Claims
- 1. A method of identifying a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent, the method comprising:
a) providing a sample comprising an efflux pump selected from the group consisting of:
i) an efflux pump comprising a polypeptide comprising SEQ ID NO:4 (gene PA1874), ii) an efflux pump comprising a polypeptide comprising SEQ ID NO: 12 (gene PA4142), iii) an efflux pump comprising a polypeptide comprising SEQ ID NO:18 (gene PA2389), b) contacting the sample with a test compound; and c) measuring activity of the efflux pump, wherein a change in the activity of the efflux pump in the presence of the test compound relative to the activity of the efflux pump in a control sample in the absence of the test compound, indicates that the compound is a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent.
- 2. The method of claim 1 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO:4 (gene PA1874), further comprises at least one of: a polypeptide comprising SEQ ID NO:6 (gene PA1875), a polypeptide comprising SEQ ID NO:8 (gene PA1876), and
- 3. The method of claim 1 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO:12 (gene PA4142), further comprises at least one of: a polypeptide comprising SEQ ID NO:14 (gene PA4143), and a polypeptide comprising SEQ ID NO:16 (gene PA4144).
- 4. The method of claim 1 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO:18 (gene PA2389), further comprises at least one of: a polypeptide comprising SEQ ID NO:20 (gene PA2390), and a polypeptide comprising SEQ ID NO:22 (gene PA2391).
- 5. A method of identifying a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent, the method comprising:
a) providing a sample comprising a polypeptide selected from the group consisting of:
i) a polypeptide comprising SEQ ID NO:8 (gene PA1876), ii) an efflux pump comprising a polypeptide comprising SEQ ID NO: 14 (gene PA4143), iii) an efflux pump comprising a polypeptide comprising SEQ ID NO:20 (gene PA2390), b) contacting the sample with a test compound; and c) measuring the ATPase activity of the polypeptide, wherein a change in the ATPase activity of the polypeptide in the presence of the test compound relative to the activity of the polypeptide in a control sample in the absence of the test compound, indicates that the compound is a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent.
- 6. The method of claim 1 wherein the sample comprises cells expressing the polypeptide.
- 7 The method of claim 1 wherein the sample comprises cells harboring an expression vector encoding the polypeptide.
- 8. The method of claim 1 wherein the cells are grown in a biofilm.
- 9. The method of claim 1 wherein the cells are grown planktonically.
- 10. The method of claim 1 wherein the sample comprises vesicles containing the efflux pump or a membrane system containing the efflux pump.
- 11. The method of claim 1 wherein the method comprises measuring the activity of two or more efflux pumps selected from the group consisting of:
i) an efflux pump comprising a polypeptide comprising SEQ ID NO:4 (gene PA1874), ii) an efflux pump comprising a polypeptide comprising SEQ ID NO: 12 (gene PA4142), and iii) an efflux pump comprising a polypeptide comprising SEQ ID NO:18 (gene PA2389).
- 12. A method of identifying a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent, the method comprising:
a) providing a sample comprising a glucosyltransferase polypeptide selected from the group consisting of:
i) a polypeptide comprising SEQ ID NO:2 (gene PA1163), ii) a polypeptide comprising SEQ ID NO:27, iii) a polypeptide comprising SEQ ID NO:28, iv) a polypeptide comprising SEQ ID NO:29, and v) a polypeptide comprising SEQ ID NO:32, b) contacting the sample with a test compound; and c) measuring activity of the glucosyltransferase polypeptide, wherein a change in the activity of the glucosyltransferase polypeptide in the presence of the test compound relative to the activity of the glucosyltransferase polypeptide in a control sample in the absence of the test compound, indicates that the compound is a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent.
- 13. The method of claim 12 wherein the glucosyltransferase polypeptide is contacted with the test compound in the presence of an antimicrobial agent.
- 14. The method of claim 12 wherein the sample comprises cells expressing the glucosyltransferase polypeptide.
- 15. The method of claim 12 wherein the sample comprises cells harboring an expression vector encoding the glucosyltransferase polypeptide.
- 16. The method of claim 14 wherein the cells are grown as a biofilm.
- 17. The method of claim 14 wherein the cells are grown planktonically.
- 18. The method of claim 12 wherein activity is measured by measuring cyclic-b-(1,3)-glucan formation.
- 19. A method of identifying a candidate compound for altering the sensitivity of a microoganism to an antimicrobial agent, the method comprising:
a) providing a sample comprising cells expressing a gene encoding a glucosyltransferase polypeptide selected from the group consisting of:
i) a polypeptide comprising SEQ ID NO:2 (gene PA1163), ii) a polypeptide comprising SEQ ID NO:27, iii) a polypeptide comprising SEQ ID NO:28, iv) a polypeptide comprising SEQ ID NO:29, and v) a polypeptide comprising SEQ ID NO:32, b) contacting the sample with a test compound; and c) measuring the expression of a gene encoding a glucosyltransferase polypeptide in the cells, wherein a change in the expression of the gene encoding the glucosyltransferase polypeptide in the presence of the test compound relative to the expression of the gene encoding the glucosyltransferase polypeptide in a control sample in the absence of the test compound, indicates that the compound is a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent.
- 20. The method of claim 19, wherein the cells are contacted with the test compound in the presence of an antimicrobial agent.
- 21. The method of claim 19 wherein the expression of the gene is measured by measuring mRNA expression.
- 22. The method of claim 19 wherein expression of the gene is measured by measuring polypeptide expression.
- 23. The method of claim 19 wherein the cells contacted with the test compound are growing as a biofilm.
- 24. A method of identifying a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent, the method comprising:
a) providing a sample of cells expressing an efflux pump selected from the group consisting of:
i) an efflux pump comprising a polypeptide comprising SEQ ID NO: 4 (gene PA1874), ii) an efflux pump comprising a polypeptide comprising SEQ ID NO: 12 (gene PA4142), iii) an efflux pump comprising a polypeptide comprising SEQ ID NO: 18 (gene PA2389), b) contacting the cells with a test compound; and c) measuring expression of the efflux pump in the cells wherein a change in the expression of the efflux pump in the presence of the test compound relative to the expression of the efflux pump in a control sample in the absence of the test compound, indicates that the test compound is a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent.
- 25. The method of claim 24 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO: 4 (gene PA1874), further comprises at least one of: a polypeptide comprising SEQ ID NO:6 (gene PA1875), a polypeptide comprising SEQ ID NO:8 (gene PA1876), and a polypeptide comprising SEQ ID NO:10 (gene PA1877).
- 26. The method of claim 24 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO:12 (gene PA4142), further comprises at least one of: a polypeptide comprising SEQ ID NO:14 (gene PA4143), and a polypeptide comprising SEQ ID NO: 16 (gene PA4144).
- 27. The method of claim 24 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO:18 (gene PA2389), further comprises at least one of: a polypeptide comprising SEQ ID NO:20 (gene PA2390), and a polypeptide comprising SEQ ID NO:22 (gene PA2391).
- 28. The method of claim 24 wherein the cells are contacted with the test compound in the presence of an antimicrobial agent.
- 29. The method of claim 24 wherein expression is measured by measuring polypeptide expression.
- 30. The method of claim 24 wherein expression is measured by measuring mRNA expression.
- 31. The method of claim 24 wherein the cells are grown as a biofilm.
- 32. The method of claim 24 wherein the cells are grown planktonically.
- 33. A purified polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:4 (gene PA1874), SEQ ID NO:6 (gene PA1875), SEQ ID NO:8 (gene PA1876), SEQ ID NO:10 (gene PA1877), SEQ ID NO:12 (gene PA4142), SEQ ID NO:14 (gene PA4143), SEQ ID NO:16 (gene PA4144), SEQ ID NO:18 (gene PA2389), SEQ ID NO:20 (gene PA2390), SEQ ID NO:22 (gene PA2391).
- 34. A purified polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO:2 (gene PA1163), SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:31.
- 35. A method of identifying a gene that functions in biofilm-related resistance to an antimicrobial agent, the method comprising:
(a) providing a library of clones of a selected microbial strain that have been subjected to mutagenesis; (b) growing each of a plurality of clones from the library as a biofilm; (c) identifying a clone having altered biofilm-related resistance to the antimicrobial agent relative the to biofilm-related resistance of the selected microbial stain; (d) isolating from the identified clone a gene having a mutation.
- 36. The method of claim 35, wherein said antimicrobial agent is an antibiotic.
- 37. The method of claim 35 wherein the mutagenesis comprises random mutagenesis.
- 38. The method of claim 35 wherein the selected strain exhibits at least a two-fold change in resistance to the anti-microbial agent.
- 39. The method of claim 35 wherein the microorganism is a bacterial microorganism.
- 40. The method of claim 35 wherein the microorganism is a fungal microorganism.
- 41. The method of claim 35 wherein the mutagenesis comprises chemical mutagenesis.
- 42. A method of identifying a compound that increases the sensitivity of a microorganism to a selected antimicrobial agent, the method comprising:
(a) providing a sample comprising a microorganism in a biofilm; (b) contacting the sample with a test compound and the selected antimicrobial agent; and (c) measuring the sensitivity of the microorganism in the sample to the selected antimicrobial agent, wherein an increase in the sensitivity of the microorganism to the selected antimicrobial agent relative to the sensitivity of the microorganism in a biofilm to the selected antimicrobial agent in the absence of the test compound indicates that the test compound is a compound that increases the sensitivity of microorganism in a biofilm to the selected antimicrobial agent.
- 43. The method of claim 42, wherein the antimicrobial agent is selected from the group consisting of aminoglycosides, macrolides, tetracyclines, penicillins, P-lactam antibiotics (including cephalosporins, β-lactam/β-lactamase combinations), quinolones (including fluoroquinolones), glycopeptides, sulfonamides, sulfones, oxazolidinones, streptogramins.
- 44. The method of claim 42, wherein the microorganism is selected from the group consisting of Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Pseudomonas syringae, Pseudomonas aureofaciens, Pseudomonas fragi, Fusobacterium nucleatum, Treponema denticola, Porphyromonas gingivalis, Moraxella catarrhalis, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudo tuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Pasteurella multocida, Pasteurella haemolytica, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio paramaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Gardnerella vaginalis, Bacteroides spp., Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycrobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus spp., Enterococcus spp., Desulfvibrio spp., Actinomyces spp., Erwinia spp., Xanthomonas spp., Xylella spp., Clavibacter spp., Desulfomonas spp., Desulfovibrio spp., Desulfococcus spp., Desulfobacter spp., Desulfobulbus spp., Desulfosarcina spp., Deslfuromonas spp., Bacillus spp., Streptomyces spp., Clostridium spp., Rhodococcus spp., Thermatoga spp., Sphingomonas spp., Zymomonas spp., Micrococcus spp., Azotobacter spp., Norcardia spp., Brevibacterium spp., Alcaligenes spp., Microbispora spp., Micromonospora spp., Methylobacterium organophilum, Pseudomonas reptilivora, Pseudomonas carragienovora, Pseudomonas dentificans, Corynebacterium spp., Propionibacterium spp., Xanothomonas spp., Methylobacterium spp., Chromobacterium spp., Saccharopolyspora spp., Actinobacillus spp., Alteromonas spp., Aeronomonas spp., Agrobacterium tumefaciens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus warneri, Staphylococcus cohnii, Staphylococcus saprophyticus, Staphylococcus capitis, Staphylococcus lugdunensis, Staphlyococcus intermedius, Staphylococcus hyicus, Staphylococcus saccharolyticus and Rhizobium spp.
- 45. The method of claim 42 wherein the biofilm is associated with an abiotic surface.
- 46. The method of claim 42 wherein the biofilm is associated with a biotic surface.
- 47. The method of claim 42 wherein the biofilm is not associated with a surface.
- 48. The method of claims 42 wherein the microorganism has a mutation that reduces the expression or activity of a polypeptide selected from the group consisting of: SEQ ID NO:4 (gene PA1874), SEQ ID NO:6 (gene PA1875), SEQ ID NO:8 (gene PA1876), SEQ ID NO:10 (gene PA1877), SEQ ID NO:12 (gene PA4142), SEQ ID NO:14 (gene PA4143), SEQ ID NO:16 (gene PA4144), SEQ ID NO:18 (gene PA2389), SEQ ID NO:20 (gene PA2390), SEQ ID NO:22 (gene PA2391).
- 49. The method of claims 42 wherein the microorganism has a mutation that reduces the expression or activity of a polypeptide selected from the group consisting of: SEQ ID NO:2 (gene PA1163), SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:31.
- 50. The method of claim 49 wherein the mutation is an insertion the gene encoding the polypeptide.
- 51. The method of claim 49 wherein the mutation is a deletion the gene encoding the polypeptide.
- 52. A method of identifying a candidate compound for altering the sensitivity of a microoganism to an antimicrobial agent, the method comprising:
a) providing a sample comprising cells harboring a reporter gene comprising a nucleotide sequence encoding a detectable protein operably linked to an expression control sequence comprising a nucleotide sequence selected from the group consisting of:
i) SEQ ID NO:23 (expression control for PA1874), ii) SEQ ID NO:24 (expression control for PA4242), iii) SEQ ID NO:25 (expression control for PA2389), and iv) SEQ ID NO:26 (expression control for PA1163), b) measuring the expression of the reporter gene in the cells in the presence of a test compound, wherein a change in the expression of the reporter gene in the presence of the test compound relative to the expression of the reporter gene in a control sample in the absence of the test compound, indicates that the compound is a candidate compound for altering the sensitivity of a microorganism to an antimicrobial agent.
- 53. The method of claim 52 wherein expression of the reporter gene is measured by measuring the expression of the detectable protein.
- 54. A method of identifying a candidate compound for inhibiting the growth of a microorganism, the method comprising:
a) providing a sample comprising an efflux pump selected from the group consisting of:
i) an efflux pump comprising a polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:4 (gene PA1874), ii) an efflux pump comprising a polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO: 12 (gene PA4142), iii) an efflux pump comprising a polypeptide comprising SEQ ID NO:18 (gene PA2389), b) contacting the sample with a test compound; and c) measuring activity of the efflux pump, wherein a change in the activity of the efflux pump in the presence of the test compound relative to the activity of the efflux pump in a control sample in the absence of the test compound, indicates that the compound is a candidate compound for inhibiting the growth of a microorganism.
- 55. The method of claim 54 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO:4 (gene PA1874), further comprises at least one of: a polypeptide comprising SEQ ID NO:6 (gene PA1875), a polypeptide comprising SEQ ID NO:8 (gene PA1876), and a polypeptide comprising SEQ ID NO:10 (gene PA1877).
- 56. The method of claim 54 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO:12 (gene PA4142), further comprises at least one of: a polypeptide comprising SEQ ID NO:14 (gene PA4143), and a polypeptide comprising SEQ ID NO:16 (gene PA4144).
- 57. The method of claim 54 wherein the efflux pump comprising a polypeptide comprising SEQ ID NO:18 (gene PA2389), further comprises at least one of: a polypeptide comprising SEQ ID NO:20 (gene PA2390), and a polypeptide comprising SEQ ID NO:22 (gene PA2391).
- 58. The method of claim 54 wherein the sample comprises cells expressing the polypeptide.
- 59. The method of claim 54 wherein the cells are grown in a biofilm.
- 60. The method of claim 54 wherein the cells are grown planktonially.
- 61. A method of identifying a candidate compound that inhibits growth of a microorganism, the method comprising:
a) providing a sample comprising a glucosyltransferase polypeptide selected from the group consisting of:
i) a polypeptide comprising SEQ ID NO:2 (gene PA1163), ii) a polypeptide comprising SEQ ID NO:27, iii) a polypeptide comprising SEQ ID NO:28, iv) a polypeptide comprising SEQ ID NO:29, and v) a polypeptide comprising SEQ ID NO:32, b) contacting the sample with a test compound; and c) measuring activity of the glucosyltransferase polypeptide, wherein a change in the activity of the glucosyltransferase polypeptide in the presence of the test compound relative to the activity of the glucosyltransferase polypeptide in a control sample in the absence of the test compound, indicates that the compound is a candidate compound inhibiting the growth of a microorganism.
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional Application No. 60/323,241, filed Sep. 18, 2001, the entirety of which is incorporated by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60323241 |
Sep 2001 |
US |