Methods of reducing microbial resistance to drugs
View Patent ↗The instant methods and compositions represent an advance in controlling drug resistance in microbes. AcrAB-like efflux pumps have been found to control resistance to drugs, even in highly resistant microbes. Accordingly, methods of treating infection, methods of screening for inhibitors of AcrAB-like efflux pumps, and methods of enhancing antimicrobial activity of drugs are provided. Pharmaceutical composition comprising an inhibitor of an AcrAB-like efflux pump and an antimicrobial agent are also provided.
1. A method of screening for compounds which reduce antibiotic resistance in a highly antibiotic resistant microbe comprising an AcrAB or AcrAB-like efflux pump, wherein the microbe comprises at least two of the following traits: i) at least one chromosomal mutation in a gene encoding an antibiotic target that renders the microbe resistant to one or more antibiotics; ii) a second mutation, to the same gene or a different gene than in (i), that further increases antibiotic resistance, and iii) increased expression of at least one efflux pump, comprising: contacting the microbe with a test compound and measuring the effect of the test compound on the activity of the AcrAB or AcrAB-like efflux pump, wherein compounds which inhibit the activity of the AcrAB or AcrAB-like efflux pump are identified as compounds which reduce antibiotic resistance in the microbe.
2. The method of claim 1 , wherein the microbe comprises all three of the traits.
3. The method of claim 1 , wherein the microbe is highly resistant to fluoroquinolones.
4. The method of claim 1 , wherein the at least one chromosomal mutation is present in a gene selected from the group consisting of: gyrase and topoisomerase.
5. The method of claim 1 , wherein the microbe is a Gram negative bacterium.
6. The method of claim 1 , wherein the microbe further comprises functional porin channels.
7. The method of claim 1 , wherein the microbe is contacted with test compounds selected from a library of test compounds.
8. The method of claim 1 , wherein the activity of the AcrAB or AcrAB-like efflux pump is determined by measuring efflux of an indicator compound which is a substrate of the efflux pump.
9. The method of claim 1 , wherein the activity of the AcrAB or AcrAB-like efflux pump is determined by measuring growth of the microbe in an antibiotic.
10. The method of claim 1 , wherein the efflux pump is AcrAB.
11. A method of screening for compounds which specifically inhibit the activity of an AcrAB or AcrAB-like efflux pump comprising:
i) contacting a microbe comprising an AcrAB or AcrAB-like efflux and a non-AcrAB or non-AcrAB-like efflux pump and at least two of the following traits: i) at least one chromosomal mutation in a gene encoding a antibiotic target that renders the microbe resistant to one or more antibiotics; ii) a second mutation, to the same gene or a different gene than in (i), that further increases antibiotic resistance, and iii) increased expression of at least one efflux pump, with a test compound and an indicator compound;
ii) testing the ability of the test compound to inhibit the activity of the AcrAB or AcrAB-like efflux pump;
iii) testing the ability of the test compound to inhibit the activity of the non-AcrAB or non-AcrAB efflux pump;
iv) and identifying compounds which inhibit the activity of the AcrAB or AcrAB-like efflux pump relative to the non-AcrAB or non-AcrAB-like efflux pump to thereby identify compounds which specifically inhibit the activity of the AcrAB or AcrAB-like efflux pump.
12. The method of claim 11 , wherein the microbe is highly antibiotic resistant.
13. The method of claim 11 , wherein the microbe is highly resistant to fluoroquinolones.
14. The method of claim 11 , wherein the at least one mutation is present in a gene selected from the group consisting of: gyrase and topoisomerase.
15. The method of claim 11 , wherein the microbe is a Gram negative bacterium.
16. The method of claim 11 , wherein the microbe further comprises functional porin channels.
17. The method of claim 11 , wherein the microbe is contacted with test compounds selected from a library of test compounds.
18. The method of claim 11 , wherein the activity of the AcrAB or AcrAB-like efflux pump is determined by measuring efflux of an indicator compound which is a substrate of the efflux pump.
19. The method of claim 11 , wherein growth of the microbe in an antibiotic is measured.
20. The method of claim 11 , wherein the efflux pump is AcrAB.
21. A method of treating an infection in a subject caused by a microbe comprising an AcrAB or AcrAB-like efflux pump and at least two of the following traits: i) at least one chromosomal mutation in a gene encoding a antibiotic target that renders the microbe resistant to one or more antibiotics; ii) a second mutation, to the same gene or a different gene than in (i), that further increases antibiotic resistance, and iii) increased expression of at least one efflux pump, comprising: contacting the microbe with an antibiotic to which the microbe is resistant and an inhibitor of an acrAB or acrAB-like efflux pump such that the infection in the subject is treated.
22. The method of claim 21 , wherein the subject is treated prophylactically.
23. The method of claim 21 , wherein the subject is treated therapeutically.
24. The method of claim 21 , wherein the microbe is highly resistant to fluoroquinolones.
25. The method of claim 21 , wherein the at least one mutation is present in a gene selected from the group consisting of: gyrase and topoisomerase.
26. The method of claim 21 , wherein the microbe is a Gram negative bacterium.
27. The method of claim 21 , wherein the microbe further comprises functional porin channels.