The effect of UV-C radiation (254 nm) on candidate microbial source tracking phages infecting a...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/php.12223

This article is protected by copyright. All rights reserved.

Received Date : 15-Aug-2013

Accepted Date : 04-Dec-2013

Article type : Research Article

Inactivation of Bacteriophage Infecting Bacteroides Strain GB124

Using UV-B Radiation

David Diston * (1), James E. Ebdon (2) and Huw D. Taylor (2)

1. Bundesamt für Gesundheit, Schwarzenburgstrasse 165, Köniz, 3003 Bern, Switzerland

2. Environment & Public Health Research Unit (EPHRU), School of Environment & Technology, University

of Brighton, Cockcroft Building, Lewes Road,

Brighton, BN2 4GJ, UK

* Corresponding author. Tel.: +41 31 325 40 69; fax: +41 31 322 95 74; Email:

[email protected]

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ABSTRACT

UV-B radiation (280-320 nm) has long been associated with the inactivation of micro-organisms in the

natural environment. Determination of the environmental inactivation kinetics of specific indicator

organisms [used as tools in the field of microbial source tracking (MST)] is fundamental to their

successful deployment, particularly in geographical regions subject to high levels of solar radiation.

Phage infecting Bacteroides fragilis host strain GB124 (B124 phage) have been demonstrated to be

highly specific indicators of human faecal contamination, but to date, little is known about their

susceptibility to UV-B radiation. Therefore, B124 phage (n = 7) isolated from municipal wastewater

effluent, were irradiated in a controlled laboratory environment using UV-B collimated beam

experiments. All B124 phage suspensions possessed highly similar first order log-linear inactivation

profiles and the mean fluence required to inactivate phage by 4-log10 was 320 mJ/cm2. These findings

suggest that phage infecting GB124 are likely to be inactivated when exposed to the levels of UV-B solar

radiation experienced in a variety of environmental settings. As such, this may limit the utility of such

methods for determining more remote inputs of faecal contamination in areas subject to high levels of

solar radiation.

INTRODUCTION

Whether delivered naturally via solar radiation (UV-A and UV-B), or artificially through disinfection

technologies (UV-C), the inactivating effect of UV radiation on microorganisms has been widely studied

and previously reported (1-4), and is thought to be the primary means of environmental viral

degradation in many systems (3). The influence of UV-A and UV-B radiation on microorganisms in the

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environment is particularly important when the organisms studied are indicators of faecal

contamination, or surrogates for infectious agents of significance to human health, as is often the case

in microbial source tracking (MST) and quantitative microbial risk assessment (QMRA) studies. MST and

QMRA methods have been developed to quantify and identify human and non-human inputs of faecal

microbial pollution in environmental waterbodies, allowing effective remediation efforts of degraded

waterbodies and the production of accurate risk assessments (5,6).

When using MST and QMRA tools, it is important to consider the likelihood of environmental surrogate

degradation leading to false-negative results for human faecal material, and assumed pathogen

absence. Of particular importance is any difference in the inactivation tendencies of indicator and

pathogen, and it is desirable that the chosen indicator display greater resistance to inactivation than the

pathogen(s) of concern (7). A pre-requisite of successful MST application is a robust understanding of

the environmental behaviour (and survival) of the MST target or targets (8).

Little information is available in the literature regarding the susceptibility of MST indicators to UV

radiation, and those reported focus mainly on UV-C radiation (9,10). Importantly, no research has been

conducted investigating the role of UV-B (the germicidal portion of solar UV) on the majority of indicator

organisms commonly used in MST studies.

To date, a wide range of culture-dependant genotypic and phenotypic MST methods have been

proposed. One such approach involves the detection and enumeration of phage infecting certain

Bacteroides spp. strains, as they have been shown to be highly human-specific and have been employed

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successfully in MST studies in varied geographical locations (11-16). A key advantage of Bacteroides

phage is the relatively low cost of the assay method used and its ease of implementation in even

modestly equipped laboratories. Moreover, internationally recognised standard methods (17-19) have

improved the quality of phage studies by facilitating allowing ready data comparison between studies.

Research into the use of bacteriophage infecting Bacteroides spp. as MST tools has been designated as a

‘high priority critical research need’ by the US Environmental Protection Agency (20).

However, the successful application of Bacteroides phage detection methods (like many MST methods)

in environmental waters is influenced by the resistance of the organisms to environmental disinfecting

agents (8). At present, no information has been published regarding the inactivation characteristics of

phage infecting GB124 during UV-B irradiation (the germicidal portion of sunlight) and for these phage

to be used effectively as MST tools, elucidation of their response to UV-B irradiation is required.

This following research sheds light on this important knowledge gap, by determining the response of

phage infecting Bacteroides fragilis strain GB124 (21,22) to UV-B radiation under controlled laboratory

conditions (23). The work represents the first experimental exposure of bacteriophage infecting B.

fragilis strain GB124 to UV-B irradiation in order to assess whether they may be used as reliable

indicators of human faecal pollution (and potentially as surrogates of specific viral pathogens) in natural

and engineered water systems that receive significant exposure to solar radiation.

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MATERIALS AND METHODS

Preparation of bacteriophage suspensions and assay method

Bacteriophage used in MST studies are likely to be a taxonomically diverse group rather than a single

morphotype/genotype (24). Therefore, in order to gain adequate information on the range of possible

responses to UV-B irradiation, seven suspensions of bacteriophage capable of infecting B. fragilis strain

GB124 from an existing laboratory collection of 60 (single plaque isolates) were selected, based on

preliminary UV-B/UV-C screening tests (data not shown) and variations in morphology (as visualised

using transmission electron microscopy). All selected phage were members of the siphoviridae family,

with dsDNA.

Working phage suspensions used in irradiation experiments were prepared by diluting stock phage

suspension in buffer [19.5 mM Na2HPO4, 22 mM KH2PO4, 85.5 mM NaCl, 1 mM MgSO4, 0.1 mM CaCl2

(25)], to achieve a final concentration of 105 PFU/ml. In order to reduce particle shielding and to

minimize the presence of phage inhibitors/photosensitizers, seeded buffer solutions, rather than sterile

sewage or river water, were used in irradiation experiments.

Following UV exposure, all irradiated samples were stored in light-tight glass tubes, in the dark at 4 °C.

Prior to analysis, a dilution series of each irradiated suspension was created and assayed using the

double-agar method for Bacteroides strain GB124, as described previously (19,22). Two dilutions were

selected for assay [based on levels of fluence observed to inactivate phage during preliminary screening

experiments (data not shown)], with each dilution assayed in triplicate. Plates containing more than 300

plaque forming units (PFU) were not included in the analysis (as individual plaques could not be clearly

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distinguished), and as it was necessary to ascertain the point at which total inactivation occurred (i.e.,

evidence of ‘tailing’), no lower exclusion limit was used. The lower limit of sensitivity of the assay was <

1 PFU/100μl and a total of 882 double agar assays were conducted (126 for each phage suspension).

UV irradiation and experimental design

All samples were irradiated using a custom-built UV collimated-beam (CB) apparatus with two UV-B low-

pressure low-intensity Hg 15W UV bulbs (UVP, UK). Irradiation using collimated-beam apparatus (26),

rather than environmental irradiation, was chosen in order to allow precise control of delivered fluences

(27) and all UV irradiation procedures were carried out in accordance with standard methods (28). The

fluence rate at the base of the collimating tube was measured using a recently calibrated IL1400A

radiometer equipped with a SED005/U sensor and a WBS320 wide band UV-IR filter (International Light

Technologies, Massachusetts USA). A UV-visible domed Teflon diffuser was also fitted to the equipment

and a spectral response curve of the sensor/filter unit (International Light) was used to determine the

calibration factor to be used for UV-B radiation.

Initial tests were carried out to characterise UV bulb output and to ascertain the point at which stability

was reached. The bulbs were labelled sequentially, corresponding to their position in the box, and to

ensure continuity the same bulbs were used in the same positions for all experiments. These

experiments were repeated on three consecutive days and it was demonstrated that UV bulb output

(fluence rate) was stable after approximately 30 minutes (data not shown). During sample irradiation,

bulbs were switched on one hour prior to commencement of inactivation to ensure consistency of UV

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output. The irradiation chamber temperature was monitored throughout the experiments, and was

shown to remain between 18 and 22°C throughout the experiments.

The spectral output of the UV-B lamps is shown in figure 1. The UV-B bulb emitted a range of

wavelengths, with a peak at 300 to 325 nm (outputs above 400 nm were blocked by the filter mounted

on the detector head). As this bulb had a wider output spectrum than monochromatic bulbs, its output

is referred to as ‘UV-B’ rather than as a specific wavelength (i.e., 302 nm, as described by

manufacturers). No UV-C radiation is delivered by the UV-B bulb. Bulbs with similar properties to those

used in this study have been used by several authors for microorganism irradiation studies (29-31).

Phage suspensions were equilibrated to room temperature prior to irradiation. Samples were stirred in

situ using a magnetic stirrer, protected from UV light by the shutter, for 10 seconds before UV exposure,

ensuring a fully-mixed sample (stirring continued throughout UV exposure). Prior to each exposure, the

fluence rate at the centre of the beam (E0) was measured and entered into the UVCalc spreadsheet

(kindly provided by Professor J.R. Bolton) and the required exposure time was calculated. Exposure

times were adjusted to compensate for Petri, Reflection, Water and Divergence factors (28). UV-B bulb

output tended to vary by ± 4 μW/cm2 between pre-exposure readings and exposure times were

adjusted to correct for this.

Seven fluences were chosen to capture bacteriophage inactivation: 0, 50, 100, 150, 200, 250 and 300

mJ/cm2 (based on the results of screening experiments; data not shown). Each exposure was repeated

three times using a new test suspension and samples were irradiated in a random order, thereby

reducing potential errors from fluctuation of UV bulb output.

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Statistical analyses

Pearson product-moment correlation was carried out to determine significance of relationship using

Microsoft Excel (v.12), whilst regression analysis and production of linear rate constants (k) were carried

out using Minitab (v.15). Identification of the phage inactivation kinetics was carried out using the

GInaFiT inactivation model fitting tool v.1.5 (32).

RESULTS AND DISCUSSION

UV-B inactivation kinetics

UV-B inactivation profiles of seven phage infecting Bacteroides strain GB124 in liquid suspension are

presented in figure 2. A highly significant (P = 0.000) relationship was observed between log10 PFU/100

µl values for B124 phage and UV-B irradiation fluence, with phage suspensions displaying minimal range:

Pearson product-moment correlations ranged from -0.98 % (B124-1, -10 and -54) to -0.99 % (B124-12, -

21, -29 and -35), with a mean value of -0.99 % and 0.007 mJ/cm2 SD. The data showed no ‘shouldering’

or ‘tailing’, and the relationship between log10 inactivation and fluence, as identified using GInaFiT

modelling software, was linear positive (y = 0.0144x - 0.3141). No deviation from first order kinetics was

observed. All phage suspensions displayed a similar response to increasing fluence. Fluences required to

inactivate phage by 4-log10 ranged from 288 mJ/cm2 (B124-10) to 402 mJ/cm2 (B124-54). The mean

fluence required was 320 mJ/cm2 (range = 114 mJ/cm2; SD = 38.59 mJ/cm2) and the inactivation curves

were not statistically different from each other (t test; P = 0.529). Therefore, the null hypothesis that the

relationship between fluence and log10 PFU/100 µl occurred as a result of chance, can be rejected.

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Owing to the complexity of aquatic environmental systems, extrapolation of laboratory findings to the

environmental is problematic. Using the CB apparatus detailed in this study, environmental exposure

times necessary to deliver required fluences may be calculated (table 1). These calculations use example

environmental UVB fluence rates of 0.022 mW/cm2 (33) and 0.20 mW/cm2 (34) and assume that

microorganisms will be exposed to a constant fluence with no virus reactivation occurring. In river

systems, constant fluence is unlikely, because microorganisms move within the water column and

sunlight intensity changes with respect to time of day and ambient meteorological conditions.

Moreover, as most UV-B is delivered during a four hour period (two hours either side of solar noon) a

constant fluence cannot be assumed (3).

Data from this study indicated that a UV-B fluence of between 288 mJ/cm2 and 402 mJ/cm2 is required

to achieve a 4-log10 inactivation of B124 phage. Given the fluence rate of 0.022 mW/cm2, this would

equate to environmental exposure times of between 3 hours 38 minutes and 5 hours 4 minutes. As this

is the peak exposure, it is unlikely that 4-log10 inactivation could be achieved during a single diurnal

cycle. However, if a higher fluence rate were used in the calculation (i.e., 0.20 mW/cm2 as recorded by

Jung et al. (34), 4-log10 inactivation would occur within as little as 35 minutes. Based on a fluence rate of

0.20 mW/cm2, the potential for environmental inactivation of B124 phage within a single diurnal UV-B

exposure cycle is evident. The findings are significant as they suggest that the persistence (and thus

successful detection) of viable of B124 phage, particularly those present in more remote inputs of

human faecal material (with longer transit times) may be severely limited in regions subject to high

levels of solar radiation.

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In determining the possibility of B124 phage inactivation in a water body by UV-B radiation, it is also

useful to determine daily fluence loads. Examples given in the literature include 10 kJ/m2 in January and

55 kJ/m2 in June in Baltimore, USA (35), and 570 to 611 kJ/m2 in September at the Red Sea (36). A 4-log10

reduction of B124 phage could theoretically be achieved during a single day based on these total fluence

loads, as this requires between 2.8 kJ/m2 to 4 kJ/m2. This may have important ramifications for the use

of GB124 phages as a tool for MST and QMRA in latitudes at which solar UV-B levels are higher.

Therefore, local climatic conditions should be assessed when using B124 phages in river systems, and

factors such as cloud cover should be taken into account as this is pivotal in controlling the amount of

UV-B radiation reaching the water surface.

In pilot tests for this study, river water samples were taken downstream of a Wastewater Treatment

Works (WwTW) in a habitat subject to relatively extended water retention times (the Pevensey Levels

wet grasslands, East Sussex, UK). Phages infecting GB124 were detected in non-UV disinfected WwTW

final effluent discharged to the wetland environment (approximately 3.70 log10 PFU/100ml; data not

shown), but were not discovered at any points in the receiving drainage system (the closest monitoring

point was approximately 1 km downstream of the WwTW discharge point). Retention times in this area

may be greater than five days, and it is likely that sufficient UV-B would be delivered to the water

column inactivate B124 phage.

In the calculations above it has been assumed that phage virions are exposed to the full UV-B fluence

throughout the water column. However, it has been shown that UV-B radiation is attenuated through

the water column (37) depending on the chemical, microbial and physical characteristics of the water,

and this may extend phage survival. As WwTW effluents are typically nutrient rich and more turbid than

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surrounding waters, UV-B penetration into the receiving water column will not be as great as into

waters with a lower nutrient status/turbidity, thus protecting the phage from inactivation. Following

discharge from WwTW, there are numerous physiochemical and biological factors that may influence

the survival of phage and enteric viruses in the environment. These include adsorption to sediments

(38,39), UV-B attenuation through the water column (36), deposition into bed-sediments (40-42), and

phage coagulation, (43).

As UV-B fluences are not constant throughout the day, it is important to recognise that viral inactivation

rates that are dependent on UV-B irradiation also vary diurnally (44). Therefore, the method and time of

water sampling may influence the density of phages present. Samples taken after the period of greatest

inactivation (i.e., two hours post solar-noon) may contain fewer inactivated B124 phages than those

taken two hours before solar noon. Also, in winter, less solar UV-B is delivered to the water surface, so

levels of UV-induced phage-inactivation will be reduced. The location of sample collection point may

also influence the levels of inactivated phages recorded. For example, if a slow moving river (in which

horizontal mixing of water columns between the river edge and the centre of the river is limited) is

sampled, the difference between UV-B exposure in the centre (i.e., exposed section) of the river will be

greater than the UV-B exposure on the banks (which may be shaded for part of the day).

Relationship between UV-B and UV-C inactivation of phage infecting GB124

Data for UV-C inactivation of the phage suspensions using the same sample handling, irradiation

protocol and phage assays methods, has previously been reported by the authors (9) and these are

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reproduced in table 2. The similarity between the inactivation kinetics of all phage suspensions during

UV-B and UV-C irradiation was statistically significant, with all phage showing linear positive inactivation

curves. Fluences required for 4-log10 inactivation of each phage during UV-B and UV-C radiation were

positively correlated (R2 = 0.73 %, P = 0.062) with an average ratio of 9:1 (UV-B requirement:UV-C

requirement). This ratio varied from 8:1 (B124-35) to 10:1 (B124-10), indicating that nearly ten times the

amount of UV-B radiation was required for a 4-log10 reduction in phage titre compared with that

required using UV-C radiation. Other studies show lower UV-B:UV-C ratios; approximately 367 mJ/cm2 is

required to inactivate murine norovirus by 4-log10 using UV-B radiation; the UV-C fluence requirement to

inactivate caliciviruses (of which murine norovirus is a member) by 4-log10 has been reported as 61.9 -

119 mJ/cm2 (45,46) giving a UV-B:UV-C ratio of between 3.0:1 and 5.9:1. Other studies have

demonstrated higher UV-B:UV-C ratios than that recorded here for B124 phage; for MS2 phage, 702

mJ/cm2 is required to inactivate MS2 phage (in the presence of TiO2) by ~4-log10 using UV-B (47),

whereas the UV-C fluence requirement has been reported as being 19 - 36 mJ/cm2 (46,48) giving a UV-

B:UV-C ratio of between 19.5:1 and 36.9:1.

A previously proposed model suggested that approximately 41 times the fluence of UV-C would

be required to inactivate viruses by the same magnitude when using UV-B (49); for the Vaccina virus, 33

times more UV-B was required than UV-C to achieve the same level of inactivation by the same authors

(3). This factor has been used in biodefense calculations and influenza epidemic risk assessments (50).

For phage infecting GB124, this estimate proves to be inaccurate. Data for B124 phage indicate that

around 9 times more UV-B than UV-C is required to achieve the same levels of inactivation.

Furthermore, other data agree with this lower estimate of UV-B: UV-C ratio requirement (45,46). It is

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important to note that differences in irradiation conditions, organism preparation and handling will

account for some degree of discrepancy, but further work is required to test the proposed UV-B: UV-C

model.

The difference in UV-B/UV-C fluence requirements to inactivate B124 phages is most probably because

of the greater efficiency of UV photon absorption by DNA at wavelengths close to 250 nm, than at 302

nm, and therefore a greater degree of cyclobutane pyrimidine dimer (CPD) formation probably occurs

(i.e., disruption in base pairing). It has been demonstrated that CPD formation during UV-B irradiation is

around 90% less efficient than dimer formation during UV-C irradiation (51,52), and as this is similar to

the observed ratio for B124 phages, it may be postulated that the main mechanism of UV-B inactivation

of B124 phages is via the production of CPDs. However, the role of protein damage, as reported by

Bosshard et al. (53) and Eischeid and Linden (54), is yet to be fully understood. Further work should

include an investigation into the timing and magnitude of B124 phage DNA and protein damage during

UV exposure.

CONCLUSIONS

The principal findings from this study may be summarised as follows:

1. B124 phages are inactivated by UV-B fluences that are recorded in the natural environment, though

the rate of inactivation will be highly dependent on a number of external factors, including water

retention time, composition of the water matrix, and temporal and geographical variations in UV

fluence. If such environmental factors are underestimated, then there is a chance that there may be

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a mis-diagnosis of risk during MST and potentially QMRA studies, resulting in negative health

impacts for resource users. It is important to note that B124 phage counts (and other

microorganisms dependent on culture assay) may be negatively influenced by sampling time;

2. The UV-B inactivation kinetics of B124 phages are highly homogeneous, and their behaviour during

UV-B irradiation may predict how they are inactivated during UV-C irradiation;

3. The UV-B/UV-C fluence ratio for B124 phages appears to be lower than that suggested by previously

published models, but is similar to the UV-B/UV-C ratio reported for other microorganisms (MS2

phages and caliciviruses).

This study represents an important step towards a fuller elucidation of the ecological inactivation

kinetics of an important phage group currently used in MST studies. Further in situ and collimated beam

inactivation experiments using selected pathogens and B124 phages may provide greater insights into

the suitability of these phages to act as surrogates of waterborne viruses in both natural and engineered

systems, in support of improved QMRA models. Furthermore, a greater variety of microorganisms of

human health significance should be tested against UV-B and UV-C radiation in parallel, to further

previously published models. This field of study is likely to support improved human health protection

in a wide variety of environments.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the cooperation of Southern Water in providing access to WwTW,

Dr W.A.M Hijnen (Kiwa Water Research Ltd., The Netherlands) for statistical advice, Mr Ian Mayor-Smith

for access and information relating to Southern Water's wastewater treatment works, and Professor J.R.

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Bolton (Bolton Photosciences) for providing the UVCalc spreadsheet and advice on the construction of

collimated beam apparatus.

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TABLES

Table 1. Exposure times for target fluences using UV-B fluence rate values detailed in the literature

(33,34)

Target fluence (mJ/cm2)

Time required with fluence rate of 0.022 mW/cm2 (33)

Time required with fluence rate of 0.2 mW/cm2 (34)

50 37m 53s 4m 10s

100 1h 15m 45s 8m 20

150 1h 53m 38s 12m 30s

200 2h 31m 31s 16m 40s

250 3h 9m 24s 23m 59s

300 3h 47m 16s 33m 30s

Table 2. Relationship between 4-log10 reduction fluence for phage suspensions during UV-B and UV-C

irradiation experiments [UV-C data from (9)]

Phage suspension ID (B124-)

UV-C 4-log10

inactivation fluence (mJ/cm2)

UV-B 4-log10 inactivation fluence (mJ/cm2)

UV-B:UV-C ratio

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1 35 325 9.19:1

10 29 288 10.00:1

12 33 294 8.90:1

21 34 310 9.07:1

29 40 323 8.15:1

35 37 301 8.05:1

54 41 402 9.90:1

FIGURE CAPTIONS

Figure 1. Spectral output of UV-B bulbs - unspecified power unit on y axis, x axis shows wavelength in

nm (courtesy and copyright UVP, UK)

Figure 2 Dose-response curves of phages B124-1, B124-10, B124-12, B124-21, B124-29 B124- 35 and

B124-54 during UV-B collimated beam inactivation experiments. Data points show the mean of 9 values.

Error bars represent one standard deviation above and below the mean.

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The effect of UV-C radiation (254 nm) on candidate microbial source tracking phages infecting a...

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