Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila.

Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

Validation of a microbead-based format for spoligotyping of Legionella pneumophila.

A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.

Legionella pneumophila sequence type 1/Paris pulsotype subtyping by spoligotyping.

Endemic strains of Legionella pneumophila sequence type 1 (ST1), in particular the ST1/Paris pulsotype, are dispersed worldwide and represent about 10% of culture-proven clinical cases of Legionnaires' disease in France. The high rate of isolation of this strain from both clinical and environmental samples makes identification of the source of infection difficult during epidemiological investigations. The full-length genome sequence of this strain was recently determined, and it revealed the presence of a CRISPR/cas complex. The aim of this study was to develop and evaluate a spoligotyping tool based on the diversity of this CRISPR locus that would allow the accurate subtyping of the L. pneumophila serogroup 1 ST1/Paris pulsotype. The CRISPR loci of 28 L. pneumophila ST1/Paris pulsotype isolates were sequenced, and 42 different spacers regions were characterized. A membrane-based spoligotyping method was developed and used to determine the subtypes of 406 L. pneumophila isolates, including 233 with the ST1/Paris pulsotype profile that were collected in France from 2000 to 2011. A total of 46 different spoligotypes were detected, and 41 of these were specifically identified in the ST1/Paris pulsotype isolates. In 27 of 33 epidemiological investigations, the environmental source of contamination was confirmed by comparing spoligotypes of clinical isolates with those of environmental isolates. With an index of discrimination of 79.72% (95% confidence interval, 75.82 to 83.63), spoligotyping of the L. pneumophila ST1/Paris pulsotype has the potential to be a useful complementary genotyping tool for discriminating isolates with undistinguishable pulsed-field gel electrophoresis (PFGE) and ST genotypes, which could help to identify environmental sources of infection.