WO2009143614A1

WO2009143614A1 - Linezolid resistance mutations in gram-positive bacteria

Info

Publication number: WO2009143614A1
Application number: PCT/CA2009/000724
Authority: WO
Inventors: Marc Quellette; Jie Feng
Original assignee: Universite Laval
Priority date: 2008-05-26
Filing date: 2009-05-26
Publication date: 2009-12-03
Also published as: EP2283122A4; CA2630253A1; EP2283122A1; CA2725983A1

Abstract

Linezolid is a member of a novel class of antibiotics, with resistance already being reported. We used whole-genome sequencing on three independent Streptococcus pneumoniae strains made resistant to linezolid in vitro in a step-by-step fashion. Analysis of the genome assemblies revealed mutations in the 23S rRNA gene in all mutants including, notably, G2576T, a previously recognized resistance mutation. Mutations in an additional 31 genes were also found in at least one of the three sequenced genomes. We concentrated our efforts on three new mutations that were found in at least two independent mutants. All three mutations were experimentally confirmed to be involved in antibiotic resistance. Mutations upstream of the ABC transporter genes SP_1114 and SP_2075 were correlated with increased expression of these genes and neighboring genes of the same operon. The hypothetical protein SP_0373 contains an RNA methyltransferase domain, and mutations within that domain were found in all S. pneumoniae linezolid-resistant strains. Interestingly, the SP_0373 ortholog was also mutated in a linezolid-resistant clinical Staphylococcus aureus isolate.

Description

LINEZOLID RESISTANCE MUTATIONS IN GRAM-POSITIVE

BACTERIA

FIELD OF THE INVENTION

The present invention relates to markers of antibiotic resistance. More specifically, the present invention relates to nucleic acid sequences, polypeptide sequences, methods of detection and reagents for assessing antibiotic resistance or susceptibility profile of a bacteria.

BACKGROUND OF THE INVENTION

Linezolid is an oxazolidinone ring containing antibiotic that represents a novel class of synthetic antibiotic. It is highly effective against a number of Gram-positive pathogens such as Staphylococcus aureus and its methicillin resistant version (MRSA), Streptococcus pneumoniae, enterococci and their vancomycin resistant versions (VRE), and several others (reviewed in (Vara Prasad, 2007)). Linezolid binds to the 5OS subunit of the bacterial ribosome via interaction with the 23S rRNA. In doing so, it blocks the formation of protein synthesis initiation complexes. Recent cross-linking experiment have shown that oxazolidinone antibiotics interact with the A site of the bacterial ribosome and possibly interfere with the placement of the aminoacyl-tRNA (Leach et al., 2007).

Resistance to linezolid is relatively infrequent and is usually due to point mutations in target genes. It was first studied in Gram-positive pathogens selected for resistance in vitro and mutations were first noted in domain V of the 23S rRNA. Several mutations have been pinpointed in the 23S rRNA, but the most common mutation is G2576T when using the E. coli numbering system (see (Meka & Gold, 2004, Woodford et al., 2007) for reviews). The same mutations were also observed in clinical isolates of S. aureus and VRE resistant to linezolid. There are several copies of the 23SrRNA genes (4 to 6) in most Gram-positive pathogens and in general the level of resistance correlates with the number of mutated copies of 23S rRNA. The domain V of the 23S rRNA is also the binding site of other type of antibiotics such as chloramphenicol, and linezolid resistant bacteria are consequently often cross- resistant to other class of antibiotics.

In addition to mutation in the 23S rRNA, a 6 base pair deletion in the ribosomal protein L4 was described in two clinical isolates of S. pneumoniae that were resistant to chloramphenicol and not susceptible to linezolid (Wolter et al., 2005). Recently, it was found that a (integrated) plasmid 23S rRNA methyltransferase coded by the cfr gene (for chloramphenicol-florfenicol resistance) could also confer resistance to linezolid in S. aureus (Toh et al., 2007, Mendes et al., 2008). There are other linezolid resistant strains, however, that do not seem to carry out one of the main known mutations (Carsenti-Dellamonica et al., 2005, Richter et al., 2007 and Venkata et al. 2004) and it is possible that other mutations or genes may also contribute to linezolid resistance. Recently, whole genome sequencing has revealed to be a powerful method to detect mutations linked to drug resistance or mode of action of antibiotics (Andries et al., 2005, Albert et al., 2005, Manjunatha et al., 2006, Mwangi et al., 2007). We used whole genome sequencing of Streptococcus pneumoniae strains selected independently for linezolid resistance in vitro and found known and novel mutations involved in resistance to this novel drug.

SUMMARY OF THE INVENTION

The present invention provides markers of antibiotic resistance. More specifically, the present invention provides nucleic acid sequences, polypeptide sequences, methods of detection and reagents for identifying an antibiotic resistant bacteria and assessing the antibiotic resistance or susceptibility profile of a bacteria.

Some aspects of the present invention relate to methods for determining an antibiotic resistance or susceptibility profile of bacteria. The method may comprise detecting a mutation in one of the genes listed in Table 3 or in the gene product or detecting modulation in expression of some genes. An embodiment of such method includes detecting the specific mutation(s) listed in Table 3.

The Applicant has found a particularly good correlation between antibiotic resistance and mutations in the SP_0373, SP_2075 and/or SP_1114 genes and in the corresponding gene product or in the promoter region of _2075 and/or SP_1114 genes. The present invention thus relates to a method for determining an antibiotic resistance or susceptibility profile of a bacteria which may comprise detecting the SP_0373 gene, the SP_2075 gene and/or the SP_1114 gene or a corresponding SP_0373, SP_1114 or SP_2075 orthologue or detecting the expression product of said gene.

The present invention more specifically relates to a method for determining an antibiotic resistance or susceptibility profile of a bacteria, the method may comprise detecting (individually or collectively) a mutation in either the SP_0373 gene, in the SP_2075 gene and/or in the SP_1114 gene or in a gene encoding a corresponding orthologue. Alternatively, the method may comprise detecting the gene product associated with said mutation.

The Applicant has particularly shown that mutations in the promoter region of the SP_2075 and/or SP_1114 genes increase expression of genes under the control of such mutated promoter.

Accordingly, methods of the present invention also comprise measuring the expression level a gene product which is under the control of a SP_1114 or SP_2075 promoter, wherein an increased expression of the gene product with reference to a normal expression level is indicative of an increased antibiotic resistance and /or an antibiotic resistant bacteria.

The methods described herein are particularly useful for identifying a Gram-positive bacteria resistant to an antibiotic such as linezolid, chloramphenicol, florfenicol and/or tiamulin. The methods described herein are also particularly useful for determining the susceptibility or resistance profile of a Gram-positive bacteria toward an antibiotic such as linezolid, chloramphenicol, florfenicol and/or tiamulin.

Other aspects of the invention relate to a protein of Table 3 having at least one mutation in comparison with the original sequence protein sequence found in accession No. NC_003098 or NC_003028. In accordance with the present invention, the mutation may be for example an amino acid substitution or deletion. The mutation of the protein may be associated with resistance to an antibiotic such as linezolid, chloramphenicol, florfenicol and/or tiamulin. These mutated proteins may especially be useful for developing tools (e.g. antibodies and reagents) for their specific detection and thus identifying bacteria carrying these mutations.

The invention thus encompasses any of the mutated proteins of Table 3 carrying one or more of the listed mutations. Other aspects of the invention also relate to a nucleic acid capable of encoding the mutated proteins of Table 3, a synthetic vector comprising such nucleic acid, amino acid sequence comprising a peptide derived from the mutated protein and antibodies capable of specific binding to the mutated proteins of Table 3.

Other aspects of the present invention relate to molecules which are capable of specific binding to a mutated protein described herein. Exemplary embodiments of such molecules include antibodies, aptamers, etc. Nucleic acids capable of encoding a desired mutated protein or mutated protein fragment (peptide) described herein is encompassed herewith. Synthetic vector comprising the nucleic acid described herein is encompassed herewith.

Another aspect of the present invention provides for an isolated cell which may comprise the synthetic vector, the mutated proteins or the peptide of the present invention. While an additional aspect provide for antibodies (or antigen binding fragment) capable of specific binding to a desired mutated protein or mutated protein fragment of the present invention.

The antibody or an antigen binding fragment described herein may preferentially bind to the mutated protein over a protein which does not comprise the mutation(s).

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

Figure 1 is a histogram illustrating the expression of ABC genes of S. pneumoniae as determined by Real time quantitative PCR. The RNA of the mutant 1974M2 was prepared and the genes SP_1114, SP_1115, SP_2075 and SP_2073 were ~ 10 fold more expressed when compared to wild type (w.t.) cells. Since a different mutation was found upstream of the ABC gene SP_2075 we also monitored its expression in 1974M1 and it was also shown to be overexpressed;

Figure 2 is a Table summarizing the Strains and plasmids used herein;

Figure 3 is a Table illustrating the susceptibility levels of S. pneumoniae isolates;

Figure 4: is a Table listing the mutations found in S. pneumoniae isolates selected for linezolid resistance;

Figure 5: is the continuation of the Table of Figure 4;

Figure 6 is a Table illustrating the chronological appearance of mutations at different levels of linezolid resistance;

Figure 7 is a Table illustrating the functional analysis of Streptococcus pneumoniae genes in antibiotic resistance;

Figure 8 is a further Table illustrating the functional analysis of Streptococcus pneumoniae genes in antibiotic resistance ; Figure 9 is a Table illustrating the mutations in linezolid resistant Sthaphylococcus aureus clinical isolates;

Figure 10 is a Table listing the SEQIDNOs., the identity of the sequence and the position of the mutation; Figure 11 : is the continuation of the Table of Figure 9, and;

Figure 12: is a further continuation of the Table of Figure 9.

DETAILED DESCRIPTION OF THE INVENTION

The Applicant has found a good correlation of antibiotic resistance and mutations in the SP_0373, SP_2075 and SP_1114 genes, these genes and orthologues as well as nucleic acids, corresponding mutated proteins and peptides represent good candidates in the development of methods, tests, kits, reagents and assays for the identification of antibiotic resistant strains. The Applicant has also found unique mutations in other genes which may be found useful for the same purposes.

The present invention therefore provides in one aspect thereof, methods for determining an antibiotic resistance or susceptibility profile of a bacteria. The method may comprise detecting a mutation in the SP_0373 gene, in the SP_2075 gene and/or in the SP_1114 gene or in a gene corresponding to an orthologue of another bacteria species. Methods of the present invention also encompass detecting the expression product associated with the mutation. It is to be understood herein that detection of an expression product such as RNA may be performed directly or indirectly. For example, the RNA may be isolated and detected with a probe (e.g., northern blot, Rnase protection assay, etc.). Alternatively, the RNA may be converted into DNA (e.g., cDNA), optionally amplified and detected using conventional methods such as by the use of DNA intercalating agent including ethidium bromide or SYBR Green™, by a probe labeled with a fluorescent dye, or dyes capable of fluorescent resonance energy transfer (FRET), a reporter molecule (etc. enzymes, biotin, etc.), a radioactive label or else.

A mutation which is of particular interest for the purpose of determining antibiotic resistance or susceptibility is any mutation in the SP_0373 gene associated with a reduced ability of the mutated SP_0373 to putatively methylate RNA.

Another mutation which is of particular interest for the purpose of determining antibiotic resistance or susceptibility is any mutation in the SP_2075 gene or in the SP_1114 gene which may be located in the promoter region. Especially a mutation in the promoter region which may allow an increased expression of a gene product regulated by such promoter. In accordance with the present invention a corresponding gene product is for example the SP_2075 gene product. Alternatively, a corresponding gene product is for example the SP_1114 gene product. Further corresponding gene product also encompass another gene product encoded by another gene located on the same operon or locus as SP_1114 or SP_2075. An example of a gene located on the same operon as SP_2075 is the SP_2073 gene.

The term "gene product" as used herein includes RNA and protein and any compounds derived from protein or RNA including for example, cDNA, DNA, complements, peptides (modified or not) etc.

The present method may thus allow one to determine the antibiotic resistance or susceptibility profile of a bacteria of the Streptococcus pneumoniae species. However, as indicated herein mutations in corresponding gene orthologues may be identified in other bacteria species and more particularly in Gram-positive bacteria which may allow one to determine the antibiotic resistance or susceptibility profile of bacteria of other species.

Such method has been applied successfully to bacteria of the Staphylococcus aureus species wherein the orthologue of SP_0373, namely SAV1444 also showed mutations associated with linezolid resistance. The method may also be applied to Staphylococcus aureus which are methicillin resistant.

The method may also be applied to Staphylococcus aureus having vancomycin resistance such as in the case of VISA or VRSA strains.

The method may also be applied to bacteria of the Enterococcus genus especially by detecting mutation in a corresponding orthologue of SP_0373, SP_1114 and/or SP_2075. In accordance with the present invention, the method may be applied to bacteria of the Enterococcus genus having vancomycin resistance. Exemplary embodiments of bacteria of the Enterococcus genus include for example, Enterococcus faecalis and Enterococcus faecium.

Corresponding SP_0373 orthologues of enterococci includes NP_814882 (locus_tag="EF1152) Enterococcus faecalis V583 and ZP_00604632 (locus_tag="Ef_0734) Enterococcus faecium DO. A bacterium carrying one or more of the mutations listed herein may thus be identified as being linezolid resistant. Especially, the presence of the mutations described herein may be indicative of a pathogen having a linezolid minimum inhibitory concentration of more than 8μg/ml.

Examples of methods for determining an antibiotic resistance or susceptibility profile of a bacterium, may comprise detecting a nucleic acid sequence having one or more of the mutation described herein. Detection may be performed, by using a probe or primer capable of specific recognition of the mutated region of the gene of coding sequence (or their complement). Of course any or all of the probes described herein may be attached to a solid support each probe being assigned an addressable position on the support, thus generating an array. Such array may be used to identify the presence of the mutation(s) in the pathogen.

The method of the present invention thus comprise determining the presence of one or more of the new mutations associated with antibiotic resistance identified herein. However, the method of the present invention will also benefit from additionally detecting known mutations. Therefore in accordance with the present invention, the presence of the mutation at position 2576 and/or 2503 (with reference to E.coli numbering) of a 23S rRNA gene or gene product is also determined.

Another aspect of the present invention involves a method of determining an antibiotic susceptibility or an antibiotic resistance profile of a bacteria which may comprise measuring the expression level of a gene product in the SP_1114 or

SP_2075 locus or measuring the expression level of a gene product which is under the control of a SP_1114 or SP_2075 promoter, whereby an increased expression of the gene product (e.g. RNA and/or protein) is associated with an increase in antibiotic resistance.

In yet another aspect, the present invention relates to methods for restoring or increasing antibiotic susceptibility (especially to linezolid) in antibiotic resistant bacteria (especially a linezolid resistant bacteria).

In an embodiment of the invention the method may comprise inactivating or lowering the expression of a gene product under the control of a SP_1114 promoter. In an additional embodiment, the method may comprise providing a bacteria with a SP_0373 protein which does not have one or all of the mutation in SP_0373 listed in Table 3. In yet a further aspect, the present invention relates to a method for identifying a compound which may be capable of impairing the growth of an antibiotic resistant bacteria, the method may comprise contacting the bacteria with a compound (which may be obtained from a library) and evaluating the doubling time of the bacteria in the presence of the compound, wherein a reduced doubling time in the presence of the compound may be indicative of a compound which is useful for impairing the growth of the bacteria.

In accordance with the present invention, the bacteria may carry one or more of the new mutations described herein. Also in accordance with the present invention, the bacteria may carry one or more known mutations (e.g., 23S rRNA; G/T2576 and/or A/G2503) in addition to one or more of the new mutations described herein.

Further in accordance with the present invention, the bacteria may be identified as being resistant to one or more antibiotic such as for example, an oxazolidinone ring containing antibiotic, chloramphenicol, florfenicol and/or tiamulin. A specific example of an oxazolidinone ring containing antibiotic is linezolid. The present invention may also be applied to bacteria resistant to vancomycine, methicilline or combination thereof or to other commonly used antibiotics such as penicillin, macrolides, etc.

Alternative methods of identifying a compound capable of interfering with the growth of an antibiotic resistant bacteria encompass contacting a mutated protein of Table 3 or an orthologue thereof or a peptide (comprising the region of mutation) derived from such mutated protein or orthologue with a library of compound and isolating a compound which is capable of interacting with such mutated protein. In an embodiment of the invention, the selected compound will be chosen for its ability to interfere with the activity of the mutated protein.

In another alternative method, a library of compound may be contacted with a gene product of SP_1114, SP_1115, SP_2703, SP_2705 or of an orthologue thereof and a compound capable of interacting with such gene product may be isolated. In an embodiment of the invention, the selected compound will be chosen for its ability to interfere with the activity of the gene product.

The present invention also provides methods to increase antibiotic susceptibility of a bacteria. An exemplary embodiment of such method may comprise, for example, inactivating or lowering the expression of a gene product which is under the control of a SP_1114 promoter region. Another exemplary embodiment of such method may comprise providing a bacteria with a SP_0373 protein which does not have one or all of the mutation(s) in SP_0373 listed in Table 3.

In a further aspect, the present invention provides a method for identifying a compound which is capable of impairing the growth of bacteria characterized as being antibiotic resistant. The method may comprise contacting the antibiotic resistant bacteria in the presence of a library comprising the compound and evaluating the doubling time of the bacteria. A reduced doubling time in the presence of the compound may thus be indicative of a compound which may be useful for impairing the growth of the bacteria.

The method may especially be applied to antibiotic resistant bacteria which carry one or more mutation in the genes listed in Table 3.

The method may more particularly be applied to bacteria which are resistant to oxazolidinone ring containing antibiotic or more specifically to bacteria which are resistant to linezolid, chloramphenicol, florfenicol, tiamulin and/or ciprofloxacin. Furthermore, this method may also be suitable for bacteria which are resistant to vancomycine, methicilline or combination thereof.

Tests and kits may contains short oligonucleotides and/or probes which are specific for a nucleic acid sequence sought to be detected and which may be used for the identification of an antibiotic resistant bacteria.

Using cloning techniques well known in the art, one may generate vectors which contain a nucleic acid sequence encoding the mutated protein of Table 3 or fragments of such mutated proteins (e.g., a fragment containing a herein described mutation). These vectors may allow transcription of a mRNA and subsequent expression of the mutated protein or mutated protein fragment. Depending on the use, these vectors may also allow expression of an antisense RNA.

Other aspects of the invention relate to nucleic acid sequences or nucleic acid fragment which comprise the mutations described herein as well as complements of such nucleic acid sequences. These nucleic acids, may be used for example, for generating probes or primers which will allow specific recognition and identification of pathogens carrying such mutations. The information thus obtained may be used to identify an antibiotic resistant pathogen or may be used to determine the antibiotic susceptibility of a pathogen. The complete genome sequence of Streptococcus pneumoniae TIGR4 is available at Accession No. NC_003028 (Gl: 118090026) and the complete genome sequence of Streptococcus pneumoniae R6 is available at Accession No. NC_003098 (Gl: 15902044).

The complete genome sequence of the mu50 strain of Staphylococcus aureus is available at Accession No. NC_002758 (Gl: 57634611 ).

By convention nucleic acid sequences are usually expressed in the 5' to 3' direction (sense strand) and the first nucleotide of the coding sequence (of prokaryotes) usually starting with ATG (corresponding to the AUG start codon) is attributed position number 1.

The nucleic acid positions indicated herein also follows this convention where position 1 is attributed to the first nucleotide of the coding sequence. Thus reference to nucleotide position 490, means that it is the 490^th nucleotide from the first nucleotide of the start codon in the 5' to 3' direction.

A particular aspect of the present invention relates to a nucleic acid sequence which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to one of the nucleic acid sequence listed herein. The invention also relates to a complement of such sequence. The invention also relates to an orthologue comprising mutations associated with antibiotic resistance.

The term "at least 10 contiguous nucleotides or nucleosides" encompass 10 nucleotides or nucleosides and up to the total length of the gene, RNA, cDNA or complement thereof. For the purpose of generating probes, nucleic acids of about 10 to about 1000 (10 to 500; 20 to 100; 10 to 50) nucleotides or nucleoside long may particularly be selected. The ranges mentioned above includes any individual value found between and including such range. For example, the range "about 10 to about 1000" includes 10 and 1000 as well as any value between 10 and 1000. The same applies to any other range mentioned herein, such as "about 10 to about to about 100" which includes 10, 100 and any values found between such range, including, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95 etc.

The present invention therefore encompasses the following:

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 1743 position of a 23S rRNA gene and having a nucleotide or nucleoside at position 1743 other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 488 position of the SP_0373 coding sequence and comprising a nucleotide or nucleoside at position 488 other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 608 position of the SP_0373 coding sequence and comprising a nucleotide or nucleoside at position 608 other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 611 position of the SP_0373 coding sequence and comprising a nucleotide or nucleoside at position 611 other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising a deletion at position 267, 268, 269, 270, 271 , 272, 273 or combination thereof of the SP_0373 coding sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the -29 position of the SP_1114 gene and wherein the nucleotide or nucleoside at position -29 is other than the original nucleotide of the TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 1419 position of the SP_1114 coding sequence and comprising a nucleotide or nucleoside at the 1419 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence. A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the -46 position of SP_2075 gene and wherein the nucleotide or nucleoside at position -46 is other than the original nucleotide of the TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the -32 position of the SP_2075 gene and wherein the nucleotide or nucleoside at position -32 is other than the original nucleotide of the TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 593 position of the SP_2075 coding sequence and comprising a nucleotide or nucleoside at the 593 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 498 position of the SP_2075 coding sequence and comprising a nucleotide or nucleoside at the 498 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 1032 position of the SP_1837 coding sequence and comprising a nucleotide or nucleoside at the 1032 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 211 position of the SP_0211 coding sequence and comprising a nucleotide or nucleoside at the 211 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 108 position of the SP_0201 coding sequence and comprising a nucleotide or nucleoside at the 108 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the -52 position of the SP_0681 gene and wherein a nucleotide or nucleoside at the -52 position is other than the original nucleotide of the TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 50 position of the SP_1690 coding sequence and comprising a nucleotide or nucleoside at the 50 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 424 position of the SP_0959 coding sequence and comprising a nucleotide or nucleoside at the 424 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 277 position of the SP_1378 coding sequence and comprising a nucleotide or nucleoside at the 277 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 393 position of the SP_1471 coding sequence and comprising a nucleotide or nucleoside at the 393 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 191 position of the SP_1725 coding sequence and comprising a nucleotide or nucleoside at the 191 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence. A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the -216 position of the SP_1847 gene and comprising a nucleotide or nucleoside at the -216 position other than the original nucleotide of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the -3 position a sequence of the SP_0185 gene and comprising a nucleotide or nucleoside at the -3 position other than the original nucleotide of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 146 position of the SP_0918 coding sequence and comprising a nucleotide or nucleoside at the 146 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 430 position of the spr1536 coding sequence and comprising a nucleotide or nucleoside at the 430 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 1388 position of the SP_0611 coding sequence and comprising a nucleotide or nucleoside at the 1388 position other than the original nucleotide or nucleoside of the corresponding TIGR4 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 896 position of the Spr0380 coding sequence and comprising a nucleotide or nucleoside at the 896 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 234 position of the SprO525 coding sequence and comprising a nucleotide or nucleoside at the 234 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 406 position of the SprO947 coding sequence and comprising a nucleotide or nucleoside at the 406 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 347 position of the SPJ3981 coding sequence and comprising a nucleotide or nucleoside at the 347 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 1562 position of the SpM 130 coding sequence and comprising a nucleotide or nucleoside at the 1562 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 887 position of the SpM 417 coding sequence and comprising a nucleotide or nucleoside at the 887 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 806 position of the SpM 808 coding sequence and comprising a nucleotide or nucleoside at the 806 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising a deletion at position 208 of the SpM 824 coding sequence of the corresponding R6 sequence. A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 116 position of the Spr0102 coding sequence and comprising a nucleotide or nucleoside at the 116 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 113 position of the SprO421 coding sequence and comprising a nucleotide or nucleoside at the 113 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 514 position of the Spr1121 coding sequence and comprising a nucleotide or nucleoside at the 514 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 921 position of the Spr1195 coding sequence and comprising a nucleotide or nucleoside at the 921 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 337 position of the Spr1316 coding sequence and comprising a nucleotide or nucleoside at the 337 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising the 317 position of the Spr1465 coding sequence and comprising a nucleotide or nucleoside at the 317 position other than the original nucleotide or nucleoside of the corresponding R6 sequence.

A nucleic acid sequence or a complement thereof which may comprise at least 10 contiguous nucleotides or nucleosides and which may be capable of hybridizing to a nucleic acid sequence comprising a deletion at any or all positions located between 931 to 969 inclusively of SAV1444 or any or all positions located between 775 and 834 of SAV 1444 (i.e., any or all positions located between 775 and 969 inclusively).

The nucleic acid of the present invention may be either in the form of DNA, RNA or DNA/RNA chimera.

In an embodiment of the invention, the nucleic acid may be 100% identical or 100% complementary to the corresponding gene sequence.

A probe may usually have from about 10 to 1000 nucleotides or nucleosides long or any number therebetween. For example a probe may have from about 15 to 500, from about 15 to 30, from about 16 to 25, from about 18 to 21 nucleotides in length or any combinations therebetween.

Of course the incorporation of a label (reporter protein, chromophore, fluorophore, etc.) into the probes described herein may facilitate detection of the desired nucleic acid hybridized by such probe.

In another embodiment, the nucleic acid may be linked to a solid support. Where two or more distinct probes are attached to the same solid support, each of the probes may carry a specific address.

Some aspects of the invention relate to a protein of Table 3 having at least one mutation in comparison with the original sequence protein sequence found in accession No. NC_003098 (TIGR4) or NC_003028 (R6). In accordance with the present invention, the mutation may be for example an amino acid substitution or deletion. Further in accordance with the present invention the mutation may be associated with resistance to an antibiotic such as linezolid, chloramphenicol, florfenicol and tiamulin.

The invention thus encompasses any of the mutated proteins of Table 3 carrying one or more of the listed mutations. Other aspects of the invention also relate to a nucleic acid capable of encoding the mutated proteins of Table 3, a synthetic vector comprising such nucleic acid, amino acid sequence comprising a peptide derived from the mutated protein and antibodies capable of specific binding to the mutated proteins of Table 3. As indicated above, these mutated proteins may be particularly useful to generate tools for detecting these mutations and/or for detecting antibiotic- resistant bacteria. In another aspect, the invention provides a polypeptide which may be selected from the group consisting of:

a. A polypeptide which may comprise at least 8 amino acids of SP_0373 and an amino acid substitution at a position selected from the group consisting of Ser163, Cys 203, Gly204 and combination thereof; b. A polypeptide comprising at least 8 amino acids of an amino acid sequence 91 to 132 of SEQ ID NO:40; c. A polypeptide which may comprise at least 8 amino acids of SP_1114 and an amino acid substitution at His473, d. A polypeptide which may comprise at least 8 amino acids of SP_2075 and an amino acid substitution at a position selected from the group consisting of Leu166, Gly198 and combination thereof, e. A polypeptide which may comprise at least 8 amino acids of SAV1444 and an amino acid deletion in comparison with a non-mutated SAV1444 protein at a position selected from the group consisting of amino acid 311 , 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 , 322, 323 and combination thereof, and; f. A polypeptide which may comprise at least 8 amino acids of SAV1444 and comprising an amino acid deletion in comparison with a non- mutated SAV1444 protein at a position selected from the group consisting of amino acid 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278 and combination thereof.

In an embodiment the polypeptide may be selected, for instance, from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10.

In accordance with the present invention, the mutation in SP_0373 may be selected, for example, from the group consisting of Ser/lle163, Cys/Tyr203, Gly/Val204 and combination thereof.

In a more specific embodiment, the polypeptide may be selected, for example, from the group consisting of SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30.

In another exemplary embodiment, the polypeptide may comprise or consist in SEQ ID NO:40.

In accordance with the present invention, the mutation in SP_1114 is His/Gln473. In an embodiment of the invention, the polypeptide may comprise or consist in SEQ ID NO:32.

In accordance with the present invention, the mutation in SP_2075 may be selected, for instance, from the group consisting of Leu/Phe166, Gly/Asp198 and combination thereof.

In an exemplary embodiment of the invention, the polypeptide may comprise or consist in SEQ ID NO: 42.

In another exemplary embodiment of the invention, the polypeptide may comprise or consist in SEQ ID NO: 36, SEQ ID NO:50 or SEQ ID NO:52.

The present invention in an additional aspect, provides a nucleic acid which may be capable of encoding the (mutated) polypeptides described.

The present invention also relates to a vector comprising the nucleic acid described herein.

Additional aspects of the invention relates to an isolated cell or an isolated bacteria comprising the polypeptide, nucleic acid or vector of the present invention.

In a particular aspect, the present invention relates to a mutated SP_0373 protein or a mutated orthologue thereof, a nucleic acid capable of encoding the mutated SP_0373 or the mutated orthologue and a synthetic vector comprising such nucleic acid, amino acid sequences comprising peptides derived from the mutated SP_0373 protein or mutated orthologue and antibodies capable of specific binding to SP_0373 or to the mutated orthologue.

More particularly, the present invention relates in one aspect thereof to a mutated SP_0373 protein having an amino acid substitution or deletion in comparison with an original SP_0373 protein sequence which may be found for example at accession No. NP_344900, such mutation being associated with antibiotic resistance of a bacteria from the Streptococcus pneumoniae species.

The mutated SP_0373 protein may have a reduced ability to methylate RNA (e.g., ribosomal RNA, e.g., 23S rRNA). Exemplary embodiments of amino acid substitution in SP_0373 may be found, for example at a position selected from the group consisting of Ser163, Cys 203, Gly204 and combination thereof. In accordance with the present invention, the amino acid substitution or deletion in the mutated SP_0373 may confer resistance to an oxazolidinone ring containing antibiotic, such as for example, linezolid. It has been shown herein that such mutation in SP_0373 is associated with a linezolid MIC of higher than 8μg/ml.

Peptides are also useful in the preparation of a composition for generating antibodies which are directed to specific and desired epitope. As such, the present invention also relates to an amino acid sequence comprising at least 8 amino acids identical to a region of a mutated SP_0373 protein and having an amino acid substitution or deletion associated with antibiotic resistance.

One of the goal of the present invention is also to develop tests and kits which could be used in a clinical setting for the rapid identification of antibiotic resistant bacteria. Such tests and kits may contains antibody which are specific for a protein sought to be detected. In that respect, it would thus be useful to detect one or more of the newly described mutated proteins described herein.

As mentioned above, aspects of the invention concerns mutated SP_0373 orthologues. The Applicant has identified such mutated orthologue in another bacteria species. This mutated orthologue has also been associated with antibiotic resistance of Staphylococcus aureus.

Thus an aspect of the present invention relates to a mutated SAV1444 protein, a nucleic acid capable of encoding the mutated SAV1444, a synthetic vector comprising such nucleic acid, amino acid sequences derived from the mutated SAV1444 protein (e.g., peptides) and antibodies capable of specific binding to SAV1444.

The mutated SAV1444 protein may have an amino acid substitution or deletion in comparison with the original SAV1444 protein sequence which may be found at accession No. NP_371968, such mutation being associated with antibiotic resistance of a bacteria from the Staphylococcus aureus species.

The present invention also covers nucleic acids which may comprise one or more silent mutation or protein sequences which may comprise one or more conservative or non-conservative amino acid substitution or deletion, located outside the region which has been associated herein with antibiotic resistance. The invention also encompasses the methods, kits, antibodies, assays using those variants. Other aspects of the present invention relate to an antibody capable of specific binding to a mutated protein described herein.

An exemplary embodiment of such antibody includes, for example, an antibody which is capable of specific binding to the mutated SP_0373 protein. In some instances it would be useful to generate antibodies which do not bind with a significant manner or not at all, to a wild type SP_0373 protein and/or to a SP_0373 protein comprising one or more of the mutations described herein.

It may be useful to generate an antibody which could specifically bind to the mutated protein while not binding to a protein which does not comprise the mutation(s). Alternatively, it may also be useful to generate an antibody which could specifically bind to the mutated protein described herein and not to the original (wild type) protein. Therefore, in the case of the mutated protein of Table 3, it may be useful to generate specific antibodies with a peptide that comprises the mutated region of the desired protein.

For proteins of Table 3 that have been shown herein to be overexpressed, antibodies specific of such protein will allow evaluation of the relative amount of such protein (e.g., in comparison with a reference value or in comparison with the level of expression of such protein in a non-resistant strain) and thereby determination of the antibiotic susceptibility or resistance profile of the bacteria.

Additional aspects of the invention concerns other mutated SP_0373 orthologues which are present in other bacteria species and which may be associated with antibiotic resistance.

The Applicant has shown the presence of several other unique mutations in nucleic acids and proteins of Table 3. These mutations may be associated with antibiotic resistance and as such would also be useful to identify pathogens carrying them.

Methods of detection, antibodies, assays and kits

The present invention more particularly provides in an aspect, a method for determining an antibiotic resistance or susceptibility profile of a bacteria, which may comprise detecting :

a. A mutation at position 1743 of a 23S rRNA gene (with reference to E. coli numbering) or in a 23S rRNA gene product; b. A mutation in a SP_0373 gene, in a SPJ0373 orthologue or in a gene product thereof; c. A mutation in a SP_1114 gene, in a SP_1114 orthologue or in a gene product thereof , d. An increased expression of a SP_1114 gene product or of another gene product located on the same operon or locus; e. A mutation in a SP_2075 gene, in a SP_2075 orthologue or in a gene product thereof, and/or; f. An increased expression of a SP_2075 gene product or of another gene product located on the same operon or locus.

In accordance with the present invention, the gene product may be, for instance, a protein and the mutation an amino acid substitution or deletion.

Also in accordance with the present invention, the gene product may be, for instance, a RNA (e.g., mRNA, converted in cDNA and complement thereof) and the mutation a nucleotide or nucleoside change or deletion.

Further in accordance with the present invention, the mutation may be located in a coding or non-coding (5'-UTR, promoter, etc.) sequence of a gene and the mutation may be therefore, a nucleotide change or deletion.

In an exemplary embodiment, the mutation in the SP_0373 gene may be associated with a reduced ability of a SP_0373 protein to methylate RNA. The mutation may be for example, a deletion at a carboxy-terminal of the SP_0373 protein.

In another exemplary embodiment, the mutation may be located in the SP_1114 gene or in the SP_2075 gene and may allow for an increased expression of a corresponding gene product. Such mutation may be found, for example, in the promoter region.

It is to be understood herein that the gene product is either the SP_2075 protein, RNA, cDNA or complement thereof, the SP_1114 protein, RNA, cDNA or complement thereof, or another gene product located on the same operon or locus.

In an embodiment of the present invention, detection may be performed, for example, with an antibody capable of specific binding to a protein encoded by the SP_0373 gene or orthologue, by the SP_1114 gene or orthologue or by the SP_2075 gene or orthologue. Suitable antibodies are those which may be capable of specific binding to a region of the protein comprising the mutation, thereby allowing distinction between a mutated and a non-mutated protein.

In accordance with an embodiment, the method of the present invention may comprise comparing the level of expression of the SP_1114 protein or of another protein encoded by the same operon or locus with the level of expression of a corresponding gene product of a non-antibiotic resistant strains.

In accordance with another embodiment, the method of the present invention may comprise comparing the level of expression of the SP_2075 protein or of protein encoded by the same operon or locus with the level of expression of a corresponding gene product of a non-antibiotic resistant strains. An increased expression of the SP_2075 protein or of another protein encoded by the same operon may be indicative, for example, of resistance to ciprofloxacin.

Detection may also be performed with a nucleic acid sequence capable of hybridizing with the gene or gene product or with a complement thereof.

For example, suitable nucleic acid sequence may be those which are capable of hybridizing with a portion of a gene, gene product or complement thereof comprising the mutation and thereby allowing distinction between a mutated gene, gene product or complement thereof and a non-mutated gene, gene product or complement thereof.

In accordance with an embodiment, the method of the present invention may comprise comparing the level of expression of the SP_1114 RNA (e.g., cDNA, complement, etc.) or of another RNA encoded by the same operon or locus with a level of expression of a corresponding gene product of a non-antibiotic resistant strains.

In accordance with another embodiment, the method of the present invention may comprise comparing the level of expression of the SP_2075 RNA (e.g., cDNA, complement, etc.) or of another RNA encoded by the same operon or locus with a level of expression of a corresponding gene product of a non-antibiotic resistant strains.

In methods of the present invention, it may also be useful to also detect in conjunction with the mutations described herein, mutations that are already known to correlate with antibiotic resistance such as a mutation at position 2576 and/or 2503 of a 23S rRNA gene or gene product (with reference to E. CoIi numbering).

Detection of the mutation(s) described herein or the increased expression of certain genes described herein, may be indicative of resistance to an oxazolidinone ring containing antibiotic, including for example, linezolid, chloramphenicol, florfenicol and/or tiamulin.

Detection of the mutation(s) described herein or the increased expression of certain genes described herein, may also be indicative of a linezolid minimum inhibitory concentration of more than 8μg/ml.

Antibodies and antigen binding fragment that may specifically bind to a mutated protein or peptide described herein as well as nucleic acids encoding such antibodies or antigen binding fragment are also encompassed by the present invention.

As used herein the term "antibody" means a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, a deimmunized antibody, an antigen-binding fragment, an Fab fragment; an F(ab')₂ fragment, and Fv fragment; CDRs, or a single-chain antibody comprising an antigen- binding fragment (e.g., a single chain Fv).

Peptides may be made by any procedure known to one of skill in the art, for example, by using in vitro translation or chemical synthesis procedures or by introducing a suitable expression vector into cells. Short peptides which provide an antigenic epitope but which by themselves are too small to induce an immune response may be conjugated to a suitable carrier. Suitable carriers and methods of linkage are well known in the art. Suitable carriers are typically large macromolecules such as proteins, polysaccharides and polymeric amino acids. Examples include serum albumins, keyhole limpet hemocyanin, ovalbumin, polylysine and the like. One of skill in the art may use available procedures and coupling reagents to link the desired peptide epitope to such a carrier. For example, coupling reagents may be used to form disulfide linkages or thioether linkages from the carrier to the peptide of interest. If the peptide lacks a disulfide group, one may be provided by the addition of a cysteine residue. Alternatively, coupling may be accomplished by activation of carboxyl groups.

The minimum size of peptides useful for obtaining antigen specific antibodies may vary. The minimum size must be sufficient to provide an antigenic epitope that is specific to the protein or polypeptide. The maximum size is not critical unless it is desired to obtain antibodies to one particular epitope. Typically, antigenic peptides selected from the present proteins and polypeptides will range without limitation, from 5 to about 100 amino acids in length. More typically, however, such an antigenic peptide will be a maximum of about 50 amino acids in length, and preferably a maximum of about 30 amino acids. It is usually desirable to select a sequence of about 6, 7, 8, 9, 10, 11 , 12 or 15 amino acids, up to about 20 or 25 amino acids (and any number therebetween).

Amino acid sequences comprising useful epitopes may be identified in a number of ways. For example, preparing a series of short peptides that taken together span the entire protein sequence may be used to screen the entire protein sequence. One of skill in the art may routinely test a few large polypeptides for the presence of an epitope showing a desired reactivity and also test progressively smaller and overlapping fragments to identify a preferred epitope with the desired specificity and reactivity.

To obtain polyclonal antibodies, a selected animal may be immunized with a protein or polypeptide. Serum from the animal may be collected and treated according to known procedures. Polyclonal antibodies to the protein or polypeptide of interest may then be purified by affinity chromatography. Techniques for producing polyclonal antisera are well known in the art.

In order to generate antibodies which are capable of specific binding to the mutated protein of Table 3, a peptide comprising the mutated region may be fused to a carrrier (bovine serum albumin (BSA), keyhole limpet hemocyanin (KHL), etc.) and subsequently administered to an animal usually with an adjuvant. Alternatively, the whole mutated protein or a substantial portion (comprising the mutation) may be administered to the animal. In such instance it might be advisable in the subsequent immunization steps, to administer a peptide (e.g., fused with the carrier) comprising the region of interest in order to maximize the chance of obtaining an antibody that will bind to the mutated region of the protein. Several rounds of immunization may be necessary to obtain the desired quantity and/or specificity.

The specificity of the antibody may be determined by performing binding assays with the mutated and non-mutated epitope or protein and selecting antibody (antibody population) which are capable of specific binding to the mutated epitope or protein and not (or at least substantially less) to the non-mutated epitope or protein. Alternatively, a portion of a mutated protein which significantly differs from the wild type may be used to generate antibodies that are specific for the mutated protein. This may be found particularly useful for the SP_0373 protein variant (SEQ ID NO:40) encoded by SEQ ID NO:39 having its last 42 amino acids (aa 91-132) differing from the wild type SP_0373 protein. As such, it may be useful to immunize animals with a composition comprising at least 8 amino acids (and up to 42 amino acids) of this amino acid sequence (i.e., aa 91-132) in order to obtain antibodies that are specific of this mutated protein.

Therefore, an exemplary antibody of the present invention may be capable, for example, of detecting a mutated SP_0373 protein and may be capable of specific binding to an amino acid sequence comprising at least 8 (consecutive) amino acids of amino acids 91 to 132 of SEQ ID NO:40.

Another exemplary antibody of the present invention may be an antibody capable of detecting a mutated SP_0373 protein and more particularly capable of specific binding to an amino acid sequence comprising amino acid Ile163, Tyr203, Val204 or combination thereof.

Another exemplary antibody of the present invention may be an antibody which may be capable of detecting a mutated SP_1114 protein and may more particularly be capable of specific binding to an amino acid sequence comprising amino acid Gln473.

Yet another exemplary antibody of the present invention may be an antibody which may be capable of detecting a mutated SP_2075 protein and may more particularly be capable of specific binding to an amino acid sequence comprising amino acid Phe166, Asp198 or combination thereof.

Monoclonal antibodies (MAbs) may be made by several procedures available to one of skill in the art, for example, by fusing antibody producing cells with immortalized cells and thereby making a hybridoma. The general methodology for fusion of antibody producing B cells to an immortal cell line is well within the grasp of one skilled in the art. Another example is the generation of MAbs from mRNA extracted from bone marrow and spleen cells of immunized animals using combinatorial antibody library technology.

Chimeric antibodies may include antibodies where some or all non-human constant domains have been replaced with human counterparts. This approach has the advantage that the antigen-binding site remains unaffected. However, significant amounts of non-human sequences may be present where variable domains are derived entirely from non-human antibodies.

Humanized antibodies may be constructed in which regions of a non-human MAb are replaced by their human counterparts. A preferred chimeric antibody is one that has amino acid sequences that comprise one or more complementarity determining regions (CDRs) of a non-human Mab that binds to a polypeptide of interest or to a portion thereof, grafted to human framework (FW) regions. Methods for producing such antibodies are well known in the art. Amino acid residues corresponding to CDRs and FWs are known to one of average skill in the art.

Antibodies of the invention also include human antibodies that are antibodies consisting essentially of human sequences. Human antibodies may be obtained from phage display libraries wherein combinations of human heavy and light chain variable domains are displayed on the surface of filamentous phage. Combinations of variable domains are typically displayed on filamentous phage in the form of Fab's or scFvs. The library may be screened for phage bearing combinations of variable domains having desired antigen-binding characteristics. Preferred variable domain combinations are characterized by high affinity for a polypeptide, or a portion thereof. Preferred variable domain combinations may also be characterized by high specificity for a polypeptide and little cross-reactivity to other related antigens. By screening from very large repertoires of antibody fragments, (2-10 x 10¹⁰) a good diversity of high affinity Mabs may be isolated, with many expected to have sub-nanomolar affinities for a desired polypeptide.

Alternatively, human antibodies may be obtained from transgenic animals into which un-rearranged human Ig gene segments have been introduced and in which the endogenous mouse Ig genes have been inactivated. Preferred transgenic animals contain very large contiguous Ig gene fragments that are over 1 Mb in size but human polypeptide-specific Mabs of moderate affinity may be raised from transgenic animals containing smaller gene loci. Transgenic animals capable of expressing only human Ig genes may also be used to raise polyclonal antiserum comprising antibodies solely of human origin.

Antibodies of the invention may include those for which binding characteristics have been improved by direct mutation or by methods of affinity maturation. Affinity and specificity may be modified or improved by mutating CDRs and screening for antigen binding sites having the desired characteristics. CDRs may be mutated in a variety of ways. One way is to randomize individual residues or combinations of residues so that in a population of otherwise identical antigen binding sites, all twenty amino acids may be found at particular positions. Alternatively, mutations may be induced over a range of CDR residues by error prone PCR methods. Phage display vectors containing heavy and light chain variable region gene may be propagated in mutator strains of E. coli. These methods of mutagenesis are illustrative of the many methods known to one of skill in the art.

The antibody of the present invention may further comprise a detectable label (reporter molecule) attached thereto.

There is provided also methods of producing antibodies able to specifically bind to the mutated protein of the present invention, the method may comprise:

a) immunizing a mammal with a suitable amount of the mutated protein or a polypeptide fragment comprising at least 6 (e.g., 8, 10, 12 etc. and up to the total length of the desired protein of interest) consecutive amino acids of the mutated protein and comprising the mutated region;

b) collecting the serum from the mammal; and

c) isolating the polypeptide-specific antibodies from the serum of the mammal.

The antibodies obtained by the means described herein may be useful for detecting the mutated protein in specific tissues, body fluid, culture medium, etc.

The monoclonal or polyclonal antibody of the present invention may be used in several types of immunoassays, such as ELISA, immunoprecipitations, immuno- histochemistry, radioimmunoassays, FACS etc.

Kits suitable for immunodiagnostic assays and containing the appropriate antibody packaged are also encompassed by the present invention.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings. Material and Methods

Bacterial strains. Key strains are listed in Table 1. S. pneumoniae R6 whose sequence has been determined (Hoskins et al., 2001 ) and one clinical isolate, S. pneumoniae 1974 CCRI were selected stepwise for resistance to linezolid. Pneumococci were grown in brain heart infusion broth (BHI, Difco) supplemented with 0.5% yeast extract, in Todd- Hewitt broth (Difco) or in blood agar containing 5% defibrinated sheep's blood as described previously (Munoz et at., 1992). Cultures were incubated for 16 to 24 hours in a 5% CO₂ atmosphere at 37°C. In order to select resistant cells, we subcultured R6 or 1974 onto medium containing increasing concentration gradients of linezolid as described previously for other antibiotics (Martineau et al., 2000, Soualhine et al., 2005). Mutants were selected in a stepwise fashion by picking colonies growing at the highest antibiotic concentration. Six selection cycles were required to obtain two resistant mutants (M1 and M2) for each strains of S. pneumoniae.

Minimal inhibitory concentration (MIC) to linezolid was determined by E-test (AB Biodisk) and the macrodilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). Todd Hewitt broth containing twofold concentration increments of linezolid was inoculated with a 5*10⁵ cfu/ml bacterial suspension. The MIC was recorded as the lowest dilution showing no growth. All measurements were done in triplicate.

RNA isolation

Wild-type strain and mutants were grown in the medium in absence of antibiotic. Bacterial cells were harvested for RNA isolation during the logarithmic growth phase at an OD₆oo_nm of 0.35 to 0.4 Total RNA from sensitive and resistant derivatives was isolated using the Qiagen RNeasy Mini Kit (Qiagen, California) according to the manufacturer's instructions. The RNA was digested with DNase I (Ambion). The quality and integrity of the starting RNA material were confirmed by agarose gel electrophoresis and BioAnalyzer analysis (Agilent Technologies). Real-time quantitative RT-PCR assays were carried out in a BioRad Cycler using SYBR Green I (Molecular Probes). First, cDNA was made from 100ng of total RNA with the Superscript reverse transcriptase (Invitrogene) according to the manufacturer recommendations. The reactions were carried out with a kit from BioRad in a final volume of 25 μl containing specific primers, and iQ SYBR Green Supermix. All realtime RT-PCR data were normalized with the real time amplification of the 16S rRNA. The expression data are shown relative to the data for the wild-type strain which was grown in the absence of antibiotic.

DNA Constructs

The genetic constructs used in this study are described in Table 1. Gene inactivation was done by insertional duplication mutagenesis using the non-replicative plasmid pEVP3 (Claverys et al., 1995) (a kind gift of D. Morrison, University of Illinois at Chicago) of pFF6, a pEVP3 derivative where the chloramphenicol resistance marker was replaced by a kanamycin resistance gene marker (Fani, F., Feng, J. and Ouellette, M., manuscript in preparation). PCR fragments of the selected genes were derived from strain S. pneumoniae R6 and cloned into the multiple cloning site of pEVP3. For episomal expression, the entire coding sequences of genes were PCR amplified with primers containing BamHI and Xhol restriction sites and cloned in the S. pneumoniae-E. coli shuttle vector pDL289 (Buckley et al., 1995) (a kind gift of D. Cvitkovitch, University of Toronto). The inserted genes were sequenced and plasmid DNAs (2 μg) were introduced in S. pneumoniae in which competence was induced using a competence peptide as described (Lee et al., 1999). Transformants were selected with 5μg ml^"1 of chloramphenicol and 500 μg ml^"1 for kanamycin. Gene inactivation was monitored by PCR.

Whole genome sequencing.

Genomic DNA prepared from mid-log phase cultures were used to isolate genomic DNA from S. pneumoniae strains using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. Mutants R6M1 and R6M2 were resequenced using the whole Genome sequencing approach of Nimblegen (http://nimbleqen.com/). Briefly, DNA of each mutants was co-hybridized to the DNA of wild-type cells (which are differentially labeled with fluorescent markers) on DNA tiling microarrays. Regions hybridizing differently were sequenced by a further design of sequencing by hybridization arrays. The sequencing and its analysis were performed by Nimblegen. For the whole genome of the 1974 strain and 1974 M2 were sequenced by the massively parallel GS-FLX DNA sequencing technique. Sequences and analysis were performed by the Genome Quebec Innovation center (http://www.qenomequebecplatforms.com/mcqill/home/index.aspx?l=e). Results

Generation of mutants and cross resistance.

Two independent mutants of two independent S. pneumoniae strains (R6 and 1974) were selected step by step for linezolid resistance in vitro. Cells were selected for resistance to 1 , 2, 4, 8, 16 and 32 μg/ml of linezolid. The MICs of both wild-type isolates were 0.75 (1974) and 0.5 μg/ml (R6). More work was done with the most highly resistant isolates and the two S. pneumoniae R6 mutants are named R6M1 and R6M2 while the S. pneumoniae 1974 mutants are named 1974M1 and 1974M2.

These linezolid resistant bacteria were cross-resistant to chloramphenicol, florfenicol and tiamulin but not to several other antibiotics, several of which are also interacting with the translational machinery (Table 2).

Whole genome sequencing of Streptococcus pneumoniae.

The two independent R6 mutants R6M1 and R6M2 were resequenced using the comparative genome sequencing (CGS) developed by Nimblegen (http://nimblegen.com/). The CGS technique, using tiling DNA microarrays hybridizations rapidly surveys entire microbial genomes, identifying the locations of single nucleotide polymorphisms (SNPs), insertions, or deletions. The SNPs are further characterized by array sequencing. Excluding the 23S rRNA, the GCS found 9 mutations in the highly resistant mutant R6M1 and also 9 mutations in the resistant mutant R6M2 (Table 3). The 4 copies of the 23S rRNA were also mutated at position G2576T, the most frequent mutation found in linezolid resistant Gram-positive bacteria. Since the sequence of S. pneumoniae 1974 was not known it was not possible to use the Nimblegen GCS platform. We instead used the massively parallel GS-FLX DNA sequencing platform (http://www.genomequebecplatforms.com/mcαill/home/index.aspx?l=e) and obtained the sequence (2OX coverage) both of the wild-type 1974 sequence and of the 1974M2. Again, excluding the 23 S rRNA gene, 18 mutations were found in 1974M2 when compared to the wild-type sequence. The four 23S rRNA were mutated at position A2503G and 3 out of the 4 copies at mutation A1743T (Table 3; according to the E. coli numbering). All the mutations found in 1974 M2 and R6 M1 and R6M2 that were detected by whole genome (re)sequencing were confirmed by PCR amplification of the mutated region and by DNA sequencing. For 1974M1 , we PCR amplified all the genes that were found to be mutated in 1974M2 and sequenced them to find putative mutations. We found that the 23S rRNA was mutated at the critical G2576T position in three of the four 23 S rRNA copies (Table 3). Mutations in three additional genes (not necessarily at the same position as in M2) were common between 1974 M1 and 1974M2. One was in the coding region of the hypothetical protein SP_0373 and the two other mutations were upstream of two genes encoding two ABC proteins named SP_1114 and SP_2075 (Table 3). Note that all the SP_# are according to the TIGR4 sequence.

The sequencing of three independent linezolid resistant mutants has confirmed that mutations at position 2576 of the 23S rRNA is central to the linezolid resistance genotype. In the 1974 M2 mutant, however, another region of the 23S rRNA was mutated. Only one other gene was mutated in all four mutants and this corresponded to the hypothetical protein SP_0373 (Table 3). The gene was mutated at different position for each mutant, but all changed amino acids and in the R6M1 mutant we observed a gene deletion of 7-bp. Analysis of SP_0373 indicate that it is a protein of

385 amino acids and it contains putative domains and motifs of RNA methyltransferases.

Two other regions were mutated at least in two independent mutants. One consists in the ABC protein SP_2075 where sequences upstream of the start codon were mutated. This corresponds to position -32 in 1974M1 and at position -46 for both 1974M2 and R6M2 (Table 3). Finally, there was a mutation upstream of the ABC gene SP_1114 at position -29 for both 1974 M1 and M2 mutants. In 1974M2, there was an additional mutation in the coding sequence (Table 3). Otherwise when comparing the sequence of the 3 linezolid resistant strains there was no other common mutations detected. Since mutations in the 23S rRNA gene is now well established for linezolid resistance we did not further investigate its role in resistance except for showing that mutations at this locus appears early during the selection process and while not all copies are mutated at the first selection step, further selection leads to more 23S rRNA gene copies mutated (Table 4).

While we cannot exclude that the mutation specific to single mutants are important, we concentrated on the three mutations that were found in at least two independent mutants. We first investigated by DNA sequencing whether mutation in SP_0373 is an early or late event in the emergence of linezolid resistance. In contrast to the mutations in the 23S rRNA, mutation in SP_0373 was a relatively late event being detected in cells either resistant to 16 or 32 μg/ml of linezolid (Table 4). To assess the role of SP_0373 in linezolid resistance we used gene transfection and gene inactivation in S. pneumoniae. Inactivation or overexpression of SP_0373 in a wild- type R6 background did not change the susceptibility level of the cell to linezolid (Table 5). Since the gene was mutated in the various mutants, we also transformed plasmids containing the wild-type sequences of SP_0373 in the various mutants. Interestingly, transformations of pSP_0373 in 1974M1 and 1974M2 and R6M1 and R6M2 have reverted resistance by 2-3 fold (Table 5) while transformation with the empty vector did not change the susceptibility. Not only were cells more sensitive to linezolid, they were also more sensitive to chloramphenicol, florfenicol and tiamulin (Table 5). Attempts to inactivate SP_0373 in cells with intermediate level of resistance to linezolid have so far been unsuccessful (results not shown). We have the NARSA collection of VISA isolates (Drummelsmith et al., 2007), three of which had linezolid MIC of 8, 32 and 64 μg/ml (Table 1 ). S. aureus has an orthologue of SP_0373 (SAV1444 (Ace. No. NP_371968) if the nomenclature of S. aureus Mu50 is used (Ace. No. NC_002758: Gl: 57634611 ) and most interestingly one of the isolate NRS119 with a MIC of 64 μg/ml had a 39 bp deletion in the SP_0373 orthologue SAV1444. No mutations could be observed in the two other S. aureus investigated (Table 6). NSR271 and NSR127 were selected in vitro for increased linezolid resistance, and their SAV1444 gene was resequenced. A 60bp deletion was observed in the linezolid-selected NSR271 (Table 6).

The two other mutations that were found in more than one mutant were upstream of ABC protein coding regions. The location of these mutations is suggestive of promoter up mutation and the expression of the ABC genes SP_1114, SP_2705 was measured by real-time PCR (Fig. 1 ). Interestingly the expression of SP_1114 and another gene part of the same operon SP_1115 were increased more than 10-fold in the 1974M2 compared to wild-type cells (Fig. 1 ). Similarly, SP_2075 and the SP_2703, which is on the same operon and which is also an ABC protein, were also found to be overexpressed in 1974M2. Since a different mutation was observed between 1974M1 and M2 upstream of SP_2075 we also checked the expression of 2075 in 1974M1 by RT-qPCR and the expression of the gene was also found to be increased. Thus linezolid selection has led to point mutations in non coding sequences that can increase the expression of genes coding for ABC proteins. ABC proteins are well known efflux pumps that can contribute to resistance by extruding the drug outside the cell (Ouellette et al., 1994). To test the role of these ABC proteins in linezolid resistance we also used gene transformation. Wild type cells transfected with episomal copy of wild-type SP_1114 or wild-type or mutated copies of SP_2075 were not more resistant to linezolid (Table 5). However resistant cells in which SP_1114 was inactivated became more sensitive to linezolid and to other selected translational inhibitors (Table 5). Inactivation of 2075 in resistant cells did not lead to a phenotype, however.

As indicated above, the SP-2705 gene is part of an operon containing two ABC protein genes (SP_2703 and SP_2705). These ABC proteins, also known as PatA and PatB, have been linked to resistance to a number of drugs, including fluoroquinolones (Marrer et al., 2006). This prompted us to test and subsequently verify that both the 1974M1 and the 1974M2 mutants, with increased patA and patB expression were also cross-resistant to ciprofloxacin (Table 5B). We inactivated both genes in both the CCRI 1974 (wild type)_and 1974M2 background. Consistent with the known role of PatA and PatB in ciprofloxacin resistance (Marrer et al., 2006), inactivation of either of these ABC genes led to a 10-fold decrease in ciprofloxacin resistance back to wild-type levels (Table 5B).

Discussion

Linezolid is a novel antibiotic useful in the treatment of multiresistant Gram-positive pathogens. Considerable work has been done on defining resistance mechanisms to linezolid either selected in vitro or in one clinical isolate and the G2576T mutation appears as the most important one reviewed in (Meka & Gold, 2004, Woodford et al., 2007). In three of our four mutants of S. pneumoniae we also observed the same mutation (Table 3). In 1974M2, mutation in the 23S rRNA was also observed although in this case the position differed. The A2503G mutation is interesting as this is the site of methylation by the Cfr methyltransferase that lead to linezolid resistance (Toh et al., 2007). The mutation A1743T is described here for the first time. Mutation in the 23S rRNA is key for resistance, it is one of the first mutation that occurs and as described previously, the number of mutated 23S rRNA copies correlates broadly with the level of resistance (Table 4).

Whole genome sequencing was useful to detect novel mutations implicated in linezolid resistance. Mutations in SP_0373 are universally found in highly resistant S. pneumoniae and also in one highly resistant linezolid S. aureus clinical isolate (Table 6). Mutations are not at the same amino acid and thus the nucleotide sequence of the gene will need to be done to detect rapidly the mutation. This could be done either by DNA microarray resequencing or by rapid DNA sequencing technologies such as pyrosequencing. SP_0373 is directly involved in resistance as shown by transformation experiments. However, the cellular context matters. Indeed, while the gene does not seem to be essential as we can disrupt it in a wild-type background (Table 5), it does not seem to be involved in resistance in that context. However, when we expressed it in resistant cells, which all have a mutated SP_0373, then cells became more sensitive. SP_0373 show homologies with RNA methyltransferases and thus it is possible that this gene product methylate adenine residues in the 23S rRNA and that this is important for linezolid activity but only when other residues (e.g. G2576 or A2503) are mutated. In this scenario methylation of a adenine residue is required for linezolid activity and reduced activity of the methylase by mutations or deletion is correlated to resistance.

In addition to SP_0373, the expression of two ABC transporter genes was changed. This was mediated by what would appear to be a promoter up mutation. Similarly to SP_0373, SP_1114 was only linked to resistance when the gene was inactivated in the resistant cells. Since the gene was 10-fold more expressed, its inactivation may explain the gain in sensitivity. This is the first functional description of SP_1114 and it would appear to be involved not only in resistance to linezolid but also to chloramphenicol and florfenicol (Table 5). ABC proteins are involved in drug resistance by efflux mechanisms and it is possible that SP_1114 could reduce the accumulation of drugs. Until now we were not able to show a role for SP_2075 in resistance. Either the gene products is not involve in resistance or since it is co- expressed with SP_2073, also an ABC protein, it is possible that either SP_2073 or an heterodimer SP_2703- SP_2075 are involved in resistance. These two ABC proteins have been shown to be implicated in resistance to a number of drugs, but so far not to linezolid (Robertson et al., 2005, Marrer et al., 2006).

In conclusion, using whole genome sequencing we were able to find new mutations that are involved in linezolid resistance. The strategy of sequencing several mutants was also useful since it allowed to concentrate on recurrent mutations that were shown here to be involved in resistance. It is possible that other mutations are also important for the phenotype studied. For example in 1974M2 we noticed the mutation in the 5OS ribosomal protein L4 (Table 3) a known target of linezolid resistance (Wolter et al., 2005). It is possible also that some of these other mutations are only involved indirectly in resistance such as fitness compensatory mutations. This whole genome comparison is more easily applied to in vitro isogenic isolates but it can nonetheless allow the detection of mutants that are relevant in the clinical setting as illustrated with the mutation of SP_0373(SAV1444) in one S. aureus isolate. Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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Claims

We claim:

1. A method for determining an antibiotic resistance or susceptibility profile of a bacteria, the method comprising detecting : a. A mutation at position 1743 of a 23S rRNA gene or in a 23S rRNA gene product; b. A mutation in a SP_0373 gene, in a SP_0373 orthologue or in a gene product thereof; c. A mutation in a SP_1114 gene, in a SP_1114 orthologue or in a gene product thereof , d. An increased expression of a SP_1114 gene product or of another gene product located on the same operon or locus; e. A mutation in a SP_2075 gene, in a SP_2075 orthologue or in a gene product thereof, and/or; f. An increased expression of a SP_2075 gene product or of another gene product located on the same operon or locus.

2. The method of claim 1, wherein said gene product is a protein and wherein said mutation is an amino acid substitution or deletion.

3. The method of claim 1 , wherein said gene product is a RNA and wherein said mutation is a nucleotide or nucleoside change or deletion.

4. The method of claim 1, wherein said mutation is in a coding or non-coding sequence of the gene and wherein said mutation is a nucleotide change or deletion.

5. The method of claim 1 , wherein the mutation in the SP_0373 gene is associated with a reduced ability of a SP_0373 protein to methylate RNA.

6. The method of claim 2, wherein said deletion is at a carboxy-terminal of a

SP_0373 protein.

7. The method of claim 1 , wherein the mutation in the SP_1114 gene allows for an increased expression of a corresponding gene product.

8. The method of claim 1 , wherein the mutation in the SP_2075 gene allows for an increased expression of a corresponding gene product.

9. The method of claim 7 or 8, wherein the mutation is located in the promoter region.

10. The method of claim 9, wherein the corresponding gene product is a SP_2075 gene product, a SP_1114 gene product or another gene product located on the same operon or locus.

11. The method of any one of claims 1 to 10, wherein said bacteria is a Gram- positive bacteria.

12. The method of claim 11 , wherein said Gram-positive bacteria is Streptococcus pneumoniae.

13. The method of claim 11, wherein said Gram-positive bacteria is

Staphylococcus aureus.

14. The method of claim 13, wherein said Staphylococcus aureus is methicilline resistant.

15. The method of claim 13 or 14, wherein said Staphylococcus aureus is a VISA strain.

16. The method of any one of claims 13 to 15, wherein the SP_0373 orthologue is SAV1444.

17. The method of claim 11 , wherein said Gram-positive bacteria species is a bacteria of the Enterococcus genus.

18. The method of claim 17, wherein said bacteria is vancomycine resistant.

19. The method of any one of claims 1 to 18, wherein detection is performed with an antibody capable of specific binding to a protein encoded by the SP_0373 gene or orthologue, by the SP_1114 gene or orthologue or by the SP_2075 gene or orthologue.

20. The method of claim 19, wherein the antibody is capable of specific binding to a region of the protein comprising the mutation, thereby allowing distinction between a mutated and a non-mutated protein.

21. The method of claim 20, wherein the antibody is capable of detecting a mutated SP_0373 protein and is capable of specific binding to an amino acid sequence comprising at least 8 amino acids of amino acids 91 to 132 of SEQ ID NO:40.

22. The method of claim 20, wherein the antibody is capable of detecting a mutated SP_0373 protein and is capable of specific binding to an amino acid sequence comprising amino acid Ile163, Tyr203, Val204 or combination thereof.

23. The method of claim 20, wherein the antibody is capable of detecting a mutated SP_1114 protein and is capable of specific binding to an amino acid sequence comprising amino acid Gln473.

24. The method of claim 20, wherein the antibody is capable of detecting a mutated SP_2075 protein and is capable of specific binding to an amino acid sequence comprising amino acid Phe166, Asp198 or combination thereof.

25. The method of claim 19, further comprising comparing the level of expression of the SP_1114 gene product or of another gene product located on the same operon or locus with a level of expression of a corresponding gene product of a non-antibiotic resistant strains.

26. The method of claim 19, further comprising comparing the level of expression of the SP_2075 gene product or of another gene product located on the same operon or locus with a level of expression of a corresponding gene product of a non-antibiotic resistant strains.

27. The method of any one of claims 1 to 18, wherein detection is performed with a nucleic acid sequence capable of hybridizing with the gene or gene product or with a complement thereof.

28. The method of claim 27, wherein said nucleic acid sequence is capable of hybridizing with a portion of a gene, gene product or complement thereof comprising the mutation thereby allowing distinction between a mutated gene, gene product or complement thereof and a non-mutated gene, gene product or complement thereof.

29. The method of claim 27 or 28, further comprising comparing the level of expression of the SP_1114 gene product or of another gene product located on the same operon or locus with a level of expression of a corresponding gene product of a non-antibiotic r^istant strains.

30. The method of claim 27 or 28, further comprising comparing the level of expression of the SP_2075 gene product or of another gene product located on the same operon or locus with a level of expression of a corresponding gene product of a non-antibiotic resistant strains.

31. The method of any one of claims 1 to 30, further comprising detecting a mutation at position 2576 and/or 2503 of a 23S rRNA gene or gene product.

32. The method of any one of claims 1 to 31 , wherein detection of said mutation or increased expression is indicative of resistance to an oxazolidinone ring containing antibiotic.

33. The method of any one of claims 1 to 31 , wherein detection of said mutation or increased expression is indicative of resistance to linezolid, chloramphenicol, florfenicol and/or tiamulin.

34. The method of any one of claims 1 to 31 , wherein detection of said mutation or increased expression is indicative of a linezolid minimum inhibitory concentration of more than 8μg/ml.

35. A polypeptide selected from the group consisting of: a. A polypeptide comprising at least 8 amino acids of SP_0373 and comprising an amino acid substitution at a position selected from the group consisting of Ser163, Cys 203, Gly204 and combination thereof; b. A polypeptide comprising at least 8 amino acids of an amino acid sequence 91 to 132 of SEQ ID NO:40; c. A polypeptide comprising at least 8 amino acids of SP_1114 and comprising an amino acid substitution at His473, d. A polypeptide comprising at least 8 amino acids of SP_2075 and comprising an amino acid substitution at a position selected from the group consisting of Leu166, Gly198 and combination thereof, and; e. A polypeptide comprising at least 8 amino acids of SAV1444 and comprising an amino acid deletion in comparison with a non-mutated SAV1444 protein at a position selected from the group consisting of amino acid 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,

270, 271 , 272, 273, 274, 275, 276, 277, 278, 311 , 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 , 322, 323 and combination thereof.

36. The polypeptide of claim 35, wherein said polypeptide is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10.

37. The polypeptide of claim 35, wherein the mutation in SP_0373 is selected from the group consisting of Ser/lle163, Cys/Tyr203, Gly/Val204 and combination thereof.

38. The polypeptide of claim 37, wherein said polypeptide is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30.

39. The polypeptide of claim 35, wherein said polypeptide comprises or consists in SEQ ID NO:40.

40. The polypeptide of claim 35, wherein the mutation in SP_1114 is His/Gln473.

41. The polypeptide of claim 40, wherein said polypeptide comprises or consists in SEQ ID NO:32.

42. The polypeptide of claim 35, wherein the mutation in SP_2075 is selected from the group consisting of Leu/Phe166, Gly/Asp198 and combination thereof.

43. The polypeptide of claim 42, wherein said polypeptide comprises or consists in SEQ ID NO: 42.

44. The polypeptide of claim 35, wherein said polypeptide comprises or consists in SEQ ID NO: 36.

45. A nucleic acid capable of encoding the polypeptide of any one of claims 35 to 44.

46. A vector comprising the nucleic acid of claim 45.

47. An isolated cell comprising the polypeptide of any one of claims 35 to 44, the nucleic acid of claim 45 or the vector of claim 46.

48. An isolated bacteria comprising the polypeptide of any one of claims 35 to 44, the nucleic acid of claim 45 or the vector of claim 46.

9. A kit comprising a reagent capable of specific detection of the polypeptide of claim 35 or capable of detecting overexpression of a SP_2075 gene product, a SP_1114 gene product or another gene product located on the same operon or locus.