Method for the in vitro detection of strains of Legionella pneumophila resistant to antibiotics

Patent metadata

Field	Value
Publication number	US-10106858-B2
Application number	US-201414900626-A
Country	US
Kind code	B2
Filing date	Jun 17, 2014
Priority date	Jun 27, 2013
Publication date	Oct 23, 2018
Grant date	Oct 23, 2018

Abstract

Disclosed is a method for the in vitro detection of strains of Legionella pneumophila, resistant to antibiotics, in particular to the fluoroquinolones. Also disclosed are nucleotide probes and a kit of reagents allowing the detection of Legionella pneumophila bacterial strains that are resistant to antibiotics, in particular of the fluoroquinolone type.

First claim

The invention claimed is:

1. In vitro method of detecting a bacterial strain of Legionella pneumophila that is resistant to fluoroquinolone, in a biological sample from a patient suffering from legionnaires' disease, the resistance being acquired in vivo, the method comprising:
detecting:
a mutation on at least one of positions 83 or 87 with respect to SEQ ID NO: 1 in a GyrA protein of L. pneumophila having at least 90% identity with SEQ ID NO: 1, said mutation resulting in a mutated GyrA protein, or
a nucleic acid encoding said mutated GyrA protein, the detection of said mutation or of the nucleic acid encoding said mutated GyrA protein indicating the presence of fluoroquinolone-resistant bacterial strain Legionella pneumophila in the sample.

2. The method according to claim 1 , in which the mutated GyrA protein is such that:
the amino acid at position 83 is different from T and the amino acid at position 87 can correspond to any amino acid, or
the amino acid at position 87 is different from D, and the amino acid at position 83 can correspond to any amino acid.

3. The method according to claim 1 , in which the mutated GyrA protein is such that:
the amino acid at position 83 is: I, L, W, A or V and the amino acid at position 87 can correspond to any amino acid, or
the amino acid at position 87 is: N, G, Y, H or V and the amino acid at position 83 can correspond to any amino acid, or
the amino acid at position 83 is: I, L, W, A or V, and the amino acid at position 87 is: N, G, Y, H or V.

4. The method according to claim 1 , in which said mutated GyrA protein comprises the consensus sequence:
GDX 1 X 2 VYX 3 T (SEQ ID NO: 2),

in which:
X 2 is A,
X 1 and X 3 correspond to the mutations at positions 83 and 87 respectively, and
X 1 is different from T, and X 3 is any amino acid, or

X 3 is different from D, and X 1 any amino acid.

5. The method according to claim 4 , in which:
X 1 is I, L, W, A or V, and X 3 corresponds to any amino acid, or
X 3 is N, G, Y, H or V, and X 1 corresponds to any amino acid, or
X 1 is I, L, W, A or V, and X 3 is N, G, Y, H or V.

6. The method according to claim 1 , wherein
the nucleic acid encoding said mutated GyrA protein has at least one nucleotide substitution with respect to SEQ ID NO: 3, at a position equivalent to position 7, 8, 19, 20 or 21 with respect to SEQ ID NO: 3,
and wherein nucleotides at positions 7 and 8 correspond to two nucleotides of a codon encoding an amino acid at position 83 of the GyrA protein, and
nucleotides at positions 19, 20 and 21 correspond to three nucleotides of a codon encoding an amino acid at position 87 of the GyrA protein.

7. The method according to claim 1 , wherein the detection of said mutated GyrA protein or of the nucleic acid encoding said mutated GyrA protein is carried out by a technique selected from the group consisting of: western blot, northern blot, southern blot, PCR, real-time PCR, PCR hybridization, PCR array, TMA, NASBA, LCR, DNA/RNA hybridization, DNA chip, DNA/RNA sequencing, dot-blot, and RFLP (Restriction fragment length polymorphism).

8. The method according to claim 1 , said method comprising the steps of:
a) bringing a biological sample likely to contain a target nucleic acid belonging to a fluoroquinolone-resistant bacterial strain of Legionella pneumophila , into contact with a pair of primers capable of hybridizing specifically with said target nucleic acid,
b) PCR amplification of said target nucleic acid using the pair of primers in order to obtain an amplification product, said amplification product comprising nucleic acid comprising a sequence having at least 90% identity with sequence SEQ ID NO: 3, and
c) detecting a nucleic acid encoding the mutated GyrA protein, wherein the detection of the nucleic acid encoding said mutated GyrA protein indicates the presence of a fluoroquinolone-resistant bacterial strain of Legionella pneumophila , in the sample.

9. The method according to claim 8 , wherein the detection of said amplification product is carried out using at least one nucleotide probe capable of hybridizing with the amplification product.

10. The method according to claim 1 , wherein the detection of the nucleic acid encoding said mutated GyrA protein is carried out using at least one nucleotide probe, said at least one nucleotide probe comprising a nucleic acid molecule having at least 90% identity with sequence SEQ ID NO: 3,
said at least one nucleotide probe being linked by a covalent bond to at least one marker molecule allowing detection thereof,
said marker molecule being a fluorochrome or a radioactive isotope.

11. The method according to claim 1 , wherein the detection of the nucleic acid encoding said mutated GyrA protein is carried out using at least one nucleotide, said at least one nucleotide probe comprising a nucleic acid molecule having at least 90% identity with sequence SEQ ID NO: 60 linked by a covalent bond to a fluorochrome.

12. The method according to claim 1 , further comprising carrying out PCR amplification a fragment of the gyrA gene of Legionella pneumophila with a pair of primers comprising a primer having at least 90% identity with sequence SEQ ID NO: 58, and a primer having at least 90% identity with sequence SEQ ID NO: 59, and wherein the nucleic acid encoding said mutated GyrA protein is detected.

13. The method according to claim 12 , wherein the detection the nucleic acid encoding said mutated GyrA protein is carried out using at least one nucleotide probe comprising a nucleic acid molecule having at least 90% identity with sequence SEQ ID NO: 60 linked by a covalent bond to a fluorochrome.

14. A method for determining an infection with a Legionella pneumophila bacterium that is resistant to fluoroquinolone in a patient,
or for determining or predicting the efficacy of a treatment with fluoroquinolones in a patient infected with the Legionella pneumophila bacterium,
the method comprising performing the method of claim 1 .

15. The method according to claim 2 , in which the mutated GyrA protein is such that:
the amino acid at position 83 is: I, L, W, A or V and the amino acid at position 87 can correspond to any amino acid, or
the amino acid at position 87 is: N, G, Y, H or V and the amino acid at position 83 can correspond to any amino acid, or
the amino acid at position 83 is: I, L, W, A or V, and the amino acid at position 87 is: N, G, Y, H or V.

16. The method according to claim 2 , wherein
the nucleic acid encoding said mutated GyrA protein has at least one nucleotide substitution with respect to SEQ ID NO: 3, at a position equivalent to position 7, 8, 19, 20 or 21 with respect to SEQ ID NO: 3,
and wherein the nucleotides at positions 7 and 8 correspond to two nucleotides of a codon encoding the amino acid at position 83 of the GyrA protein, and
the nucleotides at positions 19, 20 and 21 correspond to three nucleotides of a codon encoding the amino acid at position 87 of the GyrA protein.

17. The method according to claim 3 , wherein
the nucleic acid encoding said mutated GyrA protein has at least one nucleotide substitution with respect to SEQ ID NO: 3, at a position equivalent to position 7, 8, 19, 20 or 21 with respect to SEQ ID NO: 3,
and wherein the nucleotides at positions 7 and 8 correspond to two nucleotides of a codon encoding the amino acid at position 83 of the GyrA protein, and
the nucleotides at positions 19, 20 and 21 correspond to three nucleotides of a codon encoding the amino acid at position 87 of the GyrA protein.

Assignees

• Univ Joseph Fourier
• Centre Hospitalier Univ De Grenoble
• Univ Grenoble Alpes
• Centre Hospitalier Univ Grenoble Alpes

Inventors

• Maurin Max Maurice Louis
• Schneider Dominique
• Shadoud Lubana
• Jarraud Sophie
• Timsit Jean Francois
• Etienne Jerome

Classifications

• Y02A50/451

  Cross-Sectional Technologies · mapped topic

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/689 Primary

  for bacteria · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title