Oxygen and photoinactivation of Escherichia coli in UVA andsunlight

R. Khaengraeng and R.H. ReedDivision of Biomedical Sciences, Northumbria University, Newcastle upon Tyne, UK

2003/0711: received 15 August 2003, revised and accepted 11 January 2005

ABSTRACT

R. KHAENGRAENG AND R.H. REED. 2005.

Aims: To establish the influence of oxygen on Escherichia coli before, during and after exposure to UVA or

simulated sunlight.

Methods and Results: Bacterial suspensions were exposed either to UVA or simulated sunlight. Conventional

aerobic plate counts of illuminated cell suspensions were consistently lower than those obtained under conditions

where reactive oxygen species (ROS) were neutralized, either (i) by the addition of the peroxide scavenger sodium

pyruvate (0Æ05% w/v) to the medium with subsequent incubation in an anaerobic jar or (ii) by culturing on a

prereduced medium within an anaerobic cabinet, indicating that a substantial proportion of such cells are sublethally

injured. While the presence of oxygen during the growth period resulted in a greater resistance of aerobically grown

cells to simulated sunlight compared with their anaerobic counterparts, the extent of inactivation during

illumination was directly related to the dissolved oxygen content of the water.

Conclusions: The results show that, at each stage, oxygen has a marked influence on the observed colony count.

Significance and Impact of the Study: Overall, the results indicate that future studies of bacteria exposed to

UVA or sunlight should consider the effects of oxygen at every stage in the procedure, and especially during

enumeration, where the inhibitory effects of ROS must be neutralized in order to obtain a valid count. An

investigation of the effects of ROS neutralization on the counts of faecal bacteria under field conditions in natural

waters is now required to establish the significance of these finding to solar water treatment.

Keywords: anaerobiosis, E. coli, photo-oxidation, pyruvate, solar disinfection, sunlight.

INTRODUCTION

While the antimicrobial properties of light have been known

for well over a century (Downes and Blunt 1877), more

recent research has focussed on the effects of sunlight on the

survival of bacteria in natural waters. This is particularly

important with regard to the persistence of faecal indicator

bacteria, e.g. Escherichia coli (Alkan et al. 1995) and

Enterococcus faecalis (Sinton et al. 2002), and water-borne

enteric pathogens such as Shigella flexneri (Runyen-Janeckyet al. 1999) and Vibrio cholerae (Oufdou et al. 2000). Many

research studies have suggested that sunlight is one of the

most important factors responsible for the inactivation of

faecal bacteria in fresh water (e.g. Barcina et al. 1990) and in

sea water (e.g. Davies and Evison 1991; Sinton et al. 1999).The disinfection of drinking water using sunlight was first

proposed by Acra et al. (1980) for the treatment of oral

rehydration solutions and subsequently demonstrated to be

effective against a range of bacteria, using laboratory isolates

(e.g. Wegelin et al. 1994; Joyce et al. 1996; Walker et al.2004) and naturally contaminated waters (e.g. Lawand et al.1997; Sommer et al. 1997; Caslake et al. 2004). Most of the

inactivation is as a result of solar UVA radiation, accounting

for c. 70% of the negative effects of sunlight (Acra et al.1984). Solar water treatment has considerable potential as a

low-cost means of treating contaminated drinking water in

developing countries with consistently sunny climates

Correspondence to: R.H. Reed, Division of Biomedical Sciences, School of Applied

Sciences, Northumbria University, Ellison Place, Newcastle upon Tyne, Tyne and

Wear NE1 8ST, UK (e-mail: rob.reed@northumbria.ac.uk).

ª 2005 The Society for Applied Microbiology

Journal of Applied Microbiology 2005, 99, 39–50 doi:10.1111/j.1365-2672.2005.02606.x

(McGuigan et al. 1999), and was highlighted as an effective

small-scale method for the improvement of drinking water

quality on World Water Day 2001 (Anon 2001).

Although the early studies of solar disinfection took no

account of the level of aeration of the water to be treated,

subsequent research has shown that a high level of dissolved

oxygen is required for the rapid inactivation of Escherichiacoli and Enterococcus faecalis suspended in distilled water and

then exposed to sunlight (Reed 1996, 1997). Similar results

have been reported for the effects of oxygen on solar

disinfection of faecal coliforms and faecal streptococci under

field conditions (Reed et al. 2000; Meyer and Reed 2001).

Taken together, these studies have shown that the rate of

inactivation of faecal bacteria exposed to sunlight is four to

eight times faster in oxygenated water compared with hypo-

oxygenated water, demonstrating that photo-oxidation is the

principal reason for the rapid decrease in bacterial counts.

The underlying process responsible for this inactivation is

the light-dependent production of reactive forms of oxygen,

including oxygen-free radicals such as superoxide and

hydroxyl radicals (Gourmelon et al. 1994), along with toxic

derivatives such as hydrogen peroxide (Alam and Ohgaki

2002). These reactive oxygen species (ROS) are generated

mainly as a result of the absorption of light by endogenous

photosensitizers, e.g. intracellular porphyrins and flavins

(Curtis et al. 1992), although exogenous photosensitizers

may also be significant in the generation of ROS in natural

waters (Voelker et al. 1997; Davies-Colley et al. 1999).To date, most studies of solar disinfection have been

carried out using isolates of E. coli enumerated by culture on

conventional selective or nonselective agar-based media,

incubated under standard aerobic conditions (e.g. Clesceri

et al. 1998; Anon 2002). However, recent studies have

shown that, while such conditions are effective in enabling

the growth of healthy cells, they do not always allow

physiologically damaged cells to grow. Thus, suspensions of

heat-damaged E. coli O157 cells have been found to give

higher counts in growth media cultured under anaerobic

conditions, compared with aerobic incubation (Murano and

Pierson 1992; Bromberg et al. 1998). Such effects are also

modulated by the prior growth of the bacteria under aerobic

or anaerobic conditions (George et al. 1998). Similar effects

have been observed for other bacteria, including Salmonella(Xavier and Ingham 1993) and Listeria (Knabel and Thielen

1995; Gnanou Besse 2002). Other studies have demonstrated

that higher counts can be obtained for heat-injured E. coli byincorporating substances that interact with reactive forms of

oxygen into agar-based growth media; these substances

include scavengers of peroxides such as pyruvate (e.g.

Czechowitz et al. 1996) and dissimilatory enzymes such as

catalase (Mackey and Seymour 1987), supporting the

proposal that ROS may inhibit the growth of sublethally

injured cells of E. coli under aerobic conditions.

The present study was carried out to investigate the effects

of oxygen status on the inactivation of E. coli exposed to either(i) UVA or (ii) simulated full-spectrum sunlight, by compar-

ing conventional plate counts on a nonselective, agar-based

medium for cells grown, illuminated and incubated either in

the presence or the absence of oxygen. The results show that,

at each stage, i.e. during (i) initial culture, (ii) illumination and

(iii) subsequent enumeration, oxygen had a marked influence

on the final colony count, showing a positive effect during

initial culture, and a negative effect during exposure to light

and in the postillumination enumeration stage. This was

particularly evident in relation to the negative effects of

oxygen (via ROS) on cells sublethally injured by photo-

oxidation, with maximum colony counts obtained only under

conditions where toxic metabolites of oxygen were subse-

quently prevented from inhibiting growth, e.g. by using a

combination of a growth medium with added pyruvate

together with incubation in an anaerobic jar, or by cultivation

on a prereduced medium within an anaerobic cabinet.

MATERIALS AND METHODS

Bacterial strains and growth conditions

Escherichia coli NCTC8912 was obtained from the National

Collection of Type Cultures (Colindale, UK). Stock cultures

of this strain were maintained by repeated subculture on

nutrient agar (Oxoid, Basingstoke, UK) at 37�C, and used formost of the experiments described in the present study. Four

other isolates were used in comparative experiments: E. coliNU9904 and NU9906 (NU, Northumbria University Strain

Collection), both obtained from water from the River Tyne at

Howden; NU9912, obtained from a urine sample; and

NCTC12900, a nontoxigenic strain of E. coli O157. Standardexperimental cultures were prepared by loop inoculation of

20 ml of sterile nutrient broth (Merck VWR, Darmstadt,

Germany) in a Universal bottle (25 ml volume), followed by

18 h incubation at 37�C without shaking. The broth cultures

reached stationary phase at 18 h (determined spectrophoto-

metrically at 550 nm, A1cm ¼ 1Æ2) under anaerobic condi-

tions, with no oxygen detectable using an oxygen probe

(model 9010; Jenway, Dunmow, UK). Anaerobic conditions

at 18 h were also confirmed by the decolourization of a dilute

solution of the redox dye methylene blue to the broth culture.

For experiments where only fully oxygenated water was

required, suspensions of bacteria were shaken for 2 min

before illumination, to ensure that air equilibration and

oxygen saturation was achieved. To investigate the effects of

oxygen status during illumination, sterile water was sparged

either with sterile air (oxygenated conditions, with O2

measured at 7Æ5 mg l)1), sterile oxygen-free N2 gas (hypo-

oxygenated conditions, O2 at 0Æ9 mg l)1) or sterile O2 gas

(hyperoxygenated conditions, O2 at 27Æ2 mg l)1) for 1 h

40 R. KHAENGRAENG AND R.H. REED

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x

prior to the addition of E. coli (all gases were obtained from

BOC Special Gases, London, UK). The same treatments

were then continued throughout the illumination period, to

maintain the appropriate level of oxygenation during the

experiment, with measured pH values of 6Æ2 (air-sparged),

6Æ6 (N2-sparged) and 6Æ7 (O2-sparged) at the end of the

experiment, compared with an initial pH value of 6Æ1.For a direct comparison of aerobically grown and

anaerobically grown cultures, nutrient broths were sparged

either with sterile air or with sterile oxygen-free N2 gas

throughout the growth period. Cultures were first grown

overnight at 37�C to stationary phase (A550 ¼ 1Æ2) with

sparging using either air or N2, then subcultured to fresh

nutrient broth and incubated at 37�C to early log phase

(A550 ¼ 0Æ2) under the same conditions, to give suspensions

of metabolically active cells.

Preparation and illumination of cell suspensions

Broth cultures were centrifuged at 12 000 g for 1 min (MSE

Microcentaur; Fisher Scientific, Loughborough, UK), re-

suspended in sterile distilled water and recentrifuged at

12 000 g for 1 min. The resuspension and recentrifugation

was repeated, to remove all traces of the original growth

medium from the cells and thereby remove any potential

exogenous photosensitizers and/or medium components

that might restrict light penetration. The cells were then

resuspended in 5 ml of sterile distilled water at a density of

107–108 CFU ml)1, placed in a 10-ml borosilicate glass tube

and exposed to light from either (i) a UVA source (40W-R

sunlamp tubes; Philips, Croydon, UK), at a UVA irradiance

of 20 W m)2, measured with a SKU421/I UVA sensor and

SKT660 meter (Skye Instruments, Llandrindod Wells,

UK) or (ii) a 1000-W solar simulator (Model 91190 large

area light source; Oriel, Stanford, CA, USA) at a total

irradiance of 410 W m)2, measured using an SKS1110

pyranometer and SKT660 meter (Skye Instruments, Lland-

rindod Wells, UK). While the UVA source provides a broad

band of UVA light, with maximum emission in the range

360–380 nm (Khaengraeng 2004), the solar simulator pro-

vides a reasonable fit to the whole solar spectrum from UV

to infrared wavelengths (Kehoe 2001). The ambient tem-

perature during illumination was 24–26�C and the tubes

were cooled by fan throughout their exposure to the light

source, to prevent excessive temperature rises during

experimentation (the temperature during illumination never

exceeded 32�C), to minimize thermal effects (McGuigan

et al. 1998).

Enumeration

Timed samples were taken from the illuminated cell

suspensions and immediately processed in a dimly lit room

(irradiance, <5 W m)2), to avoid further photoinactivation.

The samples were subject to serial decimal dilution in

quarter-strength Ringer’s solution, then surface spread

(0Æ02–0Æ1 ml, depending on sample, dilution and time) onto

Petri plates containing freshly prepared nutrient agar

(Oxoid), as before (Reed 1997). Chemical supplements

(ROS-scavenging agents) required in particular experi-

ments, i.e. ascorbate, cysteine, dithiothreitol, histidine,

mannitol and pyruvate, were added to the nutrient agar

before autoclaving at 121�C for 15 min. In contrast, the

enzymes catalase and superoxide dismutase, were added to

autoclaved molten agar cooled to 50�C, prior to pouring

because of their heat lability at higher temperatures. All

supplements were obtained from Sigma Aldrich (Gilling-

ham, UK).

Inoculated media were incubated either aerobically or

anaerobically in a 2Æ5-l anaerobic jar (Anaerocult�, with

Anaerocult� A Sachet; Merck VWR) at 37�C under

conditions of total darkness, either in a conventional aerobic

incubator (model MIR-153; Sanyo, Osaka, Japan), or at

37�C in an anaerobic cabinet (Bugbox controlled environ-

ment workstation; Fred Baker Scientific, Runcorn, UK)

under an atmosphere of 10% H2 : 10% CO2 : 80% N2

(standard anaerobic gas mixture; BOC Special Gases,

Guildford, UK). Counts were performed using a colony

counter (Stuart Scientific, Redhill, UK) after 48 h, and then

checked at daily intervals thereafter, up to 7 d; colony

counts of appropriate dilutions were converted to CFU ml)1

by correcting for dilution and volume (Reed 1997). Samples

were counted in triplicate; counts are expressed as geometric

means of log-transformed data, with 95% confidence limits.

All graphs show the lowest detectable count (10 CFU ml)1,

log transformed to 1) as the minimum point on the y-axis.Where appropriate, paired t-tests have been used for

comparison of geometric means (McFeters et al. 1986):

statistically significant differences are noted where P < 0Æ05.The time for a 90% reduction in count (T90) was calculated

from plate counts of illuminated samples, as:

T90 ¼ t2 � t1logNt1 � logNt2

;

where Nt1 and Nt2 are initial and final counts at times t1 andt2 respectively. T90 values are quoted to the nearest minute.

RESULTS

Effect of oxygen scavengers and oxygen status onthe growth of UVA-treated and solar-illuminatedE. coli

Counts are shown in Fig. 1a for stationary phase E. coliNCTC8912 following harvesting and suspension in sterile

distilled water but prior to UVA exposure, plated onto

PHOTOINACTIVATION OF E. COLI 41

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x

nutrient agar with different amounts of added sodium

pyruvate (a scavenger of peroxides; McDonald et al. 1983)and then cultured either aerobically or in an Anaerocult�anaerobic jar for 48 h. A similar count was obtained in all

treatments, irrespective of whether the plates were incuba-

ted aerobically or anaerobically, or whether the medium was

supplemented with pyruvate or not. This pattern was also

observed in all subsequent experiments (data not shown),

confirming that the initial inoculum of active, healthy cells

was not influenced significantly either by oxygen status, or

by peroxide scavengers in the medium.

Figure 1b shows counts for the same suspension of E. coliNCTC8912 following 2 h exposure to UVA in air-equili-

brated (oxygenated) water. Overall, the counts were lower

than for the initial inoculum, confirming the inhibitory

effects of UVA exposure. However, the count obtained was

strongly dependent upon the incubation conditions: under

aerobic conditions, the addition of pyruvate at 0Æ01% w/v

and above caused a statistically significant increase (t-test:

P < 0Æ05) in the observed count, with no apparent differ-

ences between counts made with added pyruvate at 0Æ01–0Æ5% w/v. Similarly, under anaerobic conditions (anaerobic

jar), all of the pyruvate-supplemented media showed

a statistically significant higher count, compared with

the unsupplemented (pyruvate-free) anaerobic medium,

although some evidence of a reduction in efficacy was

observed at the highest concentration of pyruvate (0Æ5%w/v) under anaerobic conditions. It should also be noted

that the UVA-treated cells grown on pyruvate-supplemen-

ted medium showed a statistically significantly higher count

under anaerobic conditions than under aerobic conditions at

all levels of pyruvate between 0Æ01 and 0Æ1% w/v. Further

incubation of the plates beyond 48 h, gave no substantial

increase in colony counts on any medium, in this and in all

subsequent experiments, with only the occasional additional

colony observed on one or two plates up to 7 d. This

demonstrates that 48 h incubation was sufficient to provide a

valid count for UVA-treated cells (note that 48 h incubation

was required in order to allow colonies to grow under

anaerobic conditions to a size large enough to be counted).

The results of an experiment carried out to establish the

effect of added catalase on the same batch of cells of E. coliNCTC8912 exposed to UVA for 2 h in fully oxygenated

water are shown in Fig. 2, demonstrating a statistically

significant increase in the count at 10 U plate)1 added

catalase and above under aerobic and anaerobic conditions,

compared with their respective unsupplemented controls.

As with pyruvate, all levels of catalase supplementation gave

a significantly higher count under anaerobic conditions,

compared with the corresponding aerobic treatment. A

further experiment showed no difference between the

1

3

5

7

9

0 0·01 0·05 0·1 0·5

Pyruvate (% w/v)

Pla

te c

ount

(lo

g 10

CF

U m

l–1)

1

3

5

7

0 0·01 0·05 0·1 0·5

Pyruvate (% w/v)

Pla

te c

ount

(lo

g 10

CF

U m

l–1)

Fig. 1 Effects of sodium pyruvate on enumeration of UVA-exposed

Escherichia coli. Geometric mean counts (log10 CFU ml)1) for a

suspension of stationary phase E. coli NCTC8912 in water (a) prior to

UVA exposure and (b) following 2 h exposure to UVA, cultured on

nutrient agar under aerobic conditions (open bars) or in an anaerobic

jar (grey bars) at different levels of added sodium pyruvate, up to 0Æ5%w/v. The initial inoculum, prior to UVA exposure, represents an

overall average count of 7Æ5 · 107 CFU ml)1. Error bars represent

95% confidence limits (n ¼ 3)

1

3

5

7

0 10 50 100 500

Catalase (U plate–1)

Pla

te c

ount

(lo

g 10

CF

U m

l–1)

Fig. 2 Effects of catalase on enumeration of UVA-exposed Escherichia

coli. Geometric mean counts (log10 CFU ml)1) for a suspension of

stationary phase E. coli NCTC8912 in water following 2 h exposure to

UVA, cultured on nutrient agar under aerobic conditions (open bars)

or in an anaerobic jar (grey bars) at different levels of added catalase up

to 500 U plate)1. The initial inoculum in this experiment (0 h UVA

exposure) was 7Æ5 · 107 CFU ml)1. Error bars represent 95% con-

fidence limits (n ¼ 3)

42 R. KHAENGRAENG AND R.H. REED

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x

combined addition of pyruvate plus catalase and supple-

mentation with either component alone (data not shown).

This is consistent with the similar effect of pyruvate and

catalase, as the former detoxifies hydrogen peroxide by

reacting to form acetate, water and CO2 (Elstner and Heupel

1976) while the latter enzymically converts hydrogen

peroxide to water and O2 (Mongkolsuk and Helmann 2002).

Calculation of T90 values for the pyruvate supplementa-

tion experiment (Fig. 1) showed that aerobic incubation in

pyruvate-free medium gave the lowest T90, at 32 min, rising

to 81 min under anaerobic conditions with pyruvate

supplementation at 0Æ05 and 0Æ1% w/v. Similarly T90 values

for the catalase supplementation experiment (Fig. 2) also

gave a value of 84 min under anaerobic conditions with

500 U plate)1 added catalase, which is broadly equivalent to

the equivalent value for pyruvate-supplemented medium.

Experiments were performed by adding scavengers of

other ROS to nutrient agar, including mannitol (a scavenger

of hydroxyl-free radicals; Shen et al. 1997) at up to 1Æ0%w/v, histidine (a quencher of singlet oxygen; Singh and

Kshatriya 2002) at up to 1Æ0% w/v, superoxide dismutase

(which enzymically detoxifies superoxide-free radicals) at up

to 200 U plate)1, the antioxidant ascorbate (Potters et al.2002) at up to 0Æ5% w/v and the reducing agents cysteine

and dithiothreitol (Suh and Knabel 2000; Nebra et al. 2002)at up to 0Æ1% w/v. None of these supplements showed a

statistically significant change in the count of UVA-treated

E. coli NCTC8912 under either aerobic or anaerobic

conditions (data not shown). Consequently, it was decided

to focus only on pyruvate supplementation at 0Æ05% w/v in

all further experiments, as this amount had a positive effect

under aerobic and anaerobic conditions (Fig. 1) and because

this concentration is consistent with earlier studies (e.g.

Sartory and Howard 1992). Sodium pyruvate was selected in

preference to catalase as the latter is heat-sensitive and

cannot be added prior to autoclaving.

Figure 3 shows data for stationary phase E. coliNCTC8912 exposed to UVA for 4 h and then cultured on

nutrient agar in the presence or absence of 0Æ05% w/v

pyruvate either aerobically, or under one of three different

sets of anaerobic conditions, to see the effects on the counts

obtained. For the first two sets of anaerobic plates, following

dilution and spread plating on the laboratory bench in aerobic

conditions, the plates were then transferred either to an

anaerobic jar, which takes c. 1 h to create fully anaerobic

conditions (Anon 2000) or to an anaerobic cabinet, giving a

fully anaerobic atmosphere from the outset. The third set of

anaerobic plates was prepared entirely within the anaerobic

cabinet, by spread plating onto prereduced media (kept for

48 h in the anaerobic cabinet prior to use), with incubation in

the same cabinet, to maintain anaerobic conditions through-

out the preparation phase and culture period. As in the

previous experiment, the results show that there was a

substantial increase in the count observed for the pyruvate-

supplemented plates under aerobic conditions. A smaller

effect of pyruvate supplementation was seen for plates

prepared in air and then transferred to the anaerobic jar

and the anaerobic cabinet while no significant difference was

observed between pyruvate-supplemented and unsupple-

mented plates in the anaerobic cabinet when prereduced

plates were used (Fig. 3). These results indicate that a

combination of pyruvate and an anaerobic jar can give a count

directly equivalent to that obtained with an anaerobic cabinet

and the prereduced growth media. An anaerobic jar, with

initial dilution and plating in air, was used in all subsequent

experiments, as an anaerobic cabinet was not readily available.

Table 1 shows data for four other strains of E. coli grownto stationary phase and then exposed to UVA for 2 h and

counted aerobically and anaerobically in the presence and

absence of 0Æ05% w/v pyruvate. In general, the results were

similar to those obtained using E. coli NCTC8912 (Fig. 1),

with a high count obtained under anaerobic conditions with

0Æ05% added pyruvate. The greatest proportional increase

was seen for UVA-treated E. coli NU9912, which gave a

count of 8Æ3 · 103 CFU ml)1 under aerobic conditions and

3Æ1 · 105 CFU ml)1 anaerobically with added pyruvate.

The latter value is over 30 times higher than the former,

indicating that anaerobic incubation in a medium with added

pyruvate can give a substantially higher count for some

strains. UVA-treated E. coli NU9904 showed a lower

reduction in count when assayed under aerobic conditions

(1Æ25 · 106 CFU ml)1) and a far smaller effect either of

1

2

3

4

5

AIR AJ AC AC-pre

Pla

te c

ount

(lo

g 10

CF

U m

l–1)

Fig. 3 Effects of various combinations of ROS-neutralized conditions

on enumeration of UVA-exposed Escherichia coli. Geometric mean

counts (log10 CFU ml)1) for a suspension of stationary phase E. coli

NCTC 8912 in water following 4 h exposure to UVA, then cultured

either on unsupplemented nutrient agar (open bars) or on nutrient agar

with 0Æ05% w/v sodium pyruvate (grey bars) under aerobic conditions

(AIR), transferred from aerobic conditions to either an anaerobic jar

(AJ) or an anaerobic cabinet (AC), or prepared and cultured in an

anaerobic cabinet on prereduced nutrient agar (AC-pre). The initial

inoculum in this experiment (0 h UVA exposure) was c.

4Æ4 · 107 CFU ml)1. Error bars represent 95% confidence limits

(n ¼ 3)

PHOTOINACTIVATION OF E. COLI 43

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x

anaerobic incubation and pyruvate addition, with an increase

in count to 1Æ75 · 106 CFU ml)1, representing an increase

of less than half of the aerobic value (note also that the values

for pyruvate-supplemented medium are almost the same

under aerobic and anaerobic conditions). NU9906 showed a

substantially higher count following anaerobic incubation

than for plates incubated aerobically, but with little evidence

of any further effect of pyruvate supplementation, suggest-

ing that individual strains may respond differently to this

combination of conditions. The consequent effects on

apparent T90 values are also shown in Table 1, with UVA-

treated E. coli NU9912 showing an increase in T90 of over

100% when the data for aerobic and anaerobic plus pyruvate

counts are compared, while E. coli NU9904 showed an

increase of c. 10% under the same conditions.

Effect of pyruvate and anaerobiosis on theapparent inactivation of E. coli exposed tosimulated sunlight

Figure 4 shows a time course for stationary phase E. coliNCTC8912 illuminated in the solar simulator in fully

oxygenated water for up to 6 h. The trend line for each set

of incubation conditions is close to a linear relationship, with

no evidence of an initial plateau or �shoulder�. The differencein count between the various incubation conditions shows an

increase with exposure time, being minimal at the start of

the experiment and increasing thereafter, up to 6 h, with the

aerobic count being lowest and a combination of anaerobic

incubation plus pyruvate supplementation giving the highest

count. Thus, by the end of the illumination period, the

disparity in counts between the unsupplemented plates

incubated aerobically and the pyruvate-supplemented plates

incubated anaerobically was almost exactly 100-fold. In

contrast, pyruvate-supplementation under aerobic condi-

tions gave a count of around half of that on the same

medium under anaerobic conditions, showing that the

addition of pyruvate alone is not able to counteract all of

the inhibitory effects of ROS. Similar effects have also been

observed following exposure to natural sunlight in PET

bottles containing 1 l of bacterial suspension, with the

highest counts obtained on pyruvate-supplemented medium

incubated in an anaerobic jar (data not shown).

Calculating the apparent T90 values for each set of

incubation conditions using the data for 0 and 6 h shown in

Fig. 4 gives a value of 95 min for aerobic incubation of

unsupplemented medium, 160 min for aerobic incubation of

pyruvate-supplemented medium, 175 min for anaerobic

incubation of unsupplemented medium and 200 min for

anaerobic incubation of pyruvate-supplemented medium,

the latter being more than double the value for standard

aerobic incubation, in broad agreement with the results for

UVA-treated cells.

Effects of oxygen status during growth andillumination with simulated sunlight on theapparent inactivation of E. coli

A broth culture of E. coli NCTC8912 was grown to early log

phase under oxygenated (air-sparged) or hypo-oxygenated

Table 1 Comparative inactivation of four

different isolates of Escherichia coli. Geometric

mean counts (log CFU ml)1) of cells sus-

pended in water and exposed to UVA illu-

mination for 2 hStrain

Initial count

aerobic

Count following UVA exposure

Aerobic

Aerobic +

0Æ05% w/v

pyruvate Anaerobic

Anaerobic +

0Æ05% w/v

pyruvate

NU9904 7Æ57 6Æ10 (82) 6Æ23 (90) 6Æ14 (84) 6Æ24 (90)

NU9906 7Æ84 6Æ42 (85) 6Æ53 (92) 7Æ06 (154) 7Æ03 (148)

NU9912 7Æ54 3Æ92 (33) 5Æ04 (48) 4Æ93 (46) 5Æ49 (76)

NCTC12900 7Æ65 4Æ51 (38) 5Æ12 (47) 4Æ61 (39) 5Æ42 (54)

T90 values (min) are given within parenthesis.

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6

Time (h)

Pla

te c

ount

(lo

g 10

CF

U m

l–1)

Fig. 4 Time course for apparent inactivation of Escherichia coli

exposed to simulated sunlight and enumerated under various combi-

nations of ROS-neutralized conditions. Geometric mean counts (log10CFU ml)1) for a suspension of stationary phase E. coli NCTC8912 in

water exposed to simulated sunlight for up to 6 h and cultured either

aerobically (open symbols) or anaerobically (closed symbols) on

unsupplemented nutrient agar (circles) or on nutrient agar containing

0Æ05% w/v sodium pyruvate (squares). Error bars represent 95%

confidence limits (n ¼ 3)

44 R. KHAENGRAENG AND R.H. REED

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x

(N2-sparged) conditions and then illuminated in the solar

simulator under either hypo-oxygenated (N2-sparged), oxy-

genated (air-sparged) or hyperoxygenated (O2-sparged)

conditions. Samples of treated cells were then counted

either under aerobic conditions in unsupplemented nutrient

agar or under anaerobic conditions (anaerobic jar) in

pyruvate-supplemented medium, as the latter consistently

gave the highest counts in all the earlier experiments.

Figure 5 shows the data for log phase cells grown with N2

sparging. Comparing the results for plates of unsupple-

mented medium incubated aerobically, it is clear that

illumination with simulated sunlight in hyperoxygenated

(O2-sparged) water resulted in a rapid decrease in counts,

with no evidence of an initial delay or �plateau� when

compared with oxygenated (air-sparged) conditions, with

the hypo-oxygenated treatment showing the clearest evi-

dence of a plateau and the slowest overall decrease in counts.

Such results are consistent with the earlier observation that

the extent of photoinactivation is directly related to the level

of dissolved oxygen in the water during illumination (Curtis

et al. 1992; Reed 1997), rather than being linked to the

relatively small changes in pH observed as a result of

sparging. Similarly, the results for anaerobic enumeration

using pyruvate-supplemented medium show the same

overall trend, although in each case, the overall decrease at

any time point was less than that observed under aerobic

conditions, with greater evidence of an initial plateau. This

plateau effect is most clearly seen for the cell suspension

illuminated in hypo-oxygenated water, where the decrease

was most marked following 3 h illumination, when counted

anaerobically with pyruvate, in contrast to that observed

aerobically in unsupplemented medium. Comparing the two

sets of incubation conditions for each of the three

treatments, the smallest differences were seen for the

illuminated O2-sparged cells, while the largest disparities

were seen for N2-sparged cells where the aerobic count in

unsupplemented medium dropped below the detection limit

of 10 CFU ml)1 after 4 h but remained countable up to 6 h

under anaerobic conditions in pyruvate-supplemented

medium.

Figure 6 shows the corresponding data set for early log

phase cells of E. coli NCTC8912 grown under air-sparged

(oxygenated) conditions. In general, a similar overall trend

was seen to that observed for the cells cultured under N2,

with (i) cells illuminated under hyperoxygenated conditions

showing the fastest decrease, followed by oxygenated and

then hypo-oxygenated conditions and (ii) counts made in

pyruvate-supplemented medium under anaerobic conditions

consistently showing higher values than the corresponding

aerobic counts in unsupplemented medium. When Fig. 6 is

compared with Fig. 5, it is clear that the aerobically grown

cells were more resistant to illumination in simulated

sunlight than cells cultured under hypo-oxygenated condi-

tions, as each of the curves shown in Fig. 6 decreases at a

slower rate than that of the equivalent data set in Fig. 5.

Additionally, the differences between aerobic, unsupple-

mented counts and anaerobic, pyruvate-supplemented

counts were smaller for cells grown with air sparging,

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6

Time (h)

Pla

te c

ount

(lo

g 10

CF

U m

l–1)

Fig. 5 Time course for apparent inactivation of Escherichia coli grown

under hypo-oxygenated conditions and illuminated under different

levels of oxygen. Geometric mean counts (log10 CFU ml)1) for a

suspension of E. coli NCTC8912 grown to early log phase under hypo-

oxygenated conditions (N2-sparged), then suspended in water and

exposed to simulated sunlight for up to 6 h either under hypo-

oxygenated (N2-sparged) conditions (circles), oxygenated (air-sparged)

conditions (squares) or hyperoxygenated (O2-sparged) conditions

(triangles), and counted either on unsupplemented medium under

aerobic conditions (open symbols) or on medium supplemented with

0Æ05% w/v sodium pyruvate under anaerobic conditions (filled

symbols). Error bars represent 95% confidence limits (n ¼ 3)

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6

Time (h)

Pla

te c

ount

(lo

g 10

CF

U m

l–1)

Fig. 6 Time course for apparent inactivation of Escherichia coli grown

under oxygenated conditions and illuminated under different levels of

oxygen. Geometric mean counts (log10 CFU ml)1) for a suspension of

E. coli NCTC8912 grown to early log phase under oxygenated

conditions (air-sparged), then suspended in water and exposed to

simulated sunlight for up to 6 h either under hypo-oxygenated (N2-

sparged) conditions (circles), oxygenated (air-sparged) conditions

(squares) or hyperoxygenated (O2-sparged) conditions (triangles), and

counted either on unsupplemented medium under aerobic conditions

(open symbols) or on medium supplemented with 0Æ05% w/v sodium

pyruvate under anaerobic conditions (filled symbols). Error bars

represent 95% confidence limits (n ¼ 3)

PHOTOINACTIVATION OF E. COLI 45

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x

compared with N2 sparging, especially in the early stages of

illumination, supporting the idea that cells grown under

aerobic conditions are less sensitive to the subsequent

damaging effects of ROS, presumably as a result of their

antioxidant defence systems. These differences are also

evident when the T90 values for each treatment are

calculated (Table 2): cells under hypo-oxygenated condi-

tions had lower T90 values under all enumeration conditions,

compared with the corresponding aerobically grown cells,

with the greatest differences being observed on illumination

under hypo-oxygenated conditions and with smaller differ-

ences for the hyperoxygenated treatment. Overall, the T90

values for illuminated cells counted anaerobically in pyru-

vate-supplemented medium are 30–72% greater than the

corresponding unsupplemented aerobic values, with the

greatest proportional increases being seen for cells illumin-

ated under hypo-oxygenated conditions.

DISCUSSION

The enhanced enumeration of injured bacteria by peroxide-

degrading compounds such as catalase and pyruvate is well

established, and pyruvate is often incorporated into con-

ventional culture media for aerobic incubation of food and

water samples, e.g. Baird-Parker agar (Schoeller and

Ingham 2003), mLSA medium (Sartory 1995) and R2A

medium (Reasoner and Geldrich 1985). Bogosian et al.(2000) have shown that cold-stored Vibrio vulnificus can give

counts up to 1000-fold higher on aerobic media containing

catalase or pyruvate compared with unsupplemented con-

trols, and they attributed this increase to the degradation of

peroxides produced during autoclaving of the growth

medium. In contrast, Mizunoe et al. (1999) attributed

similar effects of pyruvate and catalase in temperature-

stressed E. coli O157 to the metabolic production of

hydrogen peroxide. The results in Figs 1–6 show that

supplementation of nutrient agar with 0Æ05% pyruvate can

give substantially higher aerobic counts for E. coli exposedto UVA or simulated sunlight, in agreement with the

observations of Kehoe (2001) for solar-illuminated bacteria,

including E. coli, S. flexneri and V. cholerae. However, the

current study also demonstrates that such counts are further

enhanced when pyruvate-supplemented plates are trans-

ferred to an anaerobic environment, indicating that pyruvate

alone is insufficient to counteract all the inhibitory effects of

oxygen.

Recent research has established that growth-arrested,

stressed bacterial cells may undergo a process of respir-

ation-induced self-destruction when subsequently cultured

under aerobic conditions (Dodd et al. 1997; Aldsworth

et al. 1999), where respiratory metabolism produces a burst

of intracellular-free radicals that is uncoupled from growth.

For example, the transfer of starved cells to a nutrient-rich

medium can lead to the rapid production of ROS,

including superoxide and peroxides (Bloomfield et al.1998), and these may overwhelm the antioxidant defence

systems of such cells, resulting in their death. Given that

bacteria such as E. coli are normal inhabitants of the colon,

an anaerobic environment, it is possible that the conven-

tional (aerobic) approach to culture represents an artificial

situation under which such cells are not enumerated. One

means of preventing such self-destruction is to culture the

organisms in the complete absence of oxygen, forcing them

to use anaerobic pathways for energy metabolism. Stephens

et al. (2000) have shown that such an approach can be

extremely effective in culturing sublethally injured

Salmonella following exposure to heat, acid or salt stress,

demonstrating that the combined effects of (i) neutraliza-

tion of extracellular ROS generated in the growth medium

together with (ii) the prevention of intracellular (respirat-

ory) ROS consistently gave the highest counts. This is in

agreement with the findings of George and Peck (1998),

who have shown that E. coli cells injured by heat treatment

are subsequently unable to grow in a medium of high redox

potential, implicating the reactive by-products of oxidative

respiratory metabolism as the principal toxic agents respon-

sible for preventing the growth of heat-damaged cells

under aerobic conditions. The present study shows that a

similar enhancement of the count of UVA-treated E. coli,can be observed under ROS-neutralized enumeration

Table 2 Comparative inactivation of

Escherichia coli grown and illuminated in

water under different levels of oxygen. T90

values for E. coli NCTC8912 cultured under

air-sparged or N2-sparged conditions, and

then exposed to simulated sunlight for 6 h

under different levels of dissolved oxygen and

enumerated either (i) on nutrient agar under

aerobic conditions or (ii) on nutrient agar plus

0Æ05% pyruvate under anaerobic conditions

Oxygen status during

illumination

T90 values (min) for cells grown

under air-sparged conditions

(oxygenated)

T90 values (min) for cells grown

under N2-sparged conditions

(hypo-oxygenated)

Aerobic

Anaerobic + pyruvate

(% increase) Aerobic

Anaerobic + pyruvate

(% increase)

Hypo-oxygenated 63 105 (67) 247 424 (72)

Oxygenated 36 49 (36) 79 107 (35)

Hyperoxygenated 27 37 (37) 46 60 (30)

The percentage increase in T90 observed under anaerobic, pyruvate-supplemented conditions is

shown in brackets for each experimental treatment.

46 R. KHAENGRAENG AND R.H. REED

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x

conditions, achieved by either (i) a combination of added

pyruvate with incubation in an anaerobic jar or (ii) prere-

duced growth medium maintained in an anaerobic cabinet.

Overall, the data for pyruvate supplementation indicate

that peroxide-based ROS are likely to account for the

predominant component of this inhibitory effect. The fact

that a positive effect of pyruvate supplementation was only

observed on aerobic plates transferred to an anaerobic

environment and not with prereduced plates maintained

under anaerobic conditions (Fig. 3) suggest that pyruvate

may protect against peroxide-based respiratory by-products

formed in the first hour or so after transfer to anaerobic

conditions from air, before the atmosphere and growth

medium are fully anaerobic.

The present study has demonstrated that a substantial

proportion of E. coli cells subjected to either UVA or

simulated sunlight become sublethally damaged, reaching a

point where they can no longer form colonies in air on an

agar-based medium. Following the approach of Zaske et al.(1980), the relative percentage of such injured cells can be

calculated by comparing the aerobic plate count for

unsupplemented medium (uninjured cells) with the equiv-

alent count for anaerobic, pyruvate-supplemented medium

(injured plus uninjured cells). On this basis, c. 99% of the

cells of E. coli NCTC 8912 subjected to simulated sunlight

for a period of 6 h were sublethally damaged, being capable

of forming colonies only under anaerobic, pyruvate-supple-

mented conditions and not under aerobic conditions

(Fig. 4). Such sublethally injured cells would be missed by

conventional aerobic counting procedures. Approximately

50% of these injured cells were able to grow aerobically in

the presence of pyruvate, which may indicate a lower level of

damage in such cells. However, pyruvate supplementation

alone was insufficient to provide a count of all of the viable

cells present under such conditions, and broadly similar

results were obtained with four other strains (Table 1),

including E. coli O157 NCTC12900.

The present research raises a question as to whether

studies on solar inactivation/disinfection may overestimate

the bactericidal effectiveness of sunlight when based solely

on enumeration by conventional aerobic plate counts, in

addition to the problems known to be caused by the use of

selective media (Block and Goswami 1995). The comparat-

ive significance of cells sublethally damaged by sunlight is

discussed in greater detail by Reed (2004), although it

should be noted also that Smith et al. (2000) have demon-

strated that sublethally damaged cells of Salmonellatyphimurium show a far lower infectivity to BALB/c mice,

and are therefore less likely to cause disease than their

undamaged counterparts.

While the current study focuses on the nonthermal effects

of UVA and solar illumination, there is often an additional

thermal component to solar disinfection under field condi-

tions, with a synergistic interaction between temperature

and light (e.g. Wegelin et al. 1994; McGuigan et al. 1998).However, the studies of George and Peck (1998) and

Bromberg et al. (1998) have demonstrated that heat-injured

cells also become oxygen sensitive during subsequent

culture. Based on such observations, together with the

current findings, we would suggest that researchers who

wish to establish the number of healthy and sublethally

damaged bacteria present in solar-treated natural waters

should consider enumeration under conditions where the

inhibitory effects of ROS-based self-destruction are avoided,

either using fully anaerobic conditions and a prereduced

growth medium, or using an anaerobic jar with a pyruvate-

supplemented medium, in order to evaluate the significance

of these findings under field conditions. A further factor in

natural waters will be the presence of exogenous com-

pounds, such as humic acids, that have the potential either

to act as photosensitizers, or to block the transmission of

inactivating solar radiation (e.g. Curtis et al. 1992).It is also worth noting that the present results may raise a

question regarding the measurement of T90 values for faecal

indicator bacteria based on conventional aerobic count data,

which is in widespread use for modelling the inactivation of

faecal bacteria in coastal water systems (e.g. Gameson and

Gould 1975; Guillaud et al. 1997). As Bloomfield et al.(1998) have pointed out, the lack of growth of sublethally

injured bacteria under aerobic conditions also provides an

alternative explanation for the so-called �viable noncultur-

able� hypothesis, as sublethally injured cells do not neces-

sarily enter a distinct physiological state, but are simply

unable to grow under conventional aerobic conditions, as a

result of self-generated ROS. It is tempting to speculate that

a count obtained under fully anaerobic conditions on a

nonselective medium under conditions where ROS are

neutralized or eliminated is likely to be more representative

of the true number of viable bacteria, as this major source of

growth inhibition has been removed.

The present study suggests that stationary phase cells

(Fig. 4) may be more resistant to solar illumination than

their log phase counterparts (c.f. data for oxygenated water

shown in Fig. 5), in agreement with earlier findings (Reed

1997). However, caution should be observed when compar-

ing these two sets of results, as the stationary phase cells

were grown without gas sparging whereas the log phase cells

were sparged with N2 during growth. A more important

point to note is that broadly similar overall responses were

observed for both stationary phase cells (Figs 1–4) and log

phase cells (Figs 5 and 6), with both types showing

enhanced counts under anaerobic conditions with added

pyruvate. This contrasts with the proposal that this

phenomenon should be most apparent in experiments using

exponentially growing cultures (Dodd et al. 1997), as suchcells will suffer the greatest imbalance between respiratory

PHOTOINACTIVATION OF E. COLI 47

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 39–50, doi:10.1111/j.1365-2672.2005.02606.x

metabolism and growth as a result of sublethal stress. The

findings of the current study may reflect the fact that the

damage caused by UVA and simulated sunlight is because of

photo-oxidation, which is likely to make such cells more

sensitive to subsequent oxidative stress, irrespective of their

growth phase. It is also worth noting that antioxidant

enzymes such as catalase may also be significantly damaged

during exposure to light (Kapuscinski and Mitchell 1981),

leading to an increased sensitivity to ROS. The enhanced

survival of log phase cells grown under aerobic conditions,

compared with those grown under anaerobic conditions,

following exposure to simulated sunlight (c.f. Figs 5 and 6)

also supports the idea that intracellular antioxidant defences

play a major role in determining the extent of sublethal

damage and death, as aerobically grown cells would be

expected to have a higher level of ROS-degrading enzymes,

e.g. catalase and superoxide dismutase.

ACKNOWLEDGEMENTS

Thanks are due to Dr K.G. McGuigan (Royal College of

Surgeons in Ireland, Dublin) for the loan of the solar

simulator, and to Dr J. Perry (Freeman Hospital, Newcastle

upon Tyne) for help with equipment (anaerobic jars

and anaerobic cabinet). Financial support was provided

by the Royal Thai Government and the Royal Society of

London.

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