Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans

Abstract

Background: Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila.

Methods: We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples.

Findings: We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment.

Interpretation: In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

Keywords: Fluoroquinolone resistance; In vivo selection; Legionella pneumophila; Legionellosis; Next generation sequencing; Resistance detection.

Fig. 1

Detection of gyrA QRDR mutations using the qPCRgyrALp assay. Melting curves and peaks obtained with the qPCRgyrALp assay are shown for the L. pneumophila Paris strain (Martínez, 2008), respiratory samples from patients #1 to #4 (Skippington and Ragan, 2011), the L. pneumophila Lorraine strain (Pendleton et al., 2013), the L. pneumophila Philadelphia strain (Rolain and Raoult, 2005), the L. pneumophila Lens strain (Rouli et al., 2012), and the L. pneumophila Paris mutant strains LPPI1 and LPPI4 harboring one (Maurin et al., 2001), and two (Binet and Maurelli, 2005) gyrA mutations, respectively (appendix p 5). The units of the y axes are expressed as fluorescence levels derived through time. Melting peaks of 59.34 ± 0.41 °C and 56.35 ± 0.42 °C are diagnostic of reference and mutant alleles of the gyrA QRDR, respectively.

Fig. 2

Clinical history and gyrA83 mutational dynamics for patients #2. The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #2 over his hospitalization stay. The number of days for relevant steps during hospitalization is given inside circles according to D0 which corresponds to the day of legionellosis diagnosis as determined by a positive urinary antigen test (UAT +). qPCR-mip + indicates a positive qPCRmip assay in respiratory samples. Results of the qPCRgyrALp assay are indicated as “neg” (no amplification of the gyrA QRDR), “wt” and “mut” (a positive PCR assay with a reference and mutant-type melting peak, respectively). The proportion of the reads corresponding to the T83I gyrA83 mutation, determined by next-generation sequencing, is shown (NGS-mut). Small arrows indicate the periods of antibiotic therapy. Lev: levofloxacin; Cp: ciprofloxacin; Azi: azithromycin; Ery: erythromycin; Amc: amoxicillin-clavulanate; Ca: ceftazidime; Tc: teicoplanin.

Fig. 3

Clinical history, and dynamics of L. pneumophila DNA load and gyrA83 mutations in respiratory samples from patient #4. (a) The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #4 over his hospitalization stay (see legend of Fig. 2). (b) Dynamics of L. pneumophila DNA load, expressed as log GU/mL clinical sample, in respiratory samples of patient # 4 over time after admission in ICU. The total, reference, and mutant gyrA QRDR DNA loads are shown by circles, triangles and squares, respectively. Data were extracted from the qPCRmip assay and NGS experiments. (c) Relative proportion of the reference (triangles) and mutant (squares) gyrA QRDR allele in respiratory samples of patient # 4 over time after admission in ICU. Data were extracted from the NGS experiments.