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Updated: Aug 1, 2026
Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens
Published on: March 10, 2020
Identification of ciprofloxacin-resistant Campylobacter jejuni by use of a fluorogenic PCR assay
D L Wilson1, S R Abner, T C Newman
1National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824, USA.
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.
Insights
This study developed a rapid PCR-TaqMan assay to detect ciprofloxacin resistance in Campylobacter jejuni. The method identifies a specific gyrA gene mutation, aiding in monitoring antimicrobial resistance development.
Area of Science:
- Microbiology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Fluoroquinolones are critical for treating severe Campylobacter jejuni infections.
- Ciprofloxacin resistance in C. jejuni is often linked to a C-->T transition in codon 86 of the gyrA gene.
- The gyrA gene encodes a subunit of DNA gyrase, a key target for fluoroquinolones.
Purpose of the Study:
- To establish a rapid, real-time PCR-based TaqMan method for identifying ciprofloxacin-resistant C. jejuni strains.
- To detect the specific C-->T transition in codon 86 of the gyrA gene associated with resistance.
- To provide a tool for monitoring the emergence and spread of fluoroquinolone resistance in C. jejuni.
Main Methods:
- Development and optimization of a PCR-based TaqMan assay.
- Utilizing real-time detection to eliminate the need for gel electrophoresis.
- Testing the assay's sensitivity using purified Campylobacter DNA.
Main Results:
- The developed TaqMan assay can rapidly identify C. jejuni strains with the specific gyrA C-->T transition.
- The assay demonstrated high sensitivity, detecting femtogram levels of DNA.
- Sequence comparison of the gyrA quinolone resistance-determining region aids in Campylobacter species identification.
Conclusions:
- The PCR-TaqMan method offers a fast and efficient way to detect ciprofloxacin resistance in C. jejuni.
- This assay is valuable for surveillance programs aimed at tracking antimicrobial resistance.
- Nucleotide sequencing of gyrA provides insights into both resistance mechanisms and Campylobacter species identification.
