Bioluminescence for USP sterility testing of pharmaceutical suspension products

Bioluminescence measurement significantly improved the accuracy, sensitivity, precision, and reliability of the current visual endpoint determination for the USP sterility test and eliminated the day 7 transfer/dilution step required for testing suspension products. Thirteen strains of bacteria and fungi (representing potential contaminants in sterile products), three pharmaceutical suspension products, and four media were used in the experiment. No interference from suspension products was encountered in the detection of microbial growth by the bioluminescence measurement. The poor fungal growth encountered was attributed to insufficient diffusion of oxygen into the medium and was circumvented by use of a large tube size (38 by 200 mm) or by vortexing the medium once during the 2-week incubation period. Bioluminescence measurement would facilitate automated handling of the sterility test endpoint readout operation. The optimum parameters of bioluminescence measurement for application in sterility testing were determined.     

Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

Comparison of the bioluminescence of<Emphasis Type="Italic">Photorhabdus</Emphasis> species and subspecies type strains

Natural bioluminescence of all recently accepted Photorhabdus species and subspecies type strains (bacterial symbionts of entomopathogenic nematodes) was measured using a commercial luminometer; optimum conditions for the measurement were described. Cultures emitted reliably measurable bioluminescence with characteristic level and kinetics for each strain. Bioluminescence of all strains was significantly higher at 37 than at 25 degrees C at the beginning of the measurement, no effect of bacterial concentration on the intensity of bioluminescence was observed. The technique can provide reliable and quick information for the determination of Photorhabdus taxons.     

Validation of an alternative microbiological method for tissue products

According to the European Pharmacopoeia sterility testing of products includes an incubation time of 14 days in thioglycollate medium and soya-bean casein medium. In this case a large period of time is needed for product testing. So we designed a study to evaluate an alternative method for sterility testing. The aim of this study was to reduce the incubation time for the routinely produced products in our tissue bank (cornea and amnion grafts) by obtaining the same detection limit, accurateness and recovery rates as the reference method described in the European Pharmacopoeia. The study included two steps of validation. Primary validation compared the reference method with the alternative method. Therefore eight bacterial and two fungi test strains were tested at their preferred milieu. A geometric dilution series from 10 to 0.625 colony forming unit per 10 ml culture media was used. Subsequent to the evaluation the second part of the study started including the validation of the fertility of the culture media and the parallel testing of the two methods by investigating products. For this purpose two product batches were tested in three independent runs. Concerning the validation we could not find any aberration between the alternative and the reference method. In addition, the recovery rate of each microorganism was between 83.33 and 100 %. The alternative method showed non-inferiority regarding accuracy to the reference method. Due to this study we reduced the sterility testing for cornea and amniotic grafts to 9 days.     

Study of the integral toxicity of aqueous media, polluted by petroleum and petroleum products using bacterial tests

A biotest kit was used to assess the integral toxicity level of aquatic medium contamination with petroleum and petroleum-based products. The integral toxicity dynamics was also monitored during biodegradation of petroleum and petroleum-based products by an association of petroleum-degrading strains including Acinetobacter sp., Mycobacterium flavescens, and Rhodococcus sp. The following bacterial tests were used: the bioluminescence (BL) test based on Photobacterium leiognathi; electro-orientation (EO), optoosmotic (OO), and growth test; as well as the reducing activity (RA) test based on the Agrobacterium radiobacter culture. No significant increase in the integral toxicity level of aquatic medium was observed when diesel fuel and kerosene contamination had been subjected to biodegradation. Although express biotests (EO, OO, RA, and BL) detected a pronounced increase in the integral toxicity of aquatic medium, long-term growth biotest revealed no statistically significant increase in the toxicity level.     

A Bioluminescence Method for the Measurement of l-Glutamate: Applications to the Study of Changes in the Release of l-Glutamate from Lateral Geniculate Nucleus and Superior Colliculus After Visual Cortex Ablation in Rats

We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of L-glutamate (L-Glu). Oxidation of L-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from less than 0.5 to 500 pmol of L-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (greater than 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.     

Kinetic analysis of a tod-lux bacterial reporter for toluene degradation and trichloroethylene cometabolism

Kinetics of toluene and trichloroethylene (TCE) degradation and bioluminescence from the bioreporter Pseudomonas putida B2 and TVA8 were investigated utilizing batch and continuous culture, respectively. Degradation was modeled using a Michaelis-Menten expression for the competition of two substrates for a single enzyme system, and bioluminescence was modeled assuming a luciferase enzyme saturational dependence on toluene as the inducer and growth substrate. During the batch experiments, bioluminescence increased at approximately 90 namp/min for initial toluene concentrations of 10 to 50 mg/L, but more slowly at higher toluene concentrations, suggesting maximum promoter induction at below 10 mg/L and toxic effects above 50 mg/L toluene. TCE degradation did not occur until toluene depletion, presumably due to competition between toluene and TCE for the toluene dioxygenase enzyme. During continuous culture, bioluminescence transiently increased, then gradually decreased in response to increasing step changes in toluene feed concentration. Bioluminescence in the CSTR appeared to be limited by growth substrate and/or inducer.     

Kinetic analysis of bacterial bioluminescence

Bioluminescence from the lux-based bacterial reporter Pseudomonas fluorescens HK44 was experimentally investigated under growth substrate-rich and limiting conditions in batch, continuous stirred tank (CSTR), and turbidostat reactors. A mechanistically based, mathematical model was developed to describe bioluminescence based on 1) production and decay of catalytic enzymes, and 2) reactant cofactor availability. In the model, bioluminescence was a function of inducer, growth substrate, and biomass concentration. A saturational dependence on growth substrate concentration accommodated dependence on cofactor availability and inducer concentration to accommodate enzyme production was incorporated in the model. Under growth substrate and inducer limiting conditions in the batch reactor and CSTR, bioluminescence was found to decrease in response to cellular energy limitations. The effective lux system enzyme decay rate was determined in independent measurements to be 0.35 hr(-1) and the model captured most of the bioluminescent behavior, except at long growth times and high cell density.     

Bioluminescent Salmonella typhimurium provides a rapid assay for measuring the efficacy of freeze-drying suspension media

A strain of Salmonella typhimurium , transformed to a bioluminescent phenotype, was used to compare three freeze-drying suspension media: inositol serum broth with and without added gelatin and sterile skimmed milk. Recovery and growth studies performed by measuring changes in bioluminescence demonstrated that of the three media tested, the routinely used inositol serum broth was the most effective freeze-drying suspension medium.     

Development of a biosensor for on-line detection of tributyltin with a recombinant bioluminescent Escherichia coli strain

A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli:: luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 micro M and 1.5 micro M TBT). High growth and dilution rates ( D=0.9 h(-1)) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 micro M, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.     

An ATP Bioluminescence Assay Applicable to Rapid Fluconazole Susceptibility Testing of Dermatophytes

An ATP bioluminescence assay as a rapid reference method for fluconazole (FLCZ) susceptibility testing of dermatophytes, as well as yeasts, was developed and evaluated by comparing it with viability, turbidity and fungal protein content-based conventional methods. FLCZ susceptibility results obtained with strains of Candida albicans and dermatophytes by the bioluminescence method in high-resolution medium were well correlated with those obtained by conventional methods currently used in clinical microbiology laboratories or reported previously, including a broth dilution method by the National Committee for Clinical Laboratory Standards (NCCLS). Thus, ATP bioluminescence assay can be used to monitor fungal growth in liquid culture media. The procedure has considerable potential for the rapid testing of FLCZ susceptibility of dermatophytes and other fungi.     

The factor stabilizing the bioluminescence of PVA-immobilized photobacteria

Immobilization of photobacteria in the cryogel of polyvinyl alcohol (PVA) was carried out. Immobilization was found to result in increased intensity and stability of bioluminescence. The elements determining the stability of bioluminescence were investigated. Selection of the strain was found to be of the highest importance. Among immobilized cells, Photobacterium phosphoreum exhibited the most intense and prolonged light emission, while Vibrio harveyi showed the least one. The technological procedures for cryogenic immobilization of photobacteria were determined. The role of the environment of gel formation in the preservation of the bioluminescence activity was determined. In the gels formed in rich medium for submerged cultivation of photobacteria, almost 100% luminescence activity was preserved, while light emission was considerably prolonged. Bioluminescence intensity of the preparations was shown to depend significantly on pH of the incubation medium. The pH shift to acidic values during prolonged incubation of immobilized cells was shown to be one of the factors of bioluminescence quenching. The stress effects of cryogenic immobilization were found to have an insignificant effect on the temperature profile of bioluminescence. Decreased reduction rate of the luciferase flavin substrate was shown to be a possible reason for bioluminescence quenching.     

Noninvasive bioluminescence imaging in small animals

There has been a rapid growth of bioluminescence imaging applications in small animal models in recent years, propelled by the availability of instruments, analysis software, reagents, and creative approaches to apply the technology in molecular imaging. Advantages include the sensitivity of the technique as well as its efficiency, relatively low cost, and versatility. Bioluminescence imaging is accomplished by sensitive detection of light emitted following chemical reaction of the luciferase enzyme with its substrate. Most imaging systems provide 2-dimensional (2D) information in rodents, showing the locations and intensity of light emitted from the animal in pseudo-color scaling. A 3-dimensional (3D) capability for bioluminescence imaging is now available, but is more expensive and less efficient; other disadvantages include the requirement for genetically encoded luciferase, the injection of the substrate to enable light emission, and the dependence of light signal on tissue depth. All of these problems make it unlikely that the method will be extended to human studies. However, in small animal models, bioluminescence imaging is now routinely applied to serially detect the location and burden of xenografted tumors, or identify and measure the number of immune or stem cells after an adoptive transfer. Bioluminescence imaging also makes it possible to track the relative amounts and locations of bacteria, viruses, and other pathogens over time. Specialized applications of bioluminescence also follow tissue-specific luciferase expression in transgenic mice, and monitor biological processes such as signaling or protein interactions in real time. In summary, bioluminescence imaging has become an important component of biomedical research that will continue in the future.     

Determination of sterilization effectiveness by measuring bacterial growth in a biological indicator through firefly luciferase determination of ATP

A bioluminescence procedure for measurement of microbial ATP allows a rapid determination of the effectiveness of autoclave sterilization. This determination is achieved faster than detection of acid production in a biological indicator via a pH indicator. Bacterial outgrowth from spores on test strips of the biological indicator was detected by measurement of ATP using the firefly luciferase reaction. A measureable increase in ATP was found after 5 hours of incubation of a biological indicator that had been treated under sterilizing conditions that produced 75% sterility of the biological indicator as measured by acid production. This is a marked improvement over the 24-48 hours of incubation currently required.     

Direct detection of rhizosphere-colonizing Pseudomonas sp. using an Escherichia coli rRNA promoter in a Tn7-lux system

Abstract A promoterless Tn7- lux system conferring bioluminescence was fused with an Escherichia coli rRNA gene promoter and compared with neo - or lac-luxCDABE analogs after introduction in Pseudomonas cells. Fusion of the ribosomal promoter with luxCDABE genes increased the bioluminescence of cells by approx. 100- to 1500-fold over the neo-lux system depending on the growth conditions and bacterial strain. When the cells were grown in suspension culture, light production and growth were strongly dependent on the nutrient composition of the medium. Root-colonizing competence was tested in nonsterile soil by autophotographic detection of bacterial bioluminescence on plant roots. The lower detection limit of the autophotographic method for roots inoculated with Pseudomonas fluorescens 2–79 was 10⁵ cfu g⁻¹ fresh root weight. The new bioluminescence marker did not require addition of supplemental nutrients or the aldehyde substrate for the luciferase enzyme and provides a simple and highly sensitive detection method for long term in situ studies on the microbial ecology of specific bacterial strains.     

Interference of Copper Sulphate in Growth of Erwinia amylovora

To understand the toxicity of copper salts on Erwinia amylovora, which are used in the control of fire blight, bacterial growth and cell metabolism was assayed with copper sulphate in the presence or absence of complex-forming compounds such as various amino acids or citrate. In minimal medium without amino acids copper sulphate strongly interfered with the growth of E. amylovora. A concentration of 15 μm CuSO₄ resulted in about 50% growth inhibition. In contrast to a strong effect of streptomycin, copper ions barely killed the cells when incubated in minimal medium for 1 h. The addition of 4 g asparagine per litre relieved a‘bacteriostatic’effect of copper ions and allowed growth of the bacteria at 2 mm CuSO₄. Other amino acids had a similar effect in the protection of E. amylovora against copper ions. This was in contrast to glycine betain, which was unable to suppress growth inhibition by CuSO₄. Presumably, the free ammonium groups of amino acids participated in the protective effect. The addition of citrate, exceeding the amount of copper-ions, was also protective. Bioluminescence of E. amylovora cells was expressed via a constitutive promoter from the lux-operon of Vibrio fischeri. The light emission is dependent on active cell metabolism. In a novel approach to determine the immediate response of E. amylovora after the addition of copper sulphate, the change of bioluminescence was determined. Addition of copper ions to MM3 medium strongly affected the bioluminescence, but no change in light production was noticed, when citrate or asparagine were present in addition to copper sulphate. A decrease of bioluminescence to 50% was observed for 50 μm CuSO₄ in the absence of amino acids.     

Real-time compensation of the inner filter effect in high-density bioluminescent cultures

Bioluminescence has recently become a popular research tool in several fields, including medicine, pharmacology, biochemistry, bioprocessing, and environmental engineering. Beginning with purely qualitative goals, scientists are now targeting more demanding applications where accurate, quantitative interpretation of bioluminescence is necessary. Using the recent advances in fiber-optic technology, bioluminescence is easily monitored in vivo and in real time. However, the convenience of this measurement is often concealing an unsuspected problem: the bioluminescence signal might be corrupted by a large error caused by the extinction of light by biological cells. Since bioluminescent cultures not only emit light but also absorb and scatter it, the measured signal is related in a complex, nonlinear, and cell-concentration-dependent manner to the "true" bioluminescence. This light extinction effect, known as the "inner filter effect," is significant in high-density cultures. Adequate interpretation of the bioluminescence signal can be difficult without its correction. Here, we propose a real-time algorithm for elimination of the inner filter effect in a bioreactor. The algorithm yields the bioluminescence which would be measured if the glowing culture was completely transparent. This technique has been successfully applied to batch and continuous cultivation of recombinant bioluminescent Escherichia coli. (c) 1993 John Wiley & Sons, Inc.     

Lampteromyces bioluminescence—1. Identification of riboflavin as the light emitter in the mushroom L. japonicus

The bioluminescence of the luminous mushroom, Lampteromyces japonicus, was studied by using the mushroom gills and also the luminous mycelia, the latter being cultured from the isolated spores and grown in a potato sucrose medium. The luminescence intensity of the mushroom gills and the cultured mycelia was measured in an aqueous suspension under various conditions. The original intensity was enhanced by exposing the luminous cells to oxygen for several hours or to acids or bases for a short period. This enhancement enabled measurement of their bioluminescence spectra which were identical to the fluorescence spectrum of riboflavin, having a maximum at 524 nm. The green fluorescent substance was extracted with cold water from the mushroom and it was identified as riboflavin by spectroscopic and chromatographic analyses. Riboflavin was concluded to be the light emitter of this mushroom.     

Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden

Bioluminescence imaging (BLI) is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r >.8, p <.001) between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.