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Letters in Applied Microhioloyy 1985, 1, 7-1 1 Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death J.G. BANKS, R.G. BOARD & J. PATON School of Biological Sciences, University of Bath, Bath. Avon BA2 7AY Received 8 January 1985 and accepted 15 January 1985 BANKS, J.G., BOARD, R.G. & PATON, J. 1985. Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death. Letters in Applied Microbiology 1, 7-1 1. Escherichia coli B/r exposed to visible light, oxygen and Rose Bengal was killed by singlet oxygen. There was no evidence of cytotoxicity when the organisms were suspended in non-illuminated dye (2 x mol/l). Incubation in oxygenated dye before exposure to light induced a quicker onset of exponential death but the eventual rate was slower than that of organisms exposed continuously to light. The concentration of intracellular adenosine triphosphate (ATP) of illuminated E. coli B/r declined before there was a demonstrable reduction in the number of viable organisms. A carotenoid containing species of Rhodotorula was not killed by the irradiated dye even with a photoflux greater than that used for the coliform. There was no diminution in the ATP content of this yeast with illumination. Thus this study has shown that ATP photometry can be used to explore the kinetics of photoinactivation of sensitive micro-organisms. Visible light, oxygen and a photosensitizer can cause photodynamic damage of micro- organisms (Foote 1976; Krinsky 1976). Two mechanisms can be involved. In one, which involves hydrogen atom/electron transfer from the sensitizer to the substrate, free radicals are formed and these react with oxygen (Foote 1976). In the other inter-system-crossing (energy transfer) from the excited sensitizer to dioxygen is implicated and singlet molecular oxygen is formed (Krinsky 1977; Ito 1978). Water soluble xanthene dyes, of which Rose Bengal (tetra- iodo-tetrachlorofluorescein) is the most effective (Dodge 1983) singlet oxygen producer, kill sensi- tive micro-organisms by the latter mechanism (Ito & Kobayashi 1977). This aggressive form of oxygen has the potential to damage many cell components. In practice it reacts most avidly with those containing high densities of electrons e.g. double bonds of unsaturated fatty acids (Korycka-Dahl & Richardson 1978) and peroxi- dation of membrane lipids is probably a major cause of cell damage (Krinsky 1979; Moss & Smith 1981). Carotenoid containing yeasts are generally less susceptible to photinactivation than are non-pigmented mutants (Maxwell et al. 1966). The pigments can protect an organism in a number of ways. They may quench (1) the triplet sensitizer-the first potentially harmful intermediate formed during photochemically induced oxidation (Krinsky 1979); (2) free radical intermediates; (3) lipid peroxidation derived from type 1 reactions or (4) singlet, oxygen. The last mentioned is probably of great- est importance. The avidity of carotenoids for singlet oxygen is 100000 fold greater (Foote 1976) than that of a commonly used singlet oxygen acceptor, histidine. Viable counting techniques, with relatively long intervals between samplings, have been used routinely in studies of the inactivation of micro-organisms (Freeman & Giese 1952; Bellin et al. 1969; Bezman et al. 1978; Banks et al. 1985). They are poorly suited to this type of work because the kinetics of inactivation are of greater interest than the extent of death. The inherent inaccuracy of counting techniques together with a requirement for a period of

Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death

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Page 1: Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death

Letters in Applied Microhioloyy 1985, 1, 7-1 1

Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death

J.G. B A N K S , R.G. B O A R D & J . PATON School of Biological Sciences, University of Bath, Bath. Avon BA2 7AY

Received 8 January 1985 and accepted 15 January 1985

BANKS, J .G. , B O A R D , R.G. & PATON, J . 1985. Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death. Letters in Applied Microbiology 1, 7-1 1.

Escherichia coli B/r exposed to visible light, oxygen and Rose Bengal was killed by singlet oxygen. There was no evidence of cytotoxicity when the organisms were suspended in non-illuminated dye (2 x mol/l). Incubation in oxygenated dye before exposure to light induced a quicker onset of exponential death but the eventual rate was slower than that of organisms exposed continuously to light. The concentration of intracellular adenosine triphosphate (ATP) of illuminated E. coli B/r declined before there was a demonstrable reduction in the number of viable organisms. A carotenoid containing species of Rhodotorula was not killed by the irradiated dye even with a photoflux greater than that used for the coliform. There was no diminution in the ATP content of this yeast with illumination. Thus this study has shown that ATP photometry can be used to explore the kinetics of photoinactivation of sensitive micro-organisms.

Visible light, oxygen and a photosensitizer can cause photodynamic damage of micro- organisms (Foote 1976; Krinsky 1976). Two mechanisms can be involved. In one, which involves hydrogen atom/electron transfer from the sensitizer to the substrate, free radicals are formed and these react with oxygen (Foote 1976). In the other inter-system-crossing (energy transfer) from the excited sensitizer to dioxygen is implicated and singlet molecular oxygen is formed (Krinsky 1977; Ito 1978). Water soluble xanthene dyes, of which Rose Bengal (tetra- iodo-tetrachlorofluorescein) is the most effective (Dodge 1983) singlet oxygen producer, kill sensi- tive micro-organisms by the latter mechanism (Ito & Kobayashi 1977). This aggressive form of oxygen has the potential to damage many cell components. In practice it reacts most avidly with those containing high densities of electrons e.g. double bonds of unsaturated fatty acids (Korycka-Dahl & Richardson 1978) and peroxi- dation of membrane lipids is probably a major cause of cell damage (Krinsky 1979; Moss & Smith 1981). Carotenoid containing yeasts are

generally less susceptible to photinactivation than are non-pigmented mutants (Maxwell et al. 1966). The pigments can protect an organism in a number of ways. They may quench (1) the triplet sensitizer-the first potentially harmful intermediate formed during photochemically induced oxidation (Krinsky 1979); (2) free radical intermediates; (3) lipid peroxidation derived from type 1 reactions or (4) singlet, oxygen. The last mentioned is probably of great- est importance. The avidity of carotenoids for singlet oxygen is 100000 fold greater (Foote 1976) than that of a commonly used singlet oxygen acceptor, histidine.

Viable counting techniques, with relatively long intervals between samplings, have been used routinely in studies of the inactivation of micro-organisms (Freeman & Giese 1952; Bellin et al. 1969; Bezman et al. 1978; Banks et al. 1985). They are poorly suited to this type of work because the kinetics of inactivation are of greater interest than the extent of death. The inherent inaccuracy of counting techniques together with a requirement for a period of

Page 2: Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death

8 J . G . Banks et al. incubation and perhaps resuscitation are further drawbacks. The good correlation of the concen- tration of intracellular adenosine triphosphate with the number of viable organisms in a popu- lation (DEustachio et al. 1968) has been exploited in studies of the action of antimicro- bial agents (Hojer et al. 1976; Thore et al. 1977; McWalter 1984).

The use of ATP photometry in studies of the photodynamic inactivation of Escherichia coli and a pigmented yeast by Rose Bengal is re- ported for the first time in this communication.

Materials and Methods

M I C R O - O R G A N I S M S

Escherichia coli B/r and a carotenoid-producing species of Rhodotorula (WXXX), isolated from a sheep’s fleece, were incubated for 18 h on a reciprocal shaker a t 30” and 20°C respectively. The former was grown in nutrient broth, the latter in YEPD (%w/v; yeast extract, 1; myco- logical peptone, 2; and D-glucose, 2) medium. Cells were harvested by centrifugation (7000 g, 15 rnin), washed twice and resuspended in phos- phate buffer a t p H 7.0 to give a concentration of ca 107-108 cells/ml.

I R R A D I A T I O N

Samples (5 ml) of the cell suspensions were dis- pensed into test tubes standing in a Perspex water bath (20°C) illuminated by four 15 W warm-white fluorescent lights. A photon flux density of 200 was used with E. coli and 315 microEinsteins (pE) m-’ s - ’ with Rhodotorula.

C O N T R O L S

High purity dinitrogen was passed through Nilox scrubbers to remove residual oxygen and then bubbled into the solutions of Rose Bengal (2 x mol/l) for 20 rnin before the addition of cells and throughout the period of irradia- tion. Test tubes painted with black enamel or covered with aluminium foil excluded light. Control cells were irradiated in buffer only.

V I A B L E C O U N T S

At appropriate times the contents of the test tubes were mixed and samples (100-500 pl)

taken. These were diluted in 0.1% w/v peptone water and pipetted (1G20 pl) onto the dried surface of Plate Count (PCA) agar. Twelve repli- cate drops were accommodated in each 9 cm Petri dish. Incubation (without illumination) was for 48 h at 25” (Rhodotorula WXXX) or 30°C (E. coli).

A T P ASSAY

The Lumac M2010 biocounter (Lumac B.V., Schaesberg, The Netherlands) and Lumac re- agents were used. To measure extracellular (background) ATP, a lumacuvette containing 100 pl of the sample was placed in the bio- counter reaction chamber a t 25°C. Lumit P M (100 pl) was added and the resultant increase in the photon flux measured as ‘relative light units’ (RLU) after a 10 s integration period. ‘Total’ ATP was measured by pre-incubation of the sample (100 pl) for 15 s with 100 p1 of Lumac ‘nucleotide releasing reagent for microbial cells’ (NRB) to allow for the release of ATP. The pro- cedure noted above for ‘background ATP was then followed; intracellular ATP was calculated from the difference between the total and extra- cellular levels.

Results

It is evident from Fig. 1 that there was a lag of ca 40 rnin before the onset of death of E. coli B/r suspended in an illuminated solution of Rose Bengal exposed to oxygen. There was an appre- ciable reduction in the concentration of intracel- M a r ATP during this lag period. The rate of reduction accelerated during the period of expo- nential death of the cells. The levelling off of ATP concentrations from ca 70 rnin onwards was due to the threshold for its detection (equivalent to ca lo4 viable cells/ml) after 70 rnin being attained. There was no change in the number of viable cells of E. coli B/r in Rose Bengal shielded from light and the concentra- tion of intracellular ATP in the cells remained relatively constant (Fig. 1). The same situations obtained when Rose Bengal was scrubbed with oxygen-free dinitrogen or when E. coli B/r was suspended in illuminated phosphate buffer. The rate of death of E. coli B/r which was suspended in Rose Bengal for 90 min before illumination resulted in a rate of death slower than that of cells which had been illuminated from the outset

Page 3: Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death

Photodynamic death and A T P loss 9 140 --•

r?-o-o-r-,

\ \ I ' 0 Jo, 'Lo

I I I I I 0 20 40 60 80

Time (min)

0

Fig. 1. Photodynamic inactivation of Escherichia coli B/r by Rose Bengal. Viable count of 0, non- illuminated; 0, illuminated cells. Intracellular ATP concentration, measured as RLU/ml in ., non- illuminated or 17, illuminated cells. Light intensity was 200 pE m-' s - ' , concentration of Rose Bengal was 2 x mol/l and temperature was 20°C. Exclusion of oxygen or omission of Rose Bengal gave results which were not significantly different from the non-illuminated controls shown above.

of the experiment (Fig. 2a). This trend was evident also in the decline in the concentration of intracellular ATP (Fig. 2b). Indeed the dimi- nution in the ATP content of the cells before a demonstrable decline in the number of viable micro-organisms was a feature of cells sus- pended in illuminated Rose Bengal.

Rhodotorula WXXX was not photoinacti- vated (Fig. 3) even though the photon flux (315 pE m-' s - l ) was larger than that used for E . coli Bjr. There were no appreciable changes in the concentration of ATP in illuminated or non- illuminated yeast cells (Fig. 3).

Discussion

The results presented above, especially those for concentration of intracellular ATP, confirm earlier observations (Banks et al. 1985) that Rose Bengal has little if any cytotoxic action on E . coli B/r. The ;renounced lag in changes in the number of viable cells in illuminated Rose Bengal before the onset of the exponential death phase is evidence therefore of progressive photo- dynamically mediated damage to one or several cellular sites. Thus a cell loses viability only when a lethal threshold in damage has been exceeded. Indeed our studies of changes in the concentration of intracellular ATP support this

0.0001 O o o l t I I , "\ol I

b '0 .

I I y--q I I 0 50 100 150 200

Time (rnin)

Fig. 2. Effect of pre-incubation of Escherichia coli B/r in non-illuminated Rose Bengal prior to photo- dynamic inactivation. (a) Viable count of 0, illumi- nated cells; 0, non-illuminated cells or A , cells illuminated after 90 min pre-incubation in non- illuminated Rose Bengal. (b) Intracellular ATP con- centration, measured as RLU/ml in 0, illuminated cells; m, non-illuminated cells or A, cells illuminated after 90 min pre-incubation in non-illuminated Rose Bengal. Light intensity, concentration of Rose Bengal and temperature of incubation were as shown in Fig. 1. No significant reduction in the concentration of ATP or viability was noted in controls where oxygen was excluded or Rose Bengal was omitted.

threshold concept because it is evident that intracellular ATP depletion occurs before there is a demonstrable reduction in the number of viable cells. It is known that the time taken to attain this hypothetical threshold is determined in part by the light intensity, PO,, extent of oxygen and dye penetration and distribution within the cell as well as by in uiuo sensitizers and the relative distances between the source of singlet oxygen and its eventual target (It0 1978). Of these, perhaps only dye penetration was reflected in the present study. Thus the lag (Fig. 2) before the onset of exponential death was diminished by exposure of E . coli B/r to the sensitizer before irradiation. This evidence sug- gests that at least in the experimental system used in this study, a critical concentration of dye determined the onset of photodynamic inac-

Page 4: Illuminated Rose Bengal causes adenosine triphosphate (ATP) depletion and microbial death

10 J . G. Banks et al.

Time Imin)

Fig. 3. Resistance of Rhodotorula WXXX to photo- dynamic inactivation by Rose Bengal. Viable count of 0, non-illuminated or 0, illuminated cells. Intracel- lular ATP concentration measured as RLU/ml in , non-illuminated or 0 , illuminated cells. Light inten- sity was 315 pE m-' s-', concentration of Rose Bengal and temperature of incubation were as shown in Fig. 1. Exclusion of oxygen or omission of Rose Bengal gave results which were not significantly differ- ent from those noted above.

tivation. Our results are not in accord with those of Knox and Dodge (1984) who noted that phototoxic damage of leaf discs was accen- tuated if the discs were stored for some time in non-illuminated Rose Bengal. They hypothe- sised that this effect was due to the accumula- tion of Rose Bengal within the tissue. In the present study accumulation appeared to be associated with the time to onset of photo- dynamic inactivation but not with the rate once death has begun. Other workers (e.g. Bezman e t al. 1978) have trapped Rose Bengal on poly- styrene spheres in studies of the photodynamic inactivation of bacteria. In this instance, the rate and extent of diffusion of singlet oxygen into a cell are presumably the factors that determine the rate of death. In this context it is notable that these workers reported slower rates of death than those noted here or by Banks et al. (1985).

In this study ATP photometry has been used successfully as an adjunct to viable counting techniques. It may find favour also as a tool for the screening of large numbers of photo- sensitizers or micro-organisms to assess photo- damage and inactivation. Similarly, it may be of use for the examination of the sensitivity of Legionel la to inhibitory substances which are formed during storage of enrichment and plating media exposed to light.

References

BANKS, J.G., BOARD, R.G., CARTER, J. & DODGE, A.D. 1985 The cytotoxic and photodynamic inactivation of micro-organisms by Rose Bengal. Journal of Applied Bacteriology 58, 391400.

BELLIN, J.S., LUTWICK, L. & JONAS, B. 1969 Effects of photodynamic action on E . coli. Archiues of Bio- chemistry and Biophysics 132, 157-164.

BEZMAN, S.A., BURTIS, P.A., IZOD, T.P.J. & THAYER, M.A. 1978 Photodynamic inactivation of E . coli by Rose Bengal immobilized on polystyrene beads. Photochemistry and Photobiology 28, 325-329.

DEUSTACHIO, A.J., JOHNSON, D.R. & LEVIN, D.G. 1968 Adenosine triphosphate content of bacteria. Federation Proceedings: Federation of American Societies for Experimental Biology 21, 761.

DODGE, A.D. 1983 Toxic oxygen species and herbicide action. In Human Welfare and the Environment, IUPAC Pesticide Chemistry ed. Miyamoto, J. & Kearney, P.C., pp. 59-66, Oxford : Pergamon Press.

FOOTE, C.S. 1976 Photosensitized oxidation and singlet oxygen : consequences in biological systems. In Free radicals in biology Vol. 2 ed. Pryor, W.A., pp. 85-133, New York: Academic Press.

FREEMAN, P.J. & GIESE, A.C. 1952 Photodynamic effects on metabolism and reproduction in yeast. Journal of Cellular and Comparative Physiology 39, 301-322.

HOJER, H., NILLSON, L., ANSEHN, S. & THORE, A. 1976 In-vitro effect of doxycycline on levels of adenosine triphosphate in bacterial cultures. Scandinavian Journal of Infectious Diseases 9, 58-61.

ITO, T. 1978 Cellular and subcellular mechanisms of photodynamic action: the '0, hypothesis as a driving force in recent research. Photochemistry and Photobiology 28,493-506.

ITO, T. & KOBAYASHI, K. 1977 A survey of in uiuo photodynamic activity of xanthenes, thiazines and acridines in yeast cells. Photochemistry and Photo- biology 26,581-587.

KNOX, J.P. & DODGE, A.D. 1984 Photodynamic damage to plant leaf tissue by Rose Bengal. Plant Science Letters 31, 3-7.

KORYCKA-DAHL, M.B. & RICHARDSON, T. 1978 Acti- vated oxygen species and oxidation of food constit- uents. C R C Critical Reviews in Food Science and Nutrition 10, 209-241.

KRINSKY, N.I. 1976 Cellular damage initiated by visible light. In Survival of vegetative microbes eds Gray, T.R.G. & Postgate, J.R., Cambridge & London: Cambridge University Press.

KRINSKY, N.I. 1977 Singlet oxygen in biological systems. Trends in Biochemical Sciences 2, 35-38.

KRINSKY, N.I. 1979 Carotenoid protection against oxidation. Pure and Applied Chemistry 51,649-660.

MAXWELL, W.A., MACMILLAN, J.D. & CHICHESTER, C.O. 1966 Function of carotenoids in protection of Rhodotorula glutinis against irradiation from a gas laser. Photochemistry and Photobiology 5, 567-577.

Moss, S.H. & SMITH, K.C. 1981 Membrane damage can be a significant factor in the inactivation of Escherichia coli by near-ultraviolet radiation. Photochemistry and Photobiology 33, 203-210.

MCWALTER, P.W. 1984 Determination of suscepti-

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Photodynamic death and A T P loss 11 bility of Staphylococcus aureus to methicillin by BROTE, L. 1977 Effects of ampicillin on intracellular luciferin-luciferase assay of bacterial adenosine tri- levels of adenosine triphosphate in bacterial cul- phosphate. Journal of Applied Bacteriology 56, 145- tures related to antibiotic susceptibility. Acta 150. Pathologica et Microbiologica Scandinavia B 85,

161-166. THORE, A., NILLSON, L., HOJER, H., ANSEHN, S. &