BACTERIAL CELL EXTRACTANT

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 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 J Cl in which R represents a mixture of the alkyls from C g H 17 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: 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.

Inventors: HARBER JOHN, HARBER, MICHAEL, JOHN (DECEASED)
Applicants: HARBER F E EXECUTOR AND LEGAL, UNIV WALES MEDICINE, UNIV WALES
Application year: 1986
Main classification: C12Q1
IPC codes: C12P19, C12Q1