WO1986007094A1 - Bacterial cell extractant - Google Patents

Bacterial cell extractant Download PDF

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Publication number
WO1986007094A1
WO1986007094A1 PCT/GB1986/000285 GB8600285W WO8607094A1 WO 1986007094 A1 WO1986007094 A1 WO 1986007094A1 GB 8600285 W GB8600285 W GB 8600285W WO 8607094 A1 WO8607094 A1 WO 8607094A1
Authority
WO
WIPO (PCT)
Prior art keywords
reagent
atp
tetraacetic acid
cells
bacterial cell
Prior art date
Application number
PCT/GB1986/000285
Other languages
French (fr)
Inventor
Michael John Harber (Deceased)
Original Assignee
University Of Wales College Of Medicine
HARBER, F., E. (executor and legal representative
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Wales College Of Medicine, HARBER, F., E. (executor and legal representative filed Critical University Of Wales College Of Medicine
Priority to DE8686903473T priority Critical patent/DE3678925D1/en
Publication of WO1986007094A1 publication Critical patent/WO1986007094A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • ATP adenosine triphosphate
  • the adenosine triphosphate (ATP) level in bacterial cultures may be used as a measure of bacterial cell numbers, this being an important consideration in the fields of medical micro- biology, food and dairy microbiology and environmental microbiology. Extracted ATP is easily measured using the firefly bioluminescence assay, and several medical applications of this methodology have been described including a detection test for bacteriuria, antimicrobial susceptibility tests and assays for antibiotics in clinical-specimens. The method also provides a valuable research tool.
  • the invention consists in a reagent for use in extracting substances such as ATP (Adenosine Triphosphate) from bacterial cells, comprising a quaternary ammonium compound.
  • substances such as ATP (Adenosine Triphosphate) from bacterial cells, comprising a quaternary ammonium compound.
  • the compound is Benzalkonium chloride or tetradecyltrimethyl-ainmonium bromide.
  • the reagent includes a calcium chelator.
  • the chelator may comprise for example ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol tetraacetic acid (EGTA) .
  • the invention consists in a method of detecting, analysing, or quantifying bacterial cells in which one of the constituents of the cells such as ATP is extracted using a reagent as defined and the extracted constituent is then detected or quantified by means of a lumin ⁇ escence assay or high performance liquid chromatography.
  • the major component oi: the reagent is benzalkonium chloride.
  • This is a quaternary ammonium compound which consists predominently of C 12 H 25 NC 9 H 13 C . but also contains C ⁇ , and C- 6 homologues. Alternatively it may be defined as a mixture of alkyldimethylbenzylammonium chlorides of the general formula
  • Benzalkonium chloride 1.0 mg/ml in water plus ethylenediamine tetraacetic acid at 10 ⁇ M .
  • EDTA ethylenediamine tetraacetic a.cid
  • the optimum concentrations of the components of the reagent were determined for the extraction of ATP from Escherichia coli growing in nutrient broth no.2 or human urine, or suspended in phosphate- buffered saline pH 7.3 (PBS) .
  • the reagent was found to be equally effective for ATP extraction over the pH range 5,0 to 8.0, and extraction was achieved within 5-10 seconds in a single, easy step.
  • the reagent has been compared with other existing extractants for extracting ATP from urinary isolates of E.coli, Klebsiella edwardsii; Pseudomonas aeruginosa, Proteus mirabilis,- Staphylococcus aureus and Streptococcus faecalis.
  • the reagent was as efficient as the other reagents for extracting ATP from bacterial suspension in PBS, and was consistently more efficient than the others for extracting ATP from broth and urine cultures.
  • the commercial reagents, plus the reagent were all more convenient to use than 2% TCA because extracts prepared with the latter reagent had to be diluted extensively prior to assay to overcome the quenching of light emission produced by this strong acid.
  • the reagent showed no loss of activity on o storage of aqueous solutions at 4 C for 6 months.
  • the reagent provides a simple, rapid method for extracting bacterial ATP and gives a more consistent extraction efficiency than commercial counterparts when used to extract ATP from bacterial cells under a variety of conditions.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A reagent for extracting an Adenosine Triphosphate from bacterial cells, comprises Benzalkonium chloride or tetradecyltrimethylammonium bromide, together with a calcium chelator in the form of ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol tetraacetic acid (EGTA).

Description

"Bacterial Cell Extractant"
The adenosine triphosphate (ATP) level in bacterial cultures may be used as a measure of bacterial cell numbers, this being an important consideration in the fields of medical micro- biology, food and dairy microbiology and environmental microbiology. Extracted ATP is easily measured using the firefly bioluminescence assay, and several medical applications of this methodology have been described including a detection test for bacteriuria, antimicrobial susceptibility tests and assays for antibiotics in clinical-specimens. The method also provides a valuable research tool.
Broadly stated the invention consists in a reagent for use in extracting substances such as ATP (Adenosine Triphosphate) from bacterial cells, comprising a quaternary ammonium compound.
Preferably the compound is Benzalkonium chloride or tetradecyltrimethyl-ainmonium bromide. According to a preferred feature of the invention the reagent includes a calcium chelator. The chelator may comprise for example ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol tetraacetic acid (EGTA) .
From another aspect the invention consists in a method of detecting, analysing, or quantifying bacterial cells in which one of the constituents of the cells such as ATP is extracted using a reagent as defined and the extracted constituent is then detected or quantified by means of a lumin¬ escence assay or high performance liquid chromatography.
In a particular preferred form of the invention the major component oi: the reagent is benzalkonium chloride. This is a quaternary ammonium compound which consists predominently of C12H25NC9H13C. but also contains C~ , and C-6 homologues. Alternatively it may be defined as a mixture of alkyldimethylbenzylammonium chlorides of the general formula
Figure imgf000004_0001
J Cl in which R represents a mixture of the alkyls from CgH17 to C.gH,7. A possible alternative is tetradecyltrimethyl-ammonium bromide. The benzalkonium chloride has been tested at a wide range of concentrations (0.1 to 10.0 μg/ml) , with added ethylenediamine tetraacetic acid in the range 1.0 to 50.0 μM, dissolved in water or buffered solutions over the pH range 5.0 to 8.8. The formulatio which was found to give the most consistently high efficiency for extracting ATP from a variety of bacterial genera was as follows:-
Benzalkonium chloride - 1.0 mg/ml in water plus ethylenediamine tetraacetic acid at 10 μM . •The ethylenediamine tetraacetic a.cid (EDTA) probably serves two functions. Firstly, it is known to increase the permeability of cell membranes and so probably contributes to the extraction process. Secondly, it inhibits ATP- degrading enzymes so that the released ATP is stable and can therefore be easily measured. The latter appears to be the more important function. Both aspects are dependent on the capacity of EDTA to chelate divalent cations, particularly calcium. The calcium chelator ethyleneglycol tetraacetic acid (EGTA) may be used instead. The optimum concentrations of the components of the reagent were determined for the extraction of ATP from Escherichia coli growing in nutrient broth no.2 or human urine, or suspended in phosphate- buffered saline pH 7.3 (PBS) . The reagent was found to be equally effective for ATP extraction over the pH range 5,0 to 8.0, and extraction was achieved within 5-10 seconds in a single, easy step.
The reagent has been compared with other existing extractants for extracting ATP from urinary isolates of E.coli, Klebsiella edwardsii; Pseudomonas aeruginosa, Proteus mirabilis,- Staphylococcus aureus and Streptococcus faecalis. The reagent was as efficient as the other reagents for extracting ATP from bacterial suspension in PBS, and was consistently more efficient than the others for extracting ATP from broth and urine cultures. The commercial reagents, plus the reagent, were all more convenient to use than 2% TCA because extracts prepared with the latter reagent had to be diluted extensively prior to assay to overcome the quenching of light emission produced by this strong acid.
The reagent showed no loss of activity on o storage of aqueous solutions at 4 C for 6 months. The reagent provides a simple, rapid method for extracting bacterial ATP and gives a more consistent extraction efficiency than commercial counterparts when used to extract ATP from bacterial cells under a variety of conditions.

Claims

1. A reagent for use in extracting substances such as ATP (Adenosine Triphosphate) from bacterial cells, comprising a quaternary ammonium compound.
2. A reagent according to Claim 1, in which the compound is Benzalkonium chloride or tetradecyltrimethyl-ammonium bromide.
3. A reagent according to Claim 1 or Claim 2, including a calcium chelator.
4. A reagent according to Claim 3, in which the chelator comprises ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol tetraacetic acid (EGTA) .
5. A method of detecting, analysing, or quantifying bacteria cells in which one of the constituents of the cells such as ATP is extracted using a reagent as defined and the extracted constituent is then detected or quantified by means of an immunological or other specific labelling technique.
PCT/GB1986/000285 1985-05-22 1986-05-21 Bacterial cell extractant WO1986007094A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DE8686903473T DE3678925D1 (en) 1985-05-22 1986-05-21 EXTRACTION AGENT FOR BACTERIA CELLS AND METHOD FOR EXTRACTING A SUBSTANCE.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB858513001A GB8513001D0 (en) 1985-05-22 1985-05-22 Bacterial cell extractant
GB8513001 1985-05-22

Publications (1)

Publication Number Publication Date
WO1986007094A1 true WO1986007094A1 (en) 1986-12-04

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1986/000285 WO1986007094A1 (en) 1985-05-22 1986-05-21 Bacterial cell extractant

Country Status (3)

Country Link
EP (1) EP0229094B1 (en)
GB (1) GB8513001D0 (en)
WO (1) WO1986007094A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0309184A2 (en) * 1987-09-22 1989-03-29 Lumac Bv Method for ATP extraction
WO1990004647A1 (en) * 1988-10-26 1990-05-03 Hans Funke Sampling device
EP0441469A1 (en) * 1990-01-19 1991-08-14 Promega Corporation Immunospecific and bioluminescent assay of cellular ATP
US5258285A (en) * 1987-05-21 1993-11-02 A/S Foss Electric Holding Method for detection of bacterial concentration in a sample
WO1995025174A1 (en) * 1994-03-16 1995-09-21 Foss Electric A/S Novel method for the conditioning of liquid samples

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2266520A1 (en) * 1974-04-02 1975-10-31 Conn Richard Disinfectant concentrate contg alkyl benzalkonium halides - as the active constituent
EP0018825A1 (en) * 1979-05-02 1980-11-12 National Research Development Corporation A process for the identification of bacteria and a kit of reagents for use in this process
GB2059990A (en) * 1979-07-24 1981-04-29 Kolehmainen S Enhancement of the sensitivity of viable microbial cell count by bioluminescent assay of ATP and its use for identification of microbes by means of a short incubation in nutrient media
GB2116709A (en) * 1982-03-09 1983-09-28 Wira & Mather Detecting surface mildew
EP0101398A1 (en) * 1982-07-21 1984-02-22 Packard Instrument Company, Inc. Method of concentrating and measuring unicellular organisms
EP0108303A1 (en) * 1982-11-01 1984-05-16 Miles Laboratories, Inc. Method for detection and isolation of a microorganism

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2266520A1 (en) * 1974-04-02 1975-10-31 Conn Richard Disinfectant concentrate contg alkyl benzalkonium halides - as the active constituent
EP0018825A1 (en) * 1979-05-02 1980-11-12 National Research Development Corporation A process for the identification of bacteria and a kit of reagents for use in this process
GB2059990A (en) * 1979-07-24 1981-04-29 Kolehmainen S Enhancement of the sensitivity of viable microbial cell count by bioluminescent assay of ATP and its use for identification of microbes by means of a short incubation in nutrient media
GB2116709A (en) * 1982-03-09 1983-09-28 Wira & Mather Detecting surface mildew
EP0101398A1 (en) * 1982-07-21 1984-02-22 Packard Instrument Company, Inc. Method of concentrating and measuring unicellular organisms
EP0108303A1 (en) * 1982-11-01 1984-05-16 Miles Laboratories, Inc. Method for detection and isolation of a microorganism

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 84, No. 15, 12 April 1976, (Columbus, Ohio, US), R.M.E. RICHARDS et al.: "Electron Microscope Study on the Effect on Benzalkonium Chloride and Edetate Disodium on Cell Envelope of Pseudomonas Aeruginosa", see page 115, Abstract No. 100095n, & J. Pharm. Sci., 1976, 76-80 (ENG.) *
Experientia, Vol. 37, No. 11, November 1981, (Basel, CH), H. FEY et al.: "Experiments for the Development of a Salmonella Elisa", see page 7 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5258285A (en) * 1987-05-21 1993-11-02 A/S Foss Electric Holding Method for detection of bacterial concentration in a sample
EP0309184A2 (en) * 1987-09-22 1989-03-29 Lumac Bv Method for ATP extraction
EP0309184B1 (en) * 1987-09-22 1993-08-04 Lumac Bv Method for atp extraction
WO1990004647A1 (en) * 1988-10-26 1990-05-03 Hans Funke Sampling device
EP0441469A1 (en) * 1990-01-19 1991-08-14 Promega Corporation Immunospecific and bioluminescent assay of cellular ATP
WO1995025174A1 (en) * 1994-03-16 1995-09-21 Foss Electric A/S Novel method for the conditioning of liquid samples

Also Published As

Publication number Publication date
EP0229094B1 (en) 1991-04-24
EP0229094A1 (en) 1987-07-22
GB8513001D0 (en) 1985-06-26

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