Methods to study and mechanisms of biofilm-related antibiotic resistance
AssigneeTrustees of Dartmouth College
InventorsGeorge A. O'Toole, Thien-Fah Mah
Life of this patent
Estimating the term…
Abstract
Disclosed are various genes encoding proteins that are shown to play a role, direct or indirect, in microbial resistance of an organism in a biofilm and homologs thereof. Also disclosed are methods of identifying a compound that modulates microbial resistance of an organism in a biofilm, and methods of identifying genes that encode proteins that play a role, direct or indirect, in biofilm resistance.
Claims61
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.
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.
7 The method of claim 1 wherein the sample comprises cells harboring an expression vector encoding the polypeptide.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
Dependencies are inferred from the claim text.