Résumé
Fluoroquinolone (FQ)-resistant Campylobacter jejuni is a serious problem worldwide that limits effective treatment of infections. The traditional detection method depends on bacterial isolation and MIC testing, or traditional PCR, which is time-consuming and hard to identify the FQ-resistant C. jejuni in a high abundance wild-type background. This study aimed to develop a rapid and accurate ddPCR assay to detect FQ-resistant C. jejuni mutants based on the crucial resistance mutation C257T (Thr-86-Ile) in gyrA. Our ddPCR gyrA assay showed high specificity and accuracy. Sanger sequencing and the qPCR assay could only recognize gyrA mutant sequences when the ratios of wild-type/mutant were 1:1 or 10:1, respectively. Our ddPCR gyrA assay was able to detect gyrA mutant sequences in the mixtures with up to at least 1000:1 wild-type/mutant ratios, which suggested a significant advantage to distinguish the low mutant signal from the wild-type background. We further monitored the occurrence of gyrA mutations under ciprofloxacin pressure using our ddPCR gyrA assay, and clearly showed that the transition of a dominant C. jejuni subpopulation from wild-type to gyrA C257T mutant, resulting in FQ-resistance. We tested 52 samples from live chickens and retail chicken meat and showed that four samples contained wild-type/mutant mixtures comprising 1.7%, 28.6%, 53.3%, and 87.0% gyrA C257T mutants, respectively. These results demonstrated that the ddPCR gyrA assay was a highly sensitive alternative method to distinguish and quantify FQ-resistant C. jejuni infections that could help guide the appropriate use of FQs in clinical practice.
Langue d'origine | English |
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Journal | Microbiology spectrum |
Volume | 10 |
Numéro de publication | 2 |
DOI | |
Statut de publication | Published - avr. 2022 |
Note bibliographique
Funding Information:This work was supported by the National Key Research and Development Program of China (2021YFD1800401), the National Natural Science Foundation of China (31873017), the China Agriculture Research System of MOF, and MARA (CARS-41-G13), and the Foreign expert's project of Hubei Province (2020BJH029). We declare no conflict of interest.
Publisher Copyright:
Copyright © 2022 Luo et al.
ASJC Scopus Subject Areas
- Physiology
- Ecology
- General Immunology and Microbiology
- Genetics
- Microbiology (medical)
- Cell Biology
- Infectious Diseases
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't