Evaluation of a bioluminescence assay for rapid antimicrobial susceptibility testing of mycobacteria

Beckers, Bram; Lang, Helmut; D, Schimke; Lammers, Alexander

doi:10.1007/bf02013394

Cited by 40 publications

(24 citation statements)

References 8 publications

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“…The bioluminescence assay detected effects on mycobacterial growth within 5 to 7 days with both inocula, but when the larger inoculum (106 CFU/ml) was used (data not presented), the breakpoints, which discriminated resistant from susceptible bacterial strains, had to be shifted toward higher concentrations of the drugs, and because of relatively high ATP levels of the inoculum, the ATP index at the breakpoint concentrations had to be higher than when the smaller inoculum (104 CFU/ml) was used. The optimal assay was obtained when the smaller inoculum was used, which is in agreement with the observations presented by Beckers et al (1). There was a pronounced discrimination between resistant and susceptible strains for isoniazid and rifampin by the bioluminescence assay (Fig.…”

Section: Discussionsupporting

confidence: 91%

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Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

Nilsson

Hoffner

Ånséhn

1988

Antimicrob Agents Chemother

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Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria.Testing of Mycobacterium tuberculosis susceptibility to antimycobacterial agents on the basis of growth on solid media requires 3 to 4 weeks of incubation (2, 3). Among these methods, the resistance ratio method on LowensteinJensen medium (2) is most used in Europe, while the plate proportional method on 7H10 agar (3) is the main method used in the United States. To achieve more rapid susceptibility testing, alternative techniques based on bacterial growth in broth cultures have been adapted. The rapid radiometric system (BACTEC) quantifies 14CO2 produced by mycobacteria growing in broth containing 14C-labeled palmitic acid (4). Results are provided after 1 week of incubation, and susceptibility is defined as a 99% reduction of the metabolic activity of a drug-exposed culture compared with that of an unexposed control culture (9). This technique is widely used, and the results are in good agreement with those of reference methods on solid media (4,(8)(9)(10)(11).The firefly bioluminescence assay of bacterial ATP has been used for studies of effects of antimicrobial agents on various bacteria (6, 7) and for rapid susceptibility testing of M. tuberculosis (1).The aim of this study was to evaluate this technique for rapid susceptibility testing of M. tuberculosis by comparing it with the resistance ratio method on Lowenstein-Jensen medium and with the rapid radiometric system (BACTEC). Antimycobacterial agents. Stock solutions of 10,000 pLg of active drug per ml were prepared from ethambutol (Cyanamid of Great Britain Ltd., Gosport, England), isoniazid (Ferrosan, Malmo, Sweden), rifampin (Ferrosan), and streptomycin (Glaxo Laboratories Ltd., Greenford, England). standard. The same inoculum was added to one drug-free vial (control A), and for determining the 1% proportion of resistance, another drug-free vial (control B) was inoculated with a 100-fold dilut...

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Section: Discussionsupporting

confidence: 91%

“…The firefly bioluminescence assay of bacterial ATP has been used for studies of effects of antimicrobial agents on various bacteria (6, 7) and for rapid susceptibility testing of M. tuberculosis (1).…”

mentioning

confidence: 99%

Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

Nilsson

Hoffner

Ånséhn

1988

Antimicrob Agents Chemother

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“…For these experiments, P. aeruginosa supernatants were added to a luminescent S. aureus strain constitutively expressing luxABCDE, and the impact of these supernatants on light production was examined. Light production has previously been used as a marker for antimicrobial activity (1,42,60,61). In this assay, decreases in light production correlate with increased antimicrobial activity.…”

Section: Vol 189 2007mentioning

confidence: 99%

Nutritional Cues ControlPseudomonas aeruginosaMulticellular Behavior in Cystic Fibrosis Sputum

2007

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The sputum (mucus) layer of the cystic fibrosis (CF) lung is a complex substrate that provides Pseudomonas aeruginosa with carbon and energy to support high-density growth during chronic colonization. Unfortunately, the CF lung sputum layer has been difficult to mimic in animal models of CF disease, and mechanistic studies of P. aeruginosa physiology during growth in CF sputum are hampered by its complexity. In this study, we performed chromatographic and enzymatic analyses of CF sputum to develop a defined, synthetic CF sputum medium (SCFM) that mimics the nutritional composition of CF sputum. Importantly, P. aeruginosa displays similar phenotypes during growth in CF sputum and in SCFM, including similar growth rates, gene expression profiles, carbon substrate preferences, and cell-cell signaling profiles. Using SCFM, we provide evidence that aromatic amino acids serve as nutritional cues that influence cell-cell signaling and antimicrobial activity of P. aeruginosa during growth in CF sputum.A key concept in bacterial pathogenesis is the ability of invading pathogens to obtain sufficient carbon and energy from the host for in vivo growth. Although Garber originally proposed the host as a growth medium over 40 years ago (12), the nutritional environment of most infection sites is poorly defined and often inadequately modeled by laboratory growth media. This lack of knowledge, combined with the limited utility of many animal models, provides significant challenges for mechanistic studies aimed at examining host nutrients as mediators of colonization and disease. To overcome these challenges, it is critical both to define the nutritional composition of key infection sites and to study bacterial physiology in the context of in vivo-relevant growth substrates.The heritable disease cystic fibrosis (CF) is an archetype for the development of nutritional models with which to study bacterial pathogenesis. A hallmark of CF disease is the accumulation of large volumes of sputum (mucus) within the lungs, which diminishes the host's ability to clear bacterial infections (17,31). The viscous CF lung sputum provides bacteria with a nutritionally rich growth environment composed of host-and bacterial-derived factors (17, 38). The opportunistic pathogen Pseudomonas aeruginosa chronically colonizes the CF lung, where it often grows to high cell densities in CF sputum (Ͼ10 9 cells/ml sputum). Although many other bacterial species persist and grow in the CF lung, chronic P. aeruginosa infection is likely the most clinically relevant, as it is correlated with declining lung function (17). Mechanistically, P. aeruginosa colonization and progression to chronic infection is poorly understood, although potential contributing factors are high-density growth and enhanced fitness of P. aeruginosa in CF sputum. P. aeruginosa fitness has been linked to nutritional components in CF sputum (39), thus necessitating the development of a versatile model that allows examination of CF sputum nutritional cues.The growth environment impacts several...

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“…The ATP is determined with the bioluminescence method after cooling the lysate. The relationship between ATP concentration and CFU/ml for serial dilutions of a sample of Mycobacterium tuberculosis H37Rv culture was determined in [76], and high correlation was observed (r = 0.993). Nevertheless, when similar measurements were conducted for eight different samples of the same strain taken following different incubation time, the observed correlation coefficient was slightly lower (r = 0.846).…”

Section: Determination Of Intracellular Atp In a Mixed Culture Of Micmentioning

confidence: 99%

Bioluminescence assay for cell viability

Lomakina

Modestova

Ugarova

2015

Biochemistry Moscow

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Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

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Evaluation of a bioluminescence assay for rapid antimicrobial susceptibility testing of mycobacteria

Cited by 40 publications

References 8 publications

Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

Rapid susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP

Nutritional Cues ControlPseudomonas aeruginosaMulticellular Behavior in Cystic Fibrosis Sputum

Bioluminescence assay for cell viability

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