ATP Bioluminescence Assay Research Articles

Post-antibiotic effect of beta-lactam antibiotics on gram-negative bacteria in relation to morphology, initial killing and MIC

The in vitro post-antibiotic effect (PAE) of cefepime, cefotaxime, ceftazidime and imipenem on reference strains of Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens were evaluated by bioluminescence assay of bacterial ATP. In parallel with the PAE determination, initial killing and morphology studies were performed. Imipenem produced greater than 1 h PAE on all strains tested, cefepime and cefotaxime on four strains and ceftazidime only on one of the strains tested. The length of the PAE on different strains did not correlate in the same way to MIC. Imipenem induced greater than 1 h PAE at 1/4-2 MIC while the cephalosporins caused greater than 1 h PAE at 4-256 x MIC. A PAE exceeding 1.2h was seen concomitantly with spheroplasts but there was not necessarily strong (greater than or equal to 99%) initial killing at the same time. The PAE duration at greater than or equal to 99% initial killing varied between 2.0 h and 5.0 h. When the cephalosporins produced less than 1 h PAEs, this was seen concomitantly with production on filaments and weak initial killing. The bioluminescence method was not jeopardized by filament formation and no negative PAE was found in contrast to the viable count method. The study showed that neither a certain multiple of MIC, the presence of spheroplasts nor strong initial killing can predict the length of PAE for beta-lactam antibiotics on gram-negative bacteria.

Pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis and Staphylococcus aureus demonstrated by studies of initial killing and postantibiotic effect and influence of Ca2+ and albumin on these drugs

The pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were investigated by studying the postantibiotic effect (PAE) and initial killing. The influence of Ca2+ and albumin on these drugs was also evaluated. The PAE was studied by use of bioluminescence assay of bacterial ATP. Daptomycin at clinically achievable concentrations produced a dose-dependent PAE on E. faecalis (0.6 to 6.7 h) and S. aureus (1.0 to 6.3 h). The long PAE of daptomycin was seen simultaneously with a potent dose-dependent initial killing assayed by viable count determination. The initial change in bacterial ATP was not as extensive as the decrease in viability. Vancomycin at corresponding concentrations produced shorter PAEs on E. faecalis (0.5 to 1.0 h) and S. aureus (1.3 to 1.8 h). This coincides with a weak non-dose-dependent initial change in viability and intracellular ATP. The MICs of vancomycin were not influenced by different Ca2+ concentrations or by the addition of albumin to the broth. The MICs of daptomycin for both strains were lowered, and the PAEs were prolonged with increasing concentrations of Ca2+ in the broth. The PAE of daptomycin was Ca2+ dependent to the same extent as the MIC was. In the presence of physiological concentrations of albumin and free Ca2+, the PAEs of daptomycin on both strains were reduced and the MICs were increased in comparison with the results obtained in pure Mueller-Hinton broth with approximately the same free Ca2+ concentration. This decrease in daptomycin activity was considered to be due to the albumin binding of daptomycin. Despite the albumin binding of daptomycin, the PAE produced on E. faecalis and S. aureus in the presence of a physiological free Ca2+ concentration was still over 6 h at clinically achievable concentrations.

Synergic post-antibiotic effect of mecillinam, in combination with other β-lactam antibiotics in relation to morphology and initial killing

The synergic in-vitro post-antibiotic effect (PAE) of mecillinam, in combination with either ampicillin, aztreonam, ceftazidime or piperacillin, on a reference strain of Escherichia coli was evaluated by bioluminescence assay of bacterial ATP. Ampicillin, ceftazidime and mecillinam alone induced a concentration dependent PAE (greater than 3 h) on E. coli, whereas aztreonam and piperacillin alone induced a short (less than 1 h) non-dose dependent PAE. At most concentrations, the combination of mecillinam and ampicillin, aztreonam, ceftazidime or piperacillin induced longer PAEs on E. coli than the sum of the individual antibiotics' PAEs. Long PAEs were seen concomitantly with the presence of spheroplasts. In addition to the synergistic PAE, the decrease in colony counts and changes in ATP values after a 2 h exposure to mecillinam, in combination with the other beta-lactam antibiotics, were more prominent than the respective values after exposure to the individual antibiotics. The change in ATP was generally less pronounced than the decrease in colony counts. This could be due to lysis of spheroplasts on agar plates, leading to an over-estimation of the initial killing when assayed by viable counting. Mecillinam, which induced long PAEs on E. coli at almost all concentrations in this study, has a high affinity for penicillin binding protein 2 (PBP 2) and induced spheroplast formation at all concentrations. However, mechanisms other than the affinity for PBP 2 and spheroplast formation are involved in the PAE of beta-lactam antibiotics on Gram-negative bacteria; since the PAE was prolonged when mecillinam was combined with ampicillin, aztreonam, ceftazidime or piperacillin, which bind preferentially to PBP 1 and 3.

Chemical enhancement of cisplatin cytotoxicity in a human ovarian and cervical cancer cell line

While many advances have been made in the chemotherapy of gynecologic cancers, treatment failures remain a major clinical problem. A growing understanding of the mechanisms of tumor cell resistance to antineoplastic drugs provides a framework for the development of chemotherapy regimens containing agents capable of modulating tumor response. Using a short-term ATP bioluminescence assay we studied the ability of two methylxanthines (caffeine, pentoxifylline) and an inhibitor of ADP-ribosyl transferase (3-aminobenzamide) to enhance cisplatin cytotoxicity in gynecologic cancer cell lines. Our findings of significantly enhanced cisplatin-induced cytotoxicity with two different analysis techniques confirms the effectiveness of these agents. These results may have future clinical significance.

Postantibiotic effect of beta-lactam antibiotics on Escherichia coli evaluated by bioluminescence assay of bacterial ATP

The in vitro postantibiotic effects (PAE) of aztreonam, ceftazidime, cefuroxime, imipenem, and piperacillin on Escherichia coli ATCC 25922 were studied by a bioluminescence assay of bacterial ATP. In parallel with the PAE investigation, viability and morphology studies were performed. The strain was exposed for 2 h to different concentrations of beta-lactam antibiotics. The antibiotic activity was eliminated by 10(-4) dilutions, and regrowth of bacteria was monitored hourly by the bioluminescence assay of bacterial ATP. The length of PAE was dose dependent for ceftazidime (0.5 to 2.6 h), cefuroxime (0.4 to 2.6 h), and imipenem (0.3 to 4.5 h). The long PAE for these antibiotics at higher concentrations was associated with a potent initial killing and the presence of spheroplasts. Aztreonam and piperacillin produced a short, non-dose-dependent PAE (0.4 to 0.95 h). Short PAEs (below 1 h) were seen concomitantly with production of filaments, except in the case of imipenem, which only produced spheroplasts. The bioluminescence method was not jeopardized by filament formation, in contrast to the viable count assay which is normally used for PAE investigations. This makes it possible to study PAE for beta-lactam antibiotics on gram-negative bacteria with bioluminescence.

The ATP‐bioluminescence method for a rapid evaluation of the microbial activity in the stone materials of monuments

AbstractThe examination of the state of conservation of works of art in stone includes the assessment of the presence of microbiological agents on the surface of the decayed monuments. These microorganisms can accelerate, via their metabolic activity, the decay process of the stone surface. At present this assessment is made with the traditional techniques for the microbiological examination of the soil, provides results only after a delay of 30 days. A bioluminescent ATP assay should provide rapid quantitation of actively growing organisms on the surface of a stone monument, and the applicability of this technique was verified on some samples of sandstone (Pietraforte) collected from a historic building (the Strozzi Palace) in Florence. These samples were evaluated for the amount of the ATP and the total number of microorganisms.The results obtained suggest that the bioluminescent assay could be suitable for detecting and quantitating the presence of microorganisms in a sample of stone.

Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes.

Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.

Postantibiotic and bactericidal effect of imipenem againstPseudomonas aeruginosa

The postantibiotic effect of imipenem on Pseudomonas aeruginosa was studied at different inocula using one ATCC strain and four clinical isolates. The postantibiotic effect was measured using two different methods: viable counts and bioluminescence assay of intracellular bacterial ATP. The postantibiotic effect could be demonstrated with both methods (viable counts 1-2 h, ATP assay 3-5 h) for all strains at an inoculum of 10(6) CFU/ml. When the inoculum was raised to 10(8) CFU/ml, no postantibiotic effect could be observed with either method using routine growth conditions. This disappearance of the postantibiotic effect coincided with a loss of bactericidal effect of imipenem when high inocula were used. Improved oxygenation of the cultures restored the bactericidal and postantibiotic effects of imipenem at high inocula.

Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing

Intracellular adenosine triphosphate (ATP) is the primary energy unit of living cells, and can be quantitated by measuring the light generated with luciferase-luciferin reagent in a luminometer. The use of an ATP-bioluminescence assay, to determine tumor cell viability after exposure to chemotherapeutic agents, has been adapted to test tumor chemosensitivity in vitro. This presentation will illustrate the method of the ATP-chemosensitivity assay (ATP-CSA) using an ovarian cancer cell line NIHL: OVCAR-3 as an example and present preliminary data on 54 56 successful in vitro ATP-CSA's from 46 patients with pelvic malignancies. Fresh human tumor specimens were generally tested for single and combined drug effects at two drug concentrations (0.2 × and 1 × peak plasma concentrations). Correlation of in vitro drug sensitivity and in vivo patient response was obtained for 23 treatment regimens in 22 patients with ovarian carcinoma. The true positive rate was 100% and the true negative rate 66.7%. Our data demonstrate (a) that the ATP-CSA, measuring total cell viability, is a feasible in vitro assay for human tumor drug testing and (b) that specific criteria of in vitro chemosensitivity for this assay need to be defined by further studies, for single and combined drug exposure at different concentrations, to permit a meaningful correlation with in vivo clinical response.

A microwave method for the extraction of cellular ATP

Quantitation of microbial cells by conventional microscopic counting and culturing techniques is labor intensive and time consuming. These techniques have recently been facilitated by the bioluminescence assay of cellular ATP [1]. The cells have to be lysed first to release cellular ATP which in turn can be measured with the firefly luciferase/luciferin-bioluminescence reaction [2]. The conventional method used for extracting microbial ATP is to add boiling buffer solution to the cells [3], destroying cell integrity and releasing cellular ATP for assay. Another accepted approach is to lyse the cells by specific chemicals [4] allowing the cells to liberate certain cellular material (i.e., ATP) for further analysis. Both methods have their limitations: the former is time consuming and tedious, the latter involves the use of specific lysing agents. Hence, we have developed a method which employs microwave energy to induce temperature shock and cell lysing. The resulting cellular ATP remains unaltered for quantitation. This microwave method for cell lysing is fast, simple, with high throughput, and has many potential applications. Overnight cultures of a broad range of microbial types (from the American Type Culture Collection, Rockville, MD) were used in this experiment. When a very low level of microbial cells is present, and microbial ATP content is less than 0.05 pg in the sample, the cells can be concentrated by filtration [5]. Isotonic buffers such as Tris buffer (0.02 M Tris, 0.002 M EDTA, pH 7.75) or Industrial butterfield's buffer (0.0003 M phosphate, pH 7.3) were used in the boiling buffer extraction. The sample was boiled for 5 rain, then vortexed and immediately chilled in an ice bath (~< 4°C). Duplicate aliquots (0.2-0.6 ml) of the extractants were taken for luminescence measurements. 0.1 ml of the chemical extractant (PICOEX T M B from Packard

THE ACTION OF NISIN ON BEER SPOILAGE LACTIC ACID BACTERIA

Within one minute of adding nisin to suspensions of Lactobacilli clumping of cells was observed. This happened with all the strains assayed, irrespective of their sensitivity or resistance to nisin. There was, however, no evidence of cell lysis. Two different assays for cell viability were used to show that the vast majority of cells of sensitive strains were killed within one minute of contact with nisin. The time needed to kill all the cells depended on the concentrations of both nisin and cells. One strain was more resistant and only 50–60% of the cells died within one minute of nisin addition. Using an ATP-bioluminescence assay it was shown that the addition of nisin to both Lactobacilli and Pediococci caused a rapid fall in intracellular ATP levels, which was reflected in a simultaneous appearance of ATP in the extracellular medium. Actively growing cells lost ATP more rapidly than did those in stationary phase. The amount of intracellular ATP lost was affected both by the concentration of nisin used and the sensitivity of the bacteria. It appears that an initial effect of nisin is to make the cell membrane ‘leaky’ as happens with many antibiotics, yeast killer factors (zymocins), colicins, and other gram-positive bacteriocins.

Adenosine Triphosphate Bioluminescent Assay to Enumerate Bacterial Numbers on Fresh Fish

The microbial quality of fresh fish was determined by using a bacterial ATP bioluminescent assay. Assay procedures included differential filtration followed by enzymatic degradation of somatic ATP. This technique limited interference from non-bacterial ATP sources and thus bacterial ATP was quantitated by using the luciferin-luciferase reaction in an automated luminometer. Good correlation (r = .96) was obtained from four species of finfish when microbial counts, determined from ATP analyses, were compared to counts determined by using conventional plate count procedures.

Assessment of the sensitivity of leukaemic cells to cytotoxic drugs by bioluminescence measurement of ATP in cultured cells.

A short-term test in vitro is described, which can be used to detect resistance to cytostatic agents in leukaemic cells. Leukaemic cell suspensions were incubated with cytostatic agents and the resulting intracellular ATP concentrations were measured by a bioluminescence ATP assay. There was a clear dose-effect relationship in acute leukaemia and chronic lymphocytic leukaemia cells for drugs used in the treatment of leukaemias. A good correlation was found between the ATP content of leukaemic cells and cell viability as determined by the trypan blue dye exclusion test. Preliminary individual clinical correlations suggest a correlation between the chemosensitivity in vitro and the response patterns in vivo in leukaemia patients. This simple, fast and sensitive method may have application for determination of drug-induced cytotoxicity in leukaemic cells in vitro.

Estimation of Campylobacter spp. in broth culture by bioluminescence assay of ATP.

The luciferin-luciferase bioluminescence reaction was used to estimate cell numbers of Campylobacter jejuni and Campylobacter coli in broth cultures based on a linear relationship between cell numbers (in excess of 10(4) to 10(5] and ATP levels. The sensitivity was lower than that obtained with Escherichia coli. The calculated amount of intracellular ATP per cell of C. jejuni and C. coli ranged from 1.7 to 2.1 fg.

Bacteriuria screening by direct bioluminescence assay of ATP.

A direct bioluminescence assay for bacteriuria screening is described and compared with the MS-2 system (Abbott Laboratories, Irvine, Tex.) and the chemical strip, Gram smear, and calibrated-loop methods. A total of 973 specimens were tested. Unlike previously described bioluminescence methods, this test measures total ATP in urine without pretreatment of samples to remove somatic ATP. The result was compared with an ATP standard (20 ng/ml). A low result (less than 3% of standard) was interpreted as negative and a high result (greater than 10% of standard) as positive. Samples with intermediate results (38% of total) were incubated at 35 degrees C in thioglycolate broth (1:10). A 2% increase in ATP concentration was interpreted as positive. The sensitivity of this method for detecting greater than 10(5) pathogens per ml was 92.3% and was comparable to those of the MS-2 system (92.7%) and the Gram smear method (90.5%). The chemical strip method was less sensitive (84.0%). The direct bioluminescence method was more sensitive than were the MS-2 system and the Gram smear method for detecting low-level bacteriuria (less than 10(3) to 10(5) organisms per ml), primarily because of associated pyuria. Thioglycolate broth provided a suitable medium for ATP production, and 5% CO2 decreased bacterial ATP synthesis during log-phase growth. The direct bioluminescence assay is rapid, simple, cost-effective, and reliable for bacteriuria screening.

Continuous flow method for extraction and bioluminescence assay of ATP in baker's yeast

The intracellular ATP of baker's yeast (Saccharomyces cerevisiae) was measured using the bioluminescent firefly luciferase assay. Benzalkonium chloride and trichloro-acetic acid served in the experiments as extracting agents and optimal conditions for the extraction and assay of the intracellular ATP are reported. Using the results obtained from manually performed experiments two continuous flow systems were designed for the measurement of ATP in yeast cells during cell growth. Good correlation between the amount of cellular ATP and cell growth was found during the exponential growth phase.

Application of a bioluminescence ATP assay in brewery wastewater treatment studies

AbstractThe importance of having a rapid method for determining the viable biomass in activated‐sludge wastewater treatment plants (WWTP) for process control and development is well recognized. The firefly bioluminescence ATP assay has been proposed for this purpose. Such an assay using partially purified firefly luciferase and synthetic firefly luciferin for the bioluminescence reaction, a liquid scintillation counter in the out‐of‐coincidence mode as luminescence detector, and a sludge ATP extraction technique involving dimethyl sulfoxide at room temperature is described. Experiments with several pure bacteria cultures were done to demonstrate the feasibility of applying this assay to activated sludge to activated sludge WWTP investigation and control. The ATP content of samples taken from various points in a 350000 gal/day brewery activated‐sludge WWTP was monitored for 4.5 months. Good linear correlation between ATP and mixed‐liquor suspended solids, return sludge suspended solids, and effluent suspended solids were observed. Percentage viabilities of the various sludge samples were derived from the ATP results.

Stability of reagents for use in an automated ATP bioluminescence assay system

Stability of reagents for use in an automated ATP bioluminescence assay system

A Firefly Bioluminescence ATP Assay Method for Rapid Detection and Enumeration of Brewery Microorganisms

A rapid, sensitive firefly bioluminescence ATP assay using a liquid scintillation counter for measurement of assay light output is described. The use of partially purified luciferase and synthetic D-luciferin in this assay permits the detection of as little as 10−16 moles ATP and as few as 10 yeast cells. The assay light output is directly proportional to the quantity of ATP from at least 10 to 104 fmol and is on scale throughout this range. The method developed for detection and enumeration of viable microorganisms, which requires only a few minutes per sample, involves extraction of the cellular ATP with DMSO at room temperature and estimation of the ATP by the bioluminescence assay—for dilute suspensions, prior concentration and isolation of the cells by membrane filtration is necessary. The ATP contents of a selection of yeast and bacteria determined using this technique are reported. A linear relation between quantity of ATP and number of yeast cells is demonstrated, and the changes in intracellular yeast ATP pools during fermentation of wort by ale and lager yeast are presented. A number of other possible applications of this ATP assay in brewing research, development, and microbiological quality control are evaluated and/or discussed.