaqMan PCR. Primers JL238 and JL239, along with TaqMan probe TAQ1, were used for the identification of C. jejuni chromosomal DNA. The TaqMan PCR concentrations were as follows: 1× TaqMan buffer (Perkin-Elmer), 0.2 mM (each) dNTP (0.4 mM dUTP), 0.5 pmol of each primer/µl, 200 nM TaqMan probe, 0.05 U of Amplitaq Gold polymerase (Perkin-Elmer)/µl, 0.01 U of Amperase UNG (Perkin-Elmer)/µl, 4.5 mM MgCl₂, 0.05% gelatin, 0.01% Tween 20. The PCR thermocycling parameters were as follows. Initial denaturation was at 95°C for 10 min, and the annealing and polymerization steps were combined at 60°C for 1 min and were followed by denaturation for 30 s. The 50-µl PCR samples were cycled 40 times. Prior to the initial denaturation, all TaqMan reaction mixtures were incubated at 50°C for 2 min in the presence of Amperase UNG in an effort to prevent PCR product carryover. Fluorescence emissions were monitored in real time with an ABI Prism 7700 sequence detection system (Perkin-Elmer).

	RESULTS

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Primer and probe design. Nucleotide sequence analysis was conducted on the QRDR of gyrA from 28 wild-type C. jejuni strains and 18 C. jejuni isolates that were able to grow on BASB medium containing ciprofloxacin at a concentration of 16 µg/ml. QRDR sequence alignment of DNA from selected Campylobacter strains is presented in Fig. 1. TaqMan primers (JL238 and -239) and probes (TAQ1, -2, and -3) were designed based on similar alignments (data not shown) of DNA from the Campylobacter strains listed in Table 1. A phylogram of this 300-bp QRDR alignment is presented in Fig. 2.

View larger version (87K): [in this window] [in a new window]   FIG. 1.   Multiple sequence alignment of selected Campylobacter isolates. The alignment represents a 200-bp fragment of gyrA that includes the QRDR. Codon 86 in C. jejuni is positioned at nucleotides 42 to 44 (underlined). Nucleotide positions within primer and probe sequences that are not conserved among C. jejuni strains are also underlined. Cj, C. jejuni; Cc, C. coli; Cl, C. lari; Cf, C. fetus.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Phylogram analysis of Campylobacter isolates. The phylogram is based on a 300-bp DNA fragment of the Campylobacter QRDR in gyrA. Nodes indicate a common ancestor. The lengths of the horizontal lines represent the degree of relatedness between individual strains. As, Aeromonas salmonicida; Pa, Pseudomonas aeruginosa; Ec, E. coli; Ecl, E. cloacae; Kp, K. pneumoniae; Hp, H. pylori; Cu, C. upsaliensis; Chynt, C. hyointestinalis; Ch, C. hyoilei; Ecarotovora, E. carotovora. Other abbreviations are as defined for Fig. 1.

View larger version (89K): [in this window] [in a new window]   FIG. 3.   Agarose gel electrophoresis of PCRs. The first tier (from the top) of the gel represents the amplicons produced from the QRDR sequencing primers. The second tier shows the amplification products produced with primers JL238 and JL239. Markers (100 bp) were loaded in the first lanes. Each lane was loaded with 20 µl of a 50-µl PCR mixture. The PCR conditions were as specified in the text with the exceptions that Platinum Taq DNA polymerase (Life Technologies, Gibco BRL, Grand Island, N.Y.) was the enzyme used, the concentration of each dNTP was 0.2 mM, and the MgCl2 concentration was 1.5 mM. TaqMan probes and buffer, Amperase UNG, Tween 20, and gelatin were not included in these reactions.

Detection of C. jejuni chromosomal DNA. The results of TaqMan assays are presented in Table 1. Data are reported as C_T (threshold cycle) for a R_n of 0.2 U, where R_n is the difference in normalized reporter fluor signal between a PCR tube with sample DNA and a no-template control. The threshold level (defined in our experiments as a R_n of 0.2 U) is used to indicate a positive reaction and was adjusted in order to enhance the linear relationship between DNA mass and threshold cycle (the cycle at which reporter emissions reach the threshold level). When 10 ng of chromosomal DNA (roughly equivalent to 10⁷ genomes of C. jejuni [35]) was used, a positive reaction identifying the DNA to be of C. jejuni origin was indicated before 18 PCR cycles in all reaction mixtures containing C. jejuni DNA. Reaction mixtures containing chromosomal DNA from other species produced R_ns that were less than 0.2 U after 40 PCR cycles.

View larger version (44K): [in this window] [in a new window]   FIG. 4.   Standard curve of initial DNA mass in a TaqMan reaction versus the threshold cycle. Ten-fold serial dilutions of C. jejuni chromosomal DNA were performed, and equal aliquots of each dilution were used in TaqMan reactions using TAQ2 as the probe. The starting quantity of DNA in these reactions is plotted versus the threshold cycle. (Inset) Gel electrophoresis analysis of 20 µl of each of the 50-µl TaqMan PCR mixtures. Left lane, 100-bp marker. NTC, no-template control.

AD of C. jejuni chromosomal DNA conferring ciprofloxacin resistance and susceptibility. AD is an endpoint PCR assay in which reporter fluor emissions, after the final PCR cycle, indicate whether a reaction mixture contains chromosomal DNA of a wild-type strain of C. jejuni, a strain that carries the CT transition in codon 86 of gyrA, or no C. jejuni DNA. A series of no-template control, allele 1-specific (codon 86, ATA), and allele 2-specific (codon 86, ACA) standard reaction mixtures are prepared with each experiment in order to obtain fluorescence spectra for each type of reaction. The spectra of unknown chromosomal samples are compared to those for these reference reactions, and detection system algorithms are used to assign a value from approximately 0 to 1 U for the contribution of each allele-specific signal to the reaction spectra. Based on these values, unknown samples can be categorized as described above.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   Determination of a mutant or wild-type genotype by AD. AD reactions are qualified as wild-type C. jejuni DNA (codon 86, ACA), mutant C. jejuni DNA (codon 86, ATA), or lacking amplification (absence of C. jejuni DNA). A wild-type (allele 2) reaction is characterized by an allele 2-specific signal greater than 0.75 U and an allele 1-specific signal less than 0.25 U. A mutant (allele 1) reaction is characterized by an allele 1-specific signal greater than 0.90 U and an allele 2-specific signal less than 0.10 U. An AD reaction which lacks C. jejuni DNA is characterized by allele 1- and allele 2-specific signals of less than 0.05 U.

View larger version (94K): [in this window] [in a new window]   FIG. 6.   Amplification plots of wild-type and mutant C. jejuni DNA in an AD assay. Both FAM (wild-type) and TET (mutant) reporter probes are included in the AD assay reaction. A reaction with C. jejuni DNA produces both FAM and TET signals above background levels. The relative fluorescence emissions after the final PCR cycle determine if a C. jejuni sample is mutant or wild type. 33560 and 49349 are wild-type C. jejuni strains (codon 86, ACA). The C. jejuni 33292 CR2162 isolate contains a CT transition in codon 86 of gyrA and is resistant to ciprofloxacin.

	DISCUSSION

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Nucleotide sequence alignment of the QRDRs of gyrA from bacterial isolates in our culture collection allowed us to develop what appear to be C. jejuni-specific PCR primers and TaqMan probes. However, the alignment did not include all Campylobacter species or all other closely related bacteria. The oligonucleotides were utilized to develop rapid assays (3 to 4 h after DNA isolation) for identification of C. jejuni and C. jejuni isolates that carry a CT transition in codon 86 of gyrA. The TaqMan assay correctly identified all the C. jejuni strains tested, while failing to amplify the chromosomal DNA of other bacterial species. The AD assay, in all instances, discriminated between wild-type C. jejuni and those C. jejuni strains that carry a CT transition in codon 86 of gyrA.

Phylogram analysis of the multiple sequence alignment supported the use of gyrA sequence comparison for tentative identification of Campylobacter to the species level (14, 18). The placement of C. hyoilei 51729 among the C. coli isolates within the phylogram is in agreement with the recent reclassification of C. hyoilei as C. coli (34).

In summary, the TaqMan assay is very sensitive; it can detect 1 fg of C. jejuni chromosomal DNA, which is roughly equivalent to a single C. jejuni genome, and can repeatedly detect 10 fg of chromosomal DNA. The assay monitors amplification of the PCR product in real time, eliminating the need for gel electrophoresis in diagnostic settings. The assay is also quantitative for C. jejuni because of the linear relationship between initial DNA mass and the PCR threshold cycle. The development of the AD variation of the TaqMan assay offers the potential for rapid and sensitive identification of C. jejuni isolates resistant to high levels of ciprofloxacin (MIC 16 µg/ml).

Ciprofloxacin, a fluoroquinolone, was introduced into medicinal practice in the 1990s and, like its parent compound, nalidixic acid, targets bacterial DNA gyrase and DNA topoisomerase IV. Quinolone antibiotics inhibit DNA synthesis in susceptible bacteria presumably by binding to a topoisomerase-DNA intermediate rendering it incapable of ligating double-stranded breaks in chromosomal DNA (8, 9). Mechanisms of resistance to ciprofloxacin have been characterized in many pathogenic bacteria by chromosomal mutations in the genes encoding the subunits of DNA gyrase (gyrA and gyrB) and DNA topoisomerase IV (parC and parE) (13, 19, 27, 33).

By analyzing the QRDR of gyrA, Wang et al. first described mutations in ciprofloxacin-resistant C. jejuni mutants (36). Four of the mutant strains in their study (three of which were clinical isolates) had CT transitions at the second position of codon 86 (nucleotide position 256) in gyrA and ciprofloxacin MICs ranging from 16 to 64 µg/ml. These data were supported by subsequent studies of C. jejuni clinical isolates in Greece (6), Germany (18), Sweden (12), and Spain (29) that showed that 27 of 28 strains with high levels of resistance to ciprofloxacin also had the same CT transition in the QRDR of gyrA. The single exception was a Spanish isolate that encoded a substitution of Lys for Thr at codon 86.

The 17 published QRDR sequences (12, 36, 37) for C. jejuni strains resistant to high levels of ciprofloxacin (MIC 16 µg/ml) show 100% identity to the TAQ3 mutant probe used in our assay. We have also sequenced the gyrA QRDRs of 12 C. jejuni laboratory isolates that were able to grow on BASB medium containing ciprofloxacin at a concentration of 16 µg/ml. All 12 possessed a sequence identical to that of TAQ3. Multiple sequence alignment of the QRDRs of the wild-type C. jejuni strains in Table 1 showed that each maintained a cytosine at nucleotide position 256 in codon 86 and were in effect matches for our TAQ2 wild-type probe. The predominance of the CT transition in codon 86 of gyrA among C. jejuni strains resistant to high levels of ciprofloxacin indicates a functional role for gyrA in conferring high-level ciprofloxacin resistance. The chromosomal sequence match in these strains with TAQ3 supports the feasibility of our AD approach for identifying C. jejuni isolates resistant to high levels of ciprofloxacin.

C. jejuni strains that have mismatches with both TAQ2 and TAQ3 probes have been identified (Fig. 1) (29, 36). However, the chromosomal DNA of such strains should be amplified with primers JL238 and JL239, and TAQ2 and TAQ3 probes should anneal (although with less affinity) to this DNA. Indeed, the TaqMan and AD assays performed with chromosomal DNA of C. jejuni 49349 (a strain known to contain nucleotide mismatches in both TAQ2 and TAQ3) indicated relatively high levels of both FAM and TET emissions and had the ability to correctly identify this strain as susceptible to high levels of ciprofloxacin. These data in combination with the proper identification of C. jejuni E961009 despite a nucleotide mismatch in the JL238 primer, suggest that single-base substitutions at most positions in the primers and probes will not affect the specificity of the TaqMan and AD assays.

The above-mentioned Spanish C. jejuni isolate (29) with the ThrLys substitution encoded by codon 86 also contains a mismatch with the TAQ2 and TAQ3 probes. This mismatch is at the same position in both probes but is at the only position where TAQ2 and TAQ3 differ. Given the hypothesis that the strain is conserved at all other primer and probe sequences, we predict that the mutant will produce a fluorescence spectrum in an AD assay that will be difficult to qualify as wild type or mutant. The isolate should register as C. jejuni and should warrant further sequence analysis.

The AD assay was performed using standards and samples with known concentrations of DNA, and the DNA in a reaction tube was of a single genotype. Experiments will need to be performed, and perhaps software will need to be modified, in order to adapt the AD assay for environmental and clinical specimens in complex mixtures. One possible approach for this adaptation would be to run a TaqMan assay using the TAQ1 probe, which appears to be more highly conserved in Campylobacter than TAQ2, in parallel with an AD assay using TAQ2 and TAQ3. The TaqMan assay could be used to detect C. jejuni DNA in a sample and determine its concentration. These data could then be used to help interpret the end point results of the AD assay. Recent innovations in the ability to perform TaqMan assays in the field (4) provide more optimism for the prospect of rapid environmental sampling for resistant bacteria. The information obtained from rapid and sensitive assays for the detection of antibiotic-resistant pathogens should allow for early informed decisions for the treatment of infections to be made and should also have an impact on shaping policies regarding the use of antibiotics.