Legionella pneumophila, a Gram-negative bacterium, is the main etiological agent of legionnaires' disease, a pneumonia potentially serious in humans. The fluoroquinolones and the macrolides are first-line antibiotics in the treatment of legionnaires' disease. However, therapeutic failures are frequent and mortality remains high, on average 10 to 15%, and more than 30% in immunosuppressed patients.
To date, these therapeutic failures have never been associated with acquired antibiotic-resistance in L. pneumophila. In fact, numerous studies carried out in vitro have not made it possible to show acquired resistance to fluoroquinolones or macrolides in the natural strains of L. pneumophila isolated in humans or isolated from the environment (Baltch A L et al., Antimicrob. Agents Chemother., 1998, 42(12):3153-6; Baltch A L et al., Antimicrob. Agents Chemother., 1995, 39(8):1661-6; Garcia M T et al., Antimicrob. Agents Chemother., 2000; 44(8): 2176-8; Higa F. et al., J. Antimicrob. Chemother. 2005; 56(6):1053-7; Jonas D. et al., J. Antimicrob. Chemother., 2001; 47(2):147-52; Onody C. et al., J. Antimicrob. Chemother., 1997; 39(6):815-6).
Several authors consider that the acquisition of resistance to antibiotics, in particular to the fluoroquinolones, in L. pneumophila is a non-existent phenomenon (Roig J. and Rello J., J. Antimicrob. Chemother., 2003; 51(5):1119-29 and Fields B S. et al., Clin. Microbiol. Rev. 2002; 15(3): 506-26).
The macrolides and fluoroquinolones are therefore considered as reliable antibiotics in the treatment of legionnaires' disease (Roig J. and Rello J., J. Antimicrob. Chemother., 2003; 51(5):1119-29 and Fields B S. et al., Clin. Microbiol. Rev. 2002; 15(3): 506-26).
Only rifampicin is advised against in monotherapy due to the very high risk of selecting resistant mutants in most bacteria.
The fluoroquinolones, broad-spectrum antibiotics, form part of the large family of the quinolones, synthetic antibiotics. The fluoroquinolones are so-called second-generation quinolones, in which fluorine has been added in order to increase the penetration of the molecules of quinolones into the cells (up to 200 times greater). The quinolones and fluoroquinolones target type II bacterial topoisomerases: the DNA gyrase constituted by the GyrA and GyrB proteins, and topoisomerase IV constituted by the parC and parE proteins. Certain mutations affecting the genes encoding these type II topoisomerases are known to induce resistance to the quinolones and fluoroquinolones in the bacteria. In Gram-negative species, in particular in Escherichia coli, these fluoroquinolone-resistance mutations mainly affect the gyrA gene encoding the GyrA protein of the DNA gyrase. The substitutions of amino acids which result therefrom are situated in the QRDR region (Quinolone Resistance Determining Region) of GyrA, comprising the amino acids at position 67 to 106 [Soussy C. J. Quinolones and gram-negative bacteria. In Antibiogram. Courvalin P, Leclercq R, and Rice L. B. Eds. ESKA Publishing, ASM Press, 2010, p261-274]. The level of sensitivity to the fluoroquinolones varies in the same bacterium depending on the amino acid or acids situated at these positions. However, the most frequent mutations responsible for fluoroquinolone resistance are those leading to a substitution at the amino acids at positions 83, 87 or more rarely 84 of the GyrA protein (according to the numbering adopted in the case of Escherichia coli), constituting DNA gyrase subunit A. The mutations gyrA83 and gyrA87 are frequently observed in vivo [Jacoby G A. Mechanisms of resistance to quinolones. Clin Infect Dis. 2005; 41 Suppl 2:S120-6], and have been reproduced in vitro [Barnard F M, Maxwell A. Interaction between DNA gyrase and quinolones: effects of alanine mutations at GyrA subunit residues Ser(83) and Asp(87). Antimicrob Agents Chemother. 2001; 45(7):1994-2000]. These positions are identical on the gyrA gene of L. pneumophila strain Paris (NC 006368.1). The inventors have previously shown [Almahmoud I, Kay E, Schneider D, Maurin M. Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila. J Antimicrob Chemother. 2009; 64(2):284-93] by in vitro selection of fluoroquinolone-resistant mutants, that the key positions for fluoroquinolone resistance in L. pneumophila strain Paris also correspond to amino acids 83 and 87 of the GyrA protein.
The strain Paris of L. pneumophila (CIP 107629T) is one of the reference strains known to be sensitive to the fluoroquinolones [Roch N, Maurin M. Antibiotic susceptibilities of Legionella pneumophila strain Paris in THP-1 cells as determined by real-time PCR assay. J Antimicrob Chemother. 2005; 55(6):866-71]. Other strains sensitive to fluoroquinolones are described, such as L. pneumophila Philadelphia (ATCC 33152), L. pneumophila Lens (CIP 108286) and L. pneumophila Lorraine (CIP 108729).
Moreover, the inventors have already described fluoroquinolone-resistant mutant strains selected beforehand in vitro and having one or more of the gyrA83 and gyrA87 mutations [Almahmoud I, Kay E, Schneider D, Maurin M. Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila. J Antimicrob Chemother. 2009; 64(2):284-93]. These are L. pneumophila 1 LPPI1 (CIP107629T, namely the strain Paris mutated at position 83 (T83I) of the gyrA gene), L. pneumophila 1 LPPI4 (CIP107629T, namely the strain Paris mutated at position 83 (T83I) and at position 87 (D87N) of the gyrA gene) and the strain L. pneumophila 1 LPPI5 (CIP107629T, namely the strain Paris mutated at position 83 (T83I) and at position 87 (D87H) of the gyrA gene).
However, it is currently accepted by the scientific community that L. pneumophila cannot acquire resistance to antibiotics, in particular to the fluoroquinolones used in clinical practice, in vivo. Several factors are usually mentioned in order to explain this finding in L. pneumophila (Fields B S. et al., Clin Microbiol Rev. 2002; 15(3):506-26):    1) Unlike E. coli, L. pneumophila essentially multiplies in intracellular medium in the free-living protozoans (amoebas) in the aquatic environment or in the alveolar macrophages in the infected patients; this specific multiplication niche would not be suitable for exchanges of antibiotic-resistance genes as has been described in the case of numerous other bacteria (enterobacteria for example);    2) Unlike certain strains of E. coli, and in particular enterohaemorrhagic E. coli, which have an animal reservoir in ruminants, there is no known animal reservoir for L. pneumophila. There would therefore be no pressure of selection by the antibiotics used in veterinary practice or in the agri-food sector;    3) Unlike certain strains of E. coli for which cases of interhuman transmission, in particular by faecal-oral transmission, have been reported, legionnaires' disease is not a disease of interhuman transmission, which would also limit the likelihood of transmission of any antibiotic-resistant mutants.
The sensitivity of the bacteria to the antibiotics can be assessed by phenotypic methods, such as the production of an antibiogram and the determination of the minimum inhibitory concentrations (MICs) of the antibiotics. These methods are not carried out routinely for bacteria with fastidious growth requirements such as L. pneumophila, due to the impossibility of isolating the bacterial strain in question in most infected patients. In the last few years, molecular methods based on the PCR and real-time PCR techniques have been developed in order to detect the antibiotic resistance mutations on the basis of isolated strains or directly in specimens (clinical or from various environments) containing the mutant strains.
All of the data currently available indicate the non-existence in vivo of antibiotic-resistant strains of L. pneumophila in patients suffering from legionnaires' disease and treated with these antibiotics.
Unlike the hypotheses formulated by the scientific community, the Inventors have demonstrated that the acquisition of fluoroquinolone resistance in L. pneumophila is possible in vivo, in patients infected with this pathogen.