JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2000, p. 3971–3978 0095-1137/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 38, No. 11
Identification of Ciprofloxacin-Resistant Campylobacter jejuni by Use of a Fluorogenic PCR Assay DAVID L. WILSON,1 SHEILA R. ABNER,2 THOMAS C. NEWMAN,3 LINDA S. MANSFIELD,1,2 AND JOHN E. LINZ1,2,4* National Food Safety and Toxicology Center,1 Department of Food Science and Human Nutrition,4 Department of Microbiology,2 and DOE Plant Research Laboratory,3 Michigan State University, East Lansing, Michigan Received 23 March 2000/Returned for modification 27 May 2000/Accepted 8 August 2000
Fluoroquinolones are one class of antimicrobial agents commonly used to treat severe Campylobacter jejuni infection. C. jejuni strains resistant to high levels of the fluoroquinolone ciprofloxacin (MIC >16 g/ml) have been predominantly characterized with a C3T transition in codon 86 of gyrA. The gyrA gene encodes one subunit of DNA gyrase, which is a primary target for fluoroquinolone antibiotics. This study establishes a rapid PCR-based TaqMan method for identifying ciprofloxacin-resistant C. jejuni strains that carry the C3T transition in codon 86 of gyrA. The assay uses real-time detection, eliminating the need for gel electrophoresis. Optimization of the assay parameters using purified Campylobacter DNA resulted in the ability to detect femtogram levels of DNA. The method should be useful for monitoring the development of ciprofloxacin resistance in C. jejuni. Compiled nucleotide sequence data on the quinolone resistance-determining region of gyrA in Campylobacter indicate that sequence comparison of this region is a useful method for tentative identification of Campylobacter isolates at the species level. 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-Barre´ 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 ⱖ16 g/ml) in C. jejuni appears to be a C3T 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 C3T 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. jejunispecific 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 C3T 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 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 C3T 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. Bacteria were grown on brucella agar (BBL Microbiology Systems, Becton Dickinson, Cockeysville, Md.) supplemented with 5% defibrinated sheep blood (Cleveland Scientific, Bath, Ohio) (BASB) at 37°C, 5% CO2, for 36 to 48 h. Cells were harvested and suspended in brucella broth for chromosomal DNA extraction. 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 g of proteinase K/ml. Cellular debris was removed
* Corresponding author. Mailing address: National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824. Phone: (517) 353-9624. Fax: (517) 432-2310. E-mail: [email protected]
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WILSON ET AL. TABLE 1. Bacterial strains used in this study Species
Strain donorg or reference
25922 H560 15046764 15046764 CR2161 19084571 19094451 197985544 4300 4300 CR2161 4132958 4132958 CR2161 4239928 E961009 E972653 E972796 E972796 CR2161 E9795412 81176 81176 CR2161 CS34 CS42 CS50 CS143 CS161 CS165 3124 3128 3130 3130 CR2161 3131 3133 3136 3383 33291 33291 CR2161 33292 33292 CR2162 33560 33560 CR2 33560 CR6 43429 43429 CR2161 43430 43433 43470 43470 CR2161 49349 UA580 550 1935 18493 1679368 1714086 1777708 1798888 1808517 17010887 17977081 3118 3119 3120 3132 3135 3384 3386 33559 43134 43473 43474 43479 43482
26 ATCC 28 ATCC 30 MSU Veterinary College This study MSU Veterinary College MSU Veterinary College MSU Veterinary College M. Konkel (WSU) This study M. Konkel (WSU) This study M. Konkel (WSU) M. Konkel (WSU) M. Konkel (WSU) M. Konkel (WSU) This study M. Konkel (WSU) C. Pickett (UK) This study F. Angulo (CDC) F. Angulo (CDC) F. Angulo (CDC) F. Angulo (CDC) F. Angulo (CDC) F. Angulo (CDC) I. Wesley (USDA) I. Wesley (USDA) I. Wesley (USDA) This study I. Wesley (USDA) I. Wesley (USDA) I. Wesley (USDA) I. Wesley (USDA) ATCC This study ATCC This study ATCC This study This study ATCC This study ATCC ATCC ATCC This study ATCC 36 MSU Veterinary College MSU Veterinary College MSU Veterinary College MSU Veterinary College MSU Veterinary College MSU Veterinary College MSU Veterinary College MSU Veterinary College MSU Veterinary College MSU Veterinary College I. Wesley (USDA) I. Wesley (USDA) I. Wesley (USDA) I. Wesley (USDA) I. Wesley (USDA) I. Wesley (USDA) I. Wesley (USDA) ATCC ATCC ATCC ATCC ATCC ATCC
Aeromonas salmonicida Enterobacter cloacae Erwinia carotovoraa Escherichia coli E. colia C. jejuni C. jejunib C. jejuni C. jejuni C. jejuni C. jejuni C. jejunib C. jejuni C. jejunib C. jejuni C. jejuni C. jejuni C. jejuni C. jejunib C. jejuni C. jejuni C. jejunib C. jejunib C. jejunib C. jejunib C. jejunib C. jejunib C. jejunib C. jejuni C. jejuni C. jejuni C. jejunib C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejunib C. jejuni C. jejunib C. jejuni C. jejunib C. jejunib C. jejuni C. jejunib C. jejuni C. jejuni C. jejuni C. jejunib C. jejunic C. jejunia C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli
Bovine Bovine Ovine Ovine Canine Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Chicken Chicken Chicken Bovine Human Human Human Human Human Bovine Bovine Bovine Human Human Bovine Human Human Human Human Porcine Porcine Porcine Monkey Porcine Porcine Porcine Porcine Porcine Porcine Porcine Chicken Human Chicken Oyster Porcine Porcine Human Human Human Human
CT for ⌬Rn ⫽ 0.2e
⬍0.2h ⬍0.2 16.3, 16.4 15.8, 15.8
Allele 1 Allele 2
15.8, 15.7 16.9, 16.0
Allele Allele Allele Allele Allele Allele
16.0, 16.0 16.1, 16.1 15.8, 15.9 16.2, 16.4 16.1, 16.1 16.5, 16.5 16.7, 16.8, 16.9, 16.2, 16.7, 16.9, 16.4,
16.8 16.8 16.9 16.2 16.6 16.6 16.2
2 2 1 2 1 2
Allele 1 Allele Allele Allele Allele Allele Allele Allele Allele Allele
2 1 1 1 1 1 1 1 2
Allele 2 16.7, 16.6
Allele 1 Allele 2 Allele 1
15.8, 16.0 16.3, 16.3
Allele 1 Allele 2
⬍0.2h ⬍0.2h NSA
Continued on following page
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TABLE 1—Continued Species
C. coli C. fetus C. hyoileid C. hyointestinalis C. lari C. lari C. laria C. upsaliensis H. pyloria K. pneumoniaea Pseudomonas aeruginosaa
11366 27374 51729 35217 43675 3121 35221 43954 UC946 M5A1 PAO1
Ovine Porcine Porcine Human Chicken Gull Canine Human
Strain donorg or reference
CT for ⌬Rn ⫽ 0.2e
NCTC/GenBank U63413 ATCC ATCC ATCC ATCC I. Wesley (USDA) ATCC/GenBank U63412 ATCC 23 7 20
⬍0.2h ⬍0.2h ⬍0.2h
The DNA sequences of these strains were acquired from GenBank. Ciprofloxacin-resistant isolates that contain a C3T transition in codon 86 of gyrA. Subspecies doylei. d Recently reclassified as a C. coli species (33). e Duplicate-analysis TaqMan assay with TAQ1 probe and 10 ng of chromosomal DNA per reaction. f Duplicate-analysis AD assay with TAQ2 and TAQ3 probes. g ATCC, American Type Culture Collection; WSU, Washington State University; UK, University of Kentucky; CDC, Centers for Disease Control and Prevention; USDA, U.S. Department of Agriculture. h ⌬Rn ⬍0.2 after 40 cycles. i NSA, no significant amplification. b c
by complexing with hexadecyltrimethyl ammonium bromide followed by phenolchloroform 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.). The QRDR (⬃400 bp) of each Campylobacter strain was amplified with primers previously described (18). Primers JL297 (5⬘ CCA TAC CTA CGG CGA TAC CG 3⬘) and JL299 (5⬘ GCC TGA AGC CGG TAC ACC GT 3⬘) were designed for the PCR amplification of the corresponding gyrA region in Escherichia coli but could also be used for amplification of Enterobacter chromosomal DNA. Reagent concentrations in the PCR mixtures were as follows: 2 to 4 ng of template/l, 0.2 mM (each) deoxynucleoside triphosphate (dNTP), 0.5 pmol of each primer/l, approximately 2 mM Mg2⫹, and 0.05 U of Pfu polymerase (Stratagene, La Jolla, Calif.)/l. Thermocycler parameters were as follows: 1 min at 94°C for denaturing, 1 min at 50°C for annealing, and 30 s at 72°C for extension. Samples were cycled 32 times. The amplification product was isolated in a 1.75% low-melting-temperature agarose gel (SeaPlaque GTG; FMC Bioproducts, Rockland, Maine) and purified with a QIAquick gel extraction kit (Qiagen, Valencia, Calif.). The same primers were used for dye terminator cycle sequencing of each amplicon. Sequencing was accomplished with an ABI 377 DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, Calif.) at the Michigan State University (MSU) Sequencing Facility. 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 g/ml. Colonies from these plates were transferred to BASB containing ciprofloxacin at a concentration of 16 g/ml to confirm the resistant phenotype. Chromosomal DNA samples of these mutants were sequenced as described above, and the presence of a C3T 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/l, 200 nM TaqMan probe, 0.05 U of Amplitaq Gold polymerase (Perkin-Elmer)/l, 0.01 U of Amperase UNG (Perkin-Elmer)/l, 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-l 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). For the discrimination between wild-type C. jejuni strains and C. jejuni strains that carry the C3T transition in codon 86 of gyrA, primers JL238 and JL239 were used in combination with TaqMan probes TAQ2 and TAQ3. The TaqMan PCR concentrations and thermocycler parameters were the same as those above except that both TaqMan probes were included in the reaction mixture, each at a concentration of 200 nM. C. jejuni 33560 CR6 or C. jejuni 33292 CR2162 (strains that carry the C3T transition in codon 86 of gyrA) chromosomal DNA was used in the allele 1 standard reactions, and C. jejuni 33292 or C. jejuni 33560 (wild-type strains) DNA was used in the allele 2 standard reactions. All AD reaction mixtures, with the exception of the no-template controls, contained 10 ng of chromosomal DNA. DNA standards were prepared using C. jejuni chromosomal DNA serially diluted in reverse-osmosis-deionized water. The passive reference dye used for normalization of the reporter fluor signal was included in the TaqMan reaction buffer. Allelic discrimination standards were prepared according to the specifications of Applied Biosystems.
RESULTS 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 g/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. Primer JL238 (20 nucleotides) showed 100% identity to 33 of 34 C. jejuni strains analyzed, the exception being C. jejuni E961009, which contained a one-base mismatch (95% identity; Fig. 1). In contrast, JL238 showed much lower identity to the analogous QRDRs in the Campylobacter coli (65% identity), Campylobacter lari (70%), Campylobacter fetus (70%), Campylobacter upsaliensis (65%), Campylobacter hyoilei (65%), Campylobacter hyointestinalis (65%), Helicobacter pylori (70%), E. coli (60%), Enterobacter cloacae (60%), Klebsiella pneumoniae (60%), and Erwinia carotovora (60%) sequences examined. Significantly, the three nucleotides at the 3⬘ end of JL238 do not match DNA of any species analyzed except C. jejuni. Primer JL239 (20 nucleotides) showed 100% identity with all C. jejuni sequences analyzed and much lower identity with
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conserved in the chromosomal region where TAQ2 and TAQ3 specifically anneal. Only C. jejuni strain 49349 contained a mismatch within the probe sequences (Fig. 1). The TAQ2 and TAQ3 probes had approximately 86% identity with C. coli sequences. Detection of C. jejuni chromosomal DNA. The results of TaqMan assays are presented in Table 1. Data are reported as CT (threshold cycle) for a ⌬Rn of 0.2 U, where ⌬Rn 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 ⌬Rn 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
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.
analogous chromosomal sequences of C. coli (50% identity), C. lari (50%), C. fetus (40%), C. upsaliensis (55%), C. hyoilei (50%), C. hyointestinalis (50%), H. pylori (45%), E. coli (65%), E. cloacae (65%), K. pneumoniae (55%), and E. carotovora (70%). These primers were sufficiently specific to distinguish between the C. jejuni strains in our collection and the other bacterial species examined by standard PCR methods (Fig. 3). The positioning of JL238 and JL239 adjacent to codon 86 (Fig. 1) was also important for the development of the AD assay. TAQ1 (26 nucleotides) was designed to identify C. jejuni and was therefore localized to a region between JL238 and JL239 with 100% identity to the C. jejuni strains in our culture collection. It was not necessary that this probe discriminate between different Campylobacter species because the primers served this purpose. TAQ1 identity with C. coli chromosomal DNA ranged from 88 to 92% among the strains analyzed. TAQ2 (28 nucleotides) and TAQ3 (29 nucleotides) were designed to distinguish between wild-type C. jejuni and strains that carry the C3T transition in codon 86 of gyrA. Both probes anneal to a region of DNA containing codon 86 of gyrA. The probes are identical except that TAQ3 is a single nucleotide longer at the 3⬘ terminus and encodes isoleucine (ATA) at codon 86, while TAQ2 encodes threonine (ACA) at this codon. The C. jejuni isolates analyzed in this study were highly
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.
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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 l of a 50-l 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.
threshold level). When 10 ng of chromosomal DNA (roughly equivalent to 107 genomes of C. jejuni ) 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 ⌬Rns that were less than 0.2 U after 40 PCR cycles. The relationship between the threshold cycle and the initial quantity of DNA in a TaqMan sample is approximately linear over at least 7 log units of DNA mass ranging from 10 ng to 10 fg (Fig. 4). This linear relationship was maintained when either TAQ1 or TAQ2 was used in a TaqMan reaction with wild-type C. jejuni DNA. One femtogram of C. jejuni DNA could be
detected by 40 PCR cycles; however, this level of detection was inconsistent. 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 C3T 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. The results of AD assays performed in this study are presented in Table 1. AD reactions characterized as allele 2 (codon 86, ACA) possess an allele 2-specific signal (FAM) greater than 0.75 U and an allele 1-specific signal (TET) less than 0.25 U. The AD reactions characterized as allele 1 (codon 86, ATA) created an allele 1-specific signal greater than 0.90 U and an allele 2-specific signal less than 0.10 U. Assays performed with chromosomal DNA of other bacterial species produced allele 1- and allele 2-specific signals of less than 0.05 U. Figure 5 demonstrates the ability to clearly distinguish between mutant, wild-type, and no-amplification reactions in an AD assay. In Fig. 6, the FAM and TET emissions are shown in real time for three C. jejuni chromosomal reactions from the AD PCR results presented in Fig. 5. Strains 33560 and 49349 are wild type, and strain 33292 CR2162 carries the C3T transition in codon 86 of gyrA. 33560 produced the greatest FAM signal at PCR cycle 40. The chromosomal sequence of this strain possessed a 100% match with TAQ2. The chromosomal sample of 33292 CR2162 produced the lowest FAM signal and contained a single mismatch, located in codon 86, with TAQ2. A comparison of TET emissions from the same reactions shows 33292 CR2162, which possesses 100% chromosomal identity with TAQ3, with the highest TET signal and 33560, possessing a single mismatch with TAQ3, with a lower TET emission. Strain 49349 is unique in our C. jejuni collection because it
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 l of each of the 50-l TaqMan PCR mixtures. Left lane, 100-bp marker. NTC, no-template control.
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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.
contains at least one nucleotide mismatch with both TAQ1 and TAQ2. Like other wild-type C. jejuni strains, 49349 possesses a mismatch with TAQ3 located in codon 86, but it also contains a mismatch with both TAQ2 and TAQ3 at nucleotide position 32 as depicted in Fig. 1. Despite this unique nucleotide substitution in 49349, the strain is qualified as wild type (i.e., codon 86 is ACA) in the AD assay (Fig. 5). This strain is unable to grow on BASB supplemented with ciprofloxacin at a concentration of 16 g/ml. DISCUSSION 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 C3T 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 C3T 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 ⱖ16 g/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 C3T transitions at the second position of codon 86 (nucleotide position 256) in gyrA and ciprofloxacin MICs ranging from 16 to 64 g/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 C3T 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
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 C3T transition in codon 86 of gyrA and is resistant to ciprofloxacin.
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strains resistant to high levels of ciprofloxacin (MIC ⱖ16 g/ 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 g/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 C3T 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 Thr3Lys 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. ACKNOWLEDGMENTS We thank Robert Walker, Irene Wesley, Michael Konkel, Carol Pickett, Joseph Madden, and Frederick Angulo for generously providing many of the strains used in this study, Frances Downes for assis-
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