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Previous Article | Next Article 
Applied and Environmental Microbiology, October 2001, p. 4630-4637, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4630-4637.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Determination of Pyrimidine Dimers in
Escherichia coli and Cryptosporidium
parvum during UV Light Inactivation, Photoreactivation, and
Dark Repair
Kumiko
Oguma,1,*
Hiroyuki
Katayama,1
Hiroshi
Mitani,2
Shigemitsu
Morita,3
Tsuyoshi
Hirata,3 and
Shinichiro
Ohgaki1
Department of Urban
Engineering,1 and Department of
Integrated Biosciences,2 University of Tokyo,
Bunkyo-ku, Tokyo, and College of Environmental Health, Azabu
University, Sagamihara, Kanagawa,3 Japan
Received 2 April 2001/Accepted 20 July 2001
 |
ABSTRACT |
UV inactivation, photoreactivation, and dark repair of
Escherichia coli and Cryptosporidium
parvum were investigated with the endonuclease sensitive site
(ESS) assay, which can determine UV-induced pyrimidine dimers in the
genomic DNA of microorganisms. In a 99.9% inactivation of E.
coli, high correlation was observed between the dose of UV
irradiation and the number of pyrimidine dimers induced in the DNA of
E. coli. The colony-forming ability of E.
coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the
method conventionally used to measure colony-forming ability. When
E. coli were exposed to fluorescent light after a 99.9%
inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA
were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor
recovery of colony-forming ability. When C. parvum were
exposed to fluorescent light after UV inactivation, UV-induced
pyrimidine dimers in the DNA were continuously repaired, while no
recovery of animal infectivity was observed. When kept in darkness
after UV inactivation, C. parvum also showed no recovery
of infectivity in spite of the repair of pyrimidine dimers. It was
suggested, therefore, that the infectivity of C. parvum
would not recover either by photoreactivation or by dark repair even
after the repair of pyrimidine dimers in the genomic DNA.
| |
INTRODUCTION |
UV light irradiation is one
of the effective disinfection methods for bacteria, viruses, and
parasites in water and wastewater. UV disinfection systems are easy to
maintain and need no additional chemical inputs. Moreover, UV
irradiation produces no hazardous by-products like many of the
conventional chlorination processes (22). Therefore, water
treatment plants that utilize UV disinfection processes have increased
in number in recent decades (14).
Inactivation of microorganisms by UV-B and UV-C (220 to 320 nm)
radiation is effected through the formation of lesions in the genomic
DNA of the organisms (10, 13). The major lesions induced
by the germicidal UV light (254 nm) are cis-syn cyclobutane pyrimidine dimers (CPD), while (6-4) photoproducts are also formed at
about 10% of CPD and other kinds of lesion are also produced at lower
ratios (13). The presence of UV-induced lesions would inhibit the normal replication of DNA and therefore result in inactivation of the microorganisms.
Some organisms, however, are known to possess the ability to repair
their DNA by mechanisms such as photoreactivation and dark repair
(10, 13). Photoreactivation is the phenomenon by which
UV-inactivated microorganisms recover activity through the repair of
pyrimidine dimers in the DNA under near-UV light (310 to 480 nm) with
the enzyme photolyase. In this study, the repair of pyrimidine dimers
in the DNA would be named photo repair, while the recovery of the
activity of the organism would be referred to photoreactivation. DNA
repair mechanisms other than photo repair, such as excision repair, are
named dark repair because they can repair the damaged DNA without
light. Photoreactivation and dark repair enable UV-inactivated
microorganisms to recover and may reduce the efficacy of UV
inactivation. Therefore, they disadvantage the UV disinfection methods.
Much attention has been paid to photoreactivation especially because it
can cause a great deal of recovery of microorganisms within a short
time. Photoreactivation may occur in organisms, especially in
UV-treated wastewater after its discharge to watersheds, because
UV-inactivated microorganisms would normally be exposed to sunlight,
including near-UV light.
The ability to perform photo repair depends on whether the organism has
the enzyme photolyase. Most strains of Escherichia coli, one
of the indicator bacteria for water quality control, are known to be
able to perform photoreactivation. This ability has been shown to occur
in bacteria, plants, and animals, but evolutionarily allied species
need not necessarily show similar photoreactivation characteristics
(10). The ability for dark repair is also known to differ
greatly from species to species. It would be necessary, therefore, to
investigate the ability of photoreactivation and dark repair in each
organism individually. This is especially so for the target
microorganisms of disinfection processes, such as indicator or
pathogenic organisms.
The presence of Cryptosporidium in water resources and water
supply systems has recently been identified as a public health risk
(7, 12, 19). This parasite is pathogenic for both humans
and animals and can be transmitted through the water supply. Outbreaks
of Cryptosporidium infection have been reported in many countries, most often associated with the consumption of drinking or
recreational water contaminated with Cryptosporidium oocysts.
Cryptosporidium oocysts show high resistance to conventional
disinfectants such as chlorine. Therefore, effective techniques for
removing or disinfecting oocysts, other than chlorination, have been
explored (23, 27). Many researchers have reported that UV
irradiation is an effective method to disinfect
Cryptosporidium oocysts (1, 6, 8, 16). However,
the ability of Cryptosporidium to perform photoreactivation
and dark repair has not been clarified yet in spite of the importance
of these phenomena. In order to evaluate the efficiency and
effectiveness of UV irradiation to Cryptosporidium, it is
essential to investigate its photoreactivation and dark repair abilities.
Some detection methods for oocysts of Cryptosporidium have
been established. Excystation and vital dye staining have commonly been
used for the determination of the viability of oocysts
(4). However, these methods only determine their viability
and do not address their ability to be infectious. The animal
infectivity assay is said to be the gold standard for the determination
of infection potential (16), but this method requires a
number of animal hosts, is highly expensive, needs skillful
technicians, and also involves a long testing time. Upton et al.
pointed out that cultured cells such as HCT-8 could be used as a host
for Cryptosporidium instead of animal hosts
(33), and many researchers have modified this cell culture
method by combining it with other enzymatic or genetic techniques
(11, 24, 30, 36). Such cell culture methods have become
attractive alternatives to the animal infectivity assay because they
were reported to be equivalent to the animal method in determining the
infectivity of Cryptosporidium parvum (16). On
the other hand, it has also been reported that the methods based on
reverse transcription-PCR detection of the specific mRNA in
Cryptosporidium are effective to investigate their
infectivity (20, 25, 31). Accurate and simple detection methods for Cryptosporidium are still being explored.
In this study, the UV inactivation, photo repair, and dark repair of
E. coli and C. parvum were investigated by an
endonuclease sensitive site (ESS) assay, which can determine the number
of UV-induced pyrimidine dimers in the genomic DNA as the number of
ESS. The ESS assay was initially proposed by Achey et al.
(2) for detecting pyrimidine dimers in nonradioactive DNA
and developed by Sutherland and Shih (32) for quantifying
the number of dimers. This assay proved to be effective not only for
the comparative study of the UV sensitivity of different kinds of
organisms but also for the quantitative investigation of DNA repair
mechanisms. ESS assay, therefore, has commonly been used in the field
of radiobiology (9, 18, 21), but only a few studies have
investigated the relationships between the number of pyrimidine dimers
in the DNA and the activity of the organism. Moreover, ESS assay has
never been applied for the investigation of UV disinfection of water.
The purpose of this study is to investigate the mechanisms of UV
inactivation, photo repair, and dark repair of E. coli and C. parvum with the ESS assay. In addition, a colony-forming
ability assay was applied to E. coli in order to clarify the
relationship between the number of pyrimidine dimers in the genomic DNA
and the colony-forming ability. For C. parvum, an animal
infectivity assay was applied in parallel with the ESS assay in order
to investigate the relationship between the number of pyrimidine dimers
and animal infectivity. It was further aimed to compare the UV
sensitivity and the photo repair ability of E. coli with
those of C. parvum quantitatively.
| |
MATERIALS AND METHODS |
Microorganisms.
As test microorganisms, E. coli
IFO 3301 and C. parvum HNJ-1 strain were used. The E. coli were picked up from a few purified colonies formed on a broth
medium (10 g of polypeptone, 5 g of yeast extract, 1.5 g of
glucose, 5 g of NaCl, 0.2 g of
MgSO4 · 7H2O,
0.05 g of MnSO4 · 4H2O, and 11 g of agar powder for 1 liter of
purified water) after 24 h of incubation at 37°C and suspended in a sterilized phosphate buffer solution (pH 7.6) to be at an initial
concentration of 2.5 × 10 to 4.0 × 107 CFU ml
1). An E. coli preparation (40 ml) was placed into a sterilized petri dish
(100-mm diameter) and used for the light irradiation procedures.
The strain of C. parvum, HNJ-1, which belongs to the group
of genotype 2 (bovine), was originally isolated from feces of a naturally infected human by M. Iseki at Osaka Medical School, Osaka, Japan. The C. parvum were passaged in SCID mice at
the Research Institute of Biosciences, Azabu University, and the
oocysts were purified from the fresh feces of several infected mice by sucrose flotation. The purified oocysts were stored at 4°C in phosphate-buffered saline (pH 7.4) till used. It was previously confirmed that the animal infectivity of the oocysts would not change
within a month in the stock suspension. The stock suspension of
purified oocysts was diluted by a sterilized phosphate buffer solution
(pH 7.6) to produce an initial concentration of 1.0 × 106 oocysts ml1. Then 40 ml of the preparation was placed into a sterilized petri dish (100-mm
diameter) and used for the light irradiation procedures. The age of the
oocysts was 5 to 15 days at the time of use. In the animal infectivity
assay, the initial concentration of the oocysts was 2.0 × 106 oocysts ml1, and 10 ml of the preparation was used for the light irradiation in a
sterilized petri dish of 60-mm diameter.
UV and fluorescent light irradiation.
Two low-pressure UV
lamps (Stanley germicidal lamp, 20 W; Toshiba) were used for the UV
irradiation procedures. The intensity of the UV at a wavelength of 254 nm was 0.24 mW cm2, which was measured by a UV
radiometer (UVR-2 UD-25; Topcon). The dose of UV was altered by
controlling the exposure time.
For the photoreactivation procedures, samples were irradiated by three
fluorescent lamps (white light fluorescent lamp, 18 W; Hitachi) within
5 min after the 99.9% inactivation by UV irradiation. A 6.0-mJ
cm2 dose of UV was used for E. coli
to achieve 99.9% reduction in the colony-forming ability, which was
determined via colony-forming ability assay (detailed below). A 2.2-mJ
cm2 dose of UV was used for C. parvum to achieve 99.9% reduction in infectivity, which was
determined via animal infectivity assay (detailed below). The intensity
of the photoreactivating light at 360 nm was 0.1 mW
cm2, as measured by a UV radiometer (UVR-2
UD-36; Topcon). Sample collection was intermittently performed during
the exposure to fluorescent light for 120 min. For the investigation of
dark repair, samples were kept in darkness for 24 h after the UV
irradiation. All samples were constantly stirred magnetically
throughout the experiment and kept in darkness except at the times for
the UV and fluorescent light irradiation procedures. Sample
temperatures were kept constant at 20°C by circulating cooling water
around the petri dishes.
In the experiment with the animal infectivity assay of C. parvum, a low-pressure UV lamp (QCGL 5W-14 97D, 5 W; Iwasaki) and a fluorescent lamp (FL15N, 15 W; Toshiba) were used for the UV inactivation and photoreactivation procedures, respectively. The conditions of UV and fluorescent light irradiation were the same as
mentioned above, except that the UV dose was set to be either 0.72 or
1.44 mJ cm2 and the intensity of the
photoreactivating light at 360 nm was 0.05 mW
cm2.
Colony-forming ability assay for E. coli
The
colony-forming ability of E. coli was investigated using
desoxycholate-acid medium (Eiken) in the dark room following standard
methods (35). All the plated samples were covered with aluminum foil and incubated at 37°C for 18 h. The number of
colonies formed after the incubation were counted, and the ratio of the colony-forming ability was calculated by equation 1,
|
(1)
|
where St is the ratio of the
colony-forming ability at light irradiation time t,
Nt is the number of colonies at light
irradiation time t, and N0
is the number of colonies before UV irradiation.
Animal infectivity assay for C. parvum
Samples of C. parvum for the infectivity tests were
successively diluted by a fixed dilution factor of five. Six-week-old SCID mice were inoculated orally with aliquots of selected series of
dilutions. After a 4-week incubation, fresh feces from each mouse were
collected, purified, stained by indirect immunofluorescent antibodies
(Hydrofluoro combo kit; Strategic Diagnostics) and then observed by
epifluorescent microscopy to determine the presence or absence of the
oocysts. The infective doses, i.e., the number of oocysts for 1 most-probable-number (MPN) of infection, were calculated from the data
set of the numbers of oocyst-positive mice, using the MPN program
proposed by Hurley and Roscoe (17). The relative
infectivity of each sample was calculated by equation 2,
|
(2)
|
where It is the relative
infectivity at light irradiation time t,
MPNt is the most probable number of
infective oocysts at light irradiation time t, and
MPN0 is the most probable number of
infective oocysts before UV irradiation.
ESS assay. (i) Principle of ESS assay.
DNA samples are
incubated with UV endonuclease, which incises a phosphodiester bond
specifically at the site of a pyrimidine dimer. This enables the
recognition of a pyrimidine dimer in the DNA as an ESS. After the
incision of nicks, the DNA strands are separated according to their
molecular lengths by electrophoresis on alkaline agarose gels. The
double-stranded DNA is denatured by the alkalinity and therefore
fragmented according to the number of nicks incised by the UV
endonuclease. By analyzing the migration patterns of DNA fragments
relative to the molecular length of standard markers, the median
molecular length of the DNA can be determined graphically and the
number of ESS per DNA molecule can be calculated theoretically
(32).
(ii) DNA extraction from microorganisms.
One milliliter of
E. coli sample was centrifuged at 5,000 × g
for 7 min, and the pellet was used for the Genomic-tip DNA extraction kit (Qiagen) following the manufacturer's protocol. A 40-ml sample of
C. parvum was centrifuged at 1,050 × g for
10 min, and the pellet was resuspended in 2 ml of sterilized phosphate
buffer (pH 7.6). This suspension of C. parvum was frozen in
liquid nitrogen for 3 min and thawed at 95°C for 5 min in order to
break the oocyst walls, following the method of Rochelle et al.
(24). This freezing-thawing treatment was repeated three
times. After the breakage of the oocyst walls, the DNA was extracted
from the sporozoites using the Genomic-tip DNA extraction kit (Qiagen)
following the manufacturer's protocol. All the DNA solutions of
E. coli and C. parvum were then concentrated by
extraction with 2-butanol (Wako). After concentration, the samples were
dialyzed overnight at 4°C against 30 mM Tris (pH 8.0)-40 mM NaCl-1
mM EDTA in order to remove any excess 2-butanol completely.
(iii) Conditions for ESS assay.
The UV endonuclease was
prepared from Micrococcus luteus following the method of
Carrier and Setlow (5). DNA solutions were incubated with
the UV endonuclease (1 µl of endonuclease solution per µg of DNA)
at 37°C for 45 min in 30 mM Tris (pH 8.0)-40 mM NaCl-1 mM EDTA in
order to incise the nicks at the site of pyrimidine dimers. The
reaction was stopped by the addition of an alkaline loading dye at a
final concentration of 100 mM NaOH, 1 mM EDTA, 2.5% Ficoll and 0.05%
bromocresol green. The treated samples were subjected to
electrophoresis at 0.5 V/cm for 16 h on 0.5% alkaline agarose
gels in an alkaline buffer containing 30 mM NaOH and 1 mM EDTA. Two
molecular length markers, 7GT (T4dC+T4dC/BglI digest
mixture; Wako) and 8GT (T4dC+T4dC/BglII digest mixture; Wako) were used as molecular length standards. After electrophoresis, the gel was dipped in ethidium bromide solution (0.5 µg/ml) overnight to stain the DNA. The images of the stained gels were photographed (Gel
Doc 1000; BioRad) and analyzed (Molecular Analyst software; BioRad).
With this analysis system, the fluorescence of the DNA was detected as
a set of pixels, and a distribution pattern of the fluorescence
intensity in relation to the migration distance was obtained. The total
count of the pixels, that is, the integrated area of the fluorescence
intensity, corresponded to the total amount of DNA in the sample
because the fluorescence intensity was previously confirmed to
correlate linearly with the DNA concentration in the conditions used in
this study. The midpoint of the DNA mass, that is, the median migration
distance of each sample, was graphically determined to be the
representative migration distance of the sample. The median value of
migration distance was converted into the median molecular length
(Lmed) of the DNA by means of the quadratic
regression curve obtained from the analysis of the molecular standards.
The average molecular length (Ln) of the DNA was obtained by equation 3 (Veatch and Okada
[34]):
|
(3)
|
The number of ESS per base was obtained by equation 4 (Freeman
et al. [9]):
|
(4)
|
where Ln(+UV) and
Ln(UV) are the
Ln of UV-irradiated and
nonirradiated samples, respectively.
For the discussion of photo repair, the ESS remaining ratio, defined as
the ratio of the number of ESS during fluorescent light irradiation to
the number of ESS before fluorescent light irradiation, was calculated
by equation 5:
|
(5)
|
where t is the time of exposure to
fluorescent light.
| |
RESULTS AND DISCUSSION |
UV inactivation of E. coli
Figure
1 shows an example of the result of the
ESS assay during UV irradiation of E. coli. UV doses of
2, 4, and 6 mJ cm2 corresponded to 90, 99, and 99.9%
inactivation of colony-forming ability, respectively. The photographic
image of the gel (Fig. 1A) was analyzed, and the distribution patterns
of the fluorescence intensity were determined as depicted in Fig. 1B,
in which the horizontal axis indicates the migration distance or the
corresponding molecular length and the vertical axis shows the
fluorescent intensity of the DNA fragments. As shown in Fig. 1B, the
median molecular length of each of the samples became shorter and
shorter with an increase of the UV dose, indicating that the DNA was
fragmented into smaller and smaller pieces with an increase of the dose
of UV irradiation.
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FIG. 1.
Example of ESS assay during UV irradiation of E.
coli. (A) Photographic image of the alkaline agarose gel after
electrophoresis. (B) Distribution patterns of DNA in relation to
molecular length standard markers. Arrows indicate the median point of
each distribution pattern. UV doses of 2, 4, and 6 mJ cm2
corresponded to 90, 99, and 99.9% inactivation of the colony-forming
ability of E. coli, respectively.
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Figure 2 shows that the number of ESS
during UV irradiation increased linearly with an increase of the UV
dose. A linear regression line between the UV dose and the number of
ESS was determined by the least-squares method. The coefficient of
determination (r2) was as high as
0.987 for eight data, indicating that the number of ESS correlated
highly with the UV dose. The yield of ESS formation was calculated to
be 4.0 × 105 per base per 1 mJ
cm2 of UV dose, which is approximately the same
as the result of Howard-Flanders (15), who reported that
the yield of pyrimidine dimers after irradiation by a low-pressure
mercury germicidal lamp was 6.4 × 105 per
base per 1 mJ cm2 of UV for E. coli
K-12 (wild type).
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FIG. 2.
Relationship between UV dose and number of ESS during UV
irradiation of E. coli. The regression on a straight
line which passes through the origin of the coordinates was determined
by the least-squares method for two independent series of experiments.
Coefficient of determination (r2) was 0.987 for eight data.
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Figure 3 shows the relationship between
the number of ESS and the ratio of colony-forming ability during UV
irradiation. A linear regression line between the number of ESS and the
logarithmic value of the ratio of colony-forming ability was determined
by the least-squares method. The coefficient of determination
(r2) was 0.991 for eight data,
indicating that the ESS assay is comparable to the conventional
colony-forming ability assay. Considering that the total genome size of
E. coli is approximately 4.6 × 106 bp in a steady-state cell, the number of ESS
necessary for 1 log inactivation of E. coli was calculated
to be roughly 736.
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FIG. 3.
Relationship between number of ESS and ratio of
colony-forming ability during UV irradiation of E. coli.
The regression on a straight line whose intercept was fixed to be 1 on
the vertical axis was determined by the least-squares method for two
independent series of experiments. Coefficient of determination
(r2) was 0.991 for eight data.
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Photoreactivation and dark repair of E. coli
Figure 4 shows an example of the result
of the ESS assay during the exposure of E. coli to
fluorescent light after 99.9% inactivation by UV irradiation. The
photographic image (Fig. 4A) was analyzed and the distribution patterns
of the fluorescence intensity were determined (Fig. 4B). The figure
shows that the median molecular length of the DNA became gradually
longer as the exposure time to fluorescent light increased. Figure
5 shows the profiles of the number of ESS
in the DNA and the ratio of colony-forming ability during fluorescent
light irradiation after UV inactivation. This figure indicates that,
during the exposure to fluorescent light, pyrimidine dimers were
continuously repaired and the colony-forming ability gradually
recovered. When kept in darkness after UV inactivation, however,
neither the repair of pyrimidine dimers nor the recovery of
colony-forming ability was observed, as shown in Fig.
6. This indicates that E.
coli did not perform dark repair under the conditions of this
study.
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FIG. 4.
Example of ESS assay during UV and fluorescent light
irradiation of E. coli. (A) Photographic image of the
alkaline agarose gel after electrophoresis. (B) Distribution patterns
of DNA in relation to molecular length standard markers. Arrows
indicate the median point of each distribution pattern. Dose of UV was
6 mJ cm2 for all samples, which corresponded to 99.9%
inactivation of the colony-forming ability of E. coli.
FL indicates exposure to fluorescent light irradiation.
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FIG. 5.
Profiles of number of ESS and ratio of colony-forming
ability during fluorescent light irradiation of E. coli
after UV inactivation.  , number of ESS/base;  , ratio of
colony-forming ability. Dose of UV for inactivation was 6 mJ
cm2. All symbols indicate the result of two independent
series of experiments. Solid and dotted lines connect the arithmetic
mean values of the number of ESS and the geometric mean values of the
colony-forming ability ratio, respectively.
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FIG. 6.
Profiles of number of ESS and ratio of colony-forming
ability in darkness after UV inactivation of E. coli.
, number of ESS/base; , ratio of colony-forming ability. Dose of
UV for inactivation was 6 mJ cm2. All symbols indicate
the result of two independent series of experiments. Solid and dotted
lines connect the arithmetic mean values of the number of ESS and the
geometric mean values of the colony-forming ability ratio,
respectively.
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Figure 7 shows the relationship between
the ESS remaining ratio and the colony-forming ability ratio of
E. coli during the exposure to fluorescent light. The
coefficient of determination (r2) was
0.799 for the linear regression line determined by the least-squares method between the ESS remaining ratio and the logarithmic value of the
colony-forming ability (number of data was 10). The intercept of the
regression line was fixed to be 1 on the vertical axis, assuming that
the ratio of colony-forming ability would be 1 when the photo repair of
ESS was completely performed. The high correlation between the ESS
remaining ratio and the colony-forming ability ratio indicated that,
during the exposure of E. coli to fluorescent light
irradiation, the repair of pyrimidine dimers in the genomic DNA did
contribute to the recovery of colony-forming ability.
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FIG. 7.
Relationship between ESS remaining ratio and
colony-forming ability ratio during fluorescent light irradiation of
E. coli. The regression on a straight line whose
intercept was fixed to be 1 on the vertical axis was determined by the
least-squares method. Coefficient of determination
(r2) was 0.799 for 10 data.
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UV inactivation of C. parvum
In the
inactivation of C. parvum by UV irradiation, high
correlation was observed between the number of ESS and the dose of UV
(r2 was 0.978 for seven data) as shown in
Fig. 8. The yield of ESS formation was
calculated to be 7.5 × 105 per base per 1 mJ
cm2 of UV dose, which is in the same order as that
calculated for E. coli (4.0 × 105
per base per 1 mJ cm2 of UV dose). This result suggests
that the oocyst wall of C. parvum is not more protective
against UV light than the cell wall of E. coli. This
might be the reason why UV irradiation is relatively effective at
disinfecting C. parvum compared to chlorination or other
chemical disinfectants that cannot penetrate the oocyst wall.
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FIG. 8.
Relationship between UV dose and number of ESS during UV
irradiation to C. parvum. The regression on a straight
line which passes through the origin of the coordinates was determined
by the least-squares method for three independent series of
experiments. Coefficient of determination
(r2) was 0.978 for seven data.
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Photoreactivation and dark repair of C. parvum
Figure 9 shows an example of the result
of the ESS assay during the exposure of C. parvum to
fluorescent light after UV inactivation. The dose of UV was 2.2 mJ
cm2, which corresponded to 99.9% inactivation of animal
infectivity. The photographic image of the gel (Fig. 9A) was analyzed
to obtain the distribution patterns of the fluorescence intensity (Fig. 9B). The 99.9% inactivation of the infectivity of C.
parvum was detected as a smear image of the DNA, and the median
molecular length of each of the samples became gradually longer with
increasing duration of exposure to fluorescent light after UV
irradiation. As shown in Fig. 10, a
gradual repair of pyrimidine dimers was observed during the exposure to
fluorescent light irradiation. The result indicated that C.
parvum had the ability to repair the pyrimidine dimers in
genomic DNA during the exposure to fluorescent light.
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FIG. 9.
Example of ESS assay during UV and fluorescent light
irradiation to C. parvum. (A) Photographic image of the
alkaline agarose gel after electrophoresis. (B) Distribution patterns
of DNA in relation to molecular length standard markers. Arrows
indicate the median point of each distribution patterns. Dose of UV was
2.2 mJ cm2 for all samples, which corresponded to 99.9%
inactivation of the infectivity of C. parvum. FL
indicates exposure to fluorescent light irradiation
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FIG. 10.
Profile of number of ESS during fluorescent light
irradiation to C. parvum after UV inactivation. Dose of
UV for inactivation was 2.2 mJ cm2, which corresponded to
99.9% inactivation of the infectivity of C. parvum. All
symbols indicate the result of two independent series of experiments.
Solid line connects the arithmetic mean values of the data at each
irradiation time.
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The ESS remaining ratio, defined as the ratio of the number of ESS
during fluorescent light irradiation to the total number of ESS induced
by UV irradiation, was calculated for E. coli and C. parvum in order to compare these microorganisms from the viewpoint of photo repair. As shown in Fig. 11,
the ratio of ESS remaining in C. parvum after 120 min of
exposure to fluorescent light irradiation was almost the same as that
in E. coli. This suggests that the photo repair ability of
C. parvum is almost the same as that of E. coli,
which clearly demonstrates the recovery of colony-forming ability
during photoreactivation.
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FIG. 11.
Profiles of ESS remaining ratio for E.
coli and C. parvum during fluorescent light
irradiation after 99.9% inactivation by UV. , E.
coli; , C. parvum. All symbols indicate the
result of two independent series of experiments. Solid and dotted lines
connect the arithmetic mean values for E. coli and
C. parvum, respectively.
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|
A further investigation was performed with the ESS assay and animal
infectivity assay to determine the photoreactivation and dark repair
ability of C. parvum. Two doses of UV, 0.72 mJ
cm2 and 1.44 mJ cm2,
were adopted for the inactivation procedures. Figure
12 shows the profiles of the number of
ESS and the relative infectivity during the exposure to fluorescent
light after UV irradiation, while Fig.
13 shows the profiles in darkness after
UV irradiation. As shown in Fig. 12, recovery of the infectivity of
C. parvum was not clearly observed during fluorescent light
irradiation in spite of the gradual but apparent repair of pyrimidine
dimers. The discrepancy between the recovery of animal infectivity and
the repair of pyrimidine dimers was also observed in the case of dark
repair, as shown in Fig. 13, where the infectivity did not recover
clearly even after the repair of pyrimidine dimers. It was also
reported by Shin et al. (28) that there was no phenotypic
evidence of either photoreactivation or dark repair of the infectivity
of C. parvum after UV inactivation.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 12.
Profiles of number of ESS and relative infectivity of
C. parvum during fluorescent light irradiation after UV
irradiation at 0.72 (A) or 1.44 (B) mJ cm2. Solid
circles, number of ESS/base; open diamonds, relative infectivity. All
symbols indicate the data from one series of experiments. The 60-min
time point in panel B was not tested.
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|
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 13.
Profiles of number of ESS and relative infectivity of
C. parvum in darkness after UV irradiation at 0.72 (A)
or 1.44 (B) mJ cm2. Solid Circles, number of ESS/base;
open diamonds, relative infectivity. All symbols indicate the data from
one series of experiments.
|
|
Many researchers have pointed out that the infectivity of
Cryptosporidium oocysts is lower than the viability of the
oocysts, as determined by 4',6'-diamidino-2-phenylindole-propidium
iodide staining test or the excystation test (1, 3, 29).
This suggests that the vitality of the oocysts would not always reflect their infectivity, because some of the viable oocysts could not infect
the hosts. This further suggests that the oocysts would not always be
infective even if their DNA were in a normal condition.
The result of this study indicated that the repair of pyrimidine dimers
in the genomic DNA did not contribute to the recovery of infectivity of
C. parvum. This suggests that UV irradiation produces not
only pyrimidine dimers in the DNA of C. parvum but also
other kinds of damage in the DNA or other parts of the cell. The damage
other than pyrimidine dimers would not be repaired by either
photoreactivation or dark repair and should play an important role in
infection. Further investigation of such damage would lead to
clarification of the mechanisms by which UV irradiation inactivates
C. parvum.
Conclusions.
The ESS assay was conducted to determine
UV-induced pyrimidine dimers in the genomic DNA of E. coli
and C. parvum. The following conclusions were obtained.
(i) The ESS assay was a useful method for the quantitative
investigation of the UV inactivation, photo repair, and dark repair of
E. coli and C. parvum.
(ii) In UV inactivation of E. coli, the number of pyrimidine
dimers induced in the DNA was highly correlated with the dose of UV
used as well as with colony-forming ability.
(iii) In E. coli, pyrimidine dimers induced in the DNA by UV
irradiation were continuously repaired during the exposure to fluorescent light irradiation, while they were not repaired in darkness. The number of pyrimidine dimers remaining in the DNA during
fluorescent light irradiation was highly correlated with colony-forming ability.
(iv) In UV inactivation of C. parvum, the number of
pyrimidine dimers induced in the DNA was highly correlated with the
dose of UV used.
(v) In C. parvum, pyrimidine dimers induced in the DNA by UV
irradiation were continuously repaired by exposure to fluorescent light
irradiation. The repair of pyrimidine dimers was observed even in
darkness. The ability of C. parvum to repair pyrimidine dimers during exposure to fluorescent light was almost the same as that
of E. coli. The animal infectivity of C. parvum,
however, did not recover after either exposure to fluorescent light or storage in darkness, indicating that C. parvum would not
recover infectivity either by photoreactivation or by dark repair even after the repair of pyrimidine dimers.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Urban Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Phone: (81)3-5841-6242. Fax: (81)3-5841-8533. E-mail: oguma{at}env.t.u-tokyo.ac.jp.
| |
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Applied and Environmental Microbiology, October 2001, p. 4630-4637, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4630-4637.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
