Arch Microbiol (1992) 157:242-248
Archives of
Microbiology
,© Springer-Verlag 1992
Lethal and mutational effects of solar and U V radiation on Staphylococcus aureus Robin M. Chapple, Barbara Inglis, and Peter R. Stewart Biochemistry Department, Faculty of Science, Australian National University, Canberra ACT 2601, Australia Received May 1, 1991/Accepted September 8, 1991
Abstract. Strains of Staphylococcus aureus, an opportunistic pathogen commonly found on human skin, were exposed to sunlight and UV C radiation, and the lethal and mutational effects measured. Sunlight killed cells with an inactivation constant of 3 x 10 -5 per joule per square metre; UV C was much more lethal, giving an inactivation constant of approximately 0.1 per joule per square metre. Some strains tested showed a sensitivity to sunlight that was dependent on the growth phase of the cells, exponentially growing cells showing a greater sensitivity. Mutational effects of irradiation were measured by the appearance of mutants sensitive to methicillin following irradiation of a multiresistant strain. Mutants appeared at a frequency of 10- 3; this high frequency of mutation in the region of the mec gene has also been observed when multiresistant strains are subjected to nutritional or thermal stress. Mutants showed the same chromosomal alteration (seen in pulse-field gel electrophoresis of SmaI-digested DNA) whether induced by solar or UV C irradiation. Key words: Solar radiation - UV A, UV B and UV C radiation - Sunlight - Mutagenesis - Staphylococcus aureus - Pulse-field gel electrophoresis
Staphylococcus aureus colonises 20- 50 % of humans, and is often found on the skin of these carriers. It is therefore likely to be exposed to the genetic and physiological effects of solar radiation. Since this bacterium is an opportunistic pathogen, environmental factors which alter its population size on the skin or its virulence may be expected to trigger or to influence its impact on the host. Environmental effects of this sort may help explain why its transition from commensal to parasite often occurs unexpectedly.
Offprint requests to: P. R. Stewart
The energy reaching the skin as sunlight is largely in the visible/UV range of frequencies (57% between 300 nm and 700 nm) with the remainder predominantly in the infrared (Watson-Munro 1973; Thorington 1980). Ultraviolet radiation can be conveniently divided into far UV, or UV C (200-290 nm), and near UV, containing UV B (290 320 nm) and UV A (320 400 nm). At comparable energy intensities UV C would be the most damaging to living cells, but because incident sunlight contains little or no energy in this band, the UV A and UV B bands are considered to have the most significant biological impact (Jagger 1981; Calkins et al. 1987). The intensity of UV B radiation is rising in southern latitudes, and wavelengths below 290 nm are being detected in incoming radiation, as ozone is depleted from the atmosphere (Caldwell et al. 1989; Roy et al. 1989). A comparative study of several bacteria showed that S. aureus is as susceptible as Escherichia coli to killing
by UV C radiation, and that staphylococci show similar photobiological responses to E. coli, but with some differences in SOS responses (Chang et al. 1985; Guillobel and Leitao 1988). Similar genes to those mediating UV response in E. coli have been identified in S. aureus (Thompson and Hart 1981). Despite the likely exposure of staphylococci to sunlight, we know little about the response of these bacteria to solar radiation; also little is known about the effects of expected increases in natural UV radiation on any type of bacteria. Most of what is known about the effects on bacteria of sunlight and UV irradiation is derived from extensive studies with gram-negative enteric bacteria, particularly E. coli (Eisenstark 1989), organisms which may rarely be exposed to solar radiation in their normal environments. In this study we demonstrate the lethality of sunlight to S. aureus, and compare this with the effects of UV C irradiation from a germicidal lamp. We also demonstrate that a cluster of antimicrobial resistance genes near the mec locus (encoding methicillin resistance), which occurs in many clinical isolates and is known to be unstable under conditions of nutritional and temperature stress (Inglis et al. 1990b), is also destabilized by solar radiation.
243 The inactivation constant per energy unit is also given, to allow comparison of inactivation by UV C and solar radiation, sources with differing power levels.
Methods
Bacterial strains and culture conditions The strains and isolates of S. aureus used and their origins are listed in Table 1. Luria broth (LB) cultures were grown unshaken in tubes at 37 °C overnight (final A6oo nm approximately 6) and used directly (stationary culture), or to inoculate fresh LB at a 1/100 dilution. These fresh cultures were incubated at 37 °C until they reached an A6oo nm of approximately 1 (about 3 h), then held on ice until required (exponential culture). One ml samples of cultures were centrifuged (12000 g, 2 min), then washed once and resuspended in the same volume of saline. Serial dilutions were prepared in saline. For construction of killing curves, 10 gl aliquots of serial dilutions were spotted (in triplicate) onto LB agar, allowed to absorb, and irradiated. Antibiotic resistance of strains was determined as described previously (Matthews et al. 1987).
UV C (254 nm) irradiation Plates spotted with dilutions of cell suspensions were exposed to a Philips mercury lamp (Austra Violet 2030) at a distance of 70 cm (estimated incident power 8.5 W m -2 with peak output at 254 nm) for specified times at room temperature, and survivors scored after incubation of the plates at 37 °C overnight under low intensity lighting from a tungsten lamp.
Plates spotted with dilutions of cell suspensions as above were exposed to Canberra's (latitude 35 °S) mid-to-late summer sunlight in the early afternoon (lids removed). The incident power at the surface of the plate was 160 210 W m 2 as measured by a light meter (LI-COR model LI 185B) measuring primarily in the visible range. Under these conditions, the surface temperature of the plates was 22 28 °C. Exposures were terminated by replacing plate lids and removing the plates to shade. Exposure through filters was achieved by placing the filters parallel to and 15 cm above the plates. The filters used were: photocopy paper (density 80 g m 2); perspex (3 mm thick); window glass (3 mm thick). Following exposure, plates were incubated at 37 °C overnight with low intensity illumination as described above, and scored for survivors. Absorbance spectra of filters were obtained by mounting sections in the cell housing of a Varian Techtron 635 split-beam spectrophotometer, and measuring their absorbance versus air.
Units of sensitivity to solar and UV C radiation In the descriptions which follow, the rate of cell death is indicated by the inactivation constant k (k = - 1 / t lnNt/No, where N = number of cells per ml at time = 0 and time = t seconds).
used
Saline dilutions (100 gl) of the multiresistant strain ANS46 were spread onto LB agar and exposed to sufficient sunlight or UV C to give a reduction of 4 logs in cell number per ml. Plates carrying 100-200 survivor colonies after incubation at 37 °C overnight were replica plated onto LB agar containing 10 pg ml- 1 methicillin (Mc) then onto LB agar (no antibiotic). Putative methicillin sensitive (Mc s) colonies were streaked onto LB agar containing 10 pg m l - 1 methicillin to check their sensitivity. Contaminants which may have arrived on the plate whilst it was open to the air (appearing at an average of one per plate per 10 rain exposure to sunlight) were detected by biochemical testing of putative Mc Sstrains: catalase and coagulase production, growth and fermentation on mannitol-salt agar. The pattern of Sinai restriction endonuclease fragments found in putative Mc s variants of ANS46 was also used diagnostically. Thus, Mc s variants showing four or more chromosomal changes compared with ANS46 were excluded as possibly not derived from ANS46.
In situ digestion of genomic DNA, pulse-field gel electrophoresis, Southern blotting, and hybridization These procedures were carried out as described previously (Inglis et al. 1990b).
Solar irradiation
Table 1. Strains of Staphylococcus aureus
Selection of methicillin-sensitive mutants
Results
The lethal effect of UV C (254 nm) irradiation E x p o s u r e of cells to a p p r o x . 8 . 5 W m - 2 o f 2 5 4 n m r a d i a t i o n r e s u l t e d in t h e k i n e t i c s of k i l l i n g s h o w n in Fig. 1. T h e s e n s i t i v i t y (k = 1.0) d i d n o t d e p e n d o n w h e t h e r the cells w e r e in s t a t i o n a r y o r e x p o n e n t i a l p h a s e of g r o w t h a n d is c o m p a r a b l e w i t h k i l l i n g o f S. aureus b y U V C seen in o t h e r studies, i n c i d e n t e n e r g y o f 21 J m - 2 g i v i n g a 1 l o g d r o p in cell n u m b e r s , c o m p a r e d w i t h 2 x 102 J m - 2 at 265 n m ( H o l l a e n d e r 1943) a n d 2 0 J m - 2 at 254 n m ( C h a n g et al. 1985). T h e initial lag of a few s e c o n d s b e f o r e e x p o n e n t i a l k i l l i n g o c c u r r e d w a s seen for all s t r a i n s tested, a n d m a y reflect m o r e t h a n o n e U V C s e n s i t i v e t a r g e t p e r cell, o r t h e a c t i v a t i o n of r e p a i r s y s t e m s at l o w e n e r g y i n p u t , as f o u n d in E. coli ( H o l l a e n d e r 1943; P e a k 1970). A l t e r n a t e l y , t h e lag m a y reflect t h e t e n d e n c y o f S. aureus cells to a g g r e g a t e , w h e r e b y s o m e c o l o n i e s d e r i v e f r o m a c l u s t e r o f cells r a t h e r t h a n f r o m a single cell.
Strain
Characteristics
Reference
ANS46
Clinical isolate resistant to many antibiotics, including methicillin Clinical isolate resistant to many antibiotics, including methicillin Non-clinical isolate, antibiotic-sensitive Acriflavine-induced deletion mutant of ANS46, Mc s Stress-induced deletion mutants of ANS46, Mc s Sunlight-induced mutants of ANS46, Mc s UV C-induced mutants of ANS46, Mc s
Matthews et al. 1987
SK604 L8 ANS62 V5, QI, Q3, T40 U21, U23 U27, U28
Lyon et al. 1984 Inglis et al. 1990a Matthews et al. 1987 Inglis et al. 1990b This study This study
244
SURVIVIN(
SURVIVIN
FRACTIOI~
FRACTIOI
4
~
12
16
lv
20
20
TIME (S)
40
60
TIME (min)
Fig. 1. Killing curve for U V C i r r a d i a t i o n o f S. aureus strains A N S 4 6 (e), L8 (m) a n d V5 (A). Cells were irradiated as e x p o n e n t i a l (solid) or s t a t i o n a r y (unshaded) g r o w t h p h a s e cultures
Fig. 2. Killing curve for solar irradiation o f S . aureus strains A N S 4 6 ( e ) , L8 (m) a n d V5 (A). Cells were irradiated as e x p o n e n t i a l (solid) or s t a t i o n a r y (unshaded) g r o w t h p h a s e cultures
One strain (L8, an antibiotic sensitive non-clinical isolate) was more sensitive to UV C irradiation than other strains tested, though this sensitivity is not general for antibiotic sensitive strains (results not shown).
Because of this, cells were exposed in parallel at the same time on the same day when comparing strains or effects of filters. Thus sunlight also kills S. aureus, though the cells have a sensitivity approximately 3 x 10 -4 that seen with UV C on a radiant energy basis, and approximately 5 x 10 -3 oil an exposure time basis. These results are summarised in Table 2. Some strains tested (LS, Fig. 2; SK604, not shown) displayed the same sensitivity to sunlight whether cells were from actively growing or stationary phase cultures, while others (ANS46, Fig. 2 and its derivative ANS62, not shown) were significantly more sensitive when from
The lethal effect of sunlight Exposure of the cells to sunlight on a cloud-free day gave the killing kinetics shown in Fig. 2. In this experiment, the strains showed an inactivation constant of approximately 5 x 10 -3, although in thirteen repetitions there was substantial day-to-day variation (10 .6 to 10-z).
Table 2. Sensitivity o f strains to U V C a n d solar r a d i a t i o n
Strain
I n a c t i v a t i o n c o n s t a n t k (s-1)
Inactivation constant k per unit energy (J 1 m 2)
ANS46 SK604 L8 ANS62 V5 Mean
UV C 0.74 0.74 1.65 0.92 0.74 0.96
UV C 8 . 7 x 10 -2 8.7 x 1 0 - z 19 x 10 . 2 11 x l 0 - z 8.7 x 10 - 2 11 x l 0 - 2
Solar 5 . 6 x 10 - 3 3.0 x 10 3 6.5 x 10 . 3 5.6 x 10 - 3 5.6 x 10 - 3 5.3 x 10 - 3
Solar 3 . 0 x 10 -5 1.6 x 1 0 - s 3.5 x 10 -5 3.0 x 10 -5 3.0 x 1 0 - 5 2.8 x 10 - 5
Sensitivities h a v e been calculated as described in the M e t h o d s section, u s i n g e x p o n e n t i a l p h a s e cultures. T h e a r e a s covered by 10 p,1 cell s u s p e n s i o n s placed o n the plates h a v e been a s s u m e d to be 10 m m in diameter, or 0.00025 m z
245 exponential phase cultures. A greater sensitivity of exponential phase cultures to UV A radiation has been seen in E. coli (Peak 1970). The early lag or delay periods on the killing curves were also seen with solar radiation, suggesting again cell clumping, multiple UV targets or activation of repair systems at low doses. With regard to radiation damage and repair, certain wavelengths in sunlight may induce systems that are able to protect against, or repair damage caused by, other spectral bands i.e. photoprotection occurs (Jagger 1981). At higher fluences or extended exposure, repair systems may be destroyed. The mec region of the chromosome in S. aureus encodes resistance to a range of antimicrobial agents (Inglis et al. 1988), and also maps near the locus mit, believed to confer resistance to UV in this bacterium (Wyman et al. 1974; Tam and Pattee 1986). Mutants of ANS46 (see Table 1) from which extensive segments of D N A in the mec region have been deleted (Inglis et al. 1990b) were examined to determine whether resistance to the lethal effects of sunlight may also have been lost. The five deletants tested (ANS62, V5, Table 2; others not shown) showed similar sensitivity to ANS46 when exponential phase cultures were irradiated. A difference was observed when stationary phase cultures were irradiated: ANS62, deleted for 55 kb around the mec gene, behaves like ANS46 when exposed to sunlight; V5, which has a deletion of 70 kb, no longer shows a difference in the response of stationary and exponential phase cells to sunlight (Fig. 2). This could be related to loss of part or all of the mit gene in V5, since recombination repair and excision repair genes have been shown to be involved in stationary phase cell resistance in E. coli (Tuveson and March 1980).
6
200
I 250
L 300 WAVELENGTH
I 350
400
(nm)
Fig. 3. Absorbance spectra of perspex (m) and glass (e), measured in a Varian 635 spectrophotometer. An absorbance cutoff value of 7 is used; above this, energy arriving at the detector is too low to be measured accurately. Using the paper filter, absorbance exceeded 7 units at all wavelengths measured
Table 3. Effect of filters on killing by sunlight Filter
None Glass (210-300 nm) Perspex (200-380 nm) Paper (< 200- > 400 rim)
Inactivation constant (s- 1) ANS46
L8
2.4 x 10-3 1.5 x 10-3 0.4 x 10.3 0
5.8 x l0 -3 4.1 x 10_3 1.3 x 10-3 0
Stationary phase cultures were exposed to sunlight for 45 rain. Filters were placed over the plates carrying cells as described in the 'Methods'. Inactivation constants shown were calculated from a typical experiment in which parallel cultures were exposed to sunlight through different filters
Effect of filters on killing by sunlight In an attempt to obtain a measure of the action spectrum of sunlight acting on S. aureus, cultures were exposed through a series of filters and the killing effects measured; temperatures at the plate surface were also measured. While the interpretation of these results is not straightforward, they do suggest several conclusions. Using an absorbance value of 2 as a notional point to define the cutoff limit for a filter, glass absorbs radiation at wavelengths below 300 nm, and perspex that below 380 nm (Fig. 3). Glass has only a small effect in decreasing killing by sunlight, whereas the effect of perspex is more substantial (Table 3). Complete shading, using paper, results in no kill. The temperature of the irradiated cells was raised 2 - 3 °C by the paper or perspex filters; with the glass filter the temperature at the plate surface increased by 6 - 9 °C, but did not exceed 32 °C. Thus the small degree of protection by glass from killing could be due in part to an increase in temperature, although this seems unlikely since the temperatures were always well within those in which S. aureus grows actively. In summary, we can say a major lethal effect of sunlight is due to radiation in the 300 to 380 nm window, with further effects apparent at shorter and longer wavelengths. Since the energy in solar radiation increases at longer UV
wavelengths (Watson-Munro 1973), then the observations are consistent with a greater lethality of UV B than UV A (at equal intensities) for S. aureus, as has been reported for staphylococci irradiated with UV A and UV B lamps (Faergemann and Larko 1987). Billen and Fletcher (1974) have shown that the lethality of sunlight for E. coli is due chiefly to UV B. The mutational effects o f U V C (254 rim) and sunlight When cells that carry mec (and thus are Mc') were exposed to UV C or solar radiation and the proportion of Mc ~ cells was determined amongst survivors, it was found that for a reduction in cell numbers of four logs by sunlight, the rate of appearance of Mc ~ cells was < 1 per 2100 surviving cells for stationary phase and 1 per 1600 for exponential phase cultures. For an equivalent lethal effect by 254 nm radiation, the corresponding rates were < 1 per 1400 and 1 per 3800. Five of the Mc ~ mutants obtained by irradiation with sunlight and UV C were examined by pulse-field gel electrophoresis of genomic D N A digested with the restriction endonuclease Smal (Patel et al. 1989; Inglis et al.
246
Fig. 4. Pulse-fieldgel electrophoresis of Sinai digests of chromosomal DNA from ANS46 (1), ANS62 (2) and UV-induced mutants U21 (3), U23 (4) and U27 (5), stained with ethidium bromide. Variant U28 (not shown) also gives a pattern identical to ANS62
1990b). Four of the mutants yielded genomic fragments indicating an identical deletion of approx. 55 kb of DNA from the mec region (Fig. 4). Probing of H i n d I i I digested DNA from these four mutants with MA13, a cloned fragment of DNA from the mec region of ANS46 (Inglis et al. 1990b), suggested that for all four deletions one end of the deletion occurred at the insertion site IS257.1 (Matthews et al. 1990; data not shown). The remaining mutant yielded a Sinai fragment pattern indistinguishable from that of ANS46, indicating either a point mutation or other alteration of the DNA not detectable as a change in fragment size or number; mutants of this type have been observed before (lnglis et al. 1990b).
Discussion
Our results show that though not as potent as UV C, solar radiation has lethal effects on S. aureus within a time scale that would commonly be achieved on the skin of humans exposed to sunlight. Relatively small differ-
ences were seen between the strains tested in these experiments, whether UV C or solar radiation was used. The effect of a mixed spectrum of visible and UV radiation, such as constitutes sunlight, on bacterial cells is complex. Short wavelength radiation causes the formation of pyrimidine dimers, while longer wavelength radiation promotes the repair of this damage (Jagger 1967). Furthermore, in E. coli, SOS repair mechanisms are induced by UV C (Walker 1987; Witkin 1976). An SOS-like response is induced by UV C in S. aureus (Thompson and Hart 1981) and in S. epidermidis (Guillabel and Leitao 1988; Silva and Leitao 1984); similar mutants to those in E. coli affecting these responses have been found. Repair responses are faster and occur at lower UV intensity in the staphylococci than in E. coli (Guillobel and Leitao 1988), an adaptation selected possibly because the normal habitat of the former includes exposed human skin. UV A/visible radiation protects against the mutagenicity and lethality of UV B radiation in Salmonella (Calkins et al. 1987), and prior exposure of S. aureus cells to UV B/UV A/visible light decreases the mutagenicity and lethality of UV C (Tyrrell 1980). In E. coli, radiations at wavelengths of 334, 365 and 405 nm have been shown to sensitize to radiations at other UV A wavelengths (Tyrrell and Peak 1978). However, in sunlight direct damage to DNA by UV may not be significant, since sunlight contains little radiation in the wavelengths where major absorption by DNA occurs. While the small amounts of radiation absorbed by DNA at 290 nm and above (Sutherland and Griffin 1981) could account for the mutational effects observed (Webb 1978), it is possible that the lethal and mutational effects of UV B/UV A/visible light on staphylococci are mediated primarily through the production of free radicals (Eisenstark 1989). Reactive oxygen species could react directly with DNA, and in addition have wider effects, as reported for other cellular systems (Peak et al. 1984; Eisenstark 1989; Tyrrell and Keyse 1990). Some effects of UV B/UVA/visible light and H20 2 (a source of free radicals) are common, and mutants of E. coli and Salmonella typhimurium sensitive to one are sensitive to the other (Tyrrell 1985; Eisenstark 1987). UV A is also absorbed by tRNA, leading to the arrest of protein synthesis (Favre et al. 1985); U V A also inactivates membrane transport systems (Robb et al. 1978). Our experiments show that as well as killing exposed staphylococci, solar radiation is mutagenic, in that chromosomal deletions are induced by it. The mec region in ANS46 has previously been shown to be unstable, and susceptible to deletion and other mutational change when cells are stressed by prolonged culture or growth at high temperature (Inglis et al. 1990b). In that study, it was noted that certain insertion elements (IS25 7, Tn554) may be foci for deletion of tracts of chromosomal DNA up to 250 kb in length, In the experiment reported here, the deletants examined also appear to involve the most leftward copy of IS257 as a deletion point. It is notable that all four deletion mutants examined showed the same chromosomal alteration, whether induced by solar or UV C irradiation, suggesting a common mechanism for
246
Fig. 4. Pulse-field gel electrophoresis of Sinai digests of chromosomal DNA from ANS46 (1), ANS62 (2) and UV-induced mutants U21 (3), U23 (4) and U27 (5), stained with ethidium bromide. Variant U28 (not shown) also gives a pattern identical to ANS62
1990b). Four of the mutants yielded genomic fragments indicating an identical deletion of approx. 55 kb of DNA from the mec region (Fig. 4). Probing of H i n d I I I digested DNA from these four mutants with MA13, a cloned fragment of DNA from the mec region of ANS46 (Inglis et al. 1990b), suggested that for all four deletions one end of the deletion occurred at the insertion site IS257.I (Matthews et al. 1990; data not shown). The remaining mutant yielded a Sinai fragment pattern indistinguishable from that of ANS46, indicating either a point mutation or other alteration of the DNA not detectable as a change in fragment size or number; mutants of this type have been observed before (lnglis et al. 1990b).
Discussion
Our results show that though not as potent as UV C, solar radiation has lethal effects on S. aureus within a time scale that would commonly be achieved on the skin of humans exposed to sunlight. Relatively small differ-
ences were seen between the strains tested in these experiments, whether UV C or solar radiation was used. The effect of a mixed spectrum of visible and UV radiation, such as constitutes sunlight, on bacterial cells is complex. Short wavelength radiation causes the formation of pyrimidine dimers, while longer wavelength radiation promotes the repair of this damage (Jagger 1967). Furthermore, in E. coli, SOS repair mechanisms are induced by UV C (Walker 1987; Witkin 1976). An SOS-like response is induced by UV C in S. aureus (Thompson and Hart 1981) and in S. epidermidis (Guillabel and Leitao 1988; Silva and Leitao 1984); similar mutants to those in E. coli affecting these responses have been found. Repair responses are faster and occur at lower UV intensity in the staphylococci than in E. coli (Guillobel and Leitao 1988), an adaptation selected possibly because the normal habitat of the former includes exposed human skin. UV A/visible radiation protects against the mutagenicity and lethality of UV B radiation in Salmonella (Calkins et al. 1987), and prior exposure of S. aureus cells to UV B/UV A/visible light decreases the mutagenicity and lethality of UV C (Tyrrell 1980). In E. coli, radiations at wavelengths of 334, 365 and 405 nm have been shown to sensitize to radiations at other UV A wavelengths (Tyrrell and Peak 1978). However, in sunlight direct damage to DNA by UV may not be significant, since sunlight contains little radiation in the wavelengths where major absorption by DNA occurs. While the small amounts of radiation absorbed by DNA at 290 nm and above (Sutherland and Griffin 1981) could account for the mutational effects observed (Webb 1978), it is possible that the lethal and mutational effects of UV B/UV A/visible light on staphylococci are mediated primarily through the production of free radicals (Eisenstark 1989). Reactive oxygen species could react directly with DNA, and in addition have wider effects, as reported for other cellular systems (Peak et al. 1984; Eisenstark 1989; Tyrrell and Keyse 1990). Some effects of UV B/UV A/visible light and H202 (a source of free radicals) are common, and mutants of E. coli and Salmonella typhimurium sensitive to one are sensitive to the other (Tyrrell 1985; Eisenstark 1987). UV A is also absorbed by tRNA, leading to the arrest of protein synthesis (Favre et al. 1985); U V A also inactivates membrane transport systems (Robb et al. 1978~. Our experiments show that as well as killing exposed staphylococci, solar radiation is mutagenic, in that chromosomal deletions are induced by it. The mec region in ANS46 has previously been shown to be unstable, and susceptible to deletion and other mutational change when cells are stressed by prolonged culture or growth at high temperature (Inglis et al. 1990b). In that study, it was noted that certain insertion elements (IS257, Tn554) may be foci for deletion of tracts of chromosomal DNA up to 250 kb in length, In the experiment reported here, the deletants examined also appear to involve the most leftward copy of IS257 as a deletion point. It is notable that all four deletion mutants examined showed the same chromosomal alteration, whether induced by solar or UV C irradiation, suggesting a common mechanism for
247 the two types o f radiation. It remains an open question whether different mechanisms are involved in killing and mutagenesis in S. a u r e u s . U n d e r conditions o f U V and solar radiation which bring a b o u t extensive killing o f the staphylococci, m u t a tion rates (as reflected in the generation o f deletants which have lost the m e c locus and s u r r o u n d i n g D N A ) were considerably less than those seen with nutritional or thermal stress, when deletion could a c c o u n t for wholesale conversion o f cultures to M c s at high levels of surviving cells (Inglis et al. 1990b). U n d e r conditions of nutritional or thermal stress, rates of lethal mutations m a y be slow e n o u g h to be repaired; the killing rates seen in the irradiated cultures m a y be such that only a small fraction of m u t a n t s or deletants is recovered. In the same way, sensitivity of cells to irradiation m a y be a function of p o w e r (rate of energy a b s o r p t i o n by cells) rather than of total energy absorbed. U n d e r conditions of low p o w e r radiation, repair systems m a y keep pace (particularly if mixtures of U V and visible radiation are involved; p h o t o r e p a i r of lethal d a m a g e m a y then be important), and lethality (as with mutation) m a y not be so evident. These questions are susceptible to experimental testing. The findings of this study are relevant to an understanding of the ecology of S. a u r e u s . As a n o r m a l m e m b e r o f the skin m i c r o b i o t a o f a b o u t one third o f humans, this bacterium w o u l d c o m m o n l y be exposed to unfiltered sunlight. Thus, such older remedies for S. a u r e u s and other bacterial infections as exposure of patients and their clothing and bedlinen to sunshine m a y have a s o u n d basis in fact. Yet, while the upper skin layers m a y contain the majority of S. a u r e u s cells, as is the case for coagulase-negative staphylococci (Brown et al. 1989), reservoirs o f these bacteria m a y exist deeper within the skin and be screened f r o m the h a r m f u l effects o f sunlight. These p r o b a b l y serve for later recolonization o f the b o d y surfaces. A n expected consequence o f increasing atmospheric depletion o f ozone is an increase in U V B c o m ponents o f sunlight (Caldwell et al. 1989). Since U V B appears to be m o r e lethal than U V A for S. a u r e u s (as discussed in 'Results'), a small increase in U V B levels in sunlight m a y result in a disproportionately large increase in lethality o f sunlight to S. a u r e u s . I f the staphylococci are representative o f other skin microbes, it seems likely that changes in the skin m i c r o b i o t a could be expected for individuals exposed to solar radiation where significant depletion o f atmospheric ozone has occurred. The consequences o f this remain uncertain whilst we k n o w little o f the i m p o r t a n c e o f the n o r m a l skin m i c r o b i o t a in determining susceptibility o f the host to opportunistic or virulent pathogens. Acknowledgements. We thank Keren Solomon for assistance with
these experiments. The work was supported by the Australian Research Council.
References Billen D, Fletcher MM (1974) Inactivation of dark-repair-deficient mutants of Escherichia coli by sunlight. Int J Radiat Biol 26: 73-76
Brown E, Wenzel RP, Hendley JO (1989) Exploration of the microbial anatomy of n0rmal human skin by using plasmid profiles of coagulase-negative staphylococci: search for the reservoir of resident skin flora. J Infect Dis 160:644 650 Caldwell MM, Madronich S, Bjorn LO, Ilyas M (1989) Ozone reduction and increased solar ultraviolet radiation. In: United Nations Environment Program, Environmental Effects Panel Report, pp 1 10 Calkins J, Selby C, Enoch HG (1987) Comparison of UV action spectra for lethality and mutation in Salmonella typhimurium using a broad band source and monochromatic radiations. Photochem Photobiol 45 : 631-636 Chang JCH, Ossoff SF, Lobe DC, Dorfman MH, Dumais CM, Qualls RG, Johnson JD (1985) UV inactivation of pathogenic and indicator microorganisms. Appl Environ Microbiol 49: 1361-1365 Eisenstark A (1987) Mutagenic and lethal effects of near-ultraviolet radiation (290-400 nm) on bacteria and phage. Environ Mol Mutagen 10:317 337 Eisenstark A (1989) Bacterial genes involved in response to nearultraviolet radiation. Adv Genet 26:99-147 Faergemann J, Larko O (1987) The effect of UV-light on human skin microorganisms. Acta Derm Venereol (Stockh) 67:69-72 Favre A, HajnsdorfE, Caldeira de Araujo A (1985) Mutagenesis and growth delay induced in Escherichia coli by near-ultraviolet radiations. Biochimie 67:335-342 Guillobel H, Leitao AC (1988) Characterization of Staphylococcus epidermidis mutants sensitive to ultraviolet radiation. Mutat Res 193:1-10 HollaenderA (1943) Effect of long ultraviolet and short visible radiation (3500 to 4900 ~) on Escherichia coli. J Bacteriol 46: 531-541 Inglis B, Matthews PR, Stewart PR (1988) The expression in Staphylococcus aureus of cloned DNA encoding methicillin resistance. J Gen Microbiol 134:1465-1469 Inglis B, Heding I, Merrylees M, Stewart PR (1990a) Bacteriophage 604: a marker phage for multiresistant Staphylococcus aureus in Australia. Epidemiol Infect 104: 2ll 218 Inglis B, Matthews PR, Stewart PR (1990b) Induced deletions within a cluster of resistance genes in the mec region of the chromosome of Staphylococcus aureus. J Gen MicrobioI 136: 2231-2239 Jagger J (1967) Introduction to research in ultraviolet photobiology. Prentice-Hall, Englewood Cliffs, New Jersey Jagger J (1981) Near-UV radiation effects on microorganisms. Photochem Photobiol 34:761-768 Lyon BR, Iuorio JL, May JW, Skurray RA (1984) Molecular epidemiology of multiresistant Staphylococcus aureus in Australian hospitals. J Med MicrobioI 17:79 89 Matthews PR, Inglis B, Stewart PR (1990) Clustering of resistance genes in the mec region of the chromosome of Staphylococcus aureus. In: Novick R (ed) Molecular biology of the Staphylococci. VCH Publishing, New York, pp 69-83 Matthews PR, Reed KC, Stewart PR (1987) The cloning of chromosomal DNA associated with methicillin and other resistances in Staphylococcus aureus. J Gen Microbiol 133:1919-1929 Patel AH, Foster TJ, Pattee PA (1989) Physical and genetic mapping of the protein A gene in the chromosome of Staphylococcus aureus 8325-4. J Gen Microbiol 135:1799-1807 Peak JG, Peak M J, MacCross M (1984) DNA breakage caused by 334 nm ultraviolet light is enhanced by naturally occurring nucleic acid components and nucleotide coenzymes. Photochem Photobiol 39:713-716 Peak MJ (1970) Some observations on the lethal effects of nearultraviolet light on Escherichia coli, compared with lethal effects of far-ultraviolet light. Photochem Photobiol 12:1-8 Robb FT, Hauman JH, Peak MJ (1978) Similar spectra for the inactivation by monochromatic light of two distinct leucine transport systems in Escherichia coli. Photochem Photobiol 27 : 465-469
248 Roy CR, Gies HP, Elliott G (1989) The ARL solar ultraviolet radiation measurement programme. Transactions of the Menzies Foundation 15:71-76 Silva BS, Leitao AC (1984) UV-induction of SOS responses in Staphylococcus epidermidis: characteristics of the process. Photochem Photobiol 39:781-785 Sutherland JC, Griffin JP (1981) Absorption spectrum of DNA for wavelengths greater than 300 nm. Radiat Res 86:399-409 Tam JE, Pattee PA (1986) Characterization and genetic mapping of a mutation affecting apurinic endonuclease activity in Staphylococcus aureus. J Bacteriol 168:708-714 Thompson JK, Hart MGR (1981) Novel patterns of ultraviolet mutagenesis and weigle reactivation in Staphylococcus aureus and phage fll. J Gen Microbiol 124:147-157 Thorington L (1980) Actinic effects of light and biological implications. Photochem Photobiol 32:117-129 Tuveson RW, March ME (1980) Photodynamic and sunlight inactivation of Escherichia coli strains differing in near-UV sensitivity and recombination proficiency. Photochem Photobiol 31: 287-289 Tyrrell RM (1980) Mutation induction by and mutational interaction between monochromatic wavelength radiations in the near-ultraviolet and visible ranges. Photochem Photobiol 31: 37-46
Tyrrell RM (1985) A common pathway for protection of bacteria against damage by solar UV A (334nm, 365 nm) and an oxidizing agent (H202). Mutat Res 145:129 136 Tyrrell RM, Keyse SM (1990) New trends in photobiology (invited review); the interaction of UV A radiation with cultured cells. J Photochem Photobiol 4:349-361 Tyrrell RM, Peak MJ (1978) Interactions between UV radiation of different energies in the inactivation of bacteria. J Bacteriol 136: 437-440 Walker GC (1987) The SOS response of Escherichia coli. In: Neidhardt FC et al. (eds) Escherichia coli and Salmonella typhimurium: cellular and molecular biology. Am Soc Microbiol, Washington DC, pp 1346-1357 Watson-Munro CN (1973) Report of the Committee on Solar Energy Research in Australia, 17. Australian Academy of Science, Canberra WebbRB (1978) Near-UV mutagenesis: photoreactivation of 365-nm-induced mutational lesions in Escherichia coli WP2s. J Bacteriol 133:860-866 Witkin EM (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 40:869-907 Wyman L, Goering RV, Novick RP (1974) Genetic control of chromosomal and plasmid recombination in Staphylococcus aureus. Genetics 76:681-702
Archives of
Microbiology
,© Springer-Verlag 1992
Lethal and mutational effects of solar and U V radiation on Staphylococcus aureus Robin M. Chapple, Barbara Inglis, and Peter R. Stewart Biochemistry Department, Faculty of Science, Australian National University, Canberra ACT 2601, Australia Received May 1, 1991/Accepted September 8, 1991
Abstract. Strains of Staphylococcus aureus, an opportunistic pathogen commonly found on human skin, were exposed to sunlight and UV C radiation, and the lethal and mutational effects measured. Sunlight killed cells with an inactivation constant of 3 x 10 -5 per joule per square metre; UV C was much more lethal, giving an inactivation constant of approximately 0.1 per joule per square metre. Some strains tested showed a sensitivity to sunlight that was dependent on the growth phase of the cells, exponentially growing cells showing a greater sensitivity. Mutational effects of irradiation were measured by the appearance of mutants sensitive to methicillin following irradiation of a multiresistant strain. Mutants appeared at a frequency of 10- 3; this high frequency of mutation in the region of the mec gene has also been observed when multiresistant strains are subjected to nutritional or thermal stress. Mutants showed the same chromosomal alteration (seen in pulse-field gel electrophoresis of SmaI-digested DNA) whether induced by solar or UV C irradiation. Key words: Solar radiation - UV A, UV B and UV C radiation - Sunlight - Mutagenesis - Staphylococcus aureus - Pulse-field gel electrophoresis
Staphylococcus aureus colonises 20- 50 % of humans, and is often found on the skin of these carriers. It is therefore likely to be exposed to the genetic and physiological effects of solar radiation. Since this bacterium is an opportunistic pathogen, environmental factors which alter its population size on the skin or its virulence may be expected to trigger or to influence its impact on the host. Environmental effects of this sort may help explain why its transition from commensal to parasite often occurs unexpectedly.
Offprint requests to: P. R. Stewart
The energy reaching the skin as sunlight is largely in the visible/UV range of frequencies (57% between 300 nm and 700 nm) with the remainder predominantly in the infrared (Watson-Munro 1973; Thorington 1980). Ultraviolet radiation can be conveniently divided into far UV, or UV C (200-290 nm), and near UV, containing UV B (290 320 nm) and UV A (320 400 nm). At comparable energy intensities UV C would be the most damaging to living cells, but because incident sunlight contains little or no energy in this band, the UV A and UV B bands are considered to have the most significant biological impact (Jagger 1981; Calkins et al. 1987). The intensity of UV B radiation is rising in southern latitudes, and wavelengths below 290 nm are being detected in incoming radiation, as ozone is depleted from the atmosphere (Caldwell et al. 1989; Roy et al. 1989). A comparative study of several bacteria showed that S. aureus is as susceptible as Escherichia coli to killing
by UV C radiation, and that staphylococci show similar photobiological responses to E. coli, but with some differences in SOS responses (Chang et al. 1985; Guillobel and Leitao 1988). Similar genes to those mediating UV response in E. coli have been identified in S. aureus (Thompson and Hart 1981). Despite the likely exposure of staphylococci to sunlight, we know little about the response of these bacteria to solar radiation; also little is known about the effects of expected increases in natural UV radiation on any type of bacteria. Most of what is known about the effects on bacteria of sunlight and UV irradiation is derived from extensive studies with gram-negative enteric bacteria, particularly E. coli (Eisenstark 1989), organisms which may rarely be exposed to solar radiation in their normal environments. In this study we demonstrate the lethality of sunlight to S. aureus, and compare this with the effects of UV C irradiation from a germicidal lamp. We also demonstrate that a cluster of antimicrobial resistance genes near the mec locus (encoding methicillin resistance), which occurs in many clinical isolates and is known to be unstable under conditions of nutritional and temperature stress (Inglis et al. 1990b), is also destabilized by solar radiation.
243 The inactivation constant per energy unit is also given, to allow comparison of inactivation by UV C and solar radiation, sources with differing power levels.
Methods
Bacterial strains and culture conditions The strains and isolates of S. aureus used and their origins are listed in Table 1. Luria broth (LB) cultures were grown unshaken in tubes at 37 °C overnight (final A6oo nm approximately 6) and used directly (stationary culture), or to inoculate fresh LB at a 1/100 dilution. These fresh cultures were incubated at 37 °C until they reached an A6oo nm of approximately 1 (about 3 h), then held on ice until required (exponential culture). One ml samples of cultures were centrifuged (12000 g, 2 min), then washed once and resuspended in the same volume of saline. Serial dilutions were prepared in saline. For construction of killing curves, 10 gl aliquots of serial dilutions were spotted (in triplicate) onto LB agar, allowed to absorb, and irradiated. Antibiotic resistance of strains was determined as described previously (Matthews et al. 1987).
UV C (254 nm) irradiation Plates spotted with dilutions of cell suspensions were exposed to a Philips mercury lamp (Austra Violet 2030) at a distance of 70 cm (estimated incident power 8.5 W m -2 with peak output at 254 nm) for specified times at room temperature, and survivors scored after incubation of the plates at 37 °C overnight under low intensity lighting from a tungsten lamp.
Plates spotted with dilutions of cell suspensions as above were exposed to Canberra's (latitude 35 °S) mid-to-late summer sunlight in the early afternoon (lids removed). The incident power at the surface of the plate was 160 210 W m 2 as measured by a light meter (LI-COR model LI 185B) measuring primarily in the visible range. Under these conditions, the surface temperature of the plates was 22 28 °C. Exposures were terminated by replacing plate lids and removing the plates to shade. Exposure through filters was achieved by placing the filters parallel to and 15 cm above the plates. The filters used were: photocopy paper (density 80 g m 2); perspex (3 mm thick); window glass (3 mm thick). Following exposure, plates were incubated at 37 °C overnight with low intensity illumination as described above, and scored for survivors. Absorbance spectra of filters were obtained by mounting sections in the cell housing of a Varian Techtron 635 split-beam spectrophotometer, and measuring their absorbance versus air.
Units of sensitivity to solar and UV C radiation In the descriptions which follow, the rate of cell death is indicated by the inactivation constant k (k = - 1 / t lnNt/No, where N = number of cells per ml at time = 0 and time = t seconds).
used
Saline dilutions (100 gl) of the multiresistant strain ANS46 were spread onto LB agar and exposed to sufficient sunlight or UV C to give a reduction of 4 logs in cell number per ml. Plates carrying 100-200 survivor colonies after incubation at 37 °C overnight were replica plated onto LB agar containing 10 pg ml- 1 methicillin (Mc) then onto LB agar (no antibiotic). Putative methicillin sensitive (Mc s) colonies were streaked onto LB agar containing 10 pg m l - 1 methicillin to check their sensitivity. Contaminants which may have arrived on the plate whilst it was open to the air (appearing at an average of one per plate per 10 rain exposure to sunlight) were detected by biochemical testing of putative Mc Sstrains: catalase and coagulase production, growth and fermentation on mannitol-salt agar. The pattern of Sinai restriction endonuclease fragments found in putative Mc s variants of ANS46 was also used diagnostically. Thus, Mc s variants showing four or more chromosomal changes compared with ANS46 were excluded as possibly not derived from ANS46.
In situ digestion of genomic DNA, pulse-field gel electrophoresis, Southern blotting, and hybridization These procedures were carried out as described previously (Inglis et al. 1990b).
Solar irradiation
Table 1. Strains of Staphylococcus aureus
Selection of methicillin-sensitive mutants
Results
The lethal effect of UV C (254 nm) irradiation E x p o s u r e of cells to a p p r o x . 8 . 5 W m - 2 o f 2 5 4 n m r a d i a t i o n r e s u l t e d in t h e k i n e t i c s of k i l l i n g s h o w n in Fig. 1. T h e s e n s i t i v i t y (k = 1.0) d i d n o t d e p e n d o n w h e t h e r the cells w e r e in s t a t i o n a r y o r e x p o n e n t i a l p h a s e of g r o w t h a n d is c o m p a r a b l e w i t h k i l l i n g o f S. aureus b y U V C seen in o t h e r studies, i n c i d e n t e n e r g y o f 21 J m - 2 g i v i n g a 1 l o g d r o p in cell n u m b e r s , c o m p a r e d w i t h 2 x 102 J m - 2 at 265 n m ( H o l l a e n d e r 1943) a n d 2 0 J m - 2 at 254 n m ( C h a n g et al. 1985). T h e initial lag of a few s e c o n d s b e f o r e e x p o n e n t i a l k i l l i n g o c c u r r e d w a s seen for all s t r a i n s tested, a n d m a y reflect m o r e t h a n o n e U V C s e n s i t i v e t a r g e t p e r cell, o r t h e a c t i v a t i o n of r e p a i r s y s t e m s at l o w e n e r g y i n p u t , as f o u n d in E. coli ( H o l l a e n d e r 1943; P e a k 1970). A l t e r n a t e l y , t h e lag m a y reflect t h e t e n d e n c y o f S. aureus cells to a g g r e g a t e , w h e r e b y s o m e c o l o n i e s d e r i v e f r o m a c l u s t e r o f cells r a t h e r t h a n f r o m a single cell.
Strain
Characteristics
Reference
ANS46
Clinical isolate resistant to many antibiotics, including methicillin Clinical isolate resistant to many antibiotics, including methicillin Non-clinical isolate, antibiotic-sensitive Acriflavine-induced deletion mutant of ANS46, Mc s Stress-induced deletion mutants of ANS46, Mc s Sunlight-induced mutants of ANS46, Mc s UV C-induced mutants of ANS46, Mc s
Matthews et al. 1987
SK604 L8 ANS62 V5, QI, Q3, T40 U21, U23 U27, U28
Lyon et al. 1984 Inglis et al. 1990a Matthews et al. 1987 Inglis et al. 1990b This study This study
244
SURVIVIN(
SURVIVIN
FRACTIOI~
FRACTIOI
4
~
12
16
lv
20
20
TIME (S)
40
60
TIME (min)
Fig. 1. Killing curve for U V C i r r a d i a t i o n o f S. aureus strains A N S 4 6 (e), L8 (m) a n d V5 (A). Cells were irradiated as e x p o n e n t i a l (solid) or s t a t i o n a r y (unshaded) g r o w t h p h a s e cultures
Fig. 2. Killing curve for solar irradiation o f S . aureus strains A N S 4 6 ( e ) , L8 (m) a n d V5 (A). Cells were irradiated as e x p o n e n t i a l (solid) or s t a t i o n a r y (unshaded) g r o w t h p h a s e cultures
One strain (L8, an antibiotic sensitive non-clinical isolate) was more sensitive to UV C irradiation than other strains tested, though this sensitivity is not general for antibiotic sensitive strains (results not shown).
Because of this, cells were exposed in parallel at the same time on the same day when comparing strains or effects of filters. Thus sunlight also kills S. aureus, though the cells have a sensitivity approximately 3 x 10 -4 that seen with UV C on a radiant energy basis, and approximately 5 x 10 -3 oil an exposure time basis. These results are summarised in Table 2. Some strains tested (LS, Fig. 2; SK604, not shown) displayed the same sensitivity to sunlight whether cells were from actively growing or stationary phase cultures, while others (ANS46, Fig. 2 and its derivative ANS62, not shown) were significantly more sensitive when from
The lethal effect of sunlight Exposure of the cells to sunlight on a cloud-free day gave the killing kinetics shown in Fig. 2. In this experiment, the strains showed an inactivation constant of approximately 5 x 10 -3, although in thirteen repetitions there was substantial day-to-day variation (10 .6 to 10-z).
Table 2. Sensitivity o f strains to U V C a n d solar r a d i a t i o n
Strain
I n a c t i v a t i o n c o n s t a n t k (s-1)
Inactivation constant k per unit energy (J 1 m 2)
ANS46 SK604 L8 ANS62 V5 Mean
UV C 0.74 0.74 1.65 0.92 0.74 0.96
UV C 8 . 7 x 10 -2 8.7 x 1 0 - z 19 x 10 . 2 11 x l 0 - z 8.7 x 10 - 2 11 x l 0 - 2
Solar 5 . 6 x 10 - 3 3.0 x 10 3 6.5 x 10 . 3 5.6 x 10 - 3 5.6 x 10 - 3 5.3 x 10 - 3
Solar 3 . 0 x 10 -5 1.6 x 1 0 - s 3.5 x 10 -5 3.0 x 10 -5 3.0 x 1 0 - 5 2.8 x 10 - 5
Sensitivities h a v e been calculated as described in the M e t h o d s section, u s i n g e x p o n e n t i a l p h a s e cultures. T h e a r e a s covered by 10 p,1 cell s u s p e n s i o n s placed o n the plates h a v e been a s s u m e d to be 10 m m in diameter, or 0.00025 m z
245 exponential phase cultures. A greater sensitivity of exponential phase cultures to UV A radiation has been seen in E. coli (Peak 1970). The early lag or delay periods on the killing curves were also seen with solar radiation, suggesting again cell clumping, multiple UV targets or activation of repair systems at low doses. With regard to radiation damage and repair, certain wavelengths in sunlight may induce systems that are able to protect against, or repair damage caused by, other spectral bands i.e. photoprotection occurs (Jagger 1981). At higher fluences or extended exposure, repair systems may be destroyed. The mec region of the chromosome in S. aureus encodes resistance to a range of antimicrobial agents (Inglis et al. 1988), and also maps near the locus mit, believed to confer resistance to UV in this bacterium (Wyman et al. 1974; Tam and Pattee 1986). Mutants of ANS46 (see Table 1) from which extensive segments of D N A in the mec region have been deleted (Inglis et al. 1990b) were examined to determine whether resistance to the lethal effects of sunlight may also have been lost. The five deletants tested (ANS62, V5, Table 2; others not shown) showed similar sensitivity to ANS46 when exponential phase cultures were irradiated. A difference was observed when stationary phase cultures were irradiated: ANS62, deleted for 55 kb around the mec gene, behaves like ANS46 when exposed to sunlight; V5, which has a deletion of 70 kb, no longer shows a difference in the response of stationary and exponential phase cells to sunlight (Fig. 2). This could be related to loss of part or all of the mit gene in V5, since recombination repair and excision repair genes have been shown to be involved in stationary phase cell resistance in E. coli (Tuveson and March 1980).
6
200
I 250
L 300 WAVELENGTH
I 350
400
(nm)
Fig. 3. Absorbance spectra of perspex (m) and glass (e), measured in a Varian 635 spectrophotometer. An absorbance cutoff value of 7 is used; above this, energy arriving at the detector is too low to be measured accurately. Using the paper filter, absorbance exceeded 7 units at all wavelengths measured
Table 3. Effect of filters on killing by sunlight Filter
None Glass (210-300 nm) Perspex (200-380 nm) Paper (< 200- > 400 rim)
Inactivation constant (s- 1) ANS46
L8
2.4 x 10-3 1.5 x 10-3 0.4 x 10.3 0
5.8 x l0 -3 4.1 x 10_3 1.3 x 10-3 0
Stationary phase cultures were exposed to sunlight for 45 rain. Filters were placed over the plates carrying cells as described in the 'Methods'. Inactivation constants shown were calculated from a typical experiment in which parallel cultures were exposed to sunlight through different filters
Effect of filters on killing by sunlight In an attempt to obtain a measure of the action spectrum of sunlight acting on S. aureus, cultures were exposed through a series of filters and the killing effects measured; temperatures at the plate surface were also measured. While the interpretation of these results is not straightforward, they do suggest several conclusions. Using an absorbance value of 2 as a notional point to define the cutoff limit for a filter, glass absorbs radiation at wavelengths below 300 nm, and perspex that below 380 nm (Fig. 3). Glass has only a small effect in decreasing killing by sunlight, whereas the effect of perspex is more substantial (Table 3). Complete shading, using paper, results in no kill. The temperature of the irradiated cells was raised 2 - 3 °C by the paper or perspex filters; with the glass filter the temperature at the plate surface increased by 6 - 9 °C, but did not exceed 32 °C. Thus the small degree of protection by glass from killing could be due in part to an increase in temperature, although this seems unlikely since the temperatures were always well within those in which S. aureus grows actively. In summary, we can say a major lethal effect of sunlight is due to radiation in the 300 to 380 nm window, with further effects apparent at shorter and longer wavelengths. Since the energy in solar radiation increases at longer UV
wavelengths (Watson-Munro 1973), then the observations are consistent with a greater lethality of UV B than UV A (at equal intensities) for S. aureus, as has been reported for staphylococci irradiated with UV A and UV B lamps (Faergemann and Larko 1987). Billen and Fletcher (1974) have shown that the lethality of sunlight for E. coli is due chiefly to UV B. The mutational effects o f U V C (254 rim) and sunlight When cells that carry mec (and thus are Mc') were exposed to UV C or solar radiation and the proportion of Mc ~ cells was determined amongst survivors, it was found that for a reduction in cell numbers of four logs by sunlight, the rate of appearance of Mc ~ cells was < 1 per 2100 surviving cells for stationary phase and 1 per 1600 for exponential phase cultures. For an equivalent lethal effect by 254 nm radiation, the corresponding rates were < 1 per 1400 and 1 per 3800. Five of the Mc ~ mutants obtained by irradiation with sunlight and UV C were examined by pulse-field gel electrophoresis of genomic D N A digested with the restriction endonuclease Smal (Patel et al. 1989; Inglis et al.
246
Fig. 4. Pulse-fieldgel electrophoresis of Sinai digests of chromosomal DNA from ANS46 (1), ANS62 (2) and UV-induced mutants U21 (3), U23 (4) and U27 (5), stained with ethidium bromide. Variant U28 (not shown) also gives a pattern identical to ANS62
1990b). Four of the mutants yielded genomic fragments indicating an identical deletion of approx. 55 kb of DNA from the mec region (Fig. 4). Probing of H i n d I i I digested DNA from these four mutants with MA13, a cloned fragment of DNA from the mec region of ANS46 (Inglis et al. 1990b), suggested that for all four deletions one end of the deletion occurred at the insertion site IS257.1 (Matthews et al. 1990; data not shown). The remaining mutant yielded a Sinai fragment pattern indistinguishable from that of ANS46, indicating either a point mutation or other alteration of the DNA not detectable as a change in fragment size or number; mutants of this type have been observed before (lnglis et al. 1990b).
Discussion
Our results show that though not as potent as UV C, solar radiation has lethal effects on S. aureus within a time scale that would commonly be achieved on the skin of humans exposed to sunlight. Relatively small differ-
ences were seen between the strains tested in these experiments, whether UV C or solar radiation was used. The effect of a mixed spectrum of visible and UV radiation, such as constitutes sunlight, on bacterial cells is complex. Short wavelength radiation causes the formation of pyrimidine dimers, while longer wavelength radiation promotes the repair of this damage (Jagger 1967). Furthermore, in E. coli, SOS repair mechanisms are induced by UV C (Walker 1987; Witkin 1976). An SOS-like response is induced by UV C in S. aureus (Thompson and Hart 1981) and in S. epidermidis (Guillabel and Leitao 1988; Silva and Leitao 1984); similar mutants to those in E. coli affecting these responses have been found. Repair responses are faster and occur at lower UV intensity in the staphylococci than in E. coli (Guillobel and Leitao 1988), an adaptation selected possibly because the normal habitat of the former includes exposed human skin. UV A/visible radiation protects against the mutagenicity and lethality of UV B radiation in Salmonella (Calkins et al. 1987), and prior exposure of S. aureus cells to UV B/UV A/visible light decreases the mutagenicity and lethality of UV C (Tyrrell 1980). In E. coli, radiations at wavelengths of 334, 365 and 405 nm have been shown to sensitize to radiations at other UV A wavelengths (Tyrrell and Peak 1978). However, in sunlight direct damage to DNA by UV may not be significant, since sunlight contains little radiation in the wavelengths where major absorption by DNA occurs. While the small amounts of radiation absorbed by DNA at 290 nm and above (Sutherland and Griffin 1981) could account for the mutational effects observed (Webb 1978), it is possible that the lethal and mutational effects of UV B/UV A/visible light on staphylococci are mediated primarily through the production of free radicals (Eisenstark 1989). Reactive oxygen species could react directly with DNA, and in addition have wider effects, as reported for other cellular systems (Peak et al. 1984; Eisenstark 1989; Tyrrell and Keyse 1990). Some effects of UV B/UVA/visible light and H20 2 (a source of free radicals) are common, and mutants of E. coli and Salmonella typhimurium sensitive to one are sensitive to the other (Tyrrell 1985; Eisenstark 1987). UV A is also absorbed by tRNA, leading to the arrest of protein synthesis (Favre et al. 1985); U V A also inactivates membrane transport systems (Robb et al. 1978). Our experiments show that as well as killing exposed staphylococci, solar radiation is mutagenic, in that chromosomal deletions are induced by it. The mec region in ANS46 has previously been shown to be unstable, and susceptible to deletion and other mutational change when cells are stressed by prolonged culture or growth at high temperature (Inglis et al. 1990b). In that study, it was noted that certain insertion elements (IS25 7, Tn554) may be foci for deletion of tracts of chromosomal DNA up to 250 kb in length, In the experiment reported here, the deletants examined also appear to involve the most leftward copy of IS257 as a deletion point. It is notable that all four deletion mutants examined showed the same chromosomal alteration, whether induced by solar or UV C irradiation, suggesting a common mechanism for
246
Fig. 4. Pulse-field gel electrophoresis of Sinai digests of chromosomal DNA from ANS46 (1), ANS62 (2) and UV-induced mutants U21 (3), U23 (4) and U27 (5), stained with ethidium bromide. Variant U28 (not shown) also gives a pattern identical to ANS62
1990b). Four of the mutants yielded genomic fragments indicating an identical deletion of approx. 55 kb of DNA from the mec region (Fig. 4). Probing of H i n d I I I digested DNA from these four mutants with MA13, a cloned fragment of DNA from the mec region of ANS46 (Inglis et al. 1990b), suggested that for all four deletions one end of the deletion occurred at the insertion site IS257.I (Matthews et al. 1990; data not shown). The remaining mutant yielded a Sinai fragment pattern indistinguishable from that of ANS46, indicating either a point mutation or other alteration of the DNA not detectable as a change in fragment size or number; mutants of this type have been observed before (lnglis et al. 1990b).
Discussion
Our results show that though not as potent as UV C, solar radiation has lethal effects on S. aureus within a time scale that would commonly be achieved on the skin of humans exposed to sunlight. Relatively small differ-
ences were seen between the strains tested in these experiments, whether UV C or solar radiation was used. The effect of a mixed spectrum of visible and UV radiation, such as constitutes sunlight, on bacterial cells is complex. Short wavelength radiation causes the formation of pyrimidine dimers, while longer wavelength radiation promotes the repair of this damage (Jagger 1967). Furthermore, in E. coli, SOS repair mechanisms are induced by UV C (Walker 1987; Witkin 1976). An SOS-like response is induced by UV C in S. aureus (Thompson and Hart 1981) and in S. epidermidis (Guillabel and Leitao 1988; Silva and Leitao 1984); similar mutants to those in E. coli affecting these responses have been found. Repair responses are faster and occur at lower UV intensity in the staphylococci than in E. coli (Guillobel and Leitao 1988), an adaptation selected possibly because the normal habitat of the former includes exposed human skin. UV A/visible radiation protects against the mutagenicity and lethality of UV B radiation in Salmonella (Calkins et al. 1987), and prior exposure of S. aureus cells to UV B/UV A/visible light decreases the mutagenicity and lethality of UV C (Tyrrell 1980). In E. coli, radiations at wavelengths of 334, 365 and 405 nm have been shown to sensitize to radiations at other UV A wavelengths (Tyrrell and Peak 1978). However, in sunlight direct damage to DNA by UV may not be significant, since sunlight contains little radiation in the wavelengths where major absorption by DNA occurs. While the small amounts of radiation absorbed by DNA at 290 nm and above (Sutherland and Griffin 1981) could account for the mutational effects observed (Webb 1978), it is possible that the lethal and mutational effects of UV B/UV A/visible light on staphylococci are mediated primarily through the production of free radicals (Eisenstark 1989). Reactive oxygen species could react directly with DNA, and in addition have wider effects, as reported for other cellular systems (Peak et al. 1984; Eisenstark 1989; Tyrrell and Keyse 1990). Some effects of UV B/UV A/visible light and H202 (a source of free radicals) are common, and mutants of E. coli and Salmonella typhimurium sensitive to one are sensitive to the other (Tyrrell 1985; Eisenstark 1987). UV A is also absorbed by tRNA, leading to the arrest of protein synthesis (Favre et al. 1985); U V A also inactivates membrane transport systems (Robb et al. 1978~. Our experiments show that as well as killing exposed staphylococci, solar radiation is mutagenic, in that chromosomal deletions are induced by it. The mec region in ANS46 has previously been shown to be unstable, and susceptible to deletion and other mutational change when cells are stressed by prolonged culture or growth at high temperature (Inglis et al. 1990b). In that study, it was noted that certain insertion elements (IS257, Tn554) may be foci for deletion of tracts of chromosomal DNA up to 250 kb in length, In the experiment reported here, the deletants examined also appear to involve the most leftward copy of IS257 as a deletion point. It is notable that all four deletion mutants examined showed the same chromosomal alteration, whether induced by solar or UV C irradiation, suggesting a common mechanism for
247 the two types o f radiation. It remains an open question whether different mechanisms are involved in killing and mutagenesis in S. a u r e u s . U n d e r conditions o f U V and solar radiation which bring a b o u t extensive killing o f the staphylococci, m u t a tion rates (as reflected in the generation o f deletants which have lost the m e c locus and s u r r o u n d i n g D N A ) were considerably less than those seen with nutritional or thermal stress, when deletion could a c c o u n t for wholesale conversion o f cultures to M c s at high levels of surviving cells (Inglis et al. 1990b). U n d e r conditions of nutritional or thermal stress, rates of lethal mutations m a y be slow e n o u g h to be repaired; the killing rates seen in the irradiated cultures m a y be such that only a small fraction of m u t a n t s or deletants is recovered. In the same way, sensitivity of cells to irradiation m a y be a function of p o w e r (rate of energy a b s o r p t i o n by cells) rather than of total energy absorbed. U n d e r conditions of low p o w e r radiation, repair systems m a y keep pace (particularly if mixtures of U V and visible radiation are involved; p h o t o r e p a i r of lethal d a m a g e m a y then be important), and lethality (as with mutation) m a y not be so evident. These questions are susceptible to experimental testing. The findings of this study are relevant to an understanding of the ecology of S. a u r e u s . As a n o r m a l m e m b e r o f the skin m i c r o b i o t a o f a b o u t one third o f humans, this bacterium w o u l d c o m m o n l y be exposed to unfiltered sunlight. Thus, such older remedies for S. a u r e u s and other bacterial infections as exposure of patients and their clothing and bedlinen to sunshine m a y have a s o u n d basis in fact. Yet, while the upper skin layers m a y contain the majority of S. a u r e u s cells, as is the case for coagulase-negative staphylococci (Brown et al. 1989), reservoirs o f these bacteria m a y exist deeper within the skin and be screened f r o m the h a r m f u l effects o f sunlight. These p r o b a b l y serve for later recolonization o f the b o d y surfaces. A n expected consequence o f increasing atmospheric depletion o f ozone is an increase in U V B c o m ponents o f sunlight (Caldwell et al. 1989). Since U V B appears to be m o r e lethal than U V A for S. a u r e u s (as discussed in 'Results'), a small increase in U V B levels in sunlight m a y result in a disproportionately large increase in lethality o f sunlight to S. a u r e u s . I f the staphylococci are representative o f other skin microbes, it seems likely that changes in the skin m i c r o b i o t a could be expected for individuals exposed to solar radiation where significant depletion o f atmospheric ozone has occurred. The consequences o f this remain uncertain whilst we k n o w little o f the i m p o r t a n c e o f the n o r m a l skin m i c r o b i o t a in determining susceptibility o f the host to opportunistic or virulent pathogens. Acknowledgements. We thank Keren Solomon for assistance with
these experiments. The work was supported by the Australian Research Council.
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