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Detection of ciprofloxacin-resistant Yersinia pestis by fluorogenic PCR using the LightCycler
1Department of Bacterial Diseases, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA. Luther.Lindler@na.amedd.army.mil
Abstract:
We have developed a fluorescence resonance energy transfer (FRET)-based assay to detect ciprofloxacin resistant (Cp(r)) mutants of the biothreat agent Yersinia pestis. We selected spontaneous mutants of the attenuated Y. pestis KIM 5 strain that were resistant to a ciprofloxacin (CIP) concentration of at least 1 microg/ml. DNA sequencing of gyrA encoded by 65 of these mutants revealed that all isolates contained one of four different point mutations within the quinolone resistance-determining region of gyrA. We developed a FRET-based assay that detected all of these mutations by using a single pair of fluorescent probes with sequences complementary to the wild-type Y. pestis gyrA sequence. Melting peak analysis revealed that the probe-PCR product hybrid was less stable when amplification occurred from any of the four mutant templates. This instability resulted in the PCR product obtained from the Cp(r) Y. pestis strains displaying a 4 to 11 degrees C shift in probe melting temperature. Following optimization of the reaction conditions, we were able to detect approximately 10 pg of purified wild-type template DNA or the presence of approximately 4 CFU of wild-type Y. pestis KIM 5 or Cp(r) mutants in crude lysates. Taken together, our results demonstrate the utility of FRET-based assays for detection of Cp(r) mutants of Y. pestis. This method is both sensitive and rapid.
Insights
We developed a rapid, sensitive fluorescence resonance energy transfer (FRET)-based assay to detect ciprofloxacin-resistant (Cp(r)) Yersinia pestis mutants. This assay identifies specific gyrA mutations crucial for resistance in this biothreat agent.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Yersinia pestis is a significant biothreat agent.
- Emergence of antibiotic resistance, particularly to fluoroquinolones like ciprofloxacin, poses a threat to public health.
- Rapid detection of resistant strains is critical for effective treatment and control.
Purpose of the Study:
- To develop and validate a novel assay for the rapid detection of ciprofloxacin-resistant (Cp(r)) Yersinia pestis mutants.
- To identify specific genetic mutations conferring ciprofloxacin resistance in Y. pestis.
- To assess the sensitivity and speed of the developed detection method.
Main Methods:
- Selection and characterization of spontaneous ciprofloxacin-resistant (Cp(r)) Yersinia pestis mutants.
- DNA sequencing of the gyrA gene to identify resistance mutations.
- Development of a fluorescence resonance energy transfer (FRET)-based assay using specific fluorescent probes.
- Melting peak analysis to differentiate wild-type from mutant DNA.
Main Results:
- Sixty-five Cp(r) Y. pestis mutants were isolated, all possessing one of four point mutations in the gyrA quinolone resistance-determining region.
- The FRET-based assay successfully detected all identified gyrA mutations using a single probe pair.
- Mutant templates showed a 4-11°C shift in probe melting temperature compared to wild-type.
- The assay demonstrated high sensitivity, detecting as little as 10 pg of DNA or approximately 4 CFU in crude lysates.
Conclusions:
- The developed FRET-based assay is a sensitive and rapid method for detecting ciprofloxacin-resistant Yersinia pestis mutants.
- This assay can identify key gyrA mutations associated with ciprofloxacin resistance.
- The FRET assay holds significant potential for the timely diagnosis and surveillance of Y. pestis antibiotic resistance.