Microbiology illuminated: gene engineering and bioluminescence

Review Bacteria that cause food poisoning and spoilage can be modified, by the application of recombinant DNA techniques, to express genes that code for enzymes required for bioluminescence. Such microorganisms may emit light continuously or in response to specific environmental triggers but, however light expression is controlled, it can only be produced by living cells. Consequently, antimicrobial substances (including biocides, antibiotics, environmental toxicants and bacteriophages) that compromise bacterial viability can be detected rapidly as a decrease in the luminosity of a bacterial suspension. These developments, coupled with innovative methods of detecting bacteria, challenge the central role of plate counts in the microbiology laboratory.

Microbial plate counts continue to represent the comerstone of industrial microbiology, despite the continued development of 'rapid methods'. Principally, this may be due to the fact that plate count assays have relatively simple equipment and staff requirements, the only major drawback being the assay time. The use of rapid methods that have more sophisticated staff training requirementsI or that involve substantial investment in capital equipment2 is, in the main, restricted to large laboratories and, hence, to major companies3. However, this situation may change in the near future. Microbial detection In 1987, a novel bacterial detection technique was described 4. The new technology involves the introduction, using gene engineering, of bioluminescence flux) genes into the genome of a bacteriophage, and the subsequent transduction of host bacteria to the bioluminescent phenotype. The same concept has recently been adapted to ntil~ze an alternative detection method (ice nucleation)s rather than bioluminescence (which is described below) in an otherwise analogous system. The relative benefits of ice nucleation or bioluminescence for bacterial detection assays remain to be established. The lux genes encode the enzymes that provide a bioluminescent phenotype, and originate from a number of marine bacteria. The light-emitting reaction involves an intracellular, luciferase-catalysed oxidation of the reduced form of flavin mononucleotide (FMNH,) and a long-chain aliphatic aldehyde, such as dodecanal, by molecular oxygen: FMNH2 + RCHO + Oz ~

FMN + RCOOH + H20 + light

The lux genes encode the two subanits of the luciferase enzyme (luxAB) and the multisubunit components of a

Microbiology illuminated: gene engineeringand bioluminescence Gordon S.A.B. Stewart, Stephen P. Denyer and Jay Lewington fatty acid reductase (luxCDE) 6. In effect, however, only the laxAB genes are required for the introduction of a bioluminescent phenotype, because the long-chain aldehyde can be supplied in chemical form and is freely diffusible across the procaryotic membrane~. Bacteriophage genetically engineered to contain lu:~B are not themselves bioluminescent, because they lack the intracellular biochemistry necessary for light production. However, infection of host bacteria by the phage leads to expression by the host of the phage genes and, within 30 to 50 minutes, of the additional/lax genes. The result of phage infection is bacteria capable of bioluminescence. Lambda L28, a derivative of the bacteriophage lamlxla Charon 30 that contains a 9-kb DNA segment encoding the entire lax system, can be used to detect as few as ten Escherichia coli cells 100 minutes after infection4. Similar limits could be attained for the detection of E. coli in milk using a short period of incubation at 25°C following the addition of phage. A number of lax recombinant phage have been constructed by Ulitzur and co-workers4. One such bacteriophage (P22 lax) has been used to detect as few as 100 Salmonella typhimurium cells using a simple luminometer8. The detection of specific pathogens such as Salmonella spp., Campylobacter spp. and Listeria monocytogenes requires the genetic engineering of hostspecific bacteriophage. Clearly, recombinant phage technology offers the potential for rapid, user-friendly microbiological testing. However, the required detection limits for specific pathogens in foods are of the order of one viable cell per 25 g, a level beyond the detection limit of direct recombinant phage assays. Consequently, there will still be a requirement to go through a recovery and em'ichmen~. protocol prior to assay, but same-day testing seems possible.

Injury and recovery Gordon S.A.B.Stewart is at the Departmentof Applied Biochemistryand The need for enrichment and the length of bacterial Food Science,Universityof Nottingham,Su~n Bonington,Loughborough recovery times significantly compromise current efforts LE12 5RD, UK. StephenR Denyeris at the Departmentof Pharmaceutical Sciences,UniversityPark,NottinghamNG72RD, UK. lay tewingtonis with to bring rapid, biotechnology-based methods of bacterial detection to the marketplace. Frequently, the need Amershampie, ForestFarm,Whitchurch,CardiffCF47YT,UK. Trends in Food Science & Technologylanuary 1991

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to detect pathogens or food spoilage bacteria follows the application of new food preservation methods that result in sub-lethal injury of bacteria. At present, recovery from damage is almost universally estimated by the ability of cells to form colonies on selective, as opposed to non-selective~ agar. Such a retrospective measure of sub-lethal injury gives no information about the biochemical and physiological processes of recovery. Environmental stress that directly or indirectly affects intracellular energy production can be monitored in recombinant bacteria that contain the lax genes9. Therefore, bioluminescence provides a novel method with which to study, in real time, the recovery of microorganisms such as Salmonella typhimurium from sub-lethal injury. The measurement of bioluminescence, as opposed to the plate count approach, has been validated9, and enables the use of bioluminescence data where the acquisition of viable count data would be impractical. Injury of microorganisms has been assessed by freezing, for various times, suspensions of S. typhimurium containing a constitutive expression plasmid (pSB100) containing the lax genes. Bioluminescence data showed that no injury could be observed during cooling from ambient temperature to 0°C or during the latent heat extraction period (in which the culture remained at 0°C). However, progressive injury, as measured by a real-time decrease in bioluminescence, does occur once the temperature falls below 0°C (Ref. 9). Such new techniques should enable the merits ,~f different recovery protocols to be assessed, and sh~,~mldadvance our understanding of the injury process.

Fig. 1 A lux recombinantLactococcus lactis subsp,diacetylous incubated

for four hours at 30°C with antibiotic test rings after surface inoculation onto a nutrient agar plate. BA, bacitracin (8 units); C, chloramphenicol(50 gg); NE, neomycin (30 gg); PG, penicillin G (4 units); PB, polymyxin B (250 units); S, streptomycin (25 Ilg).

acid bacteria. Figure 1 shows an image of a lax recombinant Lactococcus lactis subsp, diacetylous that has been plated onto a nutrient medium and overlaid with antibiotic test rings. After a brief incubation period, the antimicrobia[ activity of antibiotics such as penicillin G Cell viabil!ty and bacitracin can be visualized as dark zones of inhiGiven tha~, the production of light from recombinant bition. The relationship between antibiotic activity and bacteria contail~ing the lax genes depends upon a func- light emission allows the use of lax recombinant tional intracellula~' biochemistry, it can be established Lactobacillus casei or Lactococcus lactis MG1363 to that any substance ~hat impairs that biochemistry and detect low levels of antibiotics in milk in 30 to 60 minthat compromises cellolar viability will lead to a re- utes II. Another critical antimicrobial agent in the dairy duction in light emissian. A wealth of industrial industry, the bacteriophage, can also be detected within applications can focus upo~,~ this single premise; those two hours at concentrations of ~10s bacteriophage per that have particular relevance to food science and millilitre II. This compares favourably with bacteriotechnology are considered below phage contamination levels in raw milk, which can be as high as 101°bacteriophage per millilitre. Antibiotic detection Ultimately, the objective must be to transfer the lax Ulitzur et al. ~° described the use of lax genes for the genes into those bacteria that actually constitute the ferdetermination of the susceptibility of microorganisms to mentation starter culture. Such recombinant microantibiotics. The same principle can be ¢~sed to detect organisms would then reflect the characteristics of the antimicrobial residues in food and to evah~ate the bio- starter exactly, but would represent a test reagent that logical efficacy of food-grade preservative.' ~gents, par- would not be t,,tilized for any manufacturing process. ticularly food-grade antibiotics. In this respect, the Antimicrob~al agents may also be deliberately added advantage of recombinant microorganisms that contain to foods as preservatives or to specifically combat the the lax genes is that those bacteria that are either growth of pathogens. Evaluating the biological activity, particularly sensitive to specific antibiotics or particalarly in situ, of such agents, either singly or in combination, relevant to an industrial process can be studied. is extremely difficult using plate count assays, and is The presence of antibiotic residues is particularly ,~el- impossible in real time. Bioluminescence can be used to evant to the dairy industry. In cheesemaking, residual study the efficacy of preservatives in foods. For example, antibiotics active against starter culture bacteria caa 'Nisaplin', a commercial preparation of the food-grade cause failure of the starter culture, with the loss of entire antibiotic, nisin, caused a decrease in the bioluminvats of milk. Consequently, there is an opportunity to escence produced by lax recombinant Lactococcus develop rapid methods to screen for antimicrobial sub- lactis subsp, diacetylous F7/2 (Reid, C.L., Waites,W.M., stances in milk that are active specifically against lactic E thaw, R.G. and Stewart, G.S.A.B., unpublished). 8

Trends in Food Science& TechnologyJanuary1991

F~ Growth of a lux recombinant Salmonella typhimurium LT2 in increasing concentrations of gelatin as measured by bioluminescence. Gelatin gels were made with nutrient broth (no glucose) inoculated with S. typhimurium containing the lux-containing plasmid pSg230. Total light intensity was determined with a photon video camera after three hours of incubation at 22°C. Intensity of bioluminescence is colour-coded and is further represented as three-dimensional, colour-coded light-intensity profiles on the central insets. Intensity decreases in the order: white, pink, red, yellow, green, blue, violet. (a), 20% gelatin, w/v; (b), 50% gelatin, w/v; (c), 70% gelatin, w/v (Bolton, K.J., Dodd, C.E.R., Gould, G.W. and Waites, W.M., unpublished).

Unfortunately, current methods of microbial analysis is often complex, and adverse situations can lead to biodisrupt the food matrix before bacterial enumeration, with cide depletion through chemical decomposition, dilution the loss of spatial information. There is a need, therefore, and inactivation. At present, microbiological assays to to explore methods of dam generation that conserve ensure that biocides are providing adequate protection such information and, hence, enhance the confidence are retrospective. However, new assays that capitalize on the intimate in-vivo relationship between light with which food safety assessments can be made. The potential now exists to monitor the distribution emission and cell viability should provide an on-site test of preservative concentrations throughout a food matrix that takes only 15 minutes. As in m~tibiotic detection assays, where the advantage by either incorporating lux recombinant indicator bacteria of gene engineering is that the Ira" genes are inserted into into model foods and examining sections, or overlaying the indicator bacteria onto the surface of a food section the most appropriate species of bacteria, those bacteria and, in either case, evaluating the light profile. The poten- that represent the major concerns to food manufacturers tial of such an approach is illustrated by Fig. 2, which can be provided as bioluminescent derivatives that may shows, in situ and in real time, the light produced by a be used to evaluate disinfection regimes. Figure 3 shows lux recombinant derivative of Salmmlella typhimurium how lux recombinant Listeria monocytogenes can faciligrowing within a series of gelatin gels. Surface-growing tate the rapid evaluation of biocide activity ~2. Measuring Salmonella were eliminated by hypochlorite sterilization the light from bioluminescent bacteria can be used of the surface and, consequently, the images represent the light from cells growing within the gel matrix. After aiocides Decreasing concentration of biocide the cells have grown for three hours, the images clearly show the increase Control cells (no biocide) in lag time with increasing gelatin concentration. Only after 72 hours Chloroxylenol does the 70% gelatin culture attain the Chlorhexidine light output of the 20% gelatin culture gluconate at three hours. If a real food matrix 4% Ammonium, were employed instead of a homogenformulation 1 eous gel, changes in the spatial dis4% Ammonium, tribution of active bacteria would be formulation 2 easily visualized as areas of increased Hypochlorite light (or darkness) over the average. formulation Biocide monitoring Biocides are widely employed as preservatives or disinfectants for microbiological control in envimnmen:al applications, and in the food, pharmaceutical, engineering and service industries. Their application

Hypochiorite

Fig. 3 Testing biocide activity against Listeria monocytogenes by measuring cellular bioluminescence after a ;-~ninute incubation period. Each well contains 107 bioluminescent cells. The biocide concentration decreases from 1% (v/v) in the right-hand wells to zero in the wells on the extreme left.

Trenctsin Food Science & Technology January 1991

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effectively to enumerate a film of surface-attached cells ~3. Cells in an established biofilm often have an increased resistance to disinfectiont4. However, it is extremely difficult to measure antimicrobial action against biofilm cultures reproducibly, using classical plate assays. Bioluminescent bacteria should facilitate such assays and, in addition, extend detection from controlled industrial applications to the detection of environmental toxicants t'~s.

Conclusions This review has focused on only a few of the potential applications of bioluminescent bacteria in microbiology. There is a remarkable harmony between applications that have 'near-market' potential and fundamental research objectives that seek to use the new methods to further understand microbial genetics, physiology and biochemistry. With creativity and imagination, bioluminescent bacteria could be used to change aspects of microbiological practice forever.

2 3 4

S 6 7 8 9 10

11 12 13

References 1

Dodd, C.E.R.,Waites,W.M. and Stewart,G.S.A.B.in Biotechnology and Genetic EngineeringReviews7 (Tombs,M.P.,ed.), Intercept(in press)

14 lS

Eden,R. and Eden,G. (1984) ImpedanceMicrobiology, JohnWiley and Sons Jarvis,B. and Easter,M.C. (1987)J. Appl. BacterioL 63, 115S-126S Ulitzur, S. and Kuhn, J. (1987) in Bioluminescenceand Chemiluminescence New Perspectives{Schlomerich,J., Andreesen,R., Kapp,A., Ernst,M. and Woods, W.G., eds), pp. 463-472, JohnWiley Wolber,P.K.andGreen, R.L(1990) TrendsFoodSci. Technol. 1, 80-82 Meighen,E.A.(1988) Annu. Rev.Microbiol. 42, 151-176 Blissel,SJ. and Stewart,G.S.A.B.(1989) Left. Appl. MicrobioL 9, 149-152 Stewart,G.S.A.B.,Smith, AJ. and Denyer,S.P.(1989) FoodSci. TechnoL Today3, 19-22 Ellison,A., Perry,S.E.and Stewart,G.S.A.B. Int. J. Food Microbigr (in press) Ulitzer,S., Suissa,M. and Kuhn,J.C. (1989) in Rapid Methods and Automation in Microbiology and Immunology (Balows,A., Tilton, R.C. and Turano,A., eds), pp. 235-241, Brixia Academic Press Ahmad,K.A. and Stewart,G.S.A.B.J.Appl. Bacteriol. (in press) Park,S.F.,Nissen, U. and Stewart,G.S.A.B.J. Biolumin. Cbemilumin. (in press) Jassim,S.A.A., Stewart,G.S.A.B.and Denyer,S.P.Biofouling (in press) Costerton,J.W. (1984) Dev. Ind. Microbiol. 25, 363-372 Korpela,M. and Karp, M. (1988) Biotechnol. Left. 10, 383-388

Review

Recent developments

in the search for methods of assessing zinc status Antoine E. EI-Khoury

The assessment of trace element status is important for the determination of trace element requirements and for the development of strategies to achieve adequate trace element intakes in populations. Since zinc is involved in many areas of metabolism, zinc status must be considered when the nutritional status of apparently healthy individuals or of hospital patients is being studied. However, methods of assessment will vary with the subject's age and physiological state; for example, apparently healthy, growing children would be assessed in a different way than elderly osteoporotic patients receiving total parenter~I nutrition after surgery.

The studyof zinc metabolism

The difficulties of assessing zinc status are partly related to a lack of knowledge of zinc metabolism in different physiological states. A better understanding of body zinc distribution/redistribution and its metabolic significance is essential for evaluating any proposed method of assessing zinc status. Intestinal absorption and secretion of zinc have been assessed by different methods using oral' or intravenous2 administration of stable zinc isotopes. However, only one aspect of zinc metabolism was directly measured by these methods: luminal disappearance of an orally administered tracer, or luminal appearance of an intravenously administered tracer. The measurements represent the combined effects of three processes: absorption, endogenous Antoine E. EI-Khourf is with the Human Nutrition Unit, The Rowett Research excretion and faecal excretion3. Indeed, the duration of Institute, GreenburnRoad,Bucksbum,AberdeenAB2 9SB, UK. faecal collection has a significant effect on the measured

Novel methods of assessingzinc status are discussed in relation to the importance of achieving a reliable measure of zinc status in humans. Methods of assessingzinc status mend to lack specificity and sensitivity, and are often impracticable. A beger understanding of zinc metabolism would facilitate the application of more appropriate techniques for assessing zinc status. The characterization of metabolic events specific to marginal zinc deficiency is essential for a clearer understanding of zinc requirements and for the identification of foods or dietary regimesthat influence zinc status.

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©1991,ElsevierSciencePublishersLtd,(UK) 0924-2244/91/$02,e0

Trends in Food Science & Technology January 1991