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Last Updated: March 28, 2024

Claims for Patent: 4,132,599


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Summary for Patent: 4,132,599
Title: Determination of antimicrobial susceptibilities on infected urines without isolation
Abstract:Method for the quick determination of the susceptibilities of various unidentified bacteria contained in an aqueous physiological fluid sample, particularly urine, to one or more antibiotics. A bacterial adenosine triphosphate (ATP) assay is carried out after the elimination of non-bacterial ATP to determine whether an infection exists. If an infection does exist, a portion of the sample is further processed, including subjecting parts of the portion to one or more antibiotics. Growth of the bacteria in the parts are determined, again by an ATP assay, to determine whether the unidentified bacteria in the sample are susceptible to the antibiotic or antibiotics under test.
Inventor(s): Fletcher; James C. Administrator of the National Aeronautics and Space (N/A), N/A (Tantallon, MD), Picciolo; Grace L. (Tantallon, MD), Chappelle; Emmett W. (Baltimore, MD), Deming; Jody W. (Annapolis, MD), Shrock; Christian G. (Eding, MN), Vellend; Hillar (Toronto, CA), Barza; Michael J. (Boston, MA), Weinstein; Louis
Assignee:
Application Number:05/680,015
Patent Claims:1. A method for determining the general susceptibility of bacteria constituting a quantitated infection in a physiological fluid sample to antimicrobial agents without isolation of any individual bacterial strains that may be present by measurement of an ATP index indicating susceptibility, which comprises:

forming a solid containing bacteria by eliminating the liquid phase from the physiological fluid sample;

reconstituting the sample to a liquid by adding an aqueous growth medium to achieve a working inoculum;

pre-incubating the treated sample containing all the bacterial strains present in the physiological fluid to achieve log growth and dividing the sample into at least three portions;

adding sterile water to two portions and an antibiotic to each remaining portion and assaying one portion of said two portions without antibiotic for ATP;

incubating all other portions for a time sufficient for the antibiotic or antibiotics to take effect against the bacteria present, assuming the antibiotic to be effective;

assaying said all other portions for ATP; and determining the ATP index to indicate sensitivity of the bacteria to the antibiotic according to the formula

where

B.sub.t represents an ATP assay reading for the portion or portions treated with an antibiotic and allowed to incubate;

A.sub.t represents an ATP assay reading for the portion not treated with antibiotic and allowed to incubate; and

A.sub.0 represents an ATP assay reading for the portion not treated with antibiotic and not allowed to incubate.

2. A method for determining the general susceptibility of bacteria constituting a possible infection in a urine sample to antimicrobial agents without isolation of any individual bacterial strains that may be present by measurement of an ATP index indicating susceptibility, which comprises:

forming a solid containing bacterial and non-bacterial cells by eliminating the liquid phase from the urine sample;

reconstituting the sample to a liquid by adding a growth medium;

measuring out first and second portions;

storing the second portion under conditions preventing significant bacterial growth;

thereafter treating the first portion by:

adding a hydrolyzing enzyme capable of hydrolyzing ATP and a detergent capable of rupturing non-bacterial cells;

adding an agent capable of freeing bound ATP from non-bacterial cells;

forming a solid containing bacterial cells;

rupturing the bacterial cells;

assaying the treated first portion for ATP to determine the presence of an infection;

adding an aqueous growth medium to the second portion to achieve a working inoculum;

pre-incubating the treated second portion containing all the bacterial strains present in the urine sample to achieve log growth and dividing the second portion into at least three samples;

adding sterile water to two samples and an antibiotic to each remaining sample and assaying one sample of said two samples without antibiotic for ATP;

incubating the all other samples for a time sufficient for the antibiotic or antibiotics to take effect against the bacteria present, assuming the antibiotic to be effective;

assaying said all other samples for ATP; and

determining the ATP index to indicate sensitivity of the bacteria to the antibiotic according to the formula:

where

B.sub.t represents an ATP assay reading for the sample or samples treated with an antibiotic and allowed to incubate;

A.sub.t represents an ATP assay reading for the sample not treated with antibiotic and allowed to incubate; and

A.sub.0 represents an ATP assay reading for the sample not treated with antibiotic and not allowed to incubate.

3. A method for determining the general susceptibility of bacteria constituting a possible infection in a physiological fluid to antimicrobial agents without isolation of particular bacteria by measurement of an ATP index indicating susceptibility, which comprises:

assaying a portion of the fluid for ATP to determine the existence of an infection; and

dividing a remaining portion of the fluid into at least three samples before or after pre-incubation to have bateria which are all those strains present in the untreated physiological fluid present enter log phase growth;

adding sterile water to two samples and an antibiotic to each remaining sample and assaying one sample of said two samples without antibiotic for ATP;

incubating all other samples for period sufficient for significant bacterial growth in the absence of an effective antibiotic;

assaying said all other samples for ATP; and

determining the ATP index to indicate sensitivity of the bacteria to the antibiotic according to the formula:

wherein

B.sub.t represents an ATP assay reading for the sample or samples treated with an antibiotic and allowed to incubate;

A.sub.t represents an ATP assay reading for the sample not treated with antibiotic and allowed to incubate; and

A.sub.0 represents an ATP assay reading for the sample not treated with antibiotic and not allowed to incubate.

4. The method of claim 1 wherein said bacteria are derived from a body fluid selected from the group consisting of urine, ascites, lymph fluid, plasma, blood, spinal fluid, saliva and mucus.

5. The method of claim 1 wherein said solid is formed by centrifugation.

6. The method of claim 1 wherein said growth medium is trypticase soy broth.

7. The method of claim 1 wherein the working inoculum is from between 10.sup.5 and 10.sup.6 CFU/ml.

8. The method of claim 1 wherein pre-incubation is for about 1/2 hour at about 37.degree. C.

9. The method of claim 1 wherein incubation is for about 2.5 hours at about 37.degree. C.

10. The method of claim 1 wherein all ATP assays are light readings.

11. The method of claim 2 wherein the solid is a pellet formed from the urine sample by centrifuging at about 8000 rpm for about 15 minutes at 4.degree. C.

12. The method of claim 2 wherein the solid is reconstituted by adding sufficient trypticase soy broth to obtain about a twenty-times concentration of the urine sample.

13. The method of claim 2 wherein said first and second portions are at least about 0.5 ml and 1.25 ml, respectively.

14. The method of claim 2 wherein the second portion is stored at about 4.degree. C.

15. The method of claim 2 wherein the hydrolyzing enzyme and detergent are apyrase and octylphenoxyl plyethoxyethanol, respectively.

16. The method of claim 2 wherein the agent capable of freeing bound ATP is malic acid.

17. The method of claim 2 wherein the solid containing bacterial cells is a pellet formed by centrifuging at about 8000 rpm for about 15 minutes at about 4.degree. C.

18. The method of claim 2 wherein the bacterial cells are ruptured by the addition of nitric acid.

19. The method of claim 2 wherein trypticase soy broth is added to the second portion to achieve a working inoculum of from about 10.sup.5 to 10.sup.6 CFU/ml.

20. The method of claim 2 wherein pre-incubation of the treated second portion is for about 0.5 hours and at about 37.degree. C.

21. The method of claim 2 wherein incubation of the samples is for about 2.5 hours and at about 37.degree. C.

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