Identification of ciprofloxacin-resistant Campylobacter jejuni by use of a fluorogenic PCR assay

Abstract

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 microg/ml) have been predominantly characterized with a C-->T 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 C-->T 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.

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.

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.

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.

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.

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.

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 C→T transition in codon 86 of gyrA and is resistant to ciprofloxacin.