WO2004099444A2

WO2004099444A2 - Diagnostic methods

Info

Publication number: WO2004099444A2
Application number: PCT/GB2004/001985
Authority: WO
Inventors: Timothy Rutland Walsh; Matthew Beardsall Avison; Robin Anthony Howe; Mandy Wootton
Original assignee: University of Bristol
Current assignee: University of Bristol
Priority date: 2003-05-09
Filing date: 2004-05-07
Publication date: 2004-11-18
Anticipated expiration: 2005-11-09
Also published as: WO2004099444A3; GB0310794D0

Abstract

A method for detecting a strain of Staphylococcus aureus which has reduced susceptibility to vancomycin and/or teicoplanin, said method comprising detecting the level of expression of an fmtB/mrp or mrp-homologue gene or a variant thereof within said strain and relating that to the susceptibility or resistance of the strain to vancomycin and/or teicoplanin. Detection of expression is suitably carried out using immunological to detect the gene product or molecular methods to detect RNA constructs. The method allows for the detection of strains of Staphylococcus aureus that are homogeneously or heterogeneously intermediate to vancomycin and/ or teicoplanin.

Description

Diagnostic Methods

This present invention relates to a method of detecting strains of Staphylococcus aureus, which have reduced susceptibility to vancomycin and/ or teicoplanin, together with kits and reagents for use in the method. In particular the method involves detecting the level of expression of particular genes, which are known as fmtB/mrp (illustrated hereinafter for instance as SA1969 -SEQ ID NO 17) and mrp-homologues (illustrated hereinafter as SA1577 and SA 1969- SEQ ID NOS 19 and 15 respectively) .

S. aureus has long been recognised as a major cause of hospital- acquired infection. Over the last decade, methicillin resistant S. aureus (MRSA) strains have become endemic in hospitals worldwide, including the United Kingdom. Accordingly, the frequency of MRSA isolation has increased dramatically (Livermore D. M. Antibiotic resistance in staphylococci . International Journal of Antimicrobial Agents 16, S3-10 (2000) ) .

Glycopeptides, such as vancomycin, are often the therapeutic drugs of choice for serious MRSA infections. Failures of vancomycin therapy against S. aureus due to the emergence of strains that are significantly less susceptible to vancomycin (VISA - vancomycin resistant S. aureus) are, however, now well established (Hiramatsu K, et al.(1997). Journal of Antimicrobial Chemotherapy 40, 135-6, Ploy M. C. et al. , (1998) Lancet 351, 1212, Howe R. A, et al . , (1998) Lancet 351, 602., Walsh, T. R. & Howe R. A. (2002) . Annual Reviews in Microbiology. July 18) .

The resistance exhibited by S. aureus falls into three categories :

1. High-level resistance (MIC>128mg/l) where the bacterium has picked up the Enterococcal vane gene.

2. "Homogeneous" vancomycin intermediate SA (VISA) where the majority of the population can be detected using a standard laboratory inoculums. 3. "Heterogeneous" vancomycin intermediate SA" or hVISA, where approx. only 1 in a million express the low-level resistance. The first of these can be easily detected by routine susceptibility testing; the other two cannot.

The first clinical VISA, Mu50 was isolated in Japan in 1997. The VISA phenotype of Mu50 is the result of changes to the cell wall structure. In comparison to MRSA strains, Mu50 and indeed all VISA have a thickened cell wall, release more cell wall material into the culture medium and have increased rates of autolysis (Hanaki et al (1998) Journal of Antimicrobial Chemotherapy 42, 199-209, Hanaki et al (1998) . Journal of Antimicrobial Chemotherapy 42, 315-20, Cui L, et al (2000) Antimicrobial Agents and Chemotherapy . 44, 2276-85). The fine structure of the Mu50 cell wall is similar to that of MRSA strains such as N315, except that the Mu50 peptidoglycan chains shows significantly less cross-linking, and an increased content of pentapeptide chains (Cui L, et al (2000) supra., Nakel et al . (1971) . Biochemistry 10, 2170-515) . The amount of glutamine non-amidated muropeptide subunits in the Mu50 cell wall is increased (i.e. those containing D-glutamate rather than D- glutamine) and it has been proposed that the reason for decreased cross-linking is that non-amidated muropeptides are poorer substrates for transpeptidases than amidated ones (Hanaki et al (1998) J. Antimicrob. Chemo, 42, 315-20, Cui L, et al (2000) supra, Nakel et al. (1971) supra.). It is believed that these changes in cell wall thickness and cross-linking in vancomycin resistant strains are due to the modified cell wall binding vancomycin more tightly through the terminal D-alanyl-D- alanine dipeptide of the pent peptide (Hanaki et al (1998) , Hanaki et al.(1998), Cui L, et al (2000) all supra.). This action sequesters vancomycin from the surroundings but with little effect upon cell wall biosynthesis and hence viability. Vancomycin consumption in the last 10 years has risen from 15 to 40 fold to treat infections caused by the ever-increasing methicillin resistant S. aureus (MRSA) . It is well established that vancomycin-intermediate S. aureus exists, although the methods employed to detect them are generally inadequate. As a result, the frequency of occurrence of VISA is not known.

The heterogeneous form of glycopeptide resistance is thought to be more common than the homogeneous resistant form of S. aureus and may represent a pre-VISA phenotype. Current detection methods are sub-optimal for both types of resistance with the heterogeneous phenotype being the most difficult to detect.

The coding differences between S. aureus isolates, Mu50 and N315 have been analysed, however, it is not fully understood how many genes must be altered to give the VISA phenotype.

It has been suggested that in order for S. aureus to acquire vancomycin/ teicoplanin resistance it must mutate (usually by loss of function mutations) one or more key genes and in particular the open reading frames (ORFs) thereof. However, vancomycin resistance may also be caused by mutations affecting regulatory elements and therefore expression of certain genes.

The applicants have found that the expression of a particular gene, which is known as mrp or fmtB (which may therefore be referred to a fmtB/mrp and is illustrated hereinafter by SEQ ID NO 17) , as well as mrp homologues (illustrated hereinafter by SEQ ID NOs 15 and 19) , has been downregulated, usually to a significant degree, in strains with a decreased susceptibility to vancomycin. This is surprising since the proteins encoded by these genes are believed to be large outer membrane proteins . However, the differences in the expression levels are significant enough to allow these to form the basis of a diagnostic test, using for example molecular (such as polymerase chain reaction (PCR) and in particular RT-PCR) or immunological methods. In view of this finding, the fmtB/mrp and mrp homologue genes of a set of other S. aureus with different VISA phenotypes were analysed as described hereinafter.

According to the present invention, there is provided a method for detecting a strain of Staphylococcus aureus which has reduced susceptibility to vancomycin and/or teicoplanin, said method comprising detecting the level of expression of an fmtB/mrp or mrp-homologue gene or a variant thereof, within said strain and relating that to the susceptibility or resistance of the strain to vancomycin and/or teicoplanin

Using such methods provides a rapid, reproducible and sensitive method for the detection of strains with reduced susceptibility to vancomycin/teicoplanin, such that, for example, discrimination of VISA and hVISA from vancomycin/ teicoplanin sensitive SA (VSSA) is possible.

As used herein the expression "fmtB/mrp" , or "mrp" or "fiαtB" refers to genes which comprise SEQ ID NOS 2-10 as illustrated in Figure 1, or any one of SEQ ID NOS 13 or 14 of Figure 3, or 15, 17 or 19 of Figure 4, as well as variants thereof. These genes share significant identity at the nucleotide level, for instance as much as 60% identity, although the proteins they encode are far less homologous as illustrated in Figure 5. The majority sequence is shown herein as SEQ ID NO 1 in Figure 1, and therefore fmtB/mrp and mrp-homologue sequences will generally comprise variants of this sequence.

As a result of the downregulation of the gene, a resistant strain loses the function encoded by the altered gene. The precise mechanism by which the downregulation occurs is not clear, but it is possible that regulatory elements controlling its expression, such as transcription factors, are mutated so that gene expression is inhibited, or that the gene is altered so that the transcription factor no longer interacts with it. As used herein, the term "variant" used in relation to a gene or other polynucleotide sequences refers to sequences which although they show some similarities to the base sequence, include some substitution of, variation of, modification of, replacement of, deletion of, or the addition of one or more nucleic acid(s) from or to a polynucleotide sequence. The term therefore includes allelic variants and also includes a polynucleotide, which substantially hybridises to the polynucleotide sequence of the present invention. Preferably, such hybridisation occurs at, or between low and high stringency conditions. In general terms, low stringency conditions can be defined as 3 x SSC at about ambient temperature to about 55°C and high stringency condition as 0.1 x SSC at about 65°C. SSC is the name of the buffer of 0.15M NaCI, 0.015M tri-sodium citrate. 3 x SSC is three times as strong as SSC and so on.

Typically, variants have 60% or more of the nucleotides in common with the polynucleotide sequence of the present invention, more typically 65%, preferably 70%, even more preferably 80% or 85% and, especially preferred are 90%, 95%, 98% or 99% or more identity.

When comparing nucleic acid sequences for the purposes of determining the degree of identity, programs such as BESTFIT and GAP (both from Wisconsin Genetics Computer Group (GCG) software package) . BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate. Suitably, in the context of the present invention when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length. The expression "allelic variant" refers to ORFs of alternative forms of the gene found at the same locus within the Staphylococcus aureus genome.

Variants will also include analogues, which are genes that encode any mrp-like proteins, in the sense that they form outer membrane proteins. It is possible that the proteins FmtB/Mrp and Mrp-homologues share similar functions although their sequence similarity is not high. Typical sequences are illustrated hereinafter as SEQ ID NOS 11 and 12 in Figure 3 and SEQ ID NOS 16, 18 and 20 in Figure 4.

Any strain of S. aureus tested which has such an alteration in expression of one or both of said genes, is likely to be resistant to vancomycin and/or teicoplanin, and therefore treatment of a patient with such an infection should be modified accordingly. The method is particularly useful in detecting a strain of S. aureus that is fully or heterogeneously resistant to vancomycin and/ or teicoplanin.

Downregulation in fmtB/mrp and mrp-homologue expression may be detected by detecting RNA constructs encoded by these genes, for instance using molecular methods such as RT-PCR. For instance, primers designed to amplify the fmtB/mrp or mrp-homologue RNA, and in particular the messenger RNA, or preferably a region of the RNA encoded by a conserved region of the gene, will produce a significant yield of product if expression is occurring normally, but a much lower, even an undetectable amount of RNA is found in VISA/hVISA. Suitable conserved regions may be deduced from Figures 1, 3 and 4 hereinafter, but in particular include the regions found between bases 90-220, 261-306, 350-424 & 440-1610 of SEQ ID NO 1 illustrated in Figure 1.

Preferably, such a test will be conducted alongside positive and/or negative control strains, whose vancomycin or teicoplanin resistance is known, so that the relative expression levels can be judged. Examples of suitable positive control strains are the VRSA strains, Mu50 or Mu3.

The applicants have found however, that in vancomycin or teicoplanin resistant strains, the fmtB/mrp and .mrp-homologue genes are expressed at levels of less than mg/L.

Thus, where the primer is designed to amplify RNA expressed from such a gene, the absence of an amplification product, or the presence of only low levels of the amplification product will indicate that the sample is in fact a mutant and is therefore likely to show some resistance to vancomycin and/or teicoplanin.

Detection of amplification product can be carried out using any of the known methods, for example using gel electrophoresis or any of the homogenous assay systems, which detect amplification product, for example using fluorescent or other labels. In particular, assays, which allow the progress of the amplification reaction to be monitored, for example using dyes, labelled probes or primers, to allow the results to be quantified are particularly useful in determining expression levels in accordance with the invention. A particular example of a quantitative assay is the TAQMAN™ assay, which utilises a labelled probe that is hydrolysed during the amplification.

When the product is detected using gel electrophoresis, the intensity of the signal produced on the gel following visualisation of the amplification product is an indicator of the amount of product, and therefore is suggestive of the relative expression levels. As illustrated hereinafter, this can generally be assessed visually, but may be more accurately determined using a detector of the known type.

In a preferred embodiment, the amount of a protein or polypeptide, representing the gene product, such as the proteins Mrp or FmtB, can be detected as an indication of the whether the particular strain tested is likely to show some resistance to vancomycin and/or teicoplanin. The absence of such proteins or only low levels of these proteins is indicative of the presence of a strain, which may not be susceptible to vancomycin and/or teicoplanin.

The proteins or polypeptides are suitably detected using known immunological techniques such as ELISA or other methods such as lateral flow assays, or latex agglutination assays. A negative result in such an assay would ensue in the case of a mutated sequence. Thus this would provide an indication of the VISA or VSSA status.

Several tests may be performed using one such assay. Such tests are well known in the art. They may comprise direct assay formats such as the one illustrated in Figure 5, or competitive assay formats. These tests suitably use specific binding partners such as antibodies or binding fragments thereof, which are specific for FmtB/Mrp and Mrp-homologue.

In a typical assay format, a first specific binding partner, in particular an antibody or a binding fragment thereof, which specifically binds to the proteins FmtB/Mrp or Mrp-homologue, is contacted with a sample containing proteins from a strain of S. aureus suspected of being resistant to vancomycin and/or teicoplanin. In the case of direct assays, any target protein present will become bound. The presence of the complex between the first binding partner and the protein is then detected, using for example a labelled second specific binding partner.

The label may be a moiety, which is detected by an enzyme, such as horseradish peroxidase as is commonly employed in ELISA methods. Alternatively, the label may be a visible label such as a particulate label, for instance a gold or coloured latex label. The assay would be carried out in a convention manner, for example within the well on an ELISA plate, or on a membrane or the like in the case of an assay that utilises labelled antibodies, which develop a visible signal.

Where latex agglutination is used, either an antibody or antigen is attached to a latex sample. If the corresponding antigen or antibody is present, the sample agglutinates. Suitable antigens for use in these assays are the proteins themselves or epitopic fragments thereof. Specific binding partners in particular are antibodies, which may be monoclonal or polyclonal, as well as antisera. In particular, the specific binding partners comprise monoclonal antibodies or binding fragments thereof.

Where the antigen used is an epitopic fragment, the specific binding partner is suitably a monoclonal antibody, which recognises that epitope.

Preferably, one or more proteins from a strain of S. aureus, which has reduced susceptibility to vancomycin and/or teicoplanin, are included in the test as a positive control. The control proteins are suitably obtained from the VRSA strains, Mu50 or Mu3.

Antibodies used in these methods can be generated in the usual way, using the proteins or antigenic fragments. Preferably, the antibody is raised against a region of the FmtB/Mrp or Mrp- homologue protein that is conserved between strains, and is preferably at least 10 and preferably at least 20 amino acids in length. Again such regions may be determined for example from Figures 1, 3 and 4 hereinafter.

Antibodies used in these assays form a further aspect of the invention. Thus in particular there is provided an antibody, which recognises an amino acid sequence encoded by either fmtB/mrp or mrp-homologue genes . The use of these antibodies in the detection of vancomycin or teicoplanin resistant strains of Staphylococcus aureus forms a further aspect of the invention.

The applicants have further found that the presence of low levels of vancomycin or teicoplanin, for example sub-MIC concentrations, results in a further lowering of expression levels of the fmtB/mrp or mrp-homologue genes . Thus in a particular embodiment, the method of the invention is conducted (i) on a sample of the strain which is cultured in the substantial absence of vancomycin, and (ii) on a sample of the strain which is cultured in the presence of low levels of vancomycin or teicoplanin.

The latter samples may be obtained for example by culturing the strains in the presence of levels of vancomycin or teicoplanin, which are below the minimum inhibitory concentration of the antibiotic. For instance, in the case of vancomycin, the concentration may be less than 5mg/l, preferably 2mg/l or less, for example 0.5mg/l.

Conveniently, the sample for (ii) above can be obtained by placing vancomycin discs or Etest strips on a culture plate in which the target strain is cultured, and recovering analysing a sample of culture taken from the edge of any area of inhibition around the vancomycin of teicoplanin disc. Other arrangements can be envisaged, for example using columns, where vancomycin or teicoplanin is allowed to penetrate a limited distance up the column.

Clearly, if there are no areas of inhibition, the strain will be fully resistant to vancomycin. However, this methodology is useful in detecting strains, which are in the early stage of resistance development, such as hVISA. In yet a further aspect of the present invention, there is provided a diagnostic kit for detecting the presence of a mutation in a strain of S. aureus which shows reduced susceptibility to vancomycin and/or teicoplanin, said kit comprising an antibody or a binding fragment thereof, which is specific for an amino acid sequence which represents a conserved sequence within FmtB/Mrp or Mrp-homologue proteins . Particular examples of such proteins are given hereinafter.

The antibody preferably carries a label or a detectable signal thereon. The antibody is preferably located on a support, for example of membrane or "dipstick" variety.

The main advantage of the present invention is that it provides a reliable test for identifying strains of S. aureus that have a reduced susceptibility to vancomycin and/or teicoplanin. It is a simple and straightforward test giving either a positive or negative result depending upon whether a resistant strain has been identified. Where the test detects mutations in more than one open reading frame, the severity of resistance may also be detected.

A further advantage of the present invention is that it provides a rapid reproducible and sensitive method to discriminate fully and heterogeneously vancomycin and/or teicoplanin intermediate strains of S. aureus (VISA and hVISA) from vancomycin and/ or teicoplanin sensitive strains (VSSA) .

In summary, therefore, glycopeptides, such as vancomycin, are frequently the antibiotics of choice for treatment of infections caused by the now common methicillin-resistant S. aureus (MRSA) . Incidences of S. aureus with reduced susceptibility to vancomycin (VISA) have been increasing worldwide for the last five years. Complex mechanisms, producing changes in cell wall content and composition, generate the VISA phenotype, but the genetic basis of these changes had not been previously fully determined. To facilitate the genetic investigation, entire genome sequences of the archetypal VISA (Mu50) , and vancomycin sensitive MRSA strains, N315, EMRSA 16 and COL have been compared.

The difference in expression levels of the fmtB/mrp and mrp- homologue genes however, is a feature, which could not have been predicted from an analysis of the genome.

The mechanism as to how S. aureus reduces its susceptibility to vancomycin is complex, and is likely to impose VISAs with a fitness disadvantage compared to VSSAs when growing in the absence of vancomycin. This fitness disadvantage is likely to be negligible when contact with the antibiotic is made and that VISA/hVISA will out-compete its sensitive counterpart. In time, these disadvantages may be rectified and, as a consequence, the levels of vancomycin resistance seen in hospitals will rise.

The detection method of the present invention will be an important step in the control of strains of S. aureus that have reduced susceptibility to vancomycin and/ or teicoplanin.

The present invention will now be described only by way of examples in which reference shall be made to the accompanying Figures in which:-

Figure 1 shows the alignment of nucleotides sequences found in mrp genes of various vancomycin resistant phenotypes, arising from sequence analysis carried out. The mrp groups represent different glycopeptide resistant phenotypes. (Groups: 1: LA, LE, 6VSSA; Group 2: VSSA; Group 3: PC3, PCI, Mu50, 3 x VISA; Group 4: Lim 1, 2, 3, 3 x VISA, 6 x hVISA) .

Figure 2 shows the results of RT-PCR (messenger expression) of approximate 800 base pair fragments of SEQ ID NO 19 (mrp- homologue for VSSA, hVISA and VISA under different conditions. Figure 3 shows the amino acid sequences of Mrp and the corresponding nucleotide sequences found in an MRSA and vancomycin resistant strain.

Figure 4 shows the nucleotide and amino acid sequences of a number of mrp-like proteins from vancomycin susceptible strains.

Figure 5 is an illustrative diagram showing a possible immunological detection of VISA/hVISA based on repression of the fmtB/mrp transcript and therefore lack of the FmtB/Mrp with this resistant phenotype. In this diagram, the antibody is directly tagged with a marker (either fluorescence or latex or peroxidase etc.); however, for greater sensitivity an antibody against the FmtB/Mrp-antibody may be tagged instead as in a "sandwich technique" .

Example 1

Materials and Methods

Cultures were grown to mid exponential phase in Brain Heart Infusion broth (BHI) , (A600 = 0.6). Using 1ml of culture RNA was extracted using a QIAGEN RNA kit. The RNA was then quantified by reading at 260nm using a spectometer. 5μl of RNA was diluted in lOOOμl of sterile water and read at 260nm. The formula lnm=40μg/ml RNA was then employed to calculate the quantity of RNA. The RNA was then diluted to give a final concentration of 0.5μg/μl and lμl was used in each RT-PCR reaction.

The sequences targeted by the RT-PCR reactions were fragments of SEQ ID NO 17 (referred to as the fmtB/mrp gene) and SEQ ID NO 19 (referred to as the mrp-homologue gene in Table 1) of about 800 base pairs in length.

The RNA was then treated with DNase for 30 minutes at 37°C and the reaction stopped by adding a stop buffer and incubation at 65°C for 10 minutes. RT-PCR was performed using the following programme :

Stage 1 (1 cycle) 50°C for 30 minutes Stage 2 (1 cycle) 95°C for 15 minutes Stage 3 (35 cycles) 95 °C for 1 minute

55 °C for 1 minute

72°C for 1 minute

Stage 4 72°C for 5 minutes The RT-PCR products were separated and visualised by gel electrophoresis using conventional methods. Illustrative results for mrp-homologue expression are shown in Figure 2. The variance in band intensity was calculated with ImageQuant™ and the results are shown in Table 1.

Table 1 Band Intensities

These results clearly show that in the hVISA or VISA strains, the mrp and fmtB genes were significantly downregulated, and that the expression was further reduced or eliminated by the presence of sub-MIC amounts of vancomycin.

No such effects were seen with the fully susceptible strains.

Claims

1. A method for detecting a strain of Staphylococcus aureus which has reduced susceptibility to vancomycin and/or teicoplanin, said method comprising detecting the level of expression of an fmtB/mrp or mrp-homologue gene or a variant thereof within said strain and relating that to the susceptibility or resistance of the strain to vancomycin and/or teicoplanin.

2. A method according to claim 1 wherein the fmtB/mrp or mrp- homologue gene comprises any one of SEQ ID NOS 2-10 as illustrated in Figure 1, or any one of SEQ ID NOS 13 or 14 of Figure 3, or 15, 17 or 19 of Figure 4.

3. A method according to claim 1 wherein the fmtB/mrp or mrp- homologue gene is a variant of SEQ ID NO 1.

4. A method according to any one of the preceding claims wherein downregulation of fmtB/mrp or mrp-homologue expression is detected by detecting RNA constructs encoded by these genes.

5. A method according to claim 4 wherein the RNA construct is detected using RT-PCR.

6. A method according to any one of claims 1 to 3 wherein the amount of a protein or polypeptide, representing the gene product of the fmtB/mrp or mrp-homologue genes are detected.

7. A method according to claim 6 wherein the proteins or polypeptides are detected using an immunological technique.

8. A method according to any one of the preceding claims which method is repeated using at least one positive and/or negative control strains, whose vancomycin or teicoplanin resistance is known, and the results compared.

9. A method according to claim 8 wherein the positive control strain is Mu50 or Mu3.

10. A method according to any one of the preceding claims wherein expression of fmtB/mrp or mrp-homologue genes at levels of less than 4mg/L is taken as an indication that the strain has reduced susceptibility to vancomycin or teicoplanin.

11. A method according to any one of the preceding claims which is conducted (i) on a sample of the strain which is cultured in the substantial absence of vancomycin and teicoplanin, and (ii) on a sample of the strain which is cultured in the presence of low levels of vancomycin or teicoplanin.

12. A probe or primer useful in the method according to claim 4 or claim 5.

13. An antibody suitable for use in a method according to claim 7.

14. A diagnostic kit for detecting the presence of a mutation in a strain of S. aureus which shows reduced susceptibility to vancomycin and/or teicoplanin, said kit comprising an antibody or a binding fragment thereof, which is specific for an amino acid sequence which represents a conserved sequence within FmtB/Mrp or Mrp-homologue proteins .