Luminescent techniques for microbiological analysis of foods

There are many naturally bioluminescent organisms existing in nature and the mechanisms whereby some of these creatures emit light have been fully characterised1. These include the luciferin-luciferase system of bacteria, insects (fireflies and click-beetles) and the jellyfish Aequorea victoria. In essence, bioluminescence involves the conversion of chemical energy into light energy by an enzyme, commonly termed luciferase.

The luciferases from different organisms catalyse different reactions but all require oxygen. Over the years there has been great interest in developing applications based on bioluminescence2,3. A major advantage of using bioluminescent systems as analytical tools is that extremely low levels of enzyme activity can be detected by measuring the emitted light. Modern instruments are capable of detecting single photons with both temporal and spatial distribution; thus providing accurate information on the location and intensity of the light source4. Another feature of bioluminescent systems that makes them an excellent investigative tool is an almost absolute specificity for their substrates. For example, for firefly luciferase even minor changes in the structure of ATP and firefly luciferin result in a total loss of enzyme activity and this consequently results in a loss of light emission. This specificity for substrates allows the real-time measurement of luciferase activity in situ in very complex samples without the need for any pre-treatment.

ATP bioluminescence

The best known among bioluminescent methods is the ATP-assay, based on the activity of firefly luciferase (Figure 1). This assay has been used to determine biomass and is based on the principle that all living cells contain ATP and the ATP levels are proportional to the number of cells present. Thus, the amount of light emitted by the luciferase/luciferin reaction following extraction of ATP from cells provides an estimate of cell populations. This assay has been applied to estimate microbial load in a variety of foods including milk5-10, poultry11-14, meat 15, 16 and produce17. Results can be obtained within approximately 15 minutes and provide an accurate indication of whether counts have exceeded pre-determined thresholds. ATP bioluminescence has also been used to monitor the quality of processing waters during food manufacture 11, 18.

Arguably the most widely used application of this technology is ‘ATP Hygiene Monitoring’. This involves assessment of ATP levels on environmental swabs which directly relates to the cleanliness of the surface19, 20. Several commercial systems are available (Figure 2). Concern has been raised about inhibition of the luciferase in the presence of disinfectants remaining on surfaces following sanitation21-23. One elegant way to overcome this problem is to use mutant luciferases with increased resistance to the disinfectants used in industry24. In a twist to hygiene monitoring, it has been proposed that these tests can also be used to assess the potential for the presence of allergens on food contact surfaces.

Using methods for differential extraction of ATP from prokaryotic and eukaryotic cells it has been possible to develop assays for microbial cells that can be performed in minutes. However, the detection limit for these assays is high (>106 CFU/ml) and methods to improve their sensitivity have been a preoccupation of many microbiologists over the last two or three decades. To improve sensitivity, techniques involving filtration or centrifugation have been investigated and these have made it possible to detect approximately 104 CFU/ml in foods. This sensitivity can be further improved through assays of adenylate kinase, an enzyme present in cells that can be made to produce ATP by the addition of ADP in excess26. Another method to increase sensitivity involving the recycling of ATP by combining the enzyme pyruvate ortho-phosphate dikinase with luciferase/luciferin has been proposed by Sakakibara et al.27. These authors claim that their method is approximately 40 times more sensitive than that based on the luciferase/luciferin reaction alone.

Phage-based diagnostics

Another drawback of ATP bioluminescence assays is their inability to distinguish between the types of organisms present either in food or on food contact surfaces. This has been addressed in a variety of ways. Recently there has been interest in coupling ATP bioluminescence assays with immunomagnetic separation 28, 29 but the main disadvantage of this is the possibility of non-specific binding of microorganisms to the paramagnetic beads used in the technique. A commercial system has appeared that utilises host-specific bacteriophage to lyse target bacteria and the adenylate kinase that is released can be assayed using a bioluminescent method30. This forms the basis of the fastrAK™ assay supplied by Alaska Food Diagnostics.

Bacteriophages have been used in other ways to detect pathogens using a bioluminescent platform. Several researchers have used bacteriophage modified to carry luminescent or fluorescent reporter genes31. When the host cell is infected by the phage, the reporter genes are expressed and light is emitted. It has been shown that this method can be used for direct detection of bacteria on food surfaces32. As well as the structural genes encoding the enzymes involved in the bioluminescence reaction, researchers have constructed phage containing the luxI gene which encodes a signalling compound that then induces several other genes, luxCDABE, whose activation leads to bioluminescence in a reporter bacterium present on a biosensor33. It has also been proposed that biotinylated phage coupled with quantum-dot nanocomplexes can be used to detect bacteria34. We are currently investigating methods for the immobilisation of phage that will make it possible to concentrate and detect bacteria in a single step35.

Biosensors and other applications

An interesting application of bioluminescence is the ‘CANARY’ biosensor. This involves a macrophage cell that exhibits pathogen-specific antibodies on its surface36. When the target cell binds to the antibody natural signalling pathways are triggered in the cell that can be detected by aequorin, which is expressed by the macrophage (Figure 3). The biosensor is being marketed by Innovative Biosensors, Inc. and has been applied to the detection of E. coli O157:H7 in ground beef.

Bioluminescent assays have also been developed for the detection of toxicants, antibiotics and cellular metabolites.

Much recent research has been focused on in vivo imaging of bacterial pathogens that have been genetically modified to carry luciferase genes. The efficacy of treatments to prevent foodborne infections can be determined using this technique37.

As well as building on current applications, there are several new and exciting opportunities offered to microbiologists by bioluminescence.

References

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