We investigated whether a correlation may

We investigated whether a correlation may exist between a positive qPCRgyrALp assay, the presence of gyrA83 mutations in respiratory samples and severe legionellosis evolution (Table 2). A trend was noted, albeit not significant, for more frequent admission to the ICU for patients with a positive qPCRgyrALp assay. Also, patients with a positive qPCRgyrALp assay were more likely to receive a combination of a macrolide and a fluoroquinolone during their hospital stay. No significant difference was observed between the qPCRgyrALp results and the mean global hospital stay or death. A correlation between the presence of gyrA83 mutations in respiratory samples and a worse prognosis was impossible to establish because only 2 patients of 82 displayed such criterion.
Discussion It has commonly been assumed that L. pneumophila could not develop fluoroquinolone resistance (Jespersen et al., 2010; Yu et al., 2009; Pedro-Botet and Yu, 2009; Burdet et al., 2014). Very recently however, a fluoroquinolone-resistant strain of L. pneumophila was isolated in a legionellosis patient treated with ciprofloxacin (Bruin et al., 2014). This unique and preliminary observation raises the question of the dynamics and origin of fluoroquinolone resistance in L. pneumophila. It has been speculated that the environmental release of BV6 may promote the emergence of antibiotic-resistant bacterial lineages (Davies and Davies, 2010), but this has never been demonstrated for Legionella species. Here, using a combination of molecular and genomic approaches, we investigated the infection dynamics of two legionellosis patients in a cohort of 82 during their hospitalization. We demonstrated that infection occurred with a fluoroquinolone-susceptible population of L. pneumophila that evolved in vivo toward a high load of fluoroquinolone-resistant gyrA83 mutants after antibiotic therapy. We designed a qPCRgyrALp assay that allowed us to detect a signal for the gyrA QRDR in 114 samples from 62 of the 82 patients, including 63 samples collected in patients under fluoroquinolone treatment. We found gyrA83 mutations in two patients (3.1%, patients #2 and #4). We did not seek mutations in either other gyrA regions or other topoisomerase-encoding genes owing to the small amount of biological material, but such mutations may occur in vitro (Almahmoud et al., 2009). Thus, we have likely underestimated the incidence of in vivo selection of fluoroquinolone resistance in L. pneumophila. We investigated the presence of a correlation between a positive qPCRgyrALp assay and higher legionellosis severity in the 82 patients of the cohort. There was a trend for the 62 patients with a positive qPCRgyrALp assay to be more frequently admitted to the ICU, and these patients more frequently received a combination of a macrolide and a fluoroquinolone then those with a negative qPCRgyrALp assay. These findings may reflect higher L. pneumophila bacteria load in their respiratory samples, which we previously correlated with higher severity of the disease using the qPCRmip assay (Maurin et al., 2010). Indeed, there was a significant correlation between results of the qPCRgyrALP and qPCRmip assays. A gyrA83 mutation leading to the GyrA T83I substitution is likely to be clinically significant for two main reasons: first we previously demonstrated that it produced at least a four-fold increase of ciprofloxacin MIC in L. pneumophila Paris (Almahmoud et al., 2009), and second it is known to be associated with high-level fluoroquinolone resistance in other Gram-negative bacteria (Komp Lindgren et al., 2003; Jacoby, 2005). We detected a rapid increase of gyrA83 mutant alleles under fluoroquinolone treatment to 94% at D4 for patient #2 and 75% and 85% at D3 and D5, respectively, for patient #4. For this latter patient, a fluoroquinolone-susceptible strain of L. pneumophila sg1 was isolated from respiratory samples at D0. During the stay of patient #4 at ICU, we observed a major in vivo shift in the structure of the L. pneumophila population with the replacement of fluoroquinolone-susceptible by resistant cells, confirming that fluoroquinolone therapy selected for in vivo fluoroquinolone resistance during hospitalization. The two patients #2 and #4 suffered from a severe and prolonged course disease, but recovered under combined fluoroquinolone and macrolide treatment. Additional cases will be needed to establish a statistical correlation between the disease severity and the in vivo selection of gyrA QRDR mutant strains of L. pneumophila. We found by NGS a very low percentage (<0.5%) of mutated alleles in respiratory samples from control patients and in sample D3 from patient #1, and a slightly higher percentage (1.05% and 2.9%, respectively) in sample D0 from patients #2 and #4. These sequences may correspond to false positives introduced during the PCR amplification step of the NGS process itself. Alternatively, they may reflect the spontaneous occurrence of fluoroquinolone-resistance mutations in L. pneumophila, as previously described in other Gram-negative bacteria including E. coli (Komp Lindgren et al., 2003). Therefore, legionellosis patients might have been infected with a complex L. pneumophila population containing low abundance of gyrA83 mutants, with subsequent selection of this population in patients #2 and #4 under fluoroquinolone therapy. An alternative hypothesis could be the in vivo emergence of these mutants. The rapid increase in the proportion of mutated gyrA83 alleles over a few days is more compatible with the former hypothesis. In both cases however, the selective sweeps resulting in the increased abundance of the gyrA83 mutants occurred in vivo during fluoroquinolone treatment. We emphasize that patient #4 already received a three-day course of ciprofloxacin at D0.