Probes for the detection of nucleotide sequences implicated in the expression of resistance to glycopeptides, in particular in gram-positive bacteria

The invention relates to isolated polynucleotides and probes which are optionally labelled and which hybridize with polynucleotides encoding polypeptides implicated in the bacterial resistance to vancomycin, teicoplanin and to both vancomycin and teicoplanin. The invention also relates to the utilization of these polynucleotide probes for the diagnosis of resistance to the glycopeptides.

The invention relates to the polypeptides associated with the expression of 
resistance to antibiotics of the glycopeptide family, in particular in 
Gram-positive bacteria, in particular in the family of the Gram-positive 
cocci. The invention also relates to a nucleotide sequence coding for 
these polypeptides. It also relates to the use of these polypeptides and 
their nucleotide sequence as agents for the in vitro detection of 
resistance to glycopeptides. Among the Gram-positive cocci, the invention 
relates most particularly to the enterococci, the streptococci and the 
staphylococci which are of particular importance for the implementation of 
the invention. 
The glycopeptides, which include vancomycin and teicoplanin are antibiotics 
which inhibit the synthesis of the bacterial cell wall. These antibiotics 
are very much used for the treatment of severe infections due to 
Gram-positive cocci (enterococci, streptococci and staphylococci), in 
particular in light of allergy and resistance to the penicillins. In spite 
of long clinical usage of vancomycin, this antibiotic has remained active 
towards almost all of the strains up to 1986, the date at which the first 
resistant strains were isolated. Since then, resistance to the 
glycopeptides has been detected by many microbiologists in Europe and in 
the United States, in particular in strains isolated from immunodepressive 
patients, making necessary a systematic evaluation of the sensitivity of 
the microbes in hospital environments. 
The activity of the glycopeptides depends on the formation of a complex 
between the antibiotic and the precursors of the peptidoglycan, more than 
on the direct interaction with enzymes of cell wall metabolism. In 
particular, it has been observed that the glycopeptides bind to the 
terminal D-alanyl-D-alanine residues (D-ala-D-ala) of the precursors of 
the peptidoglycan. 
The recent emergence of resistance to the glycopeptides, in particular in 
the enterococci, has led to certain results being obtained with regard to 
knowledge of the factors conferring this resistance. 
For example it has been observed in a particular strain of enterococci, 
Enterococcus faecium BM4147, that the determinant of resistance to the 
glycopeptides is localized on a plasmid of 34 kb, the plasmid pIP816. This 
determinant has been cloned in E.coli (Brisson Noel et al., 1990, 
Antimicrob Agents Chemother 34, 924-927). 
According to the results hitherto obtained, the resistance to the 
glycopeptides is associated with the production of a protein of molecular 
weight of about 40 kDa, the synthesis of this protein being induced by 
sub-inhibitory concentrations of certain glycopeptides such as vancomycin. 
By carrying out a more detailed study of the resistance of certain strains 
of Gram-positive cocci towards glycopeptides, in particular vancomycin or 
teicoplanin, the inventors have observed that this resistance might be 
linked to the expression of several proteins or polypeptides encoded in 
sequences usually borne by plasmids in the resistant strains. The recent 
results obtained by the inventors also make it possible to distinguish the 
genes coding for two phenotypes of resistance, on the one hand strains 
highly resistant to the glycopeptides, and, on the other, strains with a 
low level of resistance. 
By strain with a high level of resistance is meant a strain of bacteria, in 
particular a strain of Gram-positive cocci, for which the minimal 
inhibitory concentrations (MIC) of vancomycin and teichoplaninare higher 
than 32 and 8 .mu.g/ml, respectively. The MIC of vancomycin towards 
strains with low-level resistance are included between 16 and 32 .mu.g/ml. 
These strains are apparently sensitive to teicoplanin. 
The inventors have isolated and purified, among the components necessary 
for the expression of the resistance to the glycopeptides, a particular 
protein designated VANA or VanA which exhibits a certain homology with 
D-alanine-D-alanine ligases. VanA is nonetheless functionally distinct 
from the ligases. 
In principle, a gene sequence will be designated by "van . . . " and an 
amino acid sequence by "Van . . . " 
The invention relates to polypeptides or proteins implicated in the 
expression of resistance to antibiotics of the glycopeptide family and, in 
particular, to vancomycin and/or teicoplanin as well as to the nucleotide 
sequences coding for such complexes. 
The invention also relates to nucleotide probes which can be used for the 
detection of resistance to the glycopeptides, in particular by means of 
the polymerase chain reaction (PCR), or by tests involving antibodies. 
The invention relates to a composition of polypeptides, characterized in 
that it contains at least one protein or part of a protein selected from 
the amino acid sequences identified in the list of the sequences as SEQ ID 
NO 2 (VanH), SEQ ID NO 4 (VanA), SEQ ID NO 6 (VanX) or SEQ ID NO 8 (VanC), 
or any protein or part of a protein recognized by the antibodies directed 
against VanH, VanA, VanX or VanC, or any protein or part of a protein 
encoded in a sequence hybridizing with one of the nucleotide sequences 
identified in the list of the sequences as SEQ ID NO 1, SEQ ID NO 3, SEQ 
ID NO 5 or SEQ ID NO 7 or with one of the following sequences V1 (SEQ ID 
NO:9) or V2 (SEQ ID NO:10) under stringent or only slightly stringent 
conditions: 
##STR1## 
A first particular composition according to the invention implicated in the 
expression of the resistance to the glycopeptides is characterized in that 
it comprises at least 3 proteins or any part of one or more of these 
proteins necessary to confer to Gram-positive bacteria the resistance to 
antibiotics of the glycopeptide family, in particular to vancomycin and/or 
teicoplanin or to promote this resistance, in particular in strains of the 
family of the Gram-positive cocci, these proteins or parts of proteins 
being 
a) recognized by antibodies directed against one of the sequences 
identified in the list of the sequences as SEQ ID NO 2, SEQ ID NO 4, SEQ 
ID NO 6. 
b) or encoded in genes containing a sequence identified as SEQ ID NO 1, SEQ 
ID NO 3 or SEQ ID NO 5 or hybridizing with one of these sequences or its 
complementary sequence or with the sequences V1 (SEQ ID NO:9) or V2 (SEQ 
ID NO:10), under stringent or only slightly stringent conditions. 
These sequences are also designated, respectively, by ORF3, ORF1 containing 
the gene VanH, vanA (or ORF2); they characterize the proteins responsible 
for resistance as obtained from the strain Enterococcus faecium BM4147 
described by leclerq et al (N. Engl. J. Med. 319:157-161). 
Another protein, VanC (SEQ ID NO:8), related to the D-Ala-D-Ala ligases but 
of different specificity has been characterized in Enterococcus gallinarum 
BM4173; the vanC gene (SEQ ID NO:7) possesses domains having sufficient 
homology with the vanA gene for probes corresponding to defined regions of 
vanA to make possible its detection. 
E.gallinarum is a constitutive isolate resistant to low levels of 
vancomycin (Dutka-Malen et al., Antimicrob. Agents Chemother 34 (1990b) 
1875-1879). 
By the expression "polypeptides" is meant any sequence of amino acids 
constituting proteins or being of a size less than that of a protein. 
The stringent conditions mentioned above are defined according to the usual 
conditions pertaining to the hybridization of nucleotide sequences. As an 
example, in the case of the sequences which hybridize with the sequence of 
the vanA gene (SEQ ID NO 1) it will be possible to apply the following 
conditions: 
for hybridization under conditions of high stringency: 
a reaction temperature of 65.degree. C. overnight in a solution containing 
0.1% SDS, 0.7% skimmed milk powder, 6.times.SSC (1.times.SSC=0.15M NaCl 
and 0.015M sodium citrate at pH=7.0) 
washes at 65.degree. C. in 2.times.SSC-0.1% SDS; 
for hybridization under slightly stringent conditions, the hybridization 
temperature is 60.degree. C. overnight and the temperature of the washings 
is 45.degree. C. 
The expression of resistance to glycopeptides may be expressed by the 
persistence of an infection due to microbes usually sensitive to the 
glycopeptides. 
A polypeptide or a protein is necessary for the expression of resistance to 
the glycopeptides, if its absence makes the strain which contains this 
polypeptide or this protein more sensitive to the glycopeptides and if 
this polypeptide or protein is not present in sensitive strains. 
Different levels of resistance to the glycopeptides exist in the strains of 
Gram-positive cocci, in particular. 
According to a preferred embodiment of the invention, the polypeptides 
included in the composition defined above correspond to the combination of 
the proteins identified in the list of the sequences as SEQ ID NO 2 
(VanH), SEQ ID NO 4 (VanA), SEQ ID NO 6 (VanX). 
The inventors have thus observed that the expression of resistance to the 
glycopeptides in Gram-positive bacteria requires the expression of at 
least three proteins or of polypeptides derived from these proteins. 
According to a first particular embodiment of the invention, the 
polypeptides of the composition are also characterized in that the amino 
acid sequences necessary for the expression of resistance to antibiotics 
of the glycopeptide family are under the control of regulatory elements, 
in particular of the proteins corresponding to the sequences designated by 
SEQ ID NO 12 and SEQ ID NO 14 in the list of the sequences, and which 
correspond to a regulatory sequence R and to a sensor sequence S, 
respectively. 
VanS and VanR constitute a two-component regulatory system, VanR being an 
activator of transcription and VanS stimulating the transcription 
dependent on VanR. VanS is capable of modulating the level of 
phosphorylation of VanR in response to the vancomycin present in the 
external medium and is thus involved in the control of the transcription 
of the genes for resistance to vancomycin. 
These regulatory sequences are in particular capable of increasing the 
level of resistance, to the extent to which they promote the expression of 
the proteins responsible for resistance comprised in the polypeptides of 
the invention. 
According to another advantageous embodiment of the invention, the 
polypeptides of the above composition are encoded in the sequence SEQ ID 
NO 15 identified in the list of the sequences, which represents the 
sequence coding for the 5 proteins previously described. 
Another sequence according to the invention is designated by SEQ ID NO 16 
which contains the sequence SEQ ID NO 15 as well as a sequence upstream 
from SEQ ID NO 15 coding for a transposase (encoded in the (-) strand of 
the sequence, and a sequence downstream from SEQ ID NO 15 corresponding to 
the genes vanY and vanZ and at each end reverse repeated sequences of 38 
bp. SEQ ID NO 16 constitutes a transposon, the genes of which are 
implicated at different levels in the establishment of resistance to the 
glycopeptides. 
The invention also relates to the purified proteins belonging to the 
composition and to the polypeptides described previously. In particular, 
the invention relates to the purified protein VanA, characterized in that 
it corresponds to the amino acid sequence SEQ ID NO 4 in the list of the 
sequences or a protein VanC, encoded in a gene capable of hybridizing with 
the vanA gene. 
The protein VanA contains 343 amino acids and has a calculated molecular 
mass of 37400 Da. The protein VanC contains 343 amino acids and has a 
calculated molecular mass of 37504 Da. 
Other interesting proteins in the framework of the invention correspond to 
the sequences identified as SEQ ID NO 2 (VanH), SEQ ID NO 6 (VanX), SEQ ID 
NO 12 (VanR), SEQ ID NO 14 (VanS) in the list of the sequences. 
The sequence identified by the abbreviation SEQ ID NO 2 contains the 
protein VanH encoded in the gene vanH, this protein contains 322 amino 
acids and begins with a methionine. This protein is an enzyme implicated 
in the synthesis of the peptidoglycan and has a molecular mass of 35,754 
kDA. VanH exhibits some similarities to dehydrogenases which catalyze the 
NAD.sup.+ -dependent oxidation of 2-hydroxy-carboxylic acids to form the 
corresponding 2-keto-carboxylic acids. In fact, the VanH protein might use 
NADP.sup.+ rather than NAD.sup.+. The VanH protein also contains several 
residues of reactive sites which probably participate directly in the 
binding of the substrate and in catalysis. VanH might be implicated in the 
synthesis of a substrate of the ligase VanA. This substrate of VanA might 
be a D-.alpha.-hydroxy-carboxylic acid, which might be condensed by VanA 
with D-alanine in the place of a D-amino acid, which might affect the 
binding of the precursor of the peptidoglycan with vancomycin, as a result 
of the loss of a hydrogen bond because one of the hydrogen bonds formed 
between vancomycin and N-acetyl-D-Ala-D-Ala occurs with the NH group of 
the terminal D-alanine residue. Let it be recalled that "Ala" is the 
abbreviation for "alanine". 
The inventors have been able to detect some interactions between the 
proteins VanA and VanH and have in particular been able to describe the 
following: the nature of the VanA protein (D-alanine: D-alanine ligase 
with reduced specificity for its substrate) which has made possible 
resistance to glycopeptides, implies the biosynthesis by VanA of a novel 
compound different from D-Ala-D-Ala, a peptide which may be incorporated 
into the peptidoglycans but which is not recognized by vancomycin. In 
particular, the observation of similarities between the product of the 
vanH gene and the D-specific .alpha.-keto-acid reductases has made it 
possible to determine that this compound cannot be a D-amino acid but is a 
D hydroxy acid, which when it is bound to D-alanine by VanH, can generate 
the novel depsipeptide precursor of the peptidoglycan. 
The invention also relates to any combination of these different proteins 
in a resistance complex, as well as to hybrid proteins comprising one or 
several of the above proteins, or part of these proteins, in combination 
with a defined amino acid sequence. 
Also included in the framework of the invention are nucleotide sequences 
coding for one of the amino acid sequences described above. 
A particular sequence is the nucleotide sequence of about 7.3 kb, 
corresponding to the HindIII-EcoRI restriction fragment, such as that 
obtained starting from the plasmid pIP816 described in the publication of 
Leclerq et al--1988, cited above. 
This sequence of 7.3 kb comprises the nucleotide sequence coding for the 3 
resistance proteins and the 2 regulatory proteins referred to above. This 
coding sequence is included in an internal BglII-XbaI fragment. It also 
comprises a part of the sequences coding for the transposase and the 
resolvase. 
The invention also relates to any nucleotide fragment comprising the 
above-mentioned restriction fragment as well as any part of the 
HindIII-EcoRI fragment, in particular the EcoRI-XbaI fragment of about 3.4 
kb coding for the 3 resistance proteins or the EcoRV-SacII fragment of 
about 1.7 kb coding for VanA or also HindIII-EcoRI fragment of about 3.3 
kb coding for the 2 regulatory proteins VanR and VanS. 
Another definition of a nucleotide sequence of the invention corresponds to 
a nucleotide fragment containing the following restriction sites in the 
following order, such as obtained starting from pIP816 mentioned above: 
HindIII, BglII, BglII, EcoRI, BamHI, XbaI, EcoRI. 
Another nucleotide sequence according to the invention is characterized in 
that it corresponds to a sequence selected from the sequences identified 
as SEQ ID NO 15, SEQ ID NO 17, or SEQ ID NO 16, or in that it includes 
this sequence or any part of this sequence, or also any sequence or part 
of the sequence of the complementary DNA or any sequence of RNA 
corresponding to one of these DNAs, capable, 
either of constituting a hybridization probe for the detection of 
resistance to antibiotics of the glycopeptide family, in particular to 
vancomycin and/or teicoplanin in particular in strains of the family of 
the Gram-positive cocci, 
or of coding for a sequence necessary or associated with the expression of 
resistance to antibiotics of the glycopeptide family, in particular to 
vancomycin and/or teicoplanin., in particular in strains of the family of 
the Gram-positive cocci. 
The sequence SEQ ID NO 17 codes for the 3 resistance proteins VanH, VanA 
and VanX. 
The sequence SEQ ID NO 16 includes a transposon shown in FIG. 7a; this 
transposon contains the genes necessary for the expression of resistance 
to the glycopeptides as well as the genes associated with this resistance 
implicated, for example, in the regulation of the expression of the genes 
necessary to produce the resistance phenotype or implicated in the amount 
of resistance polypeptide produced. 
A specific sequence corresponding to the above definition is one of the 
following sequences: 
##STR2## 
V1 and V2 make possible the constitution of probes, if necessary, in 
combination with other nucleotides, depending on the degree of specificity 
desired in order to detect vanA and vanC and may also be used as primers 
in polymerase chain reactions. 
Other preferred nucleotide sequences are the sequences SEQ ID NO 1, SEQ ID 
NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 18 (transposase), SEQ ID NO 20 
(resolvase), SEQ ID NO 22 (vanY), SEQ ID NO 24 (vanZ), SEQ ID NO 11 
(vanR), SEQ ID NO 13 (vanS) or a variant of one of these sequences 
provided that it codes for a protein having immunological and/or 
functional properties similar to those of the proteins encoded in the 
sequences SEQ ID NO 1 (vanA), SEQ ID NO 3 (vanH), SEQ ID NO 10 (vanX), or 
SEQ ID NO 21 (vanC), SEQ ID NO 18 (transposase), SEQ ID NO 20 (resolvase), 
SEQ ID NO 22 (vanY), SEQ ID NO 24 (vanZ), SEQ ID NO 11 (vanR), SEQ ID NO 
13 (vanS) or in that it makes possible the detection of strains resistant 
to antibiotics of the glycopeptide family. 
Variants include all of the fragments of the sequences having the following 
properties. 
These sequences code for the resistance proteins VanH, VanA and VanX. 
The nucleotide sequence designated by SEQ ID NO 1corresponds to a DNA 
fragment of 1029 bp situated between the ATG codon at position 377 and the 
TGA codon at position 1406 on the plasmid pAT214 (FIG. 6). 
The invention also relates to a nucleotide sequence coding for the sequence 
SEQ ID NO 15 corresponding to the sequence coding for the 5 proteins (2 
regulatory proteins and 3 resistance proteins), and also comprising the 
flanking sequences associated with these coding sequences, or comprising 
this sequence. 
Also included in the framework of the invention is a sequence modified with 
respect to SEQ ID NO 15, characterized in that it lacks the flanking 
sequences. These flanking sequences are the sequences shown in the 
following pages and defined as follows: 
sequence upstream from the sequence coding for R: between the bases 1 and 
1476 of the sequence shown in FIG. 5, 
sequence between the sequence coding for the sensor protein S and ORF1: 
between the bases 3347 and 3500 of the sequence shown in FIG. 5, 
sequence downstream from the sequence coding for ORF3: between the bases 
6168 and 7227 of the sequence shown in FIG. 5. 
The sequence designated by SEQ ID NO 15 is also characterized by the 
fragment bearing the restriction sites in the following order: 
BglIII-EcoRI-BamHI-EcoRI 
The location of the regulatory proteins and the resistance proteins is 
shown in FIG. 3. 
The inventors have identified upstream and downstream from the genes vanR, 
vanS, vanH, vanA and vanX, which are necessary for or associated with the 
expression of resistance to glycopeptides at a given level, genes coding 
for a transposase and a resolvase (upstream from the group previously 
mentioned) and genes vanY and vanZ, downstream from this group. The genes 
for the transposase and resolvase might be implicated in transposition 
functions and the vanY gene coding for a D,D-carboxy peptidase might be 
implicated in the metabolism of the peptidoglycan, and might contribute to 
resistance to the glycopeptides in E. faecium BM4147 even though vanR, 
vanS, vanH, vanA and vanX borne by a plasmid in a high number of copies, 
alone confer a high level of resistance. 
Let it be noted that the sequence coding for the transposase (SEQ ID NO:18) 
is located on the (-) strand of the sequence ID NO 16 which codes for 
vanR, vanS, vanH, vanA, vanX, vanY, vanZ and the resolvase. 
The invention relates not only to the DNA sequences identified in the list 
of the sequences but also to the complementary DNA sequences and the 
corresponding RNA sequences. The invention concerns in addition sequences 
which are equivalent to the former, either in terms of expression of 
proteins, polypeptides or their fragments described above, or in terms of 
the capacity to detect, for example by chain polymerization procedures, 
strains of Gram-positive bacteria exhibiting resistance to antibiotics of 
the glycopeptide family such as vancomycin or teicoplanin. 
Recombinant sequences characterized in that they comprise one of the above 
nucleotide sequences also form part of the invention. 
The invention also relates to a recombinant vector characterized in that it 
includes one of the above nucleotide sequences at a site inessential for 
its replication, under the control of regulatory elements likely to be 
implemented in the expression of the resistance to antibiotics of the 
glycopeptide family, in particular to vancomycin or teicoplanin in a 
defined host. 
Particularly advantageous recombinant vectors for the implementation of the 
invention are the following vectors: pAT214 containing the EcoRV-SacII 
fragment of 1761 bp containing a nucleotide sequence coding for the VanA 
protein; in these vectors the sequences of the invention are 
advantageously placed under the control of promoters such as the lac 
promoter. 
The invention also relates to a recombinant cell host containing a 
nucleotide sequence such as that previously described or a vector such as 
that described above under conditions which make possible the expression 
of resistance to antibiotics of the glycopeptide family, in particular 
resistance to vancomycin and/or this host being for example selected from 
the bacteria, in particular the Gram-positive cocci. 
In certain applications it is also possible to use yeasts, fungi, insect or 
mammalian cells. 
The invention also relates to a nucleotide probe characterized in that it 
is capable of hybridizing with a sequence previously described, this probe 
being labelled if necessary. These probes may or may not be specific for 
the proteins of resistance to glycopeptides. 
Labels which can be used for the requirements of the invention are the 
known radioactive labels as well as other labels such as enzymatic labels 
or chemoluminescent labels. 
Probes thus labelled may be used in hybridization tests in order to detect 
resistance to glycopeptides in Gram-positive bacteria. In this case, 
conditions of low stringency will be used. 
Nucleotide probes according to the invention may be characterized in that 
they are specific in Gram-positive bacteria for the sequences coding for a 
resistance protein to the glycopeptides, in particular to vancomycin 
and/or teicoplanin these probes being in addition universal among these 
sequences. 
By these specific probes is meant any oligonucleotide hybridizing with a 
nucleotide sequence coding for one of the proteins according to the 
invention, such as described in the preceding pages, and not exhibiting a 
cross hybridization reaction or amplification reaction (PCR) with 
sequences present in all of the sensitive strains. 
The universal character of the oligonucleotide which can be used in PCR is 
defined by their capacity to promote specifically the amplification of a 
nucleotide sequence implicated in resistance in any one strain of 
Gram-positive bacteria, resistant to the antibiotics of the glycopeptide 
family. 
The size of the nucleotide probes according to the invention may vary 
depending on the use desired. For the oligonucleotides which are used in 
PCR, recourse will be had to fragments of a length which is usual in this 
procedure. In order to construct probes, it is possible to take any part 
of the sequences of the invention, for example probe fragments of 200 
nucleotides. 
According to a particular embodiment of the invention, a nucleotide probe 
is selected for its specificity towards a nucleotide sequence coding for a 
protein necessary for the expression in Gram-positive bacteria of a high 
level of resistance to antibiotics of the glycopeptide family, in 
particular to vancomycin and teicoplanin. 
As examples, useful probes may be selected from the intragenic part of the 
vanA gene. 
Other useful probes for carrying out the invention are characterized by 
their universal character, according to the preceding definition, but are 
not specific for the resistance genes. They may also be used as primers in 
PCR, and are for example: 
##STR3## 
V1 and V2 hybridize with vanA and vanC and are capable of leading to the 
detection of proteins associated with resistance to glycopeptides in other 
micro-organisms. 
Other particular probes of the invention have the specific character of a 
nucleotide sequence coding for a protein necessary for the expression in 
Gram-positive bacteria of a low level of resistance to antibiotics of the 
glycopeptide family, in particular to vancomycin in Gram-positive 
bacteria. 
It should also be mentioned that oligonucleotide probes which might be 
derived from the sequence of the vanA gene coding for the VanA protein may 
be used indiscriminantly to detect high-level or low-level resistance. 
In a particularly preferred manner, a probe of the invention is 
characterized in that it hybridizes with a chromosomal or non-chromosomal 
nucleotide sequence of a Gram-positive strain resistant to glycopeptides, 
in particular to vancomycin and/or teicoplanin, in particular in that it 
hybridizes with a chromosomal or non-chromosomal nucleotide sequence of a 
strain of Gram-positive cocci, for example an enterococcal strain and 
preferably E. faecium 4147 or E. gallinarum. 
In order to distinguish strains with a high level of resistance from 
strains with a low level of resistance it is possible to carry out a 
hybridization test using conditions of high stringency. 
The oligonucleotides of the invention may be obtained from the sequences of 
the invention by cutting with restriction enzymes, or by chemical 
synthesis according to the standard methods. 
Furthermore, the invention relates to polyclonal or monoclonal antibodies, 
characterized in that they recognize the polypeptide(s) described above or 
an amino acid sequence described above. 
These antibodies may be obtained according to standard methods for antibody 
production. In particular, in the case of the preparation of monoclonal 
antibodies, recourse will be had to the method of Kohler and Milstein 
according to which monoclonal antibodies are prepared by cell fusion 
between myeloma cells and mouse spleen cells previously immunized with a 
polypeptide or a composition according to the invention, in conformity 
with the standard procedure. 
The antibodies of the invention can advantageously be used for the 
detection of the presence of proteins characteristic of resistance to the 
glycopeptides, in particular to vancomycin and teicoplanin. 
Particularly useful antibodies are polyclonal or monoclonal antibodies 
directed against the protein VanA or VanC. Such antibodies advantageously 
make it possible to detect strains of bacteria, in particular 
Gram-positive cocci, exhibiting high-level resistance to the antibiotics 
of the glycopeptide family. If necessary, a step entailing lysis of the 
cells of the sample undergoing detection is performed prior to the placing 
in contact of the sample with the antibodies. 
In order to carry out this detection, recourse will advantageously be had 
to antibodies labelled for example with a radioactive substance or other 
type of label. 
Hence, tests for the detection in Gram-positive bacteria of resistance to 
the glycopeptides, in particular tests making use of the ELISA procedures, 
are included in the framework of the invention. 
A kit for the in vitro diagnosis of the presence of Gram-positive strains, 
resistant to the glycopeptides, in particular to vancomycin and/or 
teicoplanin, these strains belonging in particular to the Gram-positive 
cocci for example enterococci, for example E. faecium or E. gallinarum is 
characterized in that it comprises: 
antibodies corresponding to the above definition, labelled if necessary, 
a reagent for the detection of an immunological reaction of the 
antigen-antibody type, 
if necessary, reagents to effect the lysis of the cells of the sample to be 
tested. 
Furthermore, the agents developed by the inventors offer the very useful 
advantage of being suitable for the development of a rapid and reliable 
test or kit for the detection of Gram-positive strains resistant to the 
glycopeptides by means of the polymerase chain reaction (PCR). Such a test 
makes it possible to improve the sensitivity of the existing tests which 
remain rather unreliable and, in certain cases, may make possible the 
detection of all of the representatives of the family of the genes coding 
for resistance proteins to the glycopeptides in Gram-positive bacteria. 
The carrying out of a test by means of the method of amplification of the 
genes of these proteins is done by the PCR procedure or by the RPCR 
procedure (RPCR : abbreviation for reverse polymerase chain reaction). 
The RPCR technique makes possible the amplification of the NH.sub.2 and 
COOH terminal regions of the genes it is desired to detect. 
Some specific primers make it possible to amplify the genes of the strains 
with low-level resistance. These primers are selected, for example, from 
the sequence coding for the resistance protein VanA. 
As examples, the following sequences can be used as primers for the 
preparation of probes for the detection of an amplification by means of 
the PCR or RPCR method. 
##STR4## 
X represents one of the bases A,T,C or G or also corresponds in all cases 
to inosine. 
Naturally, the invention relates to the complementary probes of the 
oligonucleotides previously described as well as possibly to the RNA 
probes which correspond to them. 
A kit for the in vitro diagnosis of the presence of strains of 
Gram-positive bacteria resistant to the glycopeptides, in particular 
resistant to vancomycin and/or teicoplanin these strains belonging in 
particular to the Gram-positive cocci, in particular that they are strains 
of enterococci, for example E. faecium or E. gallinarum, is characterized 
in that it contains: 
a nucleotide probe complying with the above specifications and if 
necessary, 
oligonucleoside triphosphates in an amount sufficient to make possible the 
amplification of the desired sequence, 
a hybridization buffer, 
a DNA polymerization agent. 
The invention also relates to a procedure for the in vitro detection of the 
presence of Gram-positive strains resistant to the glycopeptides, in 
particular to vancomycin and/or teicoplanin these strains belonging in 
particular to the family of the Gram-positive cocci, in particular in that 
they are strains of enterococci, for example E. faecium or E. gallinarum, 
characterized in that it comprises: 
a) the placing of a biological sample likely to contain the resistant 
strains in contact with a primer constituted by a nucleotide sequence 
described above, or any part of a sequence previously described, capable 
of hybridizing with a desired nucleotide sequence necessary for the 
expression of resistance to the glycopeptides, this sequence being used as 
matrix in the presence of the 4 different nucleoside triphosphates and a 
polymerization agent under conditions of hybridization such that for each 
nucleotide sequence which has hybridized with a primer, an elongation 
product of each primer complementary to the matrix is synthesized, 
b) the separation of the matrix from the elongation product obtained, this 
latter then also being capable of behaving as a matrix, 
c) the repetition of step a) so as to produce a detectable amount of the 
desired nucleotide sequences, 
d) the detection of the product of amplification of the nucleotide 
sequences. 
The detection of the elongation products of the desired sequence may be 
carried out by a probe identical with the primers used to carry out the 
PCR or RPCR procedure, or also by a probe different from these primers, 
this probe being labelled if necessary. 
Details relating to the implementation of the PCR procedures may be 
obtained from the patent applications EP 0229701 and EP 0200362. 
Other advantages and characteristics of the invention will become apparent 
in the examples which follow and from the figures.