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Antimicrob Agents Chemother. 2017 Aug 24;61(9). pii: e00628-17. doi: 10.1128/AAC.00628-17. Print 2017 Sep.

Digital PCR for Detection and Quantification of Fluoroquinolone Resistance in Legionella pneumophila.

Author information

1
Institut de Biologie et de Pathologie, Centre Hospitalier Universitaire Grenoble Alpes, Grenoble, France ahennebique@chu-grenoble.fr sboisset@chu-grenoble.fr.
2
Université Grenoble Alpes, TheREx, TIMC-IMAG, CNRS, Grenoble, France.
3
Institut de Biologie et de Pathologie, Centre Hospitalier Universitaire Grenoble Alpes, Grenoble, France.
4
Université Grenoble Alpes, Braintech Lab, CEA, INSERM, Grenoble, France.
5
Centre National de Référence des Légionelles, Institut des Agents Infectieux, Groupe Hospitalier Nord, Hospices Civils de Lyon, Lyon, France.
6
Centre International de Recherche en Infectiologie, INSERM, Université Lyon 1, CNRS, ENS, Lyon, France.
7
Réanimation, Centre Hospitalier Universitaire Grenoble Alpes, Grenoble, France.
8
Radiopharmaceutiques Biocliniques, INSERM, La Tronche, France.

Abstract

The emergence of fluoroquinolone (FQ)-resistant mutants of Legionella pneumophila in infected humans was previously reported using a next-generation DNA sequencing (NGS) approach. This finding could explain part of the therapeutic failures observed in legionellosis patients treated with these antibiotics. The aim of this study was to develop digital PCR (dPCR) assays allowing rapid and accurate detection and quantification of these resistant mutants in respiratory samples, especially when the proportion of mutants in a wild-type background is low. We designed three dPCRgyrA assays to detect and differentiate the wild-type and one of the three gyrA mutations previously described as associated with FQ resistance in L. pneumophila: at positions 248C→T (T83I), 259G→A (D87N), and 259G→C (D87H). To assess the performance of these assays, mixtures of FQ-resistant and -susceptible strains of L. pneumophila were analyzed, and the results were compared with those obtained with Sanger DNA sequencing and real-time quantitative PCR (qPCR) technologies. The dPCRgyrA assays were able to detect mutated gyrA sequences in the presence of wild-type sequences at up to 1:1,000 resistant/susceptible allele ratios. By comparison, Sanger DNA sequencing and qPCR were less sensitive, allowing the detection of gyrA mutants at up to 1:1 and 1:10 ratios, respectively. When testing 38 respiratory samples from 23 legionellosis patients (69.6% treated with an FQ), dPCRgyrA detected small amounts of gyrA mutants in four (10.5%) samples from three (13.0%) patients. These results demonstrate that dPCR is a highly sensitive alternative to quantify FQ resistance in L. pneumophila, and it could be used in clinical practice to detect patients that could be at higher risk of therapeutic failure.

KEYWORDS:

Legionella pneumophila; antibiotic resistance; digital PCR; fluoroquinolones; gyrA

PMID:
28674045
PMCID:
PMC5571300
DOI:
10.1128/AAC.00628-17
[Indexed for MEDLINE]
Free PMC Article

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