Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneunoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.
A selective method for distinguishing bacterial and nonbacterial adenosine triphosphate (ATP) in clinical bacteriological specimens was studied. The method involved incubation of samples with the detergent Triton X-100 and the ATP-hydrolyzing enzyme apyrase. The incubation selectively destroyed ATP in suspensions of various human cells while not affecting the ATP content in microbial cells. ATP remaining in the sample after incubation was extracted in boiling buffer and assayed by the firefly luciferase assay. Application of the method to 469 clinical urine specimens showed that the ATP level after treatment with Triton/apyrase was correlated to bacterial counts and that the sensitivity of the assay was sufficient for the detection of 105 bacteria/ml. The ATP levels per bacterial cell remaining in the urine specimens after treatment with Triton/apyrase were close to values observed in laboratory-grown cultures. The specificity and sensitivity of the luciferase assay for the detection of urinary bacteria and its possible use as a bacteriuria screening method are discussed.
A rapid (15 min) test for bacteriuria based on firefly luciferase analysis of bacterial ATP has been evaluated in 2,018 clinical urine specimens. The test procedure involves removal of nonbacterial ATP by treatment of urine with Triton X-100 and apyrase, extraction of bacterial ATP by boiling, and bioluminescent analysis of bacterial ATP by firefly luciferase, using a luminometer. For comparison, the widely used nitrite test was included in the study as an example of an alternative rapid chemical test. The test was set up to distinguish between specimens yielding >105 CFU/ml and specimens yielding <105 CFU/ml. A level of 13.5 nM ATP was chosen to define the limit between negative and positive results. At this discriminatory level, 92% of specimens yielding >105 CFU/ml and 88% of specimens yielding <105 CFU/ml were correctly classified with the luciferase method, whereas corresponding figures for the nitrite test were 55 and 99%, respectively. Of the 12% false luciferase positives, 20% were shown to contain >105 CFU/ml on prolonged incubation, thus reducing the false-positive rate to 10%. Of the 8% false luciferase negatives, 65% had low levels of CFU in the range of 105 to 106.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.