Batch process solar disinfection is an efcient means of

disinfecting drinking water contaminated with Shigella

dysenteriae type I
S.C. Kehoe
1
, M.R. Barer
2
, L.O. Devlin
3
and K.G. McGuigan
3
1
Department of Surgery, Royal College of Surgeons in Ireland, Dublin, Ireland,
2
Department of Infection, Immunity and Inammation,
University of Leicester, Leicester, UK, and
3
Department of Physics, Royal College of Surgeons in Ireland, Dublin, Ireland
2003/1149: received 16 December 2003, revised 17 February 2004 and accepted 24 February 2004
ABSTRACT
S. C. KEHOE, M. R. BARER, L. O. DEVLI N AND K. G. MCGUI GAN. 2004.
Aims: The mortality and morbidity rate caused by Shigella dysenteriae type I infection is increasing in the
developing world each year. In this paper, the possibility of using batch process solar disinfection (SODIS) as an
effective means of disinfecting drinking water contaminated with Sh. dysenteriae type I is investigated.
Methods: Phosphate-buffered saline contaminated with Sh. dysenteriae type I was exposed to simulated solar
conditions and the inactivation kinetics of this organism was compared with that of Sh. exneri, Vibrio cholerae and
Salmonella typhimurium.
Signicance: Recovery of injured Sh. dysenteriae type I may be improved by plating on medium supplemented
with catalase or pyruvate. Sh. dysenteriae type I is very sensitive to batch process SODIS and is easily inactivated
even during overcast conditions. Batch process SODIS is an appropriate intervention for use in developing
countries during Sh. dysenteriae type I epidemics.
Keywords: catalase, drinking water, dysentery, pyruvate, sunlight.
I NTRODUCTI ON
After virtually disappearing at the beginning of the 20th
century, epidemic Shigella dysenteriae type I reappeared in
1968 in Central America and later in Asia and Africa (Mata
et al. 1970; World Health Organisation 1988; Tuttle et al.
1995). Today, bacillary dysentery is endemic throughout the
world with 150 million cases and almost 600 000 deaths
occurring annually (Sansonetti 1999). About 95% of these
cases occur in developing countries where water quality and
sanitation is less than adequate. The low infective dose
(thought to be as little as 10 cells; Sansonetti 1999) together
with the emergence of antimicrobial resistant strains has
made it increasingly difcult to control both the spread and
treatment of this organism. In 1987, Sh. dysenteriae type I
strains resistant to all commonly available anti-microbial
agents were isolated in Bangladesh (Munshi et al. 1987).
Strains resistant to trimethorprimsulphamethoxazole and
ampicillin were isolated in both Africa and Asia (Frost et al.
1981; Central Statistics Ofce 1991).
Small improvements in water supply and sanitation
facilities in poor communities have a lower impact on
diarrhoea caused by pathogens of low infective dose such as
Sh. dysenteriae type I compared with pathogens of high
infective dose, e.g. Vibrio cholerae (Esrey et al. 1985). Zeng-
sui et al. (1989) reported that provision of good quality
drinking water supplies reduces the transmission of viral
hepatitis A, cholera and acute watery diarrhoea but does not
inuence the incidence of bacillary dysentery. It is clearly
desirable to control all potential routes of transmission of
Sh. dysenteriae type I. Batch process solar disinfection
(SODIS), which takes advantage of the most abundant
source of energy in many of these regions, natural sunlight,
may provide additional possibilities for control of bacillary
dysentery.
Correspondence to: Siobhan C. Kehoe, Department of Surgery, Royal College of
Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland (e-mail: skehoe@rcsi.ie).
2004 The Society for Applied Microbiology
Letters in Applied Microbiology 2004, 38, 410414 doi:10.1111/j.1472-765X.2004.01515.x
The SODIS technique consists of lling transparent
bottles with drinking water and exposing them to full
sunlight for up to 8 h with the subsequent inactivation of
microbial and viral pathogens (Acra et al. 1989; Sommer
et al. 1997; Kehoe et al. 2001). The bactericidal effect of
sunlight is due to optical and thermal processes and a strong
synergistic effect occurs at temperatures exceeding 45C
(McGuigan et al. 1998). In addition to direct u.v. killing,
sunlight is absorbed by endogenous (e.g. cytochromes) and
exogenous (e.g. humic substances) photosensitizers that then
react with oxygen producing highly reactive oxygen mole-
cules such as hydrogen peroxide (H
2
O
2
), singlet oxygen and
superoxides which exert a bactericidal effect (Whitelam and
Codd 1986; Farr and Kogoma 1991). As a result, oxygen
levels within the container should be at a maximum (Reed
et al. 2000; Kehoe et al. 2001). Most bacterial strains
produce catalase in response to hydrogen peroxide. How-
ever, Sh. dysenteriae type I does not produce a catalase that is
detected by standard methods and thus may be more
sensitive to batch process SODIS. Bogosian et al. (2000)
noted that H
2
O
2
-sensitive cells of V. vulnicus produced
during starvation were recovered by growth on medium
supplemented with catalase or pyruvate but not by growth
on standard medium. We supplemented medium with
catalase or pyruvate to achieve maximum plating efciency.
We show that sublethally solar injured Sh. dysenteriae type
I may be recovered on medium supplemented with either
catalase or pyruvate but not on standard medium. Sh.
dysenteriae type I is extremely sensitive to SODIS with
inactivation occurring even during overcast conditions.
MATERI ALS AND METHODS
The following bacterial strains were used in this experiment:
Sh. dysenteriae type I ATCC 13313, V. cholerae 8021 serovar
01, classical biotype, Ogawa serotype purchased from the
NCTC Collection, Colindale, London, UK, Salmonella
typhimurium C
5
Nx
r
as described by Smith et al. (2000).
Sh. exneri (M90 T; Franzon et al. 1990) was kindly
donated by P.J. Sansonetti, Institut Pasteur, Paris, France.
All experiments involving Sh. dysenteriae type I were
performed in a class 3 containment laboratory in corres-
pondence with EU regulations.
All bacterial strains were inoculated (single colony) in
100 ml of sterile nutrient broth (Oxoid CM67) and incu-
bated at 37C for 18 h to obtain a stationary phase culture.
Cells were harvested by centrifugation at 855 g for 10 min
and washed three times with HPLC analytical reagent sterile
water to completely remove the nutrients. Finally, the pellet
was resuspended in sterile phosphate-buffered saline (PBS),
pH 73 (Oxoid; BR14) to a nal concentration of
10
6
CFU ml
)1
. These organisms were found to be more
unstable when maintained in water than PBS (Kehoe 2001).
By resuspending these cells in PBS we aimed to expose them
to solar irradiation in their most stable environment. The
solar simulation apparatus described by McGuigan et al.
(1998) was used. The irradiating light source was a 150 W
Xenon arc lamp (model 66057/68806 Oriel Ltd., Stratford,
CT, USA) tted with a rear reector and u.v. collecting
optics. The light from the lamp was passed through an Air
Mass 1.0 heat-absorbing solar lter (model KG2, Melles-
Griot, Cambridge, UK), which closely approximates the
incident solar irradiation expected at sea level on the
equator. The complete continuous output spectrum of this
system is given in McGuigan et al. (1998). A low optical
irradiance of 42 mW cm
)2
, corresponding to an overcast
day in Kenya (Joyce et al. 1996) was simulated and the water
temperature was maintained at 42C for Sh. dysenteriae type
I and Sh. exneri while Salm. typhimurium and V. cholerae
were exposed to higher levels of irradiation in order to
obtain inactivation within 8 h (42 and 45C respectively and
87 mW cm
)2
). Sh. dysenteriae type I and Sh. exneri
inactivation occurred at such a high rate under these high
optical irradiances that it was necessary to reduce the optical
irradiance to 42 mW cm
)2
for calculation of inactivation
kinetics.
In the eld trials of the SODIS technique described by
Conroy et al. (2001) test subjects placed their SODIS bottles
on the roof of their dwelling or kept them inside their
dwelling in a darkened area, at room temperature. Bottles
were exposed on the roof of Maasai huts and reached water
temperatures of between 40 and 55C or were kept indoors
in the shade where water temperatures were similar to room
temperature. Consequently, in our experiments a control
solution was left in the dark at room temperature throughout
the procedure. Test samples were maintained at the
intermediate water temperature of 42C to ensure that
thermal inactivation processes did not predominate. Vol-
umes of 100 ll were taken from each bottle of the control
and irradiated groups at the beginning of each experiment
and at each sampling interval. These volumes were diluted
in a series of 10-fold dilutions and plated in triplicate on
either standard plate count agar (SPCA; Oxoid CM 463) or
agar supplemented with either catalase or pyruvate (see
below) and the CFU/ml were calculated by the method of
Hoben and Somasegeram (1982) following incubation at
37C for 18 h. First-order solar decay constants (kJ
)1
) were
calculated from the slope of the regression line L
n
(N
t
/N
0
) vs
cumulative dose in kJ, where N
0
is the number of viable
bacteria in CFU/ml at time zero and N
t
is the number of
viable bacteria in CFU/ml at time, t. Plotting values as a
function of cumulative dose as opposed to time allowed
comparison between all four organisms studied taking into
consideration the differing optical intensity and tempera-
ture. This measurement also takes into account, water
volume and dimensions of solar reactors. Each experiment
SODI S OF SHI GELLA DYSENTERI AE 411
2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 410414, doi:10.1111/j.1472-765X.2004.01515.x
was repeated at least three times. Exact statistical tests were
implemented in StatXact 5 (STATCON, Witzenhausen,
Germany). First-order decay constants were compared
using analysis of variance with general scores. Catalase
(EC 1.11.1.6, from bovine liver; Sigma, C-9322) solutions
were prepared by dilution in ice-cold phosphate buffer
(10 mM M, pH 7). Solutions were immediately lter sterilized
with 02 lm membrane lters (Sarstedt, Numbrecht, Ger-
many, 83.1826.001) and 05 ml aliquots aseptically trans-
ferred to the surface of a standard agar plate. Quantities of
406, 812, 1700, 2445, 3260 units catalase were applied to
plates and that concentration which gave optimum plating
efciency was determined. Catalase plates were prepared
approx. 1 h prior to sampling. A solution of catalase, which
had been boiled for 10 mins, acted as a control.
Pyruvate plates were prepared by addition of sodium
pyruvate (Sigma, p-8574) directly to the medium before
autoclaving. The following concentrations were examined
for plating efciency and the optimum determined; 003,
005, 007, 01 and 025%. Glacial acetic acid (003%), a
by-product of H
2
O
2
degradation by pyruvic acid acted as a
control (Zelitch 1972; Elstner and Heupel 1976).
RESULTS
The solar inactivation behaviour of the four bacteria differed
considerably (Fig. 1; Table 1). Sh. dysenteriae type I is
signicantly more sensitive to SODIS than either
Sh. exneri, V. cholerae or Salm. typhimurium (P 0015).
No change in culturability was noted in the dark control
microcosms over the course of exposure. A 6-log reduction
in CFUs of Sh. dysenteriae type I was observed after just
15-h exposure to simulated overcast conditions at equatorial
latitudes (Fig. 2). Six-hour exposure is required to inactivate
a similar concentration of Sh. exneri. The order of
sensitivity to batch process SODIS is: Sh. dysenteriae type
I > Sh. exneri > Salm. typhimurium > V. cholerae. An
optical dose of approx. 6 kJ is required to inactivate 10
6
Sh. dysenteriae type I/ml while approx. 24 and >60 kJ is
required to inactivate 10
6
Salm. typhimurium/ml and 10
6
V. cholerae/ml respectively. To put these gures in
perspective, an optical dose of 60 kJ would be achieved in
approx. 100 min under a standard equatorial solar irradiance
of 100 mW cm
)2
.
The optimum concentration of catalase and pyruvate is
406 units per plate and 005% respectively (data not shown).
10
6
10
5
10
4
10
3
10
2
10
1
10
0
0 10 20 30 40 50 60
UV dose (kJ)
C
F
U

m
l

1
Vibrio cholerae
Salmonella typhimurium
Shigella flexneri M90T
Shigella dysenteriae
Fig. 1 Solar inactivation of Sh. dysenteriae type I, Shigella exneri,
Salmonella typhimurium and Vibrio cholerae plated on standard plate
count agar (SPCA) expressed in terms of cumulative u.v. dose received
(300400 nm)
Table 1 Representative decay constants (kJ
)1
), in terms of cumulative
u.v. dose received (300400 nm), for solar disinfected Sh. dysenteriae
type I, Sh. exneri, V. cholerae 01 and Salm. typhimurium plated on
standard plate count agar (SPCA), medium supplemented with
pyruvate or medium supplemented with catalase. R
2
values in
parentheses
Decay constants (kJ
)1
)
SPCA Pyruvate Catalase
Sh. dysenteriae type I 3055 (0942) 161 (0900) 1191 (0987)
Sh. exneri 0462 (0895) 0435 (0913) 0314 (0997)
Salm. typhimurium 0168 (0952) 0171 (0969) 0171 (0986)
V. cholerae 0076 (0917) 0074 (0910)
10
7
10
6
10
5
10
4
10
3
10
2
10
1
10
0
0 1 2 3
Time (h)
C
F
U

m
l

1
SPCA
Catalase
Pyruvate
Fig. 2 Solar inactivation of Sh. dysenteriae type I, exposed to a solar
irradiance of 42 mW cm
)2
and a water temperature of 42C plated on
standard plate count agar (SPCA) (d) or medium supplemented with
catalase (406 units/plate) (s) or pyruvate (005%) ( )
412 S. C. KEHOE ET AL.
2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 410414, doi:10.1111/j.1472-765X.2004.01515.x
The inactivation of Sh. dysenteriae type I on SPCA and
plates supplemented with 005% pyruvate and 406 units
catalase are presented in Fig. 2 and Table 1. These results
show that when grown on standard plates, 6 log units of
Sh. dysenteriae type I appear to be completely inactivated
after 15-h exposure. However, when this sample was plated
on medium supplemented with pyruvate or catalase, almost
10
4
CFU ml
)1
were culturable.
Comparisons of decay constants for Sh. exneri, Salm.
typhimurium and V. cholerae when plated on standard agar
and supplemented plates are presented in Table 1 and
supplementation of medium with either catalase or pyruvate
had little effect on the culturability of these organisms.
DI SCUSSI ON
Shigella dysenteriae type I is sensitive to batch process
SODIS. When plated on SPCA 10
6
Sh. dysenteriae type
I/ml are inactivated after 15-h exposure to simulated
equatorial overcast conditions. As shown in Table 1, Salm.
typhimurium and V. cholerae have signicantly lower decay
constants. MacKenzie et al. 1992 reported that solar treat-
ment of drinking water to prevent and control the spread of
cholera is effective only under selected conditions, possibly
related to altitude and intensity of ultraviolet radiation.
However, children under 6 years of age drinking solar
disinfected water were protected from V. cholerae infection
during an outbreak in rural Kenya (Odds Ratio, 012; 95%
CI, 002065) (Conroy et al. 2001). This suggests that
drinking solar disinfected water during a Sh. dysenteriae type
I outbreak would protect against infection transmitted by
that route.
When grown on supplemented medium it takes almost
three times longer for Sh. dysenteriae type I to become
nonculturable (see Fig. 2 and Table 1) but inactivation is
still occurring at a much higher rate when compared with
Salm. typhimurium and V. cholerae although Sh. dysenteriae
type I is only exposed to overcast conditions. Pyruvate
neutralizes peroxides by a direct nonenzymatic decarboxy-
lation reaction (Mallet et al. 2002). However, it is also
thought to act as an important metabolic fuel. Therefore, the
improved plating efciency observed when irradiated
Sh. dysenteriae type I is plated on supplemented medium
may be due to either or a combination of these factors.
However, similar increases in plating efciencies were noted
when catalase was added to the medium. Catalase enzymat-
ically decomposes hydrogen peroxide and is not thought to
act as an energy reserve. Supplementation of the medium
with boiled catalase had no effect on the plating efciency of
irradiated Sh. dysenteriae type I also suggesting that catalase
does not act as an energy reserve. Since catalase and
pyruvate have a similar effect on the plating efciency of
Sh. dysenteriae type I and catalase appears to exert its effect
by enzymatic decomposition of peroxide then pyruvate is
likely to act through neutralization of peroxides rather than
acting as an energy reserve. We recommend the supple-
mentation of recovery medium with pyruvate as it may be
added to the agar prior to autoclaving and is thus evenly
distributed throughout the medium. Catalase, on the other
hand, is very unstable at room temperature and it is
therefore difcult to predict the shelf life of the plates.
Although supplementation of medium seems to have the
greatest impact on the plating efciency of Sh. dysenteriae
type I, such cells are also more susceptible to SODIS and
therefore will be inactivated at a faster rate than other
species. In addition, we have previously shown that viable
bacterial cells (Salm. typhimurium) which were exposed to
solar conditions but still culturable on standard plates are
less infective than nonexposed viable cells when adminis-
tered via the intraperitoneal route (Smith et al. 2000).
Subsequent studies showed that such culturable but irradi-
ated bacteria were also less infective when administered via
the oral route (Kehoe 2001).
Shigella dysenteriae type I is inactivated by batch process
SODIS even during equatorial overcast conditions. Batch
process SODIS is therefore an appropriate intervention for
developing countries during Sh. dysenteriae type I endemics
even where adequate sanitation is provided as improvements
in water quality and sanitation have little impact on the
epidemiology of this organism because of the low infective
dose. Studies testing the efcacy of solar/u.v. disinfection
should incorporate pyruvate into bacteriological medium.
ACKNOWLEDGEMENTS
Sincere thanks to P.J. Sansonetti, Institut Pasteur, Paris,
France for providing the Sh. exneri strain used in this
project. We thank Ronan Conroy for assistance with the
statistical analysis. This project was funded by Royal College
of Surgeons in Ireland Research Committee and Enterprise
Ireland/British Research Council Research Travel Scheme.
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