We have shown that in vivo selection of gyrA mutations

We have shown that in vivo selection of gyrA mutations responsible for fluoroquinolone-resistance in L. pneumophila occurs in legionellosis patients during fluoroquinolone treatment. A likely hypothesis is that patients were infected with L. pneumophila populations containing low doses of gyrA83 mutants. If so, it is puzzling that no fluoroquinolone resistance has been so far characterized in environmental strains of L. pneumophila, and only a single fluoroquinolone-resistant strain of this species has been isolated before in a clinical situation (Bruin et al., 2014). It should be emphasized that mutant strains of L. pneumophila have poor ability to propagate outside infected hosts because of the lack of human-to-human transmission and little opportunities to go back to their natural environmental reservoir. However, isolation of L. pneumophila from respiratory samples of legionellosis patients remains challenging, and almost impossible in patients under antibiotic therapy. We recommend the development and use of molecular tools to test the presence of resistance mutations in patients infected by L. pneumophila, especially in the case of poor response to antibiotic treatment. Our strategy should be optimized to allow more specific detection of gyrA83 and gyrA87 mutations, and other previously characterized fluoroquinolone-resistance mutations (Almahmoud et al., 2009; Jonas et al., 2003; Nielsen et al., 2000). This would allow to precisely define the prevalence of fluoroquinolone-resistant mutants in legionellosis patients and their clinical consequences. The molecular tools could also serve to assess the emergence and spread of gyrA83 mutants in environmental L. pneumophila strains. The possibility of in vivo selection for fluoroquinolone-resistance mutations in L. pneumophila fully justifies the current practice of administration of combined antibiotic therapy in severe legionellosis cases (Roig and Rello, 2003). Our study showing in vivo L. pneumophila protease activated receptor to antibiotic pressure paves the way for more intensive investigations of other intracellular bacterial species. This hidden resistance should indeed be intensively explored because it may contribute to the increasing public health threat of the gradual loss of effectiveness of antibiotic treatments. In conclusion, although fluoroquinolones and macrolides are highly active against Legionella species, death rates of 10–15% are commonly reported in legionellosis patients. No correlation can be found in the literature between treatment failures or relapses and development of antibiotic resistance in Legionella spp. However, fluoroquinolone-resistant strains of L. pneumophila have been selected in vitro, and a fluoroquinolone-resistant isolate was recently obtained from a legionellosis patient. By using state-of-the-art molecular and genomic approaches, we identified, at high frequency, fluoroquinolone resistance mutations in lower respiratory samples from two additional legionellosis patients from a cohort of 82. The detected gyrA83 mutation is associated with a significant increase in fluoroquinolones MICs in L. pneumophila. We demonstrated that selection for fluoroquinolone resistance occurred in vivo during fluoroquinolone therapy. These two patients required ICU admission and prolonged hospitalization, but recovered under combined fluoroquinolone and macrolide treatment. Although the exact clinical impact of our findings could not be statistically evaluated because only two out of 82 legionellosis patients were involved, the possibility of in vivo selection of fluoroquinolone resistance in L. pneumophila should now be considered both for prognostic evaluation and treatment optimization at least in patients with severe legionellosis.
Contributors SJ, JE, JFT, DS and MM designed the experiments. LS and IA performed the experiments. LS and SL performed the NGS experiments. SJ and JE provided the Legionella strains from species other than L. pneumophila. All authors contributed to literature search. DS and MM wrote the manuscript. LS and MM made the figures. CS and JFT provided all clinical data. All authors had access to data and commented on and approved the final version.