Polynucleotides and their use for detecting resistance to streptogramin A or to streptogramin B and related compounds

A purified peptide comprising the amino acid sequence encoded by the nucleotide sequence selected from the group consisting of SEQ ID NO:

• 1 or a fragment derived from SEQ ID NO:
• I containing 15 to 40 nucleotides, SEQ ID NO:
• 11, and SEQ ID NO. 12.

3 A purified peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO 4 or a fragment derived from SEQ ID NO:

• 4 containing at least 10 amino acids, SEQ ID NO:
• 7, and SEQ ID NO:
• 8.

4 A purified polynucleotide comprising the nucleotide sequence of SEQ ID NO. 3 or a fragment derived from SEQ ID NO:

• 3 containing 15 to 40 nucleotides.

A purified peptide comprising the amino acid sequence encoded by the nucleotide sequence selected of SEQ ID NO:

• 3 or a fragment derived from SEQ ID NO 3 containing 15 to 40 nucleotides

6 A purified peptide comprising the amino acid sequence of SEQ ID NO:

• 6 or a fragment derived from SEQ ID NO:
• 6 containing at least 10 amino acids.

8 A purified peptide comprising the amino acid sequence encoded by the nucleotide sequence selected from the group consisting of SEQ ID NO. 2 or a fragment derived from SEQ ID NO 2 containing 15 to 40 nucleotides, SEQ ID NO:

• 13, and SEQ ID NO 14

A purified peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO. 5 or a fragment derived from SEQ ID NO:

• 6 containing at least 10 amino acids, SEQ ID NO:
• 9, and SEQ ID NO:
• 10.

10 A composition of polynucleotide sequences useful for the detection of resistance to streptogramins A and/or B and related compounds comprising at least a nucleotide sequence selected from the group consisting of the polynucleotide or gene or cDNA povgaB, vgbB, and vatC.

A composition of polynucleotide sequences useful for the detection of resistance to streptogramin A or streptogramin B and related compounds comprising at least an amino acid sequence from a polypeptide encoded by a polynucleotide selected from the group consisting of the polynucleotide, gene, or cDNA vgaB, vgbB, and vatC.

A composition of polynucleotide sequences encoding resistance to streptogramins and related compounds, or inducing streptogramin resistance in Gram-positive bacteria, wherein said composition comprises at least a nucleotide sequence encoding a molecule containing ATP binding motifs conferring resistance to streptogramin A and related compounds and at least a nucleotide sequence selected from the following sequences:

• a) a nucleotide sequence encoding an acetyltransferase conferring resistance to streptogramin A and related compounds;
• and
• b) a nucleotide sequence encoding a lactonase conferring resistance to streptogramin B and related compounds.

13 A composition of polynucleotide sequences encoding resistance to streptogramins and related compounds, or inducing streptogramin resistance in Gram-positive bacteria, wherein said composition comprises at least a nucleotide sequence encoding an acetyltransferase conferring resistance to streptogramin A and related compounds and at least a nucleotide sequence selected from the following sequences

• a) a nucleotide sequence encoding a molecule containing ATP binding motifs conferring resistance to streptogramin A and related compounds;
• and
• b) a nucleotide sequence encoding a lactonase conferring resistance to streptogramin B and related compounds.

14 A composition of polynucleotide sequences encoding resistance to streptogramins and related compounds, or inducing streptogramin resistance in particular in Gram-positive bacteria, wherein said composition comprises at least a nucleotide sequence encoding a lactonase conferring resistance to streptogramin B and related compounds and at least a nucleotide sequence selected from the following sequences:

• a) a nucleotide sequence encoding a material containing ATP binding motifs conferring resistance to streptogramin A and related compounds;
• and
• b) a nucleotide sequence encoding an acetyltransferase conferring resistance to streptogramin A and related compounds.

16 A composition of claim 15, wherein the polynucleotide sequence encoding a molecule containing ATP binding motifs confers resistance to streptogramin A and related compounds in Staphylococcus aureus

18 A composition of claim 17, wherein the polynucleotide sequence encoding an acetyltransferase confers resistance to streptogramin A and related compounds in Staphylococcus cohnii.

20 A composition of claim 19, wherein the polynucleotide sequence encoding a lactonase confers resistance to streptogramin A and related compounds in Staphylococcus cohnii

21 A composition of polynucleotide sequences according to claim 12 or 13, wherein said composition comprises at least one nucleotide sequence encoding a molecule containing ATP binding motifs conferring resistance to streptogramin A and related and at least one nucleotide sequence encoding an acetyltransferase conferring to streptogramin A and related compounds.

22 A composition of polynucleotide sequences according to claim 21, wherein said composition comprises at least a vgbB nucleotide sequence encoding a molecule containing ATP binding motifs conferring resistance to streptogramin A and related compounds and at least a vatb nucleotide sequence encoding an acetyltransferase conferring resistance to streptogramin A and related compounds

24 A polynucleotide fragment comprising at least 10 nucleotides, that hybridizes under stringent conditions with a sequence according to any one of claims 1-9 or 23 or the polynucleotide complementary fragment thereof.

25 A polynucleotide fragment of claim 12, wherein said fragment corresponds to at least one of the following sequences

• 8 - Oligo I 5′-AAGTCGACTGACAATATGAGTGGTGG-3′ - Oligo II 5′-CTGCAGATGCCTCAACAGCATCGATATCC-3′

26 A polynucleotide fragment of claim 12, wherein said fragment corresponds to at least one of the following sequences:

• 9 - Oligo III 5′-ATGAATTCGCAAATCAGCAAGG-3′ - Oligo IV 5′-TCGTCTCGAGCTCTAGGTCC-3′

27 A polynucleotide fragment of claim 12, wherein said fragment corresponds to at least one of the following sequences

• 10 - Oligo V 5′-CAGCAGTCTAGATCAGAGTGG-3′ - Oligo VI 5′-CATACGGATCCACCTTTTCC-3′

28 A recombinant DNA sequence comprising at least a nucleotide sequence according to any one of claims 1, 4, or 7 under the control of regulatory elements that regulate the expression of resistance to antibiotics of the streptogramin family in a defined host.

29 A recombinant vector comprising the DNA sequence of claim 31, wherein the vector comprises a plasmid pIP1633 deposited with the C.N.C.M. under the Accession No. I-1768 or a plasmid pIP1714 deposited with the C.N.C M. under the number I-1877 on Jun. 18, 1997.

A recombinant cell host comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO 1, SEQ 11D NO. 11, SEQ ID NO 12, SEQ ID NO 3, SEQ ID NO. 2, SEQ ID NO. 13, and SEQ ID NO. 14, or the recombinant vector ot claim 32.

32 A polynucleotide comprising the full length coding sequence of a Staphylococcus streptogramin A and/or B resistant gene containing a polynucleotide sequence according to any one of claims 1, 4, or 7

33 A monoclonal or polyclonal antibodv directed against a polypeptide or a peptide fragment encoded by the polvnucleotide sequences according to any one of claims 1, 4, or 7.

34 A method of detecting the presence of bacterium harboring the polvnucleotide sequences according to any one of claims 1, 4, or 7 in a biological sample comprising

• a) contacting bacterial DNA of the biological sample with a primer or a probe according to any one of claims 24-27, which hybridizes with a nucleotide sequence encoding resistance to streptogramins,
• b) amplifying the nucleotide sequence using said primer or said probe, and
• c) detecting the hybridized complex formed between said primer or probe with the DNA

36 A kit for detecting the presence of bacterium having resistance to streptogramin A and harboring the polynucleotide sequences according to claim 1 or claim 7 in a biological sample, said kit comprising:

• a) a polynucleotide probe according to claim 24-26;
• and
• b) reagents necessary to perform a nucleic acid hybridization reaction

37 A method of screening active antibiotics for the treatment of the infections due to Gram-positive bacteria, comprising the steps of:

• a) bringing into contact a Gram-positive bacteria hazing a resistance to streptogramin A or streptogramin B and related compounds and containing the polynucleotide sequences according to any one of the claims 1, 4, or 7 with the antibiotic;
• and
• b) measunrng an activity of the antibiotic on the bacteria having a resistance to streptogramins and related compounds

A composition of a polynucleotide sequence encoding resistance to streptogramins and related compounds, or inducing resistance in Gram-positive bacteria, wherein said composition comprises a nucleotide sequence corresponding to the resistance phenotype of the plasmid pIP163j deposited with the C N.C.M under the Accession No I-1768 and of the plasmid pIP 1680 deposited with the C.N C.M under the Accession No I-1767 and of the plasmid pIP1714 deposited with the C N C M under the number I-1877 on Jun. 18, 1997

41 A method of detecting the presence of bacterium harboring the polynucleotide sequences according to claim 1 or claim 7 in a biological sample, said method comprising the steps of:

• a) contacting said sample with an antibody according to claim 36 that recognizes a polypeptide encoded by said polynucleotide sequences, and
• b) detecting said complex