JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 5 115-122 (1990)
ln vivo Bioluminescence: A Cellular Reporter for Research and Industry S. A. A. Jassim', A. Ellison, S. P. Denyer' and G. S. A. B. Stewart Department of Applied Biochemistry & Food Science, Faculty of Agricultural & Food Sciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire LEI 2 5RD, UK 'Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD. UK
The detection o f specific bacterial pathogens, indicator microorganisms and antimicrobial substances, and the recovery of microorganisms f r o m sub-lethal injury, are a l l aspects o f importance t o industry w h i c h are currently being targeted using in vivo bioluminescence. I n all instances, a key requirement f o r the application o f bioluminescence is t h e establishment o f a s t r i c t correlation between in vivo bioluminescence and cell viability, as determined by colony counting o n agar plates. Comparative studies f o r biocides (phenol, chlorhexidine diacetate. phenol thioether), f o r a virucide (hypochlorite) and f o r cellular recovery of S. typhimurium f r o m sub-lethal injury, all indicate t h a t such a correlation is valid. Furthermore, real-time measurements o f in vivo bioluminescence reveal a major population of bacterial cells t h a t retain functional intracellular biochemistry, b u t are defective i n their ability t o replicate post o f freeze injury.
Keywords: in vivo bioluminescence; biocides; virucides; sub-lethal injury
INTRODUCTION
The detection and enumeration of microorganisms, coupled with the detection and assay of antimicrobial substances, represent key industrial aspects of microbiology. The development of rapid methods to facilitate these assays is of major significance both to provide new and simple protocols and to bring microbial assays into real-time, rather than their current retrospective position. I n uiuo bioluminescence is an emerging tool for the microbiologist which may address many of these needs of industry. Genetic engineering allows the introduction of a bioluminescent phenotype into industrially important bacteria and, because the emission of light depends on bacterial viability, such constructs represent novel reagents to moni0884 3996/90/020115-08$05.00 A] 1990 by John Wiley & Sons, Ltd.
tor in situ industrial biocides, residual antibiotics, starter culture activity and the efficiency of processing parameters such as pasteurization and sterilization. Complementing the direct use of bioluminescent bacteria is the introduction of the genes that code for the biochemistry of light into bacteriophages. Lacking the enzymes for biological energy production, such engineered bacteriophages are dark. Infection of a bacterial host, however, leads to expression of the lux genes incorporated into the phage genome and subsequently the emission of light from the infected host. Specific bacterial pathogens such as Salmonella spp. and Listeria monocytogenes are being targeted using this novel bacteriophage technology. In addition specific groups of bacteria such as the enterics are being investigated to provide an indication of hyReceived 6 December 1989
116
S. A. A. JASSIM, A. ELLISON, S. P. DENYER AND G . S. A. 6.STEWART
giene status. With a total assay time of less than 1 hour and a detection limit of lo3 enterics/g this technology offers an entirely new prospect for microbiological monitoring: an on-line contribution to hazard analysis critical control point (H ACCP). Industrial aspects of in oioo bioluminescence have been the subject of several recent reviews (Stewart, 1989, 1990; Stewart, et al., 1989, 1990) which describe in detail the potential impact of this technology. The present paper therefore focuses firstly on providing validation data on the development of a rapid industrial biocide assay and, with the aid of lux' recombinant bacteriophages, extends this concept into the detection and evaluation of virucides. A second area of application focusses on the use of bioluminescence as a reporter of biochemical function during microbial stress and recovery. Although currently a research application, the ability to visualize in real-time both the genetic and physiological response of microorganisms to stress has significant implications for optimizing industrial protocols designed to resuscitate and recover bacteria from sub-lethal injury.
MATERIALS A N D M E T H O D S Bacterial strains and conditions for growth
Biocide challenge
Reaction mixtures were prepared containing varying concentrations of biocide in sterile distilled water to which were added organisms to a final density of 3 x 109/ml. At appropriate time intervals, 25 p1 samples were dispensed into microtitre tray wells containing a suitable biocide-neutralizing agent (Hugo and Denyer, 1987) and examined for light output in an Amerlite luminometer (Amersham International plc). Parallel viable counts were performed by the method of Miles et al. (1938) on culture samples treated with biocide and neutralized by a similar protocol. Virucide challenge
A recombinant derivative of bacteriophage Lambda containing the luxAB region of I.:jischeri (Ulitzur and Kuhn, 1987) was employed in this assay. The lux' Lambda phage were treated with a range of concentrations of hypochlorite solution at 20°C and at a phage concentration of 5 x 108/ml. After a virucide contact time of 15 min, 1 x lo7 phage were removed (20 pl) and mixed with 180 pl of the Lambda host E . coli W3110, at a cell density of 108/ml. The host/phage mix was adjusted to 1 ml with L-broth and incubated at 30°C for 40 min. Culture samples were assayed for bioluminescence in an Amerlite luminometer after addition of 40 pl/ ml of a 1% solution of dodecanal in ethanol.
The following recombinant bacteria were used in this study: Salmonella typhimurium LT2, transformed to a bioluminescent phenotype with either the constitutive lux expression plasmid pSB 100 (Blissett and Stewart, 1989) or a construct providing osmoregulated lux expression, pSB99 (Park et al., 1989). Escherichia coli NCTC 8 196, transformed to a bioluminescent phenotype with a plasmid providing for the expression of the entire lux operon from Vibrio jischeri. E . coli and S . typhimurium were maintained by culture in L-broth (Maniatis et al., 1982) containing, where appropriate, 30 mg/l ampicillin or 50 mg/l chloramphenicol, to maintain plasmid selection. Cultures to be used for biocide challenge were harvested after 18 h at 22"C, resuspended in and washed with sterile distilled water, recovered by centrifugation and resuspended in water to a final cell density of 6 x 108/ml.
Recovery from sub-lethal injury
Salmonella typhimurium LT2, containing either of the lux expression plasmids pSBlOO or pSB99, was grown in L-broth to an A,,, of 0.7. These cultures were subsequently diluted into 0.1% peptone water in a static water bath at 30"c. After one hour, a portion of the culture was removed for cold shock which comprised incubation at - 18°C for one hour. The cells were subsequently thawed and diluted l:lO, in parallel with the control culture, into L-broth or buffered peptone water as appropriate. When using the osmoregulated lux expression plasmid pSB99, bioluminescence was induced by the addition of NaCl to 0.3 mol/l in either Lbroth or peptone water. Bioluminescence was monitored from both the control and experimental cultures prior to, during and after recovery. Samples (1 ml) were mixed with 40 pl of a 1% solution
117
BIOLUMINESCENCE: A CELLULAR REPORTER
of dodecanal in ethanol and transferred to a Turner Designs 20 luminometer (Streptech Ins., Herts, UK). Viable counts were determined on either selective or non-selective agar. The non-selective agar was Tryptone Soy Agar supplemented with 0.3% yeast extract (TSYA). For selective agar, 0.4% sodium biselenite was added to the TSYA as directed by Mackey and Derrick (1982). RESULTS AND DISCUSSION Biocide challenge
Biocides are widely employed for microbiological control in manufacturing, environmental, engineering, service and other industries as preservatives or disinfectants. Their application is often complex
and adverse situations can lead to their depletion through chemical decomposition, dilution and inactivation. Under these circumstances, monitoring of biocide levels is essential to ensure adequate protection, and continuing biocide presence and efficacy is usually confirmed by microbiological challenge testing (Stewart et al., 1990). Currently, such tests employ conventional culture techniques for determining microbial survival and are therefore not amenable to immediate on-site testing. Bioluminescent microorganisms offer the potential for rapid biocide testing providing a clear correlation between cell viability and bioluminescence can be established. Data in support of this correlation is presented in Fig. 1 and Table 1. For E . coli NCTC 8196, the kinetics of cell death following biocide action are clearly reported by bioluminescence (Fig. 1) and can be used to estab-
\
Contact Time(min)
Contact 'Nme(min1
Figure 1. The effect of phenol concentration on [A] viable counts and [B] bioluminescence. (Phenol concentration (% w/v) 0 0.0 0.2.W 0.3, 0 0 4 , A 0.5. A 0.55. x 0.6, 0.65. 1.0)
+
118
S. A. A. JASSIM, A. ELLISON, S. P. DENYER AND G. S. A. B. STEWART
loo(
Table 1. Biocide concentration exponents determined from viable count (VC) and bioluminescent (BL) measurements over the concentration range indicated
Biocide Phenol Chlorhexidine diacetate Phenol thioether (Hatacide LP5)
Concentration range
Concentration coefficients VG BL
0.55-1 .O % W / V 5-70 pg/ml
3.21 1.89
4.05 1.92
0.001 -0.01 % w / v
4.39
4.01
lish the decimal reduction time (D-value) at specified concentrations (Fig. 2) from which the concentration exponent for individual biocides (Hugo and Denyer, 1987) can be determined (Table 1). Good agreement between bioluminescence and viable count data exists over currently used bacteriocidal concentration ranges. A concentration-dependent relationship between inhibition of bioluminescence and antimicrobial activity was also seen for the principally bacteriostatic agent 1, 2-benzisothiazolin-3-one (Fuller et a/., 1985) where emergence of full bioluminescence occurs only at sub-optimal concentrations of the compound (data not shown). In all instances, detection of bacteriocidal activity was achieved in a tenminute test and was supportive of the application of in vivo bioluminescence for rapid monitoring of biocide activity.
lo(
d
dl
10
b 1 .1
J
i
10
Concentration (oow/v)
Figure 2. Logarithmic relationship between phenol con centration and decimal reduction time (D-value) as calculated from viable count ( 0 ) and bioluminescence (0) measurements
Virucide challenge
when that host had been treated with a level of The determination and assessment of antiviral ac- hypochlorite equivalent to that carried over from tivity in potential virucides generally requires ela- phage treatment in the experimental protocol borate cell culture or electron microscopy facilities. (Table 2). The above results indicate that recombinant bacThe routine testing of such compounds would be significantly improved by the development of a teriophage containing the genes for bacterial lucirapid bioluminescent test. We have explored this ferase do offer the potential for developing rapid concept by using a recombinant bacteriophage screening assays for virucidal activity. containing expression competent luxA and /uxB Recovery f r o m sub-lethal injury genes as a model virus. Following treatment with the virucidal agent hypochlorite, lux+ Lambda phage were unable to Treatments such as heating, chilling, freezing, elicit a bioluminescent response after incubation moisture reduction, irradiation and exposure to with a non-bioluminescent E . coli host (Table 2). preservatives can produce within foods populations Control experiments confirmed that untreated of stressed or sub-lethally injured microorganisms lux' Lambda phage did produce a bioluminescent (Busta, 1976). Such sub-lethally injured cells can phenotype on transfection of the E . coli host, even retain their pathogenic traits and it is, therefore,
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BIOLUMINESCENCE: A CELLULAR REPORTER
Table 2. Destruction of LUX' Lambda phage by hypochlorite Light output (arbitrary units) Hypochlorite conc. (mg% w/v)
E. coli-phage mix following hypochlorite pretreatment of phage
E. coli-phage mix following hypochlorite pretreatment of E. coli
0.00
13.11
11.7
0.01
0.005
13.9
0.1
0.004
13.2
1
0.004
14.9
10
0.004
13.7
very important to enumerate such cells during microbial analysis. Sub-lethally injured bacteria are unable to tolerate selective growth conditions in which normal cells may easily multiply and a period of resuscitation is therefore essential, in a non-selective medium, in order to allow time for intracellular repair (Ray, 1979). Experimental methods to ensure recovery typically require overnight incubation and in consequence, severely compromise efforts to reduce the time for bacterial detection and enumeration based on emerging technologies such as DNA probes, ELISA and lux recombinant bacteriophage. In the past it has not been possible to monitor the recovery process in real-time and hence protocols for recovery are empirical and based on colony counts obtained after plating and at least overnight incubation. In uiuo bioluminescence may provide a novel real-time tool for probing the recovery of microorganisms from sub-lethal injury. In uiuo bioluminescence depends upon functional intracellular biochemistry to provide a continued supply of FMNH, (Meighen, 1988). Stress responses that directly or indirectly affect the intracellular production of FMNH, can be monitored as changes in light output/cell, since dead cells produce no light. Fig. 3 provides bioluminescence and viable count data for the freeze injury of S. typhimurium harbouring the constitutive lux expression plasmid pSB100. Fig. 3 [A, B] and [C, D] represent duplicate experiments to indicate the degree of reproducibility. Plots from control cultures have been adjusted (solid line) to take into account the absence of growth in the experimental cultures during the freezing period. Three hours into the recovery
period, the bioluminescence and viable count data reflect near-perfect correlation. Immediately after freezing, however, there is a marked discrepancy between the two data sets. In set [A, B] the viable count shows a 30-fold reduction in viable cells at all times after freezing. The bioluminescence data, however, shows only a 5-fold reduction on bioluminescence immediately after freezing but a 30-fold reduction after 3 hours. Set [C,D] gives qualitatively similar data. One important interpretation of these results is that 20% of the bacteria in set A,B and about 13% in set C,D survive the freeze-thaw cycle with functional intracellular biochemistry. Only 3% and 2% respectively, however, retain the ability to subsequently divide and replicate. Fig. 4 provides an equivalent experiment to that in Fig. 3 except that the S. typhimurium contained the osmoregulated lux expression plasmid pSB99. In Fig. 4 [A,B] the cells were osmotically upshocked at the start of the experiment, prior to the freeze-thaw cycle in the experimental culture. In Fig. 4 [C,D] the cells were osmotically upshocked at a point in time determined as immediately after freeze-thaw in the experimental culture. The increase in bioluminescence in response to osmotic upshock is clearly defined in Fig. 4 [B] as a 3 log increase over the first 60 min in the control. With this exception, the data is reflective of that in Fig. 3. A dramatic difference between bioluminescence immediately after freeze-thaw and after 3 hours is again indicative of a high percentage of cells retaining biochemical function but only a small proportion retaining replication competence. Fig. 4 [D] is more challenging, however, in that the osmotic induction of bioluminescence registered by the con-
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S. A. A. JASSIM, A. ELLISON, S. P. DENYER AND G. S. A. 6.STEWART
0
0
0
100
200
300
400
500
100
200 300 Time (min)
400
500
0
100
200
300
400
500
Figure 3. A comparison between viable plate count, [ A ] and [C], and in vivo bioluminescence, [B] and [ D ] , for constitutively bioluminescent S. typhimurium LT2 subjected to freeze injury. Graphs A/B and C/D reflect duplicate control cultures without freeze iniury-the solid line represents the control adjusted to subtract the growth experiments (0 experimental samples subjected to freezing at - 18% and obtained during the freeze period of the experimental sample samples thawed) subsequent thaw, V samples frozen,
trol culture is entirely absent from the experimental culture, subjected to the freeze-thaw cycle. A reasonable interpretation of this data could be that the cells which have been subjected to the freeze cycle do not in any detectable numbers, retain the ability to respond at the genetic and/or biochemical level to osmotic upshock. Responses would include changes in [K'] accumulation and in DNA supercoiling (Higgins et al., 1987) and where the putative defects in response are located, is not yet known. One further interesting feature is that the colony count data (Fig. 4 [C]) shows a 857-fold drop in viability, some 20-fold more severe a loss than those obtained in Fig. 3. This observation supports the concept that the additional insult of an osmotic upshock, after freezing, imparts a severe restriction on numbers of survivors.
It seems clear from the above two examples that in vivo bioluminescence can provide new insights into the complex processes of microbial recovery from sub-lethal injury and that, as a real-time probe, it should prove possible to investigate the direct effect on recovery, of media, temperature and other environmental parameters.
Acknowledgements This work was supported by a grant from the AFRC and by Amersham International plc. The authors also wish to thank Professor W. M. Waites for help and encouragement and Mrs s. Godber for typing the manuscript.
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I
0
0
100
200
300
400
500
0
100
200
300
400
500
0
100 ..
200 300 Time (min)
400
500
0
100
200 300 Time (min)
400
500
0
Figure 4. A / B A comparison between viable plate count [A] and in vivo bioluminescence [ B ] for S. typhimurum LT2 containing the osmoregulated Iuxexpression plasmid pSB99. Cells were osmotically upshocked 1 h prior to freezing. C/D As A/B expect that cells were osmotically upshocked immediately after freeze-thaw. (0control culture without freeze injury-the solid line represents a compensation for culture changes occurring during the freeze period of the experimental samples experimental samples subjected t o freezing at - 18°C and subsequent thaw, V samples frozen. sample, thawed and, for C/D. NaCl added to 0.3 mol/l
REFERENCES Blisset, S. J. and Stewart, G. S. A. B. (1989). I n viuo bioluminescent determination of apparent K , s for aldehyde in recombinant bacteria expressing lux A/B. Lett. Appl. Microbiol., 9, 149-152. Busta, F. F. (1976), Practical implications of injured microorganisms in food. J . Milk Food Technol., 39, 138. Fuller, S. J., Denyer, S. P., Hugo, W. B., Pemberton, D., Woodcock, P. M. and Buckley, A. J. (1985). The mode of action of 1,2-benzisothiazoIin-3-one on Staphylococcus aureus. Lett. Appl. Microbiol., 1, 13-15. Higgins, C. F., Cairney, J., Stirling, D., Sutherland, L. and Booth, I. R. (1987). Osmotic regulation of gene expression: ionic strength as an intracellular signal. Trends Biochem. Sci., 12, 339-344. Hugo, W. B. and Denyer, S. P. (1987). The concentration exponent of disinfectants and preservatives (biocides). In Preservatives in the Food, Pharmaceutical and Environmental
Industries. SAB Technical Series, Vol. 22, Board, R . G., Allwood, M. C. and Banks, J. G. (Eds), Blackwell Scientific Publications, Oxford, pp. 281-291. Mackey, B. M. and Derrick, C. M. (1982). The effect of sublethal injury by heating, freezing, drying and garnrna-radiation on the duration of lag phase of S. typhimurium. J . A p p l . Bact., 53, 243-251. Maniatis, T., Fritsch, E. F. and Sambrooke, J. (1982). In Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Meighen, E. A. (1988). Enzymes and genes from the lux operons of bioluminescent bacteria. Ann. Rev. Microbiol., 42, 151-176. Miles, A. A,, Misra, S. S. and Irwin, J. 0. (1938). The estimation of the bactericidal power of the blood. J . Hygiene (London), 38, 733-749. Park, S. F., Stirling, D. A,, Hulton, C. S. J., Booth, I. R., Higgins, C. F. and Stewart, G. S. A. B. (1989). A novel, non-invasive promoter probe vector: cloning of the osmoregulated proU
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promoter of Escherichia coli K12. Mol. Microhiol., 3, 101 1 -1023. Ray, B. (1979). Methods to detect stressed microorganisms. J . Food Prot., 42, 346-355. Stewart, G . S. A. B. (1989). The detection of microorganisms using in viuo bioluminescence. In Proceedings of Biotech '89. Blenheim Online Publications, Pinner, pp. 175- 182. Stewart, G. S. A. B. (1990). I n uiuo bioluminescence: new potentials for microbiology. Lett. Appl. Microbiol., 10( I), in press. Stewart, G. S. A. B., Smith, A. J. and Denyer, S. P. (1989). Genetic engineering for bioluminescent bacteria. Food Sci. &
Techno/. Today., 3, 19-22. Stewart, G. S. A. B., Jassim, S. A. A. and Denyer, S. P. (1990). Mechanisms of action and rapid biocide testing. In Mechanisms of Action ofchemical Biocides: their Study and Exploitation, S A B Technical Series 28, Denyer, S. P. and Hugo, W. B. (Eds), Blackwell Scientific Publications, Oxford, in press. Ulitzur, S. and Kuhn, J. (1987), Introduction of lux genes into bacteria, a new approach for specific determination of bacteria and their antibiotic susceptibility. In Bioluminescence and Chemiluminescence: New Perspectives. Scholmerich, J., Andreesen, R., Kapp, A,, Ernst, M. and Woods, W. G. (Eds). John Wiley, Chichester, pp. 463-472.
ln vivo Bioluminescence: A Cellular Reporter for Research and Industry S. A. A. Jassim', A. Ellison, S. P. Denyer' and G. S. A. B. Stewart Department of Applied Biochemistry & Food Science, Faculty of Agricultural & Food Sciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire LEI 2 5RD, UK 'Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD. UK
The detection o f specific bacterial pathogens, indicator microorganisms and antimicrobial substances, and the recovery of microorganisms f r o m sub-lethal injury, are a l l aspects o f importance t o industry w h i c h are currently being targeted using in vivo bioluminescence. I n all instances, a key requirement f o r the application o f bioluminescence is t h e establishment o f a s t r i c t correlation between in vivo bioluminescence and cell viability, as determined by colony counting o n agar plates. Comparative studies f o r biocides (phenol, chlorhexidine diacetate. phenol thioether), f o r a virucide (hypochlorite) and f o r cellular recovery of S. typhimurium f r o m sub-lethal injury, all indicate t h a t such a correlation is valid. Furthermore, real-time measurements o f in vivo bioluminescence reveal a major population of bacterial cells t h a t retain functional intracellular biochemistry, b u t are defective i n their ability t o replicate post o f freeze injury.
Keywords: in vivo bioluminescence; biocides; virucides; sub-lethal injury
INTRODUCTION
The detection and enumeration of microorganisms, coupled with the detection and assay of antimicrobial substances, represent key industrial aspects of microbiology. The development of rapid methods to facilitate these assays is of major significance both to provide new and simple protocols and to bring microbial assays into real-time, rather than their current retrospective position. I n uiuo bioluminescence is an emerging tool for the microbiologist which may address many of these needs of industry. Genetic engineering allows the introduction of a bioluminescent phenotype into industrially important bacteria and, because the emission of light depends on bacterial viability, such constructs represent novel reagents to moni0884 3996/90/020115-08$05.00 A] 1990 by John Wiley & Sons, Ltd.
tor in situ industrial biocides, residual antibiotics, starter culture activity and the efficiency of processing parameters such as pasteurization and sterilization. Complementing the direct use of bioluminescent bacteria is the introduction of the genes that code for the biochemistry of light into bacteriophages. Lacking the enzymes for biological energy production, such engineered bacteriophages are dark. Infection of a bacterial host, however, leads to expression of the lux genes incorporated into the phage genome and subsequently the emission of light from the infected host. Specific bacterial pathogens such as Salmonella spp. and Listeria monocytogenes are being targeted using this novel bacteriophage technology. In addition specific groups of bacteria such as the enterics are being investigated to provide an indication of hyReceived 6 December 1989
116
S. A. A. JASSIM, A. ELLISON, S. P. DENYER AND G . S. A. 6.STEWART
giene status. With a total assay time of less than 1 hour and a detection limit of lo3 enterics/g this technology offers an entirely new prospect for microbiological monitoring: an on-line contribution to hazard analysis critical control point (H ACCP). Industrial aspects of in oioo bioluminescence have been the subject of several recent reviews (Stewart, 1989, 1990; Stewart, et al., 1989, 1990) which describe in detail the potential impact of this technology. The present paper therefore focuses firstly on providing validation data on the development of a rapid industrial biocide assay and, with the aid of lux' recombinant bacteriophages, extends this concept into the detection and evaluation of virucides. A second area of application focusses on the use of bioluminescence as a reporter of biochemical function during microbial stress and recovery. Although currently a research application, the ability to visualize in real-time both the genetic and physiological response of microorganisms to stress has significant implications for optimizing industrial protocols designed to resuscitate and recover bacteria from sub-lethal injury.
MATERIALS A N D M E T H O D S Bacterial strains and conditions for growth
Biocide challenge
Reaction mixtures were prepared containing varying concentrations of biocide in sterile distilled water to which were added organisms to a final density of 3 x 109/ml. At appropriate time intervals, 25 p1 samples were dispensed into microtitre tray wells containing a suitable biocide-neutralizing agent (Hugo and Denyer, 1987) and examined for light output in an Amerlite luminometer (Amersham International plc). Parallel viable counts were performed by the method of Miles et al. (1938) on culture samples treated with biocide and neutralized by a similar protocol. Virucide challenge
A recombinant derivative of bacteriophage Lambda containing the luxAB region of I.:jischeri (Ulitzur and Kuhn, 1987) was employed in this assay. The lux' Lambda phage were treated with a range of concentrations of hypochlorite solution at 20°C and at a phage concentration of 5 x 108/ml. After a virucide contact time of 15 min, 1 x lo7 phage were removed (20 pl) and mixed with 180 pl of the Lambda host E . coli W3110, at a cell density of 108/ml. The host/phage mix was adjusted to 1 ml with L-broth and incubated at 30°C for 40 min. Culture samples were assayed for bioluminescence in an Amerlite luminometer after addition of 40 pl/ ml of a 1% solution of dodecanal in ethanol.
The following recombinant bacteria were used in this study: Salmonella typhimurium LT2, transformed to a bioluminescent phenotype with either the constitutive lux expression plasmid pSB 100 (Blissett and Stewart, 1989) or a construct providing osmoregulated lux expression, pSB99 (Park et al., 1989). Escherichia coli NCTC 8 196, transformed to a bioluminescent phenotype with a plasmid providing for the expression of the entire lux operon from Vibrio jischeri. E . coli and S . typhimurium were maintained by culture in L-broth (Maniatis et al., 1982) containing, where appropriate, 30 mg/l ampicillin or 50 mg/l chloramphenicol, to maintain plasmid selection. Cultures to be used for biocide challenge were harvested after 18 h at 22"C, resuspended in and washed with sterile distilled water, recovered by centrifugation and resuspended in water to a final cell density of 6 x 108/ml.
Recovery from sub-lethal injury
Salmonella typhimurium LT2, containing either of the lux expression plasmids pSBlOO or pSB99, was grown in L-broth to an A,,, of 0.7. These cultures were subsequently diluted into 0.1% peptone water in a static water bath at 30"c. After one hour, a portion of the culture was removed for cold shock which comprised incubation at - 18°C for one hour. The cells were subsequently thawed and diluted l:lO, in parallel with the control culture, into L-broth or buffered peptone water as appropriate. When using the osmoregulated lux expression plasmid pSB99, bioluminescence was induced by the addition of NaCl to 0.3 mol/l in either Lbroth or peptone water. Bioluminescence was monitored from both the control and experimental cultures prior to, during and after recovery. Samples (1 ml) were mixed with 40 pl of a 1% solution
117
BIOLUMINESCENCE: A CELLULAR REPORTER
of dodecanal in ethanol and transferred to a Turner Designs 20 luminometer (Streptech Ins., Herts, UK). Viable counts were determined on either selective or non-selective agar. The non-selective agar was Tryptone Soy Agar supplemented with 0.3% yeast extract (TSYA). For selective agar, 0.4% sodium biselenite was added to the TSYA as directed by Mackey and Derrick (1982). RESULTS AND DISCUSSION Biocide challenge
Biocides are widely employed for microbiological control in manufacturing, environmental, engineering, service and other industries as preservatives or disinfectants. Their application is often complex
and adverse situations can lead to their depletion through chemical decomposition, dilution and inactivation. Under these circumstances, monitoring of biocide levels is essential to ensure adequate protection, and continuing biocide presence and efficacy is usually confirmed by microbiological challenge testing (Stewart et al., 1990). Currently, such tests employ conventional culture techniques for determining microbial survival and are therefore not amenable to immediate on-site testing. Bioluminescent microorganisms offer the potential for rapid biocide testing providing a clear correlation between cell viability and bioluminescence can be established. Data in support of this correlation is presented in Fig. 1 and Table 1. For E . coli NCTC 8196, the kinetics of cell death following biocide action are clearly reported by bioluminescence (Fig. 1) and can be used to estab-
\
Contact Time(min)
Contact 'Nme(min1
Figure 1. The effect of phenol concentration on [A] viable counts and [B] bioluminescence. (Phenol concentration (% w/v) 0 0.0 0.2.W 0.3, 0 0 4 , A 0.5. A 0.55. x 0.6, 0.65. 1.0)
+
118
S. A. A. JASSIM, A. ELLISON, S. P. DENYER AND G. S. A. B. STEWART
loo(
Table 1. Biocide concentration exponents determined from viable count (VC) and bioluminescent (BL) measurements over the concentration range indicated
Biocide Phenol Chlorhexidine diacetate Phenol thioether (Hatacide LP5)
Concentration range
Concentration coefficients VG BL
0.55-1 .O % W / V 5-70 pg/ml
3.21 1.89
4.05 1.92
0.001 -0.01 % w / v
4.39
4.01
lish the decimal reduction time (D-value) at specified concentrations (Fig. 2) from which the concentration exponent for individual biocides (Hugo and Denyer, 1987) can be determined (Table 1). Good agreement between bioluminescence and viable count data exists over currently used bacteriocidal concentration ranges. A concentration-dependent relationship between inhibition of bioluminescence and antimicrobial activity was also seen for the principally bacteriostatic agent 1, 2-benzisothiazolin-3-one (Fuller et a/., 1985) where emergence of full bioluminescence occurs only at sub-optimal concentrations of the compound (data not shown). In all instances, detection of bacteriocidal activity was achieved in a tenminute test and was supportive of the application of in vivo bioluminescence for rapid monitoring of biocide activity.
lo(
d
dl
10
b 1 .1
J
i
10
Concentration (oow/v)
Figure 2. Logarithmic relationship between phenol con centration and decimal reduction time (D-value) as calculated from viable count ( 0 ) and bioluminescence (0) measurements
Virucide challenge
when that host had been treated with a level of The determination and assessment of antiviral ac- hypochlorite equivalent to that carried over from tivity in potential virucides generally requires ela- phage treatment in the experimental protocol borate cell culture or electron microscopy facilities. (Table 2). The above results indicate that recombinant bacThe routine testing of such compounds would be significantly improved by the development of a teriophage containing the genes for bacterial lucirapid bioluminescent test. We have explored this ferase do offer the potential for developing rapid concept by using a recombinant bacteriophage screening assays for virucidal activity. containing expression competent luxA and /uxB Recovery f r o m sub-lethal injury genes as a model virus. Following treatment with the virucidal agent hypochlorite, lux+ Lambda phage were unable to Treatments such as heating, chilling, freezing, elicit a bioluminescent response after incubation moisture reduction, irradiation and exposure to with a non-bioluminescent E . coli host (Table 2). preservatives can produce within foods populations Control experiments confirmed that untreated of stressed or sub-lethally injured microorganisms lux' Lambda phage did produce a bioluminescent (Busta, 1976). Such sub-lethally injured cells can phenotype on transfection of the E . coli host, even retain their pathogenic traits and it is, therefore,
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Table 2. Destruction of LUX' Lambda phage by hypochlorite Light output (arbitrary units) Hypochlorite conc. (mg% w/v)
E. coli-phage mix following hypochlorite pretreatment of phage
E. coli-phage mix following hypochlorite pretreatment of E. coli
0.00
13.11
11.7
0.01
0.005
13.9
0.1
0.004
13.2
1
0.004
14.9
10
0.004
13.7
very important to enumerate such cells during microbial analysis. Sub-lethally injured bacteria are unable to tolerate selective growth conditions in which normal cells may easily multiply and a period of resuscitation is therefore essential, in a non-selective medium, in order to allow time for intracellular repair (Ray, 1979). Experimental methods to ensure recovery typically require overnight incubation and in consequence, severely compromise efforts to reduce the time for bacterial detection and enumeration based on emerging technologies such as DNA probes, ELISA and lux recombinant bacteriophage. In the past it has not been possible to monitor the recovery process in real-time and hence protocols for recovery are empirical and based on colony counts obtained after plating and at least overnight incubation. In uiuo bioluminescence may provide a novel real-time tool for probing the recovery of microorganisms from sub-lethal injury. In uiuo bioluminescence depends upon functional intracellular biochemistry to provide a continued supply of FMNH, (Meighen, 1988). Stress responses that directly or indirectly affect the intracellular production of FMNH, can be monitored as changes in light output/cell, since dead cells produce no light. Fig. 3 provides bioluminescence and viable count data for the freeze injury of S. typhimurium harbouring the constitutive lux expression plasmid pSB100. Fig. 3 [A, B] and [C, D] represent duplicate experiments to indicate the degree of reproducibility. Plots from control cultures have been adjusted (solid line) to take into account the absence of growth in the experimental cultures during the freezing period. Three hours into the recovery
period, the bioluminescence and viable count data reflect near-perfect correlation. Immediately after freezing, however, there is a marked discrepancy between the two data sets. In set [A, B] the viable count shows a 30-fold reduction in viable cells at all times after freezing. The bioluminescence data, however, shows only a 5-fold reduction on bioluminescence immediately after freezing but a 30-fold reduction after 3 hours. Set [C,D] gives qualitatively similar data. One important interpretation of these results is that 20% of the bacteria in set A,B and about 13% in set C,D survive the freeze-thaw cycle with functional intracellular biochemistry. Only 3% and 2% respectively, however, retain the ability to subsequently divide and replicate. Fig. 4 provides an equivalent experiment to that in Fig. 3 except that the S. typhimurium contained the osmoregulated lux expression plasmid pSB99. In Fig. 4 [A,B] the cells were osmotically upshocked at the start of the experiment, prior to the freeze-thaw cycle in the experimental culture. In Fig. 4 [C,D] the cells were osmotically upshocked at a point in time determined as immediately after freeze-thaw in the experimental culture. The increase in bioluminescence in response to osmotic upshock is clearly defined in Fig. 4 [B] as a 3 log increase over the first 60 min in the control. With this exception, the data is reflective of that in Fig. 3. A dramatic difference between bioluminescence immediately after freeze-thaw and after 3 hours is again indicative of a high percentage of cells retaining biochemical function but only a small proportion retaining replication competence. Fig. 4 [D] is more challenging, however, in that the osmotic induction of bioluminescence registered by the con-
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Figure 3. A comparison between viable plate count, [ A ] and [C], and in vivo bioluminescence, [B] and [ D ] , for constitutively bioluminescent S. typhimurium LT2 subjected to freeze injury. Graphs A/B and C/D reflect duplicate control cultures without freeze iniury-the solid line represents the control adjusted to subtract the growth experiments (0 experimental samples subjected to freezing at - 18% and obtained during the freeze period of the experimental sample samples thawed) subsequent thaw, V samples frozen,
trol culture is entirely absent from the experimental culture, subjected to the freeze-thaw cycle. A reasonable interpretation of this data could be that the cells which have been subjected to the freeze cycle do not in any detectable numbers, retain the ability to respond at the genetic and/or biochemical level to osmotic upshock. Responses would include changes in [K'] accumulation and in DNA supercoiling (Higgins et al., 1987) and where the putative defects in response are located, is not yet known. One further interesting feature is that the colony count data (Fig. 4 [C]) shows a 857-fold drop in viability, some 20-fold more severe a loss than those obtained in Fig. 3. This observation supports the concept that the additional insult of an osmotic upshock, after freezing, imparts a severe restriction on numbers of survivors.
It seems clear from the above two examples that in vivo bioluminescence can provide new insights into the complex processes of microbial recovery from sub-lethal injury and that, as a real-time probe, it should prove possible to investigate the direct effect on recovery, of media, temperature and other environmental parameters.
Acknowledgements This work was supported by a grant from the AFRC and by Amersham International plc. The authors also wish to thank Professor W. M. Waites for help and encouragement and Mrs s. Godber for typing the manuscript.
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I
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Figure 4. A / B A comparison between viable plate count [A] and in vivo bioluminescence [ B ] for S. typhimurum LT2 containing the osmoregulated Iuxexpression plasmid pSB99. Cells were osmotically upshocked 1 h prior to freezing. C/D As A/B expect that cells were osmotically upshocked immediately after freeze-thaw. (0control culture without freeze injury-the solid line represents a compensation for culture changes occurring during the freeze period of the experimental samples experimental samples subjected t o freezing at - 18°C and subsequent thaw, V samples frozen. sample, thawed and, for C/D. NaCl added to 0.3 mol/l
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Techno/. Today., 3, 19-22. Stewart, G. S. A. B., Jassim, S. A. A. and Denyer, S. P. (1990). Mechanisms of action and rapid biocide testing. In Mechanisms of Action ofchemical Biocides: their Study and Exploitation, S A B Technical Series 28, Denyer, S. P. and Hugo, W. B. (Eds), Blackwell Scientific Publications, Oxford, in press. Ulitzur, S. and Kuhn, J. (1987), Introduction of lux genes into bacteria, a new approach for specific determination of bacteria and their antibiotic susceptibility. In Bioluminescence and Chemiluminescence: New Perspectives. Scholmerich, J., Andreesen, R., Kapp, A,, Ernst, M. and Woods, W. G. (Eds). John Wiley, Chichester, pp. 463-472.
