COMPOSITION AND METHOD FOR TREATING INFECTIONS CAUSED BY VANCOMYCIN-RESISTANT INFECTIOUS AGENTS IN A SUBJECT

Abstract

The disclosure is related to vancomycin resistance and, in particular, to compositions comprising vancomycin for use in inhibiting growth of a vancomycin-resistant microorganism or for use in treating a subject infected with a vancomycin-resistant pathogen. In one aspect, the disclosure provides a composition comprising vancomycin and d-alanine. The disclosure also provides means and methods for treating a subject infected with a vancomycin-resistant microorganism. The disclosure further provides bacteria with a functionally deactivated gene in the vancomycin-resistance cluster.

Claims

1. A composition comprising: vancomycin; and D-alanine amino acid.

2. The composition according to claim 1, further comprising: a therapeutically effective amount of a substance that decreases the level and/or activity of a dipeptidase enzyme encoded by a vanX gene of a vancomycin-resistant microorganism.

3. The composition according to claim 2, wherein the substance is one or more of a peptide, a peptide analog, a phosphinate peptide analog, a phosphonamidate peptide analog, a phosphonate peptide analog, an antisense molecule, a transcription factor, and/or a small RNA, and an RNAi that reduces expression of the vanX gene.

4. The composition of claim 1, further comprising: a therapeutically effective amount of a substance that decreases the level and/or activity of a ligase enzyme encoded by a vanA gene of a vancomycin-resistant microorganism.

5. A method of inhibiting growth of a vancomycin-resistant microorganism, the method comprising: contacting the vancomycin-resistant microorganism with the composition of claim 1.

6. A method of treating a subject infected, suspected to be infected or at risk of being infected with a vancomycin-resistant pathogen, the method comprising: administering the composition of claim 1 to the subject.

7. The method according to claim 5, wherein the microorganism is a gram-positive bacterium.

8. A method of inhibiting growth of a vancomycin-resistant microorganism, the method comprising: exposing the microorganism to an effective amount of vancomycin and to an effective amount of D-alanine, wherein the antibacterial effect of vancomycin on the microorganism is increased relative to its antibacterial effect on the microorganism in the absence of D-alanine.

9. A method of treating a subject infected, suspected to be infected, or at risk of infection, with a vancomycin-resistant pathogen, wherein the method comprises: administering to the subject a therapeutically effective amount of vancomycin and a therapeutically effective amount of D-alanine to treat a vancomycin-resistant pathogen.

10. A kit of parts comprising: a therapeutically effective amount of vancomycin; a therapeutically effective amount of D-alanine amino acid, and, optionally, a therapeutically effective amount of a substance that decreases the level and/or activity of a dipeptidase enzyme encoded by a vanX gene or a functionally similar nucleic acid of a vancomycin-resistant pathogen.

11. A bacterium having a vancomycin resistance cluster comprising a vanHAX operon, in which the ddl gene coding for the wild-type D-alanyl-alanine synthetase A is functionally deactivated.

12. Bacterium having a vancomycin resistance cluster comprising a vanHAX operon, of which at least the vanX gene is functionally deactivated.

13. The bacterium according to claim 11, in which the ddl gene is functionally deactivated by a deletion of at least a part of the coding nucleotide sequence of the ddl gene.

14. A method for screening for a substance that decreases the level and/or activity of a polypeptide encoded by a van gene of a vancomycin-resistant infectious agent comprising a vanHAX operon, the method comprising: using the bacterium of claim 11 to screen for the substance.

15. A method for screening for a substance that decreases the level and/or activity of a dipeptidase enzyme encoded by a vanX gene or a functionally similar nucleic acid of a vancomycin-resistant microorganism, the method comprising: comparing the growth rate of the microorganism exposed to vancomycin to the growth rate of the microorganism exposed to vancomycin, a test substance, and optionally D-alanine.

16. The composition of claim 2, wherein the substance is a cyclic thiohydroxarnic acid-based peptide analog that reduces expression of the vanX gene.

17. The method according to claim 7, wherein the microorganism is one or more microorganisms selected from the group consisting of Staphylococcus, Enterococcus, Clostridium, methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Enterococcus faecium, methicillin-resistant Enterococcus faecalis, and methicillin-resistant Clostridium difficile.

18. The method according to claim 6, wherein the pathogen is a gram-positive bacterium.

19. The method according to claim 18, wherein the pathogen is one or more pathogens selected from the group consisting of Staphylococcus, Enterococcus, Clostridium, methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Enterococcus faecium, methicillin-resistant Enterococcus faecalis, and methicillin-resistant Clostridium difficile.

20. The bacterium of claim 12, wherein the vanX gene is functionally deactivated by a deletion of at least a part of the coding nucleotide sequence of the vanX gene.

21. A method of treating a subject infected with, suspected to be infected with, or at risk of being infected with a vancomycin-resistant pathogen, the method comprising: administering the composition of claim 2 to the subject for the vancomycin-resistant pathogen.

22. A method of screening for a substance that decreases the level and/or activity of a polypeptide encoded by a van gene of a vancomycin-resistant infectious agent comprising a vanHAX operon, the method comprising: using the bacterium of claim 12 to screen for the substance.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] These and other aspects of the disclosure are further elucidated by the accompanying drawings, which form part of the present application. The drawings are not in any way meant to reflect a limitation of the scope of the disclosure unless this is clearly and explicitly indicated.

[0041] FIG. 1 shows growth of the wild-type S. coelicolor strain M145, and its mutant derivatives LAG1 (M145 ddl) and LAG2 (M145ddl suppressor mutant) on Soy Flour Medium (SFM) plates with and without vancomycin. The ddl deletion mutant (ddl) is vancomycin-dependent and only grows on media supplied with vancomycin. LAG2 constitutively expresses the vancomycin resistance cluster and grows both with and without vancomycin.

[0042] FIG. 2 shows growth of S. coelicolor M145, and its mutant derivatives LAG2 (ddl suppressor mutant), LAG3 (M145 vanX), and LAG4 (LAG2 vanX) on the following media: 1, SFM plate; 2, SFM+1 mM D-ala; 3, SFM+10 g/ml vancomycin; and 4, SFM+1 mM D-ala+10 g/ml vancomycin.

[0043] FIGS. 3A-3D show a Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of the peptidoglycan precursor.

[0044] FIG. 3A: Shows two peaks from wild-type S. coelicolor M145+vancomycin as an example, the area of the peak corresponding to a precursor terminating in D-Ala-D-Ala is shown in black, the area corresponding to a precursor terminating in D-Ala-D-Lac in grey. For each sample, the area of the peaks were pooled to 100% and the different areas are shown in percentages.

[0045] FIG. 3B: Shows S. coelicolor M145 with and without vancomycin. The ddl mutant LAG1 is only shown with vancomycin as it fails to grow in its absence. LAG2 with and without vancomycin have less than 1% wild-type (vancomycin-sensitive) peptidoglycan.

[0046] FIG. 3C: Shows the accumulation of wild-type and vancomycin-resistant precursors over time in LAG2 and LAG2vanX. At t=0, 50 mM of D-ala was added to the growth media, and samples were taken after 1, 5, 15, 30, 60, 120 and 180 minutes.

[0047] FIG. 3D: Same as FIG. 3C, but with L-Ala added to the growth media instead of D-Ala.

[0048] FIGS. 4A and 4B demonstrate the recovery of vancomycin localization by addition of D-alanine. The strains were grown in liquid NMMP media for 12 hours, after which 50 mM D-Ala was added to the media and cultures were left to grow for another hour. Fluorescence images with inverted greyscale show localization of Vanco-FL (Top) and the corresponding light image (Bottom). Vanco-FL localizes at the interaction site of vancomycinactively growing peptidoglycan, which is at the hyphal tips and forming septa in Streptomyces. Scale bar, 10 m.

[0049] FIG. 4A: Before the addition of D-Ala, hyphae of wild-type S. coelicolor M145 are stained at the tips, and the same is true for M145vanX LAG2 and LAG2vanX show little or no staining.

[0050] FIG. 4B: An hour after the addition of D-Ala, M145 and M145vanX show similar staining as before. LAG2 shows a slight recovery of localization of vanco-FL, while LAG2vanX shows a very strong recovery of vanco-FL localization.

[0051] FIG. 5 shows a model of how D-Ala influences the activity of VanA in the presence or absence of VanX. Panel A shows the normal situation. The products of VanA are both D-Ala-D-Ala and D-Ala-D-Lac. D-Ala-D-Ala is broken down by VanX. Panel B shows the situation in the presence of an excess D-Ala, which is then ligated by VanA to the D-Ala-D-Ala dipeptide but broken down by VanX. Panel C shows the situation in the absence of vanX Because of the lack of VanX, D-Ala-D-Ala accumulates and the addition of D-Ala dramatically increases the pool of D-Ala-D-Ala, thereby enhancing the percentage of wild-type cell wall precursors and thus efficacy of vancomycin.

[0052] FIG. 6 shows the growth result of wild-type M145 and of LAG2 on solid agar in 24-well plates with increasing amounts of alanine and vancomycin. D-ala was added in concentrations of 5, 10 and 50 mM and the effect on the MIC of vancomycin was assessed. As a control, L-ala was added in the same concentrations of 5, 10 and 50 mM.

[0053] FIG. 7 shows examples of VanX-inhibitors of which an effect on vanX has been demonstrated in vitro. Compound 1 is as described in Ardoz, Anhalt et al. 2000, compounds 2 and 4 are as described in Muthyala, Rastogi et al. 2014, and compounds 3a-3e are as described in Yang, Cheng et al. 2011. Compound 3a demonstrates the most in vitro inhibition. The compounds can be synthesized as described in the respective literature.

DETAILED DESCRIPTION

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure. However, other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting, unless so indicated. All publications and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The following definitions are used unless otherwise described.

[0055] As used herein, the term mutated, employed in combination with vanX, refers to loss-of-function mutations in vanX that cause the VanX peptidase to be no longer capable of hydrolyzing the peptide bond in the D-ala-D-ala dipeptide. The mutations may be small-scale mutations, such as those affecting one or a few nucleotides. These mutations may be point mutations in which a single nucleotide is exchanged for another. The point mutation occurs within the protein coding region of the vanX gene and is either a missense mutation, resulting in a codon, which codes for a different amino acid, or a nonsense mutation, resulting in a stop codon, which leads to a truncated VanX enzyme. The mutations may alternatively be insertions that add one or more extra nucleotides into the DNA. Insertions in the coding region of a gene may alter splicing of the mRNA (splice site mutation), or cause a shift in the reading frame (frameshift), both of which can significantly alter the gene product. The mutations may also be deletions that remove one or more nucleotides from the DNA, or the entire gene. These mutations can also alter the reading frame of the gene.

[0056] The term therapeutically effective amount as used herein with regard to vancomycin refers to a quantity of vancomycin that is effective in at least partially treating or curing an infection caused by a vancomycin-resistant pathogen. Amounts effective to achieve this goal will, of course, depend on the type of pathogen and associated vancomycin resistance, and the severity of the disease and the general condition, particularly the weight, of the subject to be treated.

[0057] The term therapeutically effective amount as used herein with regard to D-alanine refers to a quantity of the D-alanine amino acid that is effective in lowering the vancomycin resistance of a pathogen to a MIC suitable for preventing, curing or at least partially treating or arresting an infection caused by the pathogen. Amounts effective to achieve this goal will, of course, depend on the type of pathogen and associated vancomycin resistance, and the severity of the disease and the general condition, particularly the weight, of the subject to be treated.

[0058] The skilled person will be able to determine what an effective or therapeutically effective amount of vancomycin and D-alanine would be on the basis of routine dose response protocols and routine techniques for assessing microbial growth inhibition. The skilled person will also be able, without undue burden, to optimize the amounts of vancomycin and D-alanine with respect to each other to maximize the combinatorial effect thereof.

[0059] The term exposing as used herein refers to any means of delivering or contacting the vancomycin or D-alanine to the microorganism or pathogen. This may be directly or indirectly, e.g., applying the vancomycin and D-alanine directly to the microorganism or pathogen, or indirectly via administering the vancomycin and D-alanine to a subject within which or on which the microorganism or pathogen is present, e.g., a subject infected with a vancomycin-resistant pathogen.

[0060] The disclosure will be illustrated by means of the following experimental results and examples, which are provided by way of illustration and not of limitation. It will be understood that many variations in the methods described can be made without departing from the spirit of the disclosure and the scope of the appended claims.

D-Alanine Reduces Vancomycin Resistance

[0061] The structural analysis of the VanA enzyme revealed that it may use both lactate and alanine as a substrate (Roper et al., 2000; Wright and Walsh, 1992). D-Ala might, therefore, compete with D-Lac in the active site of the enzyme, suggesting competitive inhibition of the synthesis of the D-Ala-D-Lac dipeptide required for vancomycin resistance, in favor of the production of wild-type D-Ala-D-Ala precursors. This should increase the proportion of wild-type peptidoglycan and, hence, also the sensitivity to vancomycin.

[0062] The naturally vancomycin-resistant model organism S. coelicolor M145 was used as the initial test system. The strain was grown on solid minimal medium (MM) agar plates with increasing amounts of D-Ala and vancomycin. D-Ala was thereby added in concentrations of 5, 10 and 50 mM and the effect on the MIC of vancomycin assessed. As a first control, L-Ala was added and, as a second control, no amino acids were added. In the absence of added amino acids, the MIC of vancomycin against S. coelicolor was 128 g/ml. This was not altered when the culture media were supplemented with up to 50 mg/ml L-Ala (Table 1 and FIG. 6). Excitingly, vancomycin tolerance dropped significantly when D-Ala was added: with 10 mM D-Ala, the MIC was decreased to 32 g/ml (4-fold reduction), while 50 mM of D-Ala further reduced the MIC to 4 g/ml (32-fold reduction) (Table 1 and FIG. 6). This provided the first evidence that the addition of D-Ala, which is a food grade compound, effectively reduces VanA-based vancomycin resistance, suggesting possible competition with D-Lac at the active site of the VanA enzyme.

Creation of a Vancomycin-Independent Ddl Mutant

[0063] To study the molecular basis of this phenomenon in more detail, a strain that lacked the wild-type D-Ala-D-Ala ligase was created, so as to ensure that VanA was the only enzyme responsible for wild-type precursor biosynthesis. D-Ala-D-Ala ligase (Ddl, SC05560 in S. coelicolor) is essential for growth, as it is required for the synthesis of the D-Ala-D-Ala dipeptide (Kwun et al., 2013). An S. coelicolor ddl null mutant was created by replacing the entire coding region by the apramycin cassette (aacC4) via homologous recombination. The aacC4 gene was flanked by loxP sites, allowing the subsequent removal by expression of the Cre recombinase, resulting in a markerless deletion mutant of ddl. Since ddl is required for biosynthesis of the essential cell-wall precursor D-Ala-D-Ala, the deletion experiments were done in the presence of vancomycin, which induces the expression of the vancomycin resistance genes, thereby eliciting the production of the alternative precursor dipeptide D-Ala-D-Lac by VanA. A number of candidate ddl null mutants was obtained, all of which failed to grow in the absence of vancomycin and showed normal sporulation. One of these strains was selected for further characterization and was designated LAG1. The absence of the ddl gene in this mutant was confirmed by PCR (data not illustrated). Expectedly, the ddl mutant could only grow on SFM agar plates with vancomycin (FIG. 1). Introduction of plasmid pGWS1159, which expresses the ddl gene from its own promoter, into the ddl null mutant restored normal development and growth in the absence of vancomycin (data not shown).

[0064] To allow studying the sensitivity of VanA to inhibitory molecules regardless of the presence or absence of vancomycin, spores (10.sup.7 cfu) of the ddl mutant LAG1 were plated onto SFM agar plates lacking vancomycin, so as to select for suppressors with constitutive expression of the vancomycin resistance cluster. This yielded a small number of spontaneous suppressor mutants, which occurred at a frequency of around 10.sup.6. These likely constitutively expressed the vancomycin resistance cluster required to compensate for the absence of ddl. One of the suppressor mutants that grew well in the absence of vancomycin was selected and designated LAG2 (FIG. 1).

[0065] DNA sequencing of the vancomycin resistance gene cluster from this suppressor mutant revealed that transposase IS466A (Yamasaki et al., 2000) (SCO3469) had inserted at nt position +55 relative to the translational start of vanS, causing loss-of-function. VanS is the sensory kinase of a two-component system formed with VanR, and it has been shown previously that certain mutations in vanS will constitutively upregulate the vancomycin resistance cluster, explaining the viability of strain LAG2 in the absence of vancomycin. Using the same sensitivity assay as for the parent strain (M145), it was demonstrated that ddl suppressor mutant LAG2 had similar vancomycin resistance as the parental strain, with an MIC of 128 g/ml (Table 1). Like in wild-type cells, adding L-Ala did not affect the MIC for vancomycin, while D-Ala decreased the MIC in very similar fashion, down to 4 g/ml for 50 mM D-Ala (Table 1). Thus, while LAG2 constitutively expresses the van cluster, the strain had vancomycin resistance levels comparable to those of its parent S. coelicolor M145 (measured in terms of the MIC), and was sensitized in the same way following addition of D-Ala to the growth media.

TABLE-US-00001 TABLE 1 Effect of D-alanine on the MIC of vancomycin against S. coelicolor 0 mM 5 mM 10 mM 50 mM D-Ala D-Ala D-Ala D-Ala M145 128 32 32 4 LAG2 128 64 32 4

[0066] The MIC of vancomycin is reduced by the addition of D-alanine for the wild-type strain S. coelicolor M145 carrying inducible resistance and its mutant derivative LAG2, which constitutively expresses the van resistance genes.

Analysis of Peptidoglycan Precursors

[0067] In order to get more insights into the synthesis of vancomycin-sensitive (i.e., wild-type) or the alternate vancomycin-resistant peptidoglycan, the pool of peptidoglycan precursors was analyzed by Liquid Chromatography coupled to Mass Spectrometry (LC-MS). When cells produce wild-type PG, only the MurNac-pentapeptide with a D-Ala-D-Ala terminus are detected, while vancomycin-resistant PG precursors have a D-Ala-D-Lac terminus. To analyze the precursors produced in S. coelicolor M145 and its mutant derivatives LAG1 (ddl) and LAG2 (ddl vanS::IS466A) in the presence or absence of vancomycin, the strains were grown until exponential phase, after which the cultures were harvested, washed and the precursors extracted with 5% TCA. The supernatants were desalted, concentrated and analyzed by LC-MS (see Materials and Methods section). Wild-type precursors ending with D-Ala-D-Ala are characterized by a peak of 1994 Da and a retention time of around 7.2 minutes, while vancomycin-insensitive precursors ending with D-Ala-D-Lac are characterized by a peak of 1995 Da and a significantly higher retention time of around 8.2 minutes (FIG. 3A).

[0068] In extracts from the parental strain grown in the absence of vancomycin, only wild-type precursors were produced (FIG. 3B). Expectedly, when S. coelicolor M145 was grown in the presence of 10 g/ml vancomycin, the vast majority of the precursors (91.5%) represented the vancomycin-insensitive variant. Similarly, 95.7% of the precursors from the ddl null mutant (LAG1) grown in the presence of vancomycin contained the terminal D-Ala-D-Lac dipeptide (FIG. 3B). This indicates that VanA produces a low level of D-Ala-D-Ala. In the ddl suppressor mutant (LAG2), which constitutively expresses the van gene cluster, nearly all peptidoglycan precursors terminated with D-Ala-D-Lac (99.8% and 99.7% for cultures grown with and without vancomycin, respectively) (FIG. 3B).

[0069] To investigate what the effect would be of the addition of excess of D-Ala on the accumulation of wild-type precursors, a time-course experiment was performed to follow the effect of D-Ala addition on the ratio between wild-type and resistant precursors. Therefore, 300 ml liquid-grown NMMP cultures were supplemented with either D-Ala or L-Ala (control) at 50 mM end concentration, and 10 ml samples were collected prior to and 1, 5, 15, 30, 60, 120 and 180 minutes after the addition of either alanine stereoisomer. Samples were immediately filtered by vacuum filtration, washed in 0.9% NaCl, biomass scraped off the filter, added to a tube containing 5% TCA and samples prepared as described above. Before the addition of D-Ala or L-Ala (t=0), LAG2 did not accumulate any wild-type precursors. However, addition of 50 mM D-Ala triggered the production of small amounts of wt precursor (1%) within 1 minute. After 15 minutes, this amount had increased to 4%, which appeared to be close to the maximum, with levels of wt precursors never exceeding 5%. L-Ala did not activate the production of detectable levels of wt precursors in LAG2.

Deletion of vanX Amplifies the Effect of D-Ala on Vancomycin Sensitivity.

[0070] VanX effectively eradicates the D-Ala-D-Ala dipeptide, thereby counteracting the accumulation of wild-type precursors and supporting vancomycin resistance (Lessard and Walsh, 1999; Tan et al., 2002). Therefore, a vanX null mutant was created using a similar strategy as for ddl, replacing the coding region of vanX by the apramycin resistance cassette aacC4. The mutant was created in both the parental strain S. coelicolor M145 and in its ddl suppressor mutant LAG2, generating LAG3 (M145 vanX) and LAG4 (M145 ddl vanX vanS::IS466A), respectively.

[0071] Surprisingly, both vanX mutants showed the same morphology as the respective parental strains on SFM agar plates, but displayed strongly increased vancomycin sensitivity (FIG. 2). LAG4 produced 20% wt precursors prior to the addition of D-Ala. This strongly suggests that VanA produces a significant amount of D-Ala-D-Ala in vivo in wild-type cells, which accumulates in the absence of VanX. Consistent with this idea, the MIC of vancomycin was lower for the vanX mutant, namely 32 g/ml for LAG3 and 64 g/ml for LAG4, as compared to 128 g/ml for the parental strain M145 (Table 2).

TABLE-US-00002 TABLE 2 Effect of D-alanine on the MIC of vancomycin against S. coelicolor vanX mutants. 0 M 10 M 50 M 100 M D-Ala D-Ala D-Ala D-Ala M145 vanX 32 16 1 1 LAG2 vanX 64 32 8 2

[0072] Without D-Ala, the MIC of vancomycin against the vanX-deleted strains is lower than against the respective parental strains. Micromolar amounts of D-Ala decrease the MIC drastically, while millimolar concentrations are needed to reduce the MIC of vancomycin in the presence of vanX (see Table 1).

[0073] Importantly, after the addition of D-Ala as competitor, the percentage of wild-type precursor increased further, namely 25% after 1 and 5 minutes, 40% after 15-30 minutes, and 80% wild-type precursors 3 hours after the addition of D-Ala. In terms of the effect on the MIC, D-Ala very effectively reduced vancomycin resistance, with very low quantities of D-Ala sufficient to strongly inhibit vancomycin resistance, whereby the MIC of M145 vanX dropped to 1 g/ml in the presence of only 50 g/ml D-Ala. This means that 1000-fold lower D-Ala concentration still effected a 4-fold stronger reduction of the MIC of vancomycin as compared to M145 (Table 1).

Fluorescence Microscopy.

[0074] To visualize the differential binding of vancomycin in the presence and absence of vancomycin, and to ensure that D-Ala indeed enhances the binding of vancomycin to the cell wall, the mycelia of S. coelicolor were stained with the fluorescent dye BODIPY-FL vancomycin (Vanco-FL). In vancomycin-sensitive bacteria, vancomycin localizes in foci at sites of de novo cell wall synthesis (Daniel and Errington, 2003). In Streptomyces coelicolor, the hyphae of which grow at the apex, these sites are, in particular, the hyphal tips and cell division septa (Flrdh, 2003a).

[0075] While hyphae of S. coelicolor M145 were stained very well by vancomycin-FL, barely any fluorescent staining was seen for its mutant derivatives LAG2 or LAG2vanX, which both constitutively express vancomycin resistance (FIG. 4A). However, the addition of D-Ala to LAG2 already caused a slight recovery of Vanco-FL staining at the hyphal tips (FIG. 4B), indicative of a slight recovery of vancomycin binding, while the LAG2vanX mutant, which is hypersensitive to D-Ala and accumulates a large amount of wild-type cell wall precursors (FIGS. 4A and 4B), was stained very well by vancomycin-FL (FIGS. 4A and 4B). Taken together, the mutational, microscopy and LC-MS experiments together show that the enhanced sensitivity to vancomycin in the presence of D-Ala is due to the accumulation of wild-type cell-wall precursors, which are then incorporated into the cell wall and bound by vancomycin.

[0076] The results show that the addition of D-Ala to a vancomycin-resistant strain increases the production and incorporation of peptidoglycan precursors terminating in D-Ala-D-Ala instead of D-Ala-D-Lac and hence, vancomycin sensitivity. A new model for vancomycin resistance is shown in FIG. 5 in which the function of VanA is driven by the offered substrate, which normally consists of both D-Ala and D-Lac. When an excess of D-Ala is added to the medium, the main product of VanA is D-Ala-D-Ala. Residual D-Ala-D-Ala produced by VanA is normally cleaved by VanX but an excess of D-Ala causes a dramatic increase of the D-Ala-D-Ala pool, increasing vancomycin sensitivity. The deletion of vanX represents the effect of an inhibitor of this gene (or enzyme).

Analysis of the Effect of D-Ala on the MIC of Clinical Isolates of VRE

[0077] Having established the important role of D-Ala in enhancing the efficacy of vancomycin against vancomycin-resistant S. coelicolor cells, next, its effect on the resistance of clinical isolates of Enterococcus faecium, which were vanA-positive strains, was assessed. MIC values were calculated by testing a serial dilution of vancomycin in the presence or absence of D-Ala in triplicate (Table 3).

TABLE-US-00003 TABLE 3 Effect of D-Ala of the MIC of vancomycin against clinical isolates of vanA-positive Enterococcus faecium MIC with MIC without 50 mM D-Ala reduction D-Ala (g/ml) (g/ml) of MIC # vanA1 4096 256 16 vanA2 4096 256 16 vanA3 4096 128 32 vanA4 4096 128 32 vanA10 2048 16 128 # approximation based on 2-fold dilution steps.

[0078] The MIC was determined by measurement of the OD.sub.600 or by visual assessment, following the Clinical Laboratory and Standards Institute (CLSI) guidelines. Similarly, as seen for S. coelicolor, addition of 50 mM D-Ala to the growth media resulted in a strong increase in the efficacy of vancomycin against all clinical isolates, with reduction of 4-7 dilution steps. In the worst cases, the MIC of vancomycin was reduced from 4096 g/ml to 256 g/ml or 128 g/ml, while a decrease down to 16 g/ml in strain vanA10 was also noted. This value compares to intermediate instead of total resistance. The strains were serially diluted in a 96-well plate and growth was assayed at OD.sub.600. The steps down refer to CSLI guidelines for clinical MIC measurements.

Materials and Methods

Bacterial Strains, Culturing Conditions and Minimal Inhibitory Concentration (MIC)

[0079] Escherichia coli strains JM109 (Sambrook et al., 1989) and ET12567 (Kieser et al., 2000) were used for routine cloning procedures and for extracting non-methylated DNA, respectively. Cells of E. coli were grown in Luria-Berani broth (LB) at 37 C. Streptomyces coelicolor A3(2) M145 was the parent of all mutants described in this work. All media and routine Streptomyces techniques were carried out as described (Kieser et al., 2000). SFM (soy flour mannitol) agar plates were used for propagating S. coelicolor strains and to prepare spore suspensions. For liquid-grown cultures, S. coelicolor mycelia were grown in normal minimal media with phosphate (NMMP) supplemented with 1% (w/v) mannitol as the sole carbon source. The MIC of vancomycin against S. coelicolor M145 and its mutant derivatives were determined by growth on minimal media (MM) agar plates supplemented with 1% mannitol as the sole carbon source, and 0, 2, 4, 8, 16, 32, 64, 128, 256 or 512 g/ml vancomycin, in combination with 0, 1, 5, 10 or 50 mM of D-Ala or L-Ala. The vanX deletion mutants have been tested with 1, 5, 10, 50 and 100 M D-Ala and L-Ala.

[0080] Five vanA-positive Enterococcus faecium strains were collected in 2011 and 2014 from patients in the Erasmus University Medical Centre, Rotterdam, The Netherlands. Presence of the vanA gene was confirmed by real-time PCR with the LIGHTCYCLER 480 instrument (Roche Diagnostics, Almere, The Netherlands) with the primers vanA F1 and vanA R1, and the vanA probe. The resistance profile of these isolates was determined as MIC values using the VITEK II (BioMerieux) system AST-P586. To determine the MIC of vancomycin against E. faecium, cells were grown overnight on Trypticase Soy Agar (TSA) blood agar plates (Becton Dickinson, Breda, The Netherlands) and suspended in 0.9% NaCl until OD.sub.600 0.5 (0.05). Of this suspension, 10 l was dispensed into wells of sterile flat-bottom 96-well polystyrene tissue culture plates (Greiner Bio-One, Alphen aan Den Rijn, The Netherlands) containing serial dilutions of vancomycin in 190 l of a 1:1 mixture of Fetal Bovine Serum (FBS) (Gibco, Bleiswijk, The Netherlands) and Iscove's Modified Dulbecco's Medium (IMDM) (without phenolred, Gibco, Bleiswijk, The Netherlands), and in the presence or absence of 50 mM D-alanine (Alfa Aesar, Ward Hill, Mass., USA). Plates were incubated for 18-24 hours at 37 C. and MIC values determined visually following the CLSI guidelines, or by spectrophotometer at 600 nm.

Constructs for Gene Disruption and Complementation

Constructs for Gene Disruption

[0081] Deletion mutants were constructed according to (Swiatek et al., 2012). For deletion of ddl, the 948/+20 and +1173/+2638 regions relative to the start of ddl were amplified by PCR using primer pairs ddl LF and ddl LR, and ddl RF and ddl RR. The left and right flanks were cloned into the multi-copy vector pWHM3 (Vara et al., 1989), which is highly unstable in Streptomyces and, therefore, allows efficient gene disruption (van Wezel et al., 2005). Subsequently, the apramycin resistance cassette aac(3)IV flanked by loxP sites was cloned into the engineered XbaI site to create knock-out construct pGWS1152. The same strategy was used to create a construct for the deletion of vanX. In this case, the 1477/+30 and +572/+2035 regions relative to the start of vanX (SC03596) were PCR-amplified using primer pairs vanX LF and vanX LR, and vanX RF and vanX RR. Insertion of aac(3)IV-loxP site in the engineered XbaI site generated knock-out construct pGWS1164.

[0082] The presence of loxP sites allows the efficient removal of the apramycin resistance cassette from the chromosome following the introduction of plasmid pUWLCRE that expresses the Cre recombinase (Fedoryshyn et al., 2008).

Complementation Constructs

[0083] A construct for the genetic complementation of ddl was made by amplifying the promoter- and coding region of ddl using primers ddlcomp F and ddlcomp R (nt positions: 573/+1184 relative to the start of ddl), and inserted as an EcoRI/BamHI fragment in the low copy vector pHJL401 (Larson and Hershberger, 1986), a highly stable low-copy number vector that is well suited for genetic complementation (van Wezel et al., 2000), resulting in pGWS1159.

Fluorescence Microscopy

[0084] Samples were grown for 18 hours in liquid NMMP after which a sample was taken from the culture to stain with BODIPY FL vancomycin (Vanco-FL) as described (Daniel and Errington, 2003; Flrdh, 2003b). Equal amounts of unlabeled vancomycin and Van-FL were added to the sample to a final concentration of 1 g/ml and was incubated for 10-20 minutes at 30 C. Directly after taking the first sample, 50 mM D-Ala was added to the medium and was left to grow for another hour before imaging. Imaging was done as described previously (Willemse and van Wezel, 2009). A Zeiss observer with a Plan-Neofluar 40/0.9 lens was used, and GFP was excited with a wavelength of 488 nm and observed at 515 nm with filter BP505-550, with illumination power set to 7.5%. The images were analyzed with ImageJ, all the fluorescent images were processed identically. The final figure was made with Adobe Photoshop CS6.

Isolation of Peptidoglycan Precursors

[0085] For the precursor isolation and identification, a modification of the method described previously by Hong and colleagues (Hong et al., 2004) was used. 10 g vancomycin was added to the strains at the moment of inoculation. The strains were grown in NMMP (1% (w/v) Mannitol, 50 mM MgCl.sub.2) until mid-log phase (OD-0.3-0.4) and mycelia were harvested by centrifugation at 4 C. and washed in 0.9% NaCl. The mycelium was extracted with 5% cold trichloric acid (TCA) for 30 minutes at 4 C. This was centrifuged and the supernatant desalted on a Sephadex G-25 column (Illustra NAP-10 Columns, GE Healthcare, Pittsburgh), and concentrated by rotary evaporation. The concentrated precursors were dissolved in HPLC-grade water and separated by LC-MS using a gradient of 0-20% acetonitrile in water with 0.1% TFA. The elution was monitored at 254 nm and monitored by the sizes eluted (1193.8-1195.3).

[0086] For the measurement over time, the protocol was adjusted in the following way: 300 ml NMMP cultures were grown until exponential phase, at which point a 10 ml sample was taken (sample t=0) and 50 mM of D-Ala or L-Ala was added, followed by further sampling after 1, 5, 15, 30, 60, 120 and 180 minutes. Samples were rapidly filtered with a vacuum pump and washed with 0.9% (w/v) NaCl, mycelia scraped off the filter and transferred to 5% TCA.

[0087] For the purpose of clarity and a concise description, features are described herein as part of the same or separate aspects and preferred embodiments thereof; however, it will be appreciated that the scope of the disclosure may include embodiments having combinations of all or some of the features described.

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