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Definitions

• the present invention relates to a method for the in vitro detection of Legionella pneumophila strains resistant to antibiotics, especially fluoroquinolones.
• the invention also relates to nucleotide probes and a kit of reagents for the detection of bacterial strains Legionella pneumophila resistant to antibiotics, especially fluoroquinolone type.
• the invention finally relates to a real-time PCR technique for the implementation of the above-mentioned method for the in vitro detection of antibiotic-resistant Legionella pneumophila strains.
• Legionella pneumophila a Gram-negative bacterium
• Fluoroquinolones and macrolides are the first-line antibiotics for legionellosis.
• therapeutic failures are frequent and mortality remains high, on average 10 to 15%, and more than 30% in immunocompromised patients.
• Macrolides and fluoroquinolones are therefore considered to be reliable antibiotics in the treatment of legionellosis (Roig J. and Rello J., J. Antimicrob Chemother., 2003; 51 (5): 1119-29 and Fields BS. al, Clin Microbiol, Rev. 2002; 15 (3): 506-26).
• Fluoroquinolones broad-spectrum antibiotics, are part of the general family of quinolones, synthetic antibiotics. Fluoroquinolones are so-called second-generation quinolones in which fluorine has been added to increase the penetration of quinolone molecules into cells (up to 200-fold). Quinolones and fluoroquinolones are targeted at type II bacterial topoisomerases: gyrase DNA consisting of GyrA and GyrB proteins, and topoisomerase IV consisting of parC and parE proteins. Certain mutations affecting the genes encoding these type II topoisomerases are known to induce resistance to quinolones and fluoroquinolones in bacteria.
• these fluoroquinolone resistance mutations mainly affect the gyrA gene encoding the GyrA protein of DNA gyrase.
• the resulting amino acid substitutions are located in the QRDR region (Quinolone Resistance Determining Region) of GyrA, comprising the amino acids at position 67 to 106 [Soussy CJ Quinolones and gram-negative bacteria. In Antibiogram. Courvalin P, Leclercq R, and Rice LB Eds. ESKA Publishing, ASM Press, 2010, p261-274].
• the level of sensitivity to fluoroquinolones varies in the same bacterium depending on or amino acids located at these positions.
• the mutations responsible for resistance to the most frequent fluoroquinolones are those leading to a substitution at the amino acid level at positions 83, 87 or, more rarely 84, of the GyrA protein (according to the Escherichia coli count), constituting the subunit. unit A DNA gyrase. 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 were reproduced in vitro [Barnard FM, 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].
• the L. pneumophila strain of Paris (CIP 107629T) is one of the reference strains known to be sensitive to fluoroquinolones [Roch N, Maurin M. Antibiotic susceptibilities of Legionella pneumophila strain 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).
• pneumophila 1 LPPI4 (CIP107629T, namely the mutated Paris strain at position 83 (T83I) and at position 87 (D87N) of the gyrA gene) and L. pneumophila 1 LPPI5 strain (CIP107629T, namely the mutated Paris strain at position 83 (T83I) and at position 87 (D87H) of the gyrA gene).
• L. pneumophila multiplies predominantly intracellularly in free protozoa (amoebae) in the aquatic environment or in alveolar macrophages in infected patients; this specific niche multiplication would not be conducive to the exchange of antibiotic resistance genes as has been described in many other bacteria (enterobacteria for example); 2) Unlike some E. coli strains, including enterohaemorrhagic E. coli, which have an animal reservoir in ruminants, there is no known animal reservoir for L. pneumophila. There would be no selection pressure by antibiotics used in veterinary practice or agri-food;
• the susceptibility of bacteria to antibiotics can be assessed by phenotypic methods, such as performing an antibiogram and determining minimum inhibitory concentrations (MICs) of antibiotics. These methods are not routinely performed for fastidiously growing bacteria such as L. pneumophila because of the impossibility of isolating the bacterial strain involved in most infected patients.
• phenotypic methods such as performing an antibiogram and determining minimum inhibitory concentrations (MICs) of antibiotics.
• One aspect of the invention is a method of detecting L. pneumophila strains resistant to antibiotics.
• Another aspect of the invention is the development of nucleotide sequences of primers and probes (tandem probes) for amplifying and specifically detecting mutations involved in antibiotic resistance in species L pneumophila.
• One of the other aspects of the invention is a real-time PCR technique for determining specific mutations responsible for resistance.
• Another aspect of the invention is a kit for detecting the resistance of L. pneumophila strains to fluoroquinolones to prevent therapeutic failure and high mortality in patients with legionellosis.
• the present invention is based on an in vitro method for the detection of at least one bacterial strain of Legionella pneumophila resistant to antibiotics, especially of the fluoroquinolone type, in a biological sample, by the detection:
• SEQ ID NO: 1 represents the sequence of the wild type GyrA protein of L. Pneumophila (GyrA protein of L. pneumophila strain Paris, Accession number: YP 123696.1), encoded by the nucleotide sequence SEQ ID NO: 13.
• biological sample any collection of tissues, cells, organs, fluids, secretions or blood from the human or animal body, and their derivatives.
• antibiotic resistance is defined as the ability of a microorganism to resist the effects of antibiotics both in vitro and in vivo.
• This resistance is usually defined by measuring the minimum inhibitory concentration (MIC) of an antibiotic against a given bacterium, according to a method validated by a reference organism, such as the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in Europe [E. Matuschek, D. F. Brown J. and G. Kahlmeter. Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. Clin Microbiol Infect 2014; 20: 0255-0266], or the Standard Clinical and Laboratorty Institute (CLSI) in the United States [CLSI. M07-A9: Methods for Antimicrobial Dilution Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Ninth Edition. Flight. 32 No. 2].
• a strain is said to be resistant to an antibiotic if the MIC of this antibiotic vis-à-vis the strain is greater than a critical value defined by the same reference organism.
• the term "equivalent position” means a nucleotide of the gyrA gene or an amino acid of the GyrA protein of the same nature and function as that described in E. coli for a given position, although the numbering of this position may be different in the species considered compared to E. coli because of a different number of nucleotides (gyrA) or amino acids (GyrA) between the two species.
• identity is also understood to mean the number of identical nucleotides between two sequences of the same length, determined for each position in the sequences. In the wild strains of L. pneumophila, position 83 of the GyrA protein is occupied by threonine (T), position 84 is occupied by alanine (A) and position 87 is occupied by an aspartic acid (D).
• a bacterial strain of L. pneumophila according to the invention is said to be mutated since the position 83 of the GyrA protein is no longer occupied by a threonine (T), and / or since the position 84 is no longer occupied by an alanine (A), and / or when position 87 is no longer occupied by an aspartic acid (D).
• T threonine
• A alanine
• D aspartic acid
• a particular embodiment of the invention relates to an in vitro method for the detection of at least one bacterial strain of Legionella pneumophila resistant to antibiotics, especially of the fluoroquinolone type, in a biological sample, in which method the mutated GyrA protein is such that:
• the amino acid at position 83 is different from T and the amino acids at positions 84 and 87 can correspond to any amino acid, or
• amino acid at position 84 is different from A, and the amino acids at positions 83 and 87 may correspond to any amino acid, or
• amino acid at position 87 is different from D, and the amino acids at positions 83 and 84 may correspond to any amino acid.
• amino acids at positions 84 and 87 (or 83 and 87) or (83 and 84) may correspond to any amino acid” means that said amino acids may be both the amino acid present in a wild strain than any other amino acid. In the latter case, the amino acid considered therefore constitutes a mutation.
• a bacterial strain of L. pneumophila according to the invention having a mutated GyrA protein can therefore contain a single mutation, a double mutation or a triple mutation.
• strains comprising a single mutation are thus mutated in position 83 or in position 84 or in position 87.
• Strains comprising a double mutation are therefore mutated either at positions 83 and 84, or at positions 84 and 87, or at position 83 and 87.
• the strains comprising a triple mutation are therefore mutated jointly on the three positions 83; 84 and 87.
• the in vitro method making it possible to demonstrate at least one bacterial strain of Legionella pneumophila resistant to antibiotics, in particular of the fluoroquinolone type, applies when the mutated GyrA protein is such that :
• said amino acid at position 83 is: I, L, W, A or V and the amino acids at positions 84 and 87 may correspond to any amino acid, or - said amino acid at position 84 is: P or V and the amino acids at positions 83 and 87 can correspond to any amino acid, or
• amino acid at position 87 is: N, G, Y, H or V and the amino acids at positions 83 and 84 may correspond to any amino acid, or
• said amino acid at position 83 is: I, L, W, A or V
• said amino acid at position 84 is: P or V
• said amino acid at position 87 is any amino acid, or
• said amino acid at position 83 is: I, L, W, A or V
• said amino acid at position 87 is: N, G, Y, H or V and said amino acid at position 84 is any amino acid
• said amino acid at position 84 is: P and V
• said amino acid at position 87 is: N, G, Y, H or V
• said amino acid at position 83 is any amino acid, or
• said amino acid at position 83 is: I, L, W, A or V
• said amino acid at position 84 is: P or V
• said amino acid at position 87 is: N, G, Y, H or V.
• the amino acids at position 84 and 87 can be the amino acids of the wild-type protein, A (alanine) and D (aspartic acid), respectively, or any other amino acid, that is, the amino acids at position 84 and 87 can also be mutated.
• the amino acids at position 83 and 87 may be the amino acids of the wild-type protein, respectively T (threonine) and D (aspartic acid) or any other amino acid, that is, the amino acids at position 83 and 87 can also be mutated.
• the amino acids at position 83 and 84 may be the amino acids of the wild-type protein, respectively T (Threonine) and A (Alanine) or any other amino acid, c that is, the amino acids at position 83 and 84 can also be mutated.
• said in vitro method making it possible to demonstrate at least one bacterial strain of Legionella pneumophila resistant to antibiotics, in particular of the fluoroquinolone type, applies when the mutated GyrA protein is such than :
• said amino acid at position 83 is: an isoleucine (I), a leucine (L), a tryptophan (W), an alanine (A) or a valine (V), said amino acid at position 84 is an alanine (A ) and said amino acid at position 87 is an aspartic acid (D), or said amino acid at position 84 is: proline (P) or valine (V) and said amino acid at position 83 is threonine (T) and said amino acid at position 87 is an aspartic acid (D), or
• said amino acid at position 87 is: asparagine (N), glycine (G), tyrosine (Y), histidine (H) or valine (V) and said amino acid at position 83 is threonine (T) ) and said amino acid at position 84 is an alanine (A), or
• said amino acid at position 83 is: isoleucine (I), leucine (L), tryptophan (W), alanine (A) or valine (V), said amino acid at position 84 is: proline ( P) or valine (V) and said amino acid at position 87 is an aspartic acid (D), or
• said amino acid at position 83 is: an isoleucine (I), a leucine (L), a tryptophan (W), an alanine (A) or a valine (V), said amino acid in position 87 is: an asparagine ( N), a glycine (G), a tyrosine (Y), a histidine (H) or a valine (V) and said amino acid at position 84 is an alanine (A), or
• said amino acid at position 84 is: proline (P) or valine (V)
• said amino acid at position 87 is: asparagine (N), glycine (G), tyrosine (Y), histidine ( H) or a valine (V)
• said amino acid at position 83 is a threonine (T).
• the table below shows the set of possible mutations in amino acids, on each of the three positions 83, 84 and 87 and the corresponding codons.
• the method according to the invention allows the detection of a mutated GyrA protein which comprises the following consensus sequence: GDX ! X 2 VYX 3 T (SEQ ID NO: 2) wherein: X ls X 2 and X 3 correspond to mutated amino acids in positions 83, 84 and 87, and
• - Xi is different from T (threonine), and X 2 and X 3 are any amino acid, or
• X 2 is different from A (alanine), and X 1 and X 3 are any amino acid, or
• - X3 is different from D (aspartic acid), and Xi and X 2 are any amino acid.
• the amino acid corresponding to Xi, different from T can be an isoleucine (I), a leucine (L), a tryptophan (W), an alanine (A) or a valine (V), and X 2 and X 3 correspond to any amino acid, or
• the amino acid corresponding to X 2 is a proline (P) or a valine (V), and X 1 and X 3 correspond to any amino acid, or the amino acid corresponding to X 3 is an asparagine (N), a glycine (G), a tyrosine (Y), a histidine (H) or a valine (V), and X 1 and X 2 correspond to any which amino acid, or
• the amino acid corresponding to Xi, different from T is an isoleucine (I), a leucine (L), a tryptophan (W), an alanine (A) or a valine (V), X 2 is a proline ( P) or a valine (V) and X 3 is any amino acid, or
• the amino acid corresponding to Xi, different from T is an isoleucine (I), a leucine (L), a tryptophan (W), an alanine (A) or a valine (V),
• X 3 is an asparagine ( N), a glycine (G), a tyrosine (Y), a histidine (H) or a valine (V) and
• X 2 is any amino acid, or
• X 2 is a proline (P) or a valine (V)
• X 3 is an asparagine (N), a glycine (G), a tyrosine (Y), a histidine (H) or a valine (V)
• Xi is any amino acid, or
• the amino acid corresponding to Xi, different from T is an isoleucine (I), a leucine (L), a tryptophan (W), an alanine (A) or a valine (V), X 2 is a proline ( P) or valine (V) and X 3 is asparagine (N), glycine (G), tyrosine (Y), histidine (H) or valine (V).
• I isoleucine
• L leucine
• W tryptophan
• A alanine
• V valine
• X 2 is a proline ( P) or valine (V)
• X 3 is asparagine (N), glycine (G), tyrosine (Y), histidine (H) or valine (V).
• the method of the invention allows the detection of a mutated GyrA protein which comprises the consensus sequence GDXiX 2 VYX 3 T in which:
• - Xi is I, L, W, A or V, X 2 is A and X 3 is D, or
• X 2 is P or V
• Xi is T and X 3 is D, or
• X 3 is N, G, Y, H or V, Xi is T and X 2 is A, or
• - Xi is I, L, W, A or V, X 2 is P or V and X 3 is D, or
• - Xi is I, L, W, A or V
• X 3 is N, G, Y, H or V and X 2 is A
• X 2 is P or V
• X 3 is N, G, Y, H or V and Xi is T, or
• - Xi is I, L, W, A or V
• X 2 is P or V
• X 3 is N, G, Y, H or V.
• the method allows the detection of the sequence of the gene encoding said mutated GyrA protein.
• This sequence has at least one nucleotide substitution with respect to SEQ ID NO: 3 (GGGGATACAGCTGTTTATGACAC), in position equivalent to position 7, 8, 10, 11, 19, 20 or 21 with respect to SEQ ID NO : 3, where the nucleotides at positions 7 and 8 correspond to the first two nucleotides of the codon encoding the amino acid at position 83 of the GyrA protein of Legionella pneumophila, the nucleotides at positions 10 and 11 correspond to the first two nucleotides of the codon encoding the amino acid at position 84, and the nucleotides at positions 19, 20 and 21 correspond to the three nucleotides of the codon encoding the amino acid at position 87.
• sequence of the nucleic acid encoding said mutated GyrA protein comprises a sequence chosen from:
• SEQ ID NO: 11 (GGGGATACATCTGTTTATGACAC)
• SEQ ID NO: 12 (GGGGAT AC AC CTGTTT ATG AC AC)
• SEQ ID NO: 17 (GGGGATACAGCTGTTTATAACAC),
• SEQ ID NO: 18 (GGGGATACAGCTGTTTATTACAC),
• SEQ ID NO: 21 (GGGGATACAGCTGTTTATGGCAC),
• SEQ ID NO: 22 (GGGGATACAGCTGTTTATGCCAC),
• SEQ ID NO: 24 (GGGGATACAGCTGTTTATGAAAC),
• SEQ ID NO: 27 GGGGATCTTGCTGTTTATGACAC
• SEQ ID NO: 28 (GGGGATCTCGCTGTTTATGACAC),
• SEQ ID NO: 30 (GGGGATCTGGCTGTTTATGACAC)
• SEQ ID NO: 31 (GGGGATTGGGCTGTTTATGACAC)
• SEQ ID NO: 36 (GGGGATGTCGCTGTTTATGACAC),
• SEQ ID NO: 37 (GGGGATGTAGCTGTTTATGACAC),
• SEQ ID NO: 38 (GGGGATGTGGCTGTTTATGACAC),
• SEQ ID NO: 39 (GGGGATACACCCGTTTATGACAC),
• SEQ ID NO: 40 (GGGGATACACCAGTTTATGACAC),
• SEQ ID NO: 41 (GGGGATACACCGGTTTATGACAC),
• SEQ ID NO: 42 (GGGGATACAGTCGTTTATGACAC),
• SEQ ID NO: 45 (GGGGATACAGCTGTTTATAATAC),
• SEQ ID NO: 46 (GGGGATACAGCTGTTTATGGTAC),
• SEQ ID NO: 47 (GGGGATACAGCTGTTTATGGAAC),
• SEQ ID NO: 48 (GGGGATACAGCTGTTTATGGGAC), SEQ ID NO: 49 (GGGGAT AC AGCTGTTT ATT AT AC),
• SEQ ID NO: 50 (GGGGATACAGCTGTTTATCATAC),
• SEQ ID NO: 52 (GGGGATACAGCTGTTTATGTAAC),
• SEQ ID NO: 56 (GGGGAT ATC GCTGTTT ATG AC AC),
• the detection of the presence of said mutated GyrA protein or of the target nucleic acid encoding said mutated GyrA protein, including the detection of the mRNA encoded by the mutated gene is carried out by a chosen technique.
• the detection of the presence of said nucleic acid encoding said mutated GyrA protein is performed by real-time PCR.
• Real-time PCR uses the basic principle of classical PCR (cyclic amplification of a DNA fragment, based on an enzymological reaction) with the aim of difference an amplification measured not in final but throughout the reaction, so in real time.
• the amount of DNA is measured by means of a fluorescent marker whose emission is directly proportional to the quantity of amplicons produced. This makes it possible to obtain a kinetics of the reaction and thus the quantification of the DNA whereas the conventional PCR only gives the final measurement.
• the detection or quantification of the fluorescent signal in real time can be done using intercalators or probes.
• the intercalant agent currently used most is SYBR® Green (Roche, Meylan, France).
• probes there are 4 different technologies, allowing the measurement of a fluorescent signal: “Taqman” or hydrolysis of probes, “HybProbes” (FRET) or hybridization of 2 probes, “Molecular Beacons” or molecular beacons. “Scorpion” or scorpion primers.
• the real-time PCR is used for the detection of mutations gyrA83, 84 and / or 87, for example using two probes in tandem: a so-called anchoring probe and a so-called detection probe .
• the fluorophore located on the anchoring probe is for example LCRed-640 (Roche Diagnostic)
• the fluorophore located on the emission probe is for example fluorescein.
• a mutation affecting this fragment causes a downward shift of the melting temperature.
• the fragment tested here is delimited from the base 241 to the base 263 of gyrA of the strain L. pneumophila Paris [GGGGATACAGCTGTTTATGACAC], or from amino acid 81 to amino acid 88 of GyrA of said strain [GDTAVYDT]. It therefore includes T83, A84 and D87, which are related to fluoroquinolone resistance in L. pneumophila.
• This technique shows a change in the melting temperature in case of alteration of this DNA fragment, whatever the mutation and its position. Due to natural variations of L. pneumophila in the nucleotide sequence, which can lead to other mutations, the results obtained by the method according to the invention can be confirmed by a complementary technique such as, for example, the sequencing of the amplified DNA.
• hydrolysis probe or "Taqman”, “beacon” or “scorpion” type probe could target the same region of gyrA where mutations responsible for substitutions in positions 83, 84 and 87, without the use of an anchoring probe.
• this type of technique are presented in the literature concerning the detection of fluoroquinolone resistance in Mycobacterium tuberculosis (Chakravorty S. et al., J Clin Microbiol. 2011; 49 (3): 932-40) or in Staphylococcus aureus (Lapierre P. et al., J Clin Microbiol 2003; 41 (7): 3246-51).
• SNPs single nucleotide polymorphisms
• Some of these techniques are already commercially available, for example the Xpert TB / RIF® test (Cepheid), which detects resistance to rifampicin in Mycobacterium tuberculosis using a "Phare” probe.
• the method for detecting the presence of the target nucleic acid encoding said mutated GyrA protein comprising the steps of:
• a biological sample that may contain a target nucleic acid belonging to a bacterial strain of Legionella pneumophila antibiotic-resistant, with a pair of primers capable of hybridizing specifically with said target nucleic acid, and
• amplification product comprising a sequence having at least 90% identity with the sequence SEQ ID NO: 3,
• nucleic acid encoding said mutated GyrA protein When a nucleic acid encoding said mutated GyrA protein is detected, this indicates the presence of at least one bacterial strain Legionella pneumophila resistant to antibiotics in the sample.
• the amplification product obtained in step b) is an amplicon of 259 nucleotides.
• This 259 nucleotide amplicon corresponds to SEQ ID NO: 57 or to any sequence having an identity percentage equal to at least 90% with the sequence SEQ ID NO: 57.
• amplicon is meant any amplified DNA fragment by PCR, using two primers.
• step b) of said method for detecting a target nucleic acid encoding said mutated GyrA protein is carried out using a primer having at least 90% identity with the sequence SEQ ID NO: 58, and / or a primer having at least 90% identity with the sequence SEQ ID NO: 59.
• primer is meant a DNA fragment whose size is generally from 15 nucleotides to 25 nucleotides, capable of hybridizing to a strand of DNA as a template, at a given melting temperature, so as to allow the duplication of this strand of DNA.
• the primers used in the method according to the invention are represented by the sequences SEQ ID NO: 58 (sense primer, named LpgyrALSFw and corresponding to the following nucleic acid sequence: CCTGATGTACGTGATGGTTTAA) and SEQ ID NO: 59 (anti-virus primer).
• LpgyrALSRv and corresponding to the following nucleic acid sequence: GCATGGCAGCTGGAGCATCTCC), or any other nucleic acid sequence having at least 90% identity with said sequences SEQ ID NO: 58 and SEQ ID NO: 59.
• step b) of said method for detecting a target nucleic acid encoding said mutated GyrA protein can be carried out using at least one nucleotide probe capable of hybridizing with the target nucleic acid.
• the detection method requires the use of probes.
• the detection probe that can be used in the context of the present invention is a nucleic acid sequence represented by the sequence SEQ ID NO: 3 or by any other nucleotide sequence having at least 90% identity with the sequence SEQ ID. NO: 3 (named LpgyrALSPl).
• This detection probe is covalently linked to at least one marker molecule allowing its detection by a suitable device.
• the marker molecule is selected from a fluorochrome or a radioactive isotope.
• a fluorochrome is a chemical substance capable of emitting fluorescent light after excitation.
• a fluorochrome useful in the context of the invention may be chosen from: fluorescein, Cy2, Cy3, Cy5, Cy7, Red613, Red640, Rhodamine, Texas Red, TRITC, Alexa Fluor, this list not being exhaustive.
• the fluorochrome related to the detection probe in the context of the invention is fluorescein attached to 3 'of said probe.
• the detection method according to the invention may also comprise an anchoring probe, which coupled to the detection probe allows the detection of the amplification product obtained in step b).
• the anchoring probe according to the invention is a nucleic acid sequence represented by the sequence SEQ ID NO: 60 (called LpgyrALSP3) or by any other nucleotide sequence having at least 90% identity with the sequence SEQ ID NO: 60.
• This anchoring probe is covalently bonded to at least one marker molecule allowing its detection by a suitable device.
• the 5 'end of the anchoring probe is bonded to an acceptor fluorochrome.
• the acceptor fluorochrome may be selected from fluorescein, Cy2, Cy3, Cy5, Cy7, Red613, Red640, Rhodamine, Red Texas, TRITC, Alexa Fluor, this list not being exhaustive. More particularly, the acceptor fluorochrome may be LightCycler® Red 640 (Roche Diagnostic).
• the anchoring probe has at its 3 'end, a phosphorylation denoted "P", preventing its elongation by the DNA polymerase. .
• the anchoring probe is therefore in the form 5 '-LCRed640-TTGTTCGTATGGCTCAGCCTTTTTC-P-3'
• a melting curve of the amplification product is generated after step c).
• the present invention also aims at protecting a pair of primers allowing the amplification of a fragment of the Legionella pneumophila gyrA gene, comprising a primer having at least 90% identity with the sequence SEQ ID NO: 58 (LpgyrALSFw), and a primer having at least 90% identity with the sequence SEQ ID NO: 59 (LpgyrALSRv).
• the method according to the invention and especially the step of amplification of a fragment of the gyrA gene can be carried out with a sense primer corresponding to the sequence SEQ ID NO: 58 and an antisense primer chosen from all the sequences of nucleic acids having at least 90% identity with the sequence SEQ ID NO: 59 or with a sense primer corresponding to one of the sequences selected from the group comprising all the nucleic acid sequences having at least 90% identity with the sequence SEQ ID NO: 58 and an antisense primer corresponding to the sequence SEQ ID NO: 59 or with a sense primer corresponding to one of the sequences chosen from the group comprising all the nucleic acid sequences having at least 90 %> identity with the sequence SEQ ID NO: 58 and an antisense primer corresponding to one of the sequences selected from the group comprising all the nucleic acid sequences presenting a at least 90% identity with the sequence SEQ ID NO:
• the present invention also aims at protecting a kit of reagents allowing the detection of bacterial strains Legionella pneumophila resistant to antibiotics, in particular of the fluoroquinolone type, comprising at least one pair of primers allowing the amplification of the fragment of interest, namely the amplicon of 259 nucleotides corresponding to the sequence SEQ ID NO: 59 and at least one probe chosen from those represented by the sequences SEQ ID NO: 3 and SEQ ID NO: 60, which are the detection and anchoring probes.
• the detection method according to the invention may be used for the diagnosis of an infection with antibiotic-resistant bacteria Legionella pneumophila, including fluoroquinolone type, in a patient.
• a sample of a patient likely to present strains of L. pneumophila resistant to antibiotics the method of detection of gyrA gene mutated by real-time PCR is implemented.
• a sample may in particular be a collection of secretions from the respiratory tract, but also blood, serum, plasma, urine, tissue biopsies (examples: pulmonary or pleural biopsies), various puncture fluids (examples: cerebrospinal fluid, articular fluid, pleural fluid) and various suppurations (eg pulmonary abscess)
• the detection method according to the invention may be used for determining or predicting the efficacy of a fluoroquinolone treatment in a patient infected with the bacterium Legionella pneumophila.
• the detection method according to the invention may be implemented to ensure that the patient does not exhibit resistance to fluoroquolone antibiotics, so as to modify the treatment plan if applicable.
• FIG. 1 represents the melting curves obtained for a fluoroqumolone-sensitive L. pneumophila Paris strain (melting temperature of 59 ° C.), for a mutant gyrA83 (mp 56.6 ° C) and for a double mutant gyrA83 + gyrA87 (melt temperature 50 ° C).
• Figure 2 shows the real-time PCR amplifications of the gyrA gene in different Legionella species.
• the bacterial inoculum tested for each species is standardized beforehand.
• Figure 3 shows the melting peaks obtained for four strains of L. pneumophila and three other species of Legionella.
• Figure 4 shows the melting peaks obtained for 2 strains of L. pneumophila Paris and 21 strains belonging to other species of Legionella.
• FIG. 5 shows the LP-gyrA RT-PCR melting curves showing a melting temperature (Tm) of 59 ° C for L. pneumophila Paris, and for most respiratory samples from patients with legionellosis. Samples 1-2 and 2-2 instead show a decrease of about 4-5 ° C in the Tm suggesting a mutation of the gyrA gene.
• FIG. 6 represents the sequencing results of the gyrA gene for the L. pneumophila Paris strain sensitive to fluoroquinolones, and for the respiratory samples taken in patients 1 and 2, ie before treatment with fluoroquinolones (1-1 and 2- 1) or at different times during treatment with these antibiotics (1-2, 2-2, 2-3, and 2-4).
• Example 1 Amplification of the GGGGATACAGCTGTTTATGACAC fragment of the gyrA gene (ID: 3119437) of the L. pneumophila strain PARIS (Genbank NC 006368.1, SEQ ID NO: 13), a fragment encoding the amino acids bound to fluoroquinolone resistance.
• the primers described below make it possible to amplify and detect a fragment of the L. pneumophila gyrA gene (gyrA gene: SEQ ID NO: 1).
• the detection method is based on a real-time PCR, using FRET technology (Fluorescence Resonance Energy Transfer).
• FRET Fluorescence Resonance Energy Transfer
• two probes are used, one of which carries 3 'of a transmitting fluorochrome and the other 5' of an acceptor fluorochrome.
• the probes are selected to hybridize to their target sequences spaced from each other by 1 to 5 nucleotides.
• the donor fluorochrome emits only fluorescence background noise whereas when they are hybridized to less than 10 nucleotides apart, the proximity of the two fluorochromes allows the transfer of the energy of the fluorochrome donor to the fluorocarbon acceptor causing the fluorescent emission of the latter (FRET: Fluorescent Resonance Energy Transfer).
• the fluorescence acquisition is then measured, proportional to the amount of DNA synthesized, at the time of hybridization.
• the anchor probe contains LCRed-640 fluorophore (Sigma Aldrich, L'I
• the detection probe contains fluorescein 3 '. Fluorescence emission is detected in real time by the amplification apparatus. A melting curve is established at the end of amplification and makes it possible to determine a melting temperature characteristic of the size and the base content of the amplicon. A mutation affecting this fragment causes a downward shift of the melting temperature.
• the fragment tested here goes from the nucleotide located at position 241 to the nucleotide located at position 263 of L. pneumophila gyrA Paris.
• the primers used make it possible to obtain an amplicon of 259 bp (SEQ ID No. 57).
• the amplification is carried out under the conditions below, on a Light-Cycler type device (Roche Diagnostics, Meylan, France).
• the results are given in FIG. 1.
• the single mutant gyrA 83 has a melting temperature of 56.6 ° C.
• the double mutant gyrA $ 3 + gyrA% 7 has a melting temperature of 50 ° C.
• the unmutated Paris strain has a melting temperature of 59 ° C.
• the sensitivity and specificity of the LP-gyrA RT-PCR assay was evaluated on a large number of strains.
• Figure 2 shows that the test sensitively and specifically detects the targeted fragment of the L. pneumophila gyrA gene.
• the defined primers preferentially amplify the L. pneumophila gyrA fragment. However, for a number of amplification cycles greater than or equal to 35, an amplification signal may be observed for other species of this kind.
• Figure 3 shows that the test allows, on the basis of the melting curves, to distinguish between L. pneumophila strains (Paris, Philadelphia, Lorraine and Lens strains) and the strains of three other Legionella species.
• the fusion temperatures of Legionella strains belonging to non-pneumophila species are higher than those obtained for L. pneumophila.
• Figure 4 shows that 21 strains of Legionella belonging to species other than L. pneumophila have higher melting temperatures than that of L. pneumophila strain Paris, with the exception of strains of species L longbeachea which have a melting peak around 50 ° C. Nevertheless, they can not be confused with the melting curves of the gyrA mutants because of their profile in triple melting peaks (50 ° C, 60 ° C, 65 ° C).
• Table 1 Amplification of the L. pneumophila Paris gyrA gene from a series of dilutions of a bacterial inoculum of 8.6 ⁇ 10 7 bacteria (per test).
• Example 3 Evaluation of the Relevance of the LP-gyrA RT-PCR Test on Airway Samples of Patients With Legionellosis Due to L. pneumophila
• Figure 5 shows the melting curves obtained on the basis of samples from patients with legionellosis.
• the melting curves (1-2 and 2-2) obtained from clinical samples collected after implementation of the antibiotic treatment with a fluoroquinolone show a melting peak of about 55 ° vs. This lower peak than that of the control L. pneumopihla wild Paris (non-mutated) evokes the presence of at least one mutation in the QRDR of gyrA in the strain of L. pneumophila infecting each of these patients.
• the QRDR of the gyrA gene of the infectious L. pneumophila strain was amplified and sequenced directly from a clinical specimen because of the impossibility of isolating this strain. culture.
• Two DNA sequencing methods were used: the classical Sanger method and a high-throughput sequencing method to measure the percentage of mutants relative to the non-mutated population in the clinical sample of each of the two patients. .
• sequence 1-1 obtained from a clinical specimen collected before treatment with a fluoroquinolone, has no mutation compared to the Paris strain.
• the DNA sequence before treatment with fluoroquinolone (sequence 2-1) is wild-type (non-mutated).
• a strain of L. pneumophila could be isolated in culture from this same clinical specimen. The fact that this strain was well sensitive to fluoroquinolones was confirmed, in terms of pheno typical (antibiogram) and because of the absence of gyrA mutation after amplification and sequencing of this gene from the strain.
• High throughput sequencing data confirmed in vivo selection of L. pneumophila gyrA83 mutants in both patients during fluoroquinolone treatment.
• Figure 7 shows for patient 2 a progressive increase in the percentage of mutants compared to the non-mutated population, i.e. progressive replacement of a population of fluoroquinolone-sensitive L. pneumophila by a population that is predominantly resistant to these antibiotics.

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