L pneumophila is a Gram negative facultative

L. pneumophila is a Gram-negative facultative intracellular bacterium that multiplies in aquatic environments, especially in free-living protozoa and biofilms (Newton et al., 2010; Carratalà and Garcia-Vidal, 2010). Legionellosis is an acute pneumonia caused by inhalation of aerosols containing Legionella sp., with L. pneumophila serogroup (sg) 1 accounting for more than 90% of human infections (Newton et al., 2010; Carratalà and Garcia-Vidal, 2010). The macrolides (especially azithromycin) and the fluoroquinolones are first-line O4I1 cost for the treatment of legionellosis patients (Chidiac et al., 2012; Jespersen et al., 2010; Yu et al., 2009; Pedro-Botet and Yu, 2009; Burdet et al., 2014). However, treatment failures and relapses may occur despite administration of an appropriate antibiotic therapy (Chidiac et al., 2012; Jespersen et al., 2010; Yu et al., 2009; Pedro-Botet and Yu, 2009; Burdet et al., 2014). The mortality rate of legionellosis is 10% on average, but can be as high as 30% in immunocompromised patients (Chidiac et al., 2012; Jespersen et al., 2010; Yu et al., 2009). Acquired resistance to the macrolides and fluoroquinolones in L. pneumophila has been readily selected for in vitro (Almahmoud et al., 2009; Jonas et al., 2003; Nielsen et al., 2000). In striking contrast, no macrolide resistance (Jespersen et al., 2010; Yu et al., 2009; Pedro-Botet and Yu, 2009; Burdet et al., 2014), and only a single case of fluoroquinolone resistance has been reported in clinical strains (Bruin et al., 2014). In the latter case however, only one patient was studied with only microbiological approaches and, importantly the dynamics of fluoroquinolone-resistance during infection and hospitalization was not investigated. Here, we identified and further characterized, using molecular and genomic approaches, the in vivo selection of fluoroquinolone-resistance mutations in L. pneumophila during a large survey of 82 unrelated legionellosis cases.
Methods
Results A positive urinary antigen test for L. pneumophila sg1 (Binax Now Legionella, Binax, Portland, ME, USA) was found for all 82 investigated pneumonic patients. Legionellosis diagnosis was further confirmed in 15 of the 82 patients by isolation of a L. pneumophila strain from the first collected respiratory sample. These 15 clinical strains of L. pneumophila displayed ciprofloxacin MICs ranging from 0.25 to 0.38mg/L, whereas MICs were 0.38mg/L for the susceptible L. pneumophila reference strains (Paris, Philadelphia, Lens, and Lorraine), 1.5mg/L for the LPPI1 mutant with a single gyrA mutation, and 2mg/L for the LPPI4 and LPPI5 mutants with two gyrA mutations (appendix p 5). We found no mutation in the gyrA QRDR of the 15 clinical strains. Legionellosis was also confirmed in 61 (74.4%) of the 82 legionellosis patients (including 13 patients with a positive L. pneumophila culture), by direct detection of L. pneumophila DNA in respiratory samples (112 of the 139 collected samples, 80.6%), using the qPCRmip assay (Maurin et al., 2010). The L. pneumophila DNA load in these samples varied from 5.8×102 to 6.8×107 genome units (GU)/mL of sample. For 19 patients, L. pneumophila culture and PCR assays were negative. After standardization of the qPCRgyrALp assay, a strong amplification signal of the gyrA QRDR was detected for all tested L. pneumophila strains. Furthermore, this assay could distinguish Legionella versus non-Legionella strains, L. pneumophila versus other Legionella species, fluoroquinolone-susceptible versus resistant mutants of L. pneumophila, and single versus double mutants (Fig. 1; appendix p 3; appendix p 5). When applied to the 139 respiratory samples collected from the 82 legionellosis patients, an amplification signal corresponding to the gyrA QRDR was detected in 114 samples (82%) from 62 patients (75.6%), including 63 and 11 samples collected from patients under and without fluoroquinolone therapy, respectively (Table 1). The qPCRgyrALp assay was positive for 108 (96.4%) of the 112 samples showing a positive qPCRmip assay, but only for 7 of the other 27 qPCRmip-negative samples (25.9%, p<0.01), suggesting that the DNA load in clinical samples was critical for detection of either the mip or gyrA gene. All qPCRgyrALp-positive respiratory samples (104) collected from 58 patients gave identical results, their melting curve profiles suggesting a reference gyrA QRDR sequence (mean melting peak±SD, 59.34±0.41°C) when compared to control strains (Fig. 1, Table 1). For 4 patients however (#1 to #4), we detected melting peaks of 56.35±0.42°C (three replicates per patient) for 6 of 11 respiratory samples, suggesting the presence of gyrA QRDR mutations.