The use of the firefly bioluminescent reaction for the rapid detection and counting of mycobacterium BCG

Janaszek, W; Aleksandrowicz, J.; Sitkiewicz, Dariusz

doi:10.1016/0092-1157(87)90012-6

Cited by 14 publications

(6 citation statements)

References 13 publications

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“…This step is critical and impacts directly the reliability of the detection (Selan, Berlutti, Passariello, Thaller, & Renzini, 1992). Various methods for ATP extraction have been developed, including continuous DC voltage (Lee & Cho, 2007;Wang, Bhunia, & Lu, 2006), microwave (Tsai, 1986), ultrasound (Law, Derrick, & Higson, 2003), organic solvent (Romanova, Brovko, & Ugarova, 1997), strong acid (Contin, Nobili, & Brookes, 1995;Ishida, Yoshikawa, Nakazawa, & Kamidate, 2002;Janaszek, Aleksandrowicz, & Sitkiewicz, 1987;Narsaiah et al, 2012), and surfactant (Dukhovich, Rodriguez, Gaona, Levashova, & Ugarova, 1995). For chemical extraction, reagents as trichloroacetic acid (TCA), TriseEDTA buffer, cetyltrimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS), commercial bacterial releasing reagent (BRA) can be used at different concentration.…”

Section: Extractionsmentioning

confidence: 99%

Determination of microbial load for different beverages and foodstuff by assessment of intracellular ATP

Bottari

Santarelli

Neviani

2015

Trends in Food Science & Technology

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Section: Extractionsmentioning

confidence: 99%

Determination of microbial load for different beverages and foodstuff by assessment of intracellular ATP

Bottari

Santarelli

Neviani

2015

Trends in Food Science & Technology

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“…Initially, FFluc was used to measure ATP as an indirect method of assessing cell viability and cell numbers [11], [12]. Considering the long duplication times of mycobacteria, the advantages of using bioluminescence as a reporter to assay anti-mycobacterial agents soon became evident when compared to the more traditional colony count methods.…”

Section: Introductionmentioning

confidence: 99%

Optimisation of Bioluminescent Reporters for Use with Mycobacteria

et al. 2010

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Background Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.Methodology/Principal FindingsOne of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.Conclusions/SignificanceWhile much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

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“…In particular, bioluminescence has been harnessed as a surrogate biomarker for mycobacterial numbers and viability, 5,6 and bioluminescent mycobacterial reporter strains have been utilized for high-throughput, real-time screening of antimycobacterial agents. 7–10 More recently this powerful technology has also been applied to real-time, non-invasive in vivo imaging of mycobacterial infection.…”

Section: Introductionmentioning

confidence: 99%

Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase

Andreu

Zelmer

Sampson

et al. 2013

Journal of Antimicrobial Chemotherapy

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ObjectivesIn vivo experimentation is costly and time-consuming, and presents a major bottleneck in anti-tuberculosis drug development. Conventional methods rely on the enumeration of bacterial colonies, and it can take up to 4 weeks for Mycobacterium tuberculosis to grow on agar plates. Light produced by recombinant bacteria expressing luciferase enzymes can be used as a marker of bacterial load, and disease progression can be easily followed non-invasively in live animals by using the appropriate imaging equipment. The objective of this work was to develop a bioluminescence-based mouse model of tuberculosis to assess antibiotic efficacy against M. tuberculosis in vivo.MethodsWe used an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene (FFlucRT) to infect mice, and monitored disease progression in living animals by bioluminescence imaging before and after treatment with the frontline anti-tuberculosis drug isoniazid. The resulting images were analysed and the bioluminescence was correlated with bacterial counts.ResultsUsing bioluminescence imaging we detected as few as 1.7 × 103 and 7.5 × 104 reporter bacteria ex vivo and in vivo, respectively, in the lungs of mice. A good correlation was found between bioluminescence and bacterial load in both cases. Furthermore, a marked reduction in luminescence was observed in living mice given isoniazid treatment.ConclusionsWe have shown that an improved bioluminescent strain of M. tuberculosis can be visualized by non-invasive imaging in live mice during an acute, progressive infection and that this technique can be used to rapidly visualize and quantify the effect of antibiotic treatment. We believe that the model presented here will be of great benefit in early drug discovery as an easy and rapid way to identify active compounds in vivo.

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The use of the firefly bioluminescent reaction for the rapid detection and counting of mycobacterium BCG

Cited by 14 publications

References 13 publications

Determination of microbial load for different beverages and foodstuff by assessment of intracellular ATP

Determination of microbial load for different beverages and foodstuff by assessment of intracellular ATP

Optimisation of Bioluminescent Reporters for Use with Mycobacteria

Rapid in vivo assessment of drug efficacy against Mycobacterium tuberculosis using an improved firefly luciferase

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