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The application of a recently isolated strain of Bacteroides(GB-124) to identify human sources of faecal pollution in atemperate river catchment

James Ebdona,�, Maite Muniesab, Huw Taylora

aEnvironment & Public Health Research Unit, School of the Environment, University of Brighton, Cockcroft Building, Lewes Road,

Brighton BN2 4GJ, UKbDepartment of Microbiology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain

a r t i c l e i n f o

Article history:

Received 17 May 2006

Received in revised form

8 December 2006

Accepted 12 December 2006

Available online 1 February 2007

Keywords:

Bacteroides

Catchment

Faecal

Phages

Source

nt matter & 2007 Elsevie.2006.12.020

thor. Tel.: +44 1273 642274: je3@bton.ac.uk (J. Ebdo

a b s t r a c t

Recent work has suggested that bacteriophages infecting Bacteroides are a potential tool for

faecal source tracking, but that different host strains may be needed for different

geographic areas. This study used a recently identified strain of Bacteroides (GB-124) to

detect human sources of faecal pollution in a river catchment in southeast England (UK). A

total of 306 river water, municipal wastewater and animal samples were obtained over a 16-

month period. Bacteriophages capable of infecting GB-124 were present in all municipal

wastewaters but were not detected in faecal samples from animals, and were detected at

significantly lower levels (Po 0.001) in river waters directly downstream of a dairy farm.

This last observation was despite the presence of high levels of faecal indicator bacteria at

this site. The study suggests that GB-124 appears to be specific to human faeces. As such it

may represent an effective and low-cost method of faecal source identification.

& 2007 Elsevier Ltd. All rights reserved.

1. Introduction

According to a recent report by the UK Environment Agency,

there is a need for ‘a better understanding of how different

types of pollution move and interact, while passing through

river basins towards the sea’ (Anon, 2005). Contamination of

surface waters by point and non-point sources of faecal

pollution can degrade water quality and impact upon drinking

water supply, fishing, aquaculture and recreational pursuits.

Determining whether contamination is originating from hu-

man or animal sources is therefore essential for estimating

public health risk, facilitating remediation measures and for

resolving legal responsibility for remediation. As a result, novel

faecal source tracking (FST) techniques have been developed,

many of which have been recently reviewed (Sinton et al., 1998;

Simpson et al., 2002; Field, 2004; Meays et al., 2004).

r Ltd. All rights reserved.

; fax: +44 1273 642285.n), h.d.taylor@brighton.ac

A limitation of much of the current research into FST is that

little attention has been focussed on the provision of simple,

low-cost techniques. However, one approach that has shown

promise and that is simple and low cost involves the

detection and enumeration of bacteriophages (viruses) cap-

able of infecting bacteria of the genus Bacteroides (Jofre et al.,

1986; Tartera and Jofre, 1987; Cornax et al., 1990; Armon, 1993;

Puig et al., 1999). According to Haroun and Saheer (2002)

Bacteroides are obligate anaerobic Gram-negative bacteria that

constitute a major proportion of the bacterial flora of the

human intestinal tract.

Unfortunately, the host range of Bacteroides is not restricted

to humans, but interestingly bacteriophages infecting parti-

cular strains of B. fragilis (such as HSP40) appear to be found

almost exclusively in faecal material of human origin (Tartera

and Jofre, 1987; Lucena et al., 1996; Puig et al., 1999). It has

.uk (H. Taylor).

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WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 3 6 8 3 – 3 6 9 03684

therefore been possible to use phages of Bacteroides to

distinguish successfully human from non-human sources of

faecal pollution in surface waters (Tartera and Jofre, 1987;

Tartera et al., 1989; Puig et al., 1999). The reason for the

narrow host range of Bacteroides phages is not fully under-

stood, though Tartera et al. (1989) have suggested that one

explanation might be that both phages and host bacteria

might have co-evolved more separately in obligate anaerobes

than in facultative anaerobes. However, research has shown

that phages that are shown to attack a specific host strain of

Bacteroides in one part of the world, are not necessarily

detected in samples of similar origin in other parts of the

world (Kator and Rhodes, 1992; Grabow et al., 1993; Chung et

al., 1998; Puig et al., 1999; Payan et al., 2005). This is likely to be

attributed to regional differences in the human faecal flora.

These findings therefore suggest that different Bacteroides

host strains are needed for FST studies in different catch-

ments or geographic regions.

Recent research by Payan et al. (2005) led to the isolation of

a new and potentially useful Bacteroides strain (GB-124) from a

UK municipal wastewater (MW). Strain GB-124 is most closely

analogous to Bacteroides ovatus and was isolated from the

influent of a sewage treatment works (STW) situated in

southeast England. A detailed account of the method by

which Bacteroides GB-124 was isolated is reported by Payan et

al. (2005). Briefly, dilutions of MW were plated onto Bacteroides

bile aesculine agar (BBE) (Livingston, 1978) and incubated at

36 1C (72 1C) for 44 (72) h under anaerobic conditions

(Anaerogen, Oxoid, UK). Bacterial colonies with a dark halo

were plated for pure culture on BBE and incubated under

aerobic and anaerobic conditions. Gram-negative colonies

growing in anaerobic conditions were grown up in Bacteroides

phage recovery medium broth (BPRM, Scharlau, Spain) at

36 1C (72 1C) for 18 (72) h and exposed to faecal samples of

human and animal origin. Strain GB-124 gave zero or very low

phage counts in samples from pigs, cattle and poultry and

high counts in MW samples.

However, the study only managed to test a very limited

number of samples and no attempt was made to determine

whether GB-124 phages could be detected successfully in UK

surface waters. The work presented herein therefore follows

on from the work of Payan et al. (2005) in that it attempts

to determine whether GB-124 phages are host specific

(i.e., found exclusively in the faeces of humans) and are

present in sufficient numbers to allow their routine detection

in contaminated river waters. If this is the case then GB-124

may potentially be a useful tool for identifying and assessing

the contribution of wastewater discharges, leaking sewers

and leaking septic tanks to river waters in southeast England.

2. Materials and methods

2.1. Study catchment

For this study, a river catchment was selected in southeast

England from which river water samples were collected on at

least a monthly basis from nine water-monitoring sites. The

sites were situated along a 15 km stretch of a tributary of the

River Ouse. The river is situated in the county of East Sussex

and is 67 km in length, draining 396 km2 to its tidal limit. The

tributary catchment includes a range of potential faecal

sources and is largely rural, although the stream passes

through several villages before joining the main river channel.

Whilst agriculture is the dominant land-use, there are over 20

STW discharging partially treated MW into the river system.

In addition to river water samples, and to test the host

specificity of GB-124, 110 MW and 30 pooled samples from the

faeces of livestock, domestic and wild animals were also

obtained from across southeast England.

2.2. River water and faecal samples

A total of 166 river water samples were taken between April

2005 and August 2006. For clarity, river monitoring sites are

referred to on the basis of their proximity to known potential

faecal pollution inputs, e.g., ‘o0.5 km downstream of STW’, or

‘directly downstream of dairy farm’, etc. River water samples

were taken from the centre of the river at a depth of

approximately 30 cm below the surface using sterile poly-

ethylene sampling bottles clamped to an extendible sampling

pole. All river water and animal faecal samples were

transported to the laboratory in the dark at 4 1C, and analysed

within 4 h of sampling. All media and reagents were prepared

in accordance with the manufacturers’ instructions, unless

otherwise stated.

In the case of animal samples, faecal material was obtained

on each occasion from at least 20 animals using sterile swabs

placed into sample bags, or from approximately 500 ml of

liquid run-off from livestock sheds collected in polyethylene

bottles. In the laboratory the faecal material was mixed well

(either using a vortex mixer WhirlimixerTM, or a Seward

stomacher 400, Lab System, UK) with other samples from

the same source to form pooled samples (e.g., cattle, pig, etc.).

For each pooled sample either 1 gramme (wet weight) of

faecal matter or 1 ml of run-off was suspended in 9 ml 14

strength Ringer solution and a decimal dilution series made

before the samples were analysed for the presence of faecal

organisms.

2.3. Municipal wastewater samples

Final effluent from STW was collected by staff of the local

water supply and sewerage company in sterile 1 l polyethy-

lene sampling bottles, kept in the dark at 4 1C and transported

to the University of Brighton for analysis. Samples were

obtained from 29 STW (Table 1). MW samples from northern

England and Denmark were additionally analysed in order to

determine whether the geographical distribution of phages

infecting GB-124 was limited to the southeast of England.

2.4. Detection of faecal organisms

In addition to Bacteroides samples were tested for the

presumptive presence of faecal coliforms (FC), intestinal

enterococci (ENT) and the presence of somatic coliphages.

Somatic coliphages were selected for use within the study as

they appear to offer a good indication of the presence of

faecal contamination in surface waters (Borrego et al., 1987;

Skraber et al., 2002). The results achieved using GB-124 could

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Table 1 – Numbers of GB-124 phages in final effluent from29 Sewage treatment works (n ¼ 110)

Site Populationequivalent(pop. eq.)a

Numbers of phageinfecting GB-

124 Log10 PFU/100 ML

Brighton

Portobello

261,254 4.22–4.38

Eastbourne 119,336 3.76–4.70

Newhaven

East

55,955 2.50–3.27

Goddards

Green

49,824 3.98

Scaynes Hill 37,327 3.77–4.18

Hailsham

South

27,448 2.30–4.26

Vines Cross 23,518 2.95

Uckfield 23,163 3.15–4.04

Crowborough

Redgate

20,700 3.32

Hailsham

North

14,725 2.00–3.00

Ringmer 4805 3.74

Forest Row 4456 2.30–3.63

Barcombe 3533 2.70

Cuckfield 3186 3.78

Wadhurst

Whitegate

2675 2.00–3.00

Crowborough

St John’s

2377 2.00–2.30

Ditchling 1621 2.47–4.30

Maresfield 1542 3.47

Blackboys 1046 2.47

Hartfield 949 2.00–2.60

Kingston 946 2.30–3.32

Alfriston 769 3.07

East Hoathly 761 2.30–3.65

Rodmell 381 2.00–2.84

Poynings 371 2.30–2.84

Ansty 240 2.00

Streat (Golf

course)

28 3.90

Esholt (N.

England)

624,000 3.15

Aalborg

(Denmark)

— 2.70

Total 1,286,936

a Pop. eq supplied by Southern Water (UK), and Yorkshire Water

(UK).

WAT E R R E S E A R C H 41 (2007) 3683– 3690 3685

then be compared with the more established faecal indicator

organisms. Briefly, FC were enumerated by membrane filtra-

tion on 0.45mm pore size membranes, followed by incubation

for 24 h on mFC Agar (Difco, BDMS, UK) at 44.5 1C, in

accordance with standardised methods (Anon, 1990). ENT

were also enumerated on 0.45mm pore size membranes by

membrane filtration followed by incubation on m-Enterococcus

Agar (Difco, BDMS, UK) at 37 1C for 48 h in accordance with

standardised methods (Anon, 1984). Bacterial counts were

expressed as colony-forming units (CFU) per 100 ml of

sample.

2.5. Bacteriophage enumeration

Enumeration of somatic coliphages was carried in accordance

with standardised methods (Anon, 2000) using the host strain

Escherichia coli WG-5, and was based on a double agar plaque

count procedure similar to that described below for Bacter-

oides phage detection (Anon, 2001). Screw-topped glass

tubes (Hach, UK) containing BPRM broth were used to grow

strain GB-124 (1 ml host in 12 ml broth) to the correct

optical density (approx. 0.33 at 620 nm) for phage detection.

Once the correct optical density was reached (usually within

3 h), strain GB-124 was placed on melting ice and used within

4 h. All samples were filtered using 0.22mm polyvinylidene

difluoride (PVDF) membrane syringe filters (Millipore, US) to

remove any background bacterial contamination before

phage detection. These low protein binding membranes have

been shown to retain very low levels of phages (Tartera et al.,

1992).

On each occasion, 1 ml of the filtrate (or dilution thereof)

and 1 ml of host GB-124 were added to a sterile 10 ml

disposable test tube containing 2.5 ml of semi-solid BPRM

agar and mixed gently to avoid bubble formation. The

contents were then poured onto the surface of BPRM agar

and left to set. The plates were inverted and incubated at 36 1C

(72 1C) for 18 (72) h in anaerobic jars containing anaerobic

sachets (Anaerogen, Oxoid, UK). The presence of phages

resulted in the production of visible plaques (zones of lysis) in

a confluent lawn of the host bacterium. All samples (1 ml

volume) were analysed in at least duplicate and expressed as

the mean number of plaque forming units (PFU) per 100 ml of

sample.

3. Results

3.1. Faecal indicator organisms in municipal wastewaterand faecal samples

Levels of phages infecting Bacteroides GB-124 in MW ranged

from 2.0 Log10 PFU/100 ml in final effluent from Hailsham

North, Wadhurst Whitegate, Crowborough (St. John’s), Hart-

field, Rodmell and Ansty STW to 4.70 Log10 PFU/100 ml in final

effluent from Eastbourne STW (Table 1). The mean number of

phages infecting Bacteroides GB-124 (3.92 Log10 PFU/100 ml) in

MW (Table 2) was very similar to that reported for phages

infecting B. fragilis HSP40 (3.73 Log10 PFU/100 ml) (Tartera et al.,

1989; Lucena et al., 1996). Phages were detected in the final

effluent from all STW tested although, unlike the river water

and animal faecal samples, MW samples were not always

analysed on the day of sampling. This was due to the fact

that MW samples were independently gathered and delivered

to the University of Brighton for subsequent analysis. There-

fore, the levels of phages reported may be a slight under-

estimation of the levels present had the samples been

analysed within 4 h of sampling. However, it is unlikely that

this factor would result in a significant reduction in counts as

Puig et al. (1999) have reported good survival of Bacteroides

phages at 4 1C.

Importantly, phages infecting Bacteroides GB-124 were not

detected in any of the 30 non-human samples taken from

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Table 2 – Numbers of phages in samples from different sources (n ¼ 306)

Category (description) (Log10 PFU per 100 ml)

n Bacteroides GB-124 Somatic coliphages

Mean Range + (%) Mean Range + (%)

A (River o0.5 km d/s of STW) 24 3.65 4.30–2.48 100 4.32 4.72–3.78 100

B (River o2.0 km d/s of STW) 23 3.12 3.90–2.00 100 4.08 4.60–3.18 100

C (River 42.0 km d/s STW+farms) 90 2.38 3.63–0 56 3.85 4.81–2.30 100

D (River directly d/s of farm) 25 1.15 2.48–0 12 4.35 4.81–2.78 100

E (River close to origin) 4 oDT — 0 3.08 3.36–0 50

(Municipal wastewater) 110 3.92 4.70–2.00 100 4.61 5.01–3.74 100

(Animal samples) 30 oDT — 0 4.20 4.70–2.00 100

+ (%) ¼ Percentage positive isolates; oDT ¼ Below Detection Threshold (1 PFU per ml), STW ¼ Sewage treatment works.

Table 3 – Numbers of faecal bacteria in samples from different sources (n ¼ 306)

Category (description) (Log10 CFU per 100 ml)

n Faecal coliforms Enterococci

Mean Range + (%) Mean Range + (%)

A (River o0.5 km d/s of STW) 24 4.07 4.86–2.60 100 3.61 4.76–2.30 100

B (River o2.0 km d/s of STW) 23 3.82 4.88–2.30 100 3.74 4.84–2.00 100

C (River 42.0 km d/s STW+farms) 90 3.54 4.99–2.48 100 3.21 5.08–2.00 100

D (River directly d/s of farm) 25 4.54 5.24–3.48 100 4.40 5.08–3.38 100

E (River close to origin) 4 3.43 3.72–1.48 100 3.12 3.13–3.10 100

(Municipal wastewater) 110 7.19 7.98–6.18 100 6.37 7.11–4.97 100

(Animal samples) 30 9.08 6.08–10.24 100 7.92 8.89–5.70 100

+ (%) ¼ Percentage positive isolates; STW ¼ Sewage Treatment Works.

WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 3 6 8 3 – 3 6 9 03686

the faeces of cattle, horses, pigs, poultry, rabbits and sheep

(Table 2). This was despite high mean levels of FC and ENT in

these samples (9.08 and 7.92 Log10 CFU/100 ml, respectively)

(Table 3).

3.2. Faecal indicator organisms in river water samples

Microbial load in river water was highly variable as can be

seen in Tables 2 and 3, and Figs. 1–3. Phages infecting GB-124

were present in 100% of samples originating from sampling

points ‘o 0.5 and o 2.0 km downstream of STW’ (mean-

3.39 Log10 PFU/100 ml), but were significantly higher (Student’s

t test; Po 0.01) in those samples taken from the sampling

point ‘o0.5 km downstream of STW’ (mean ¼ 3.65 Log10 PFU/

100 ml). GB-124 phages were also present in 56% of all river

samples originating ‘42.0 km downstream of STW’ (Table 2).

Conversely, GB-124 phages were present at significantly

lower levels (Student’s t-test; Po 0.001) in samples collected

‘directly downstream of a dairy farm’ and were not recovered

in any samples taken ‘close to the river’s origin’ (Table 2 and

Fig. 1). This site is situated on a tributary of the river as it

emerges from an aquifer and is not likely to receive any

appreciable human inputs upstream, though it may be

impacted upon by avian sources and grazing sheep. Interest-

ingly, counts for FC, ENT, and somatic coliphages were

highest in river samples ‘directly downstream of dairy farm’

(mean ¼ 4.54 and 4.40 Log10 CFU/100 ml and 4.35 Log10 PFU/

100 ml, respectively) (Tables 2 and 3). This site is directly

downstream of a bridge over which dairy cattle pass four

times a day for milking. Run-off containing the faeces of

cattle was regularly observed to flow into the stream at this

site (especially after heavy rainfall).

Tables 2 and 3 also show that the mean numbers of somatic

coliphages in river samples ‘o0.5 km downstream from STW’

was twice that of FC, 5 times higher than that of ENT and 5

times higher than that of GB-124 phages. Numbers of somatic

coliphages were found to be consistently higher (several Logs)

than equivalent numbers of GB-124 phages in all sources

tested. These findings are in concordance with the results

reported by other investigators (Tartera and Jofre, 1987;

Tartera et al., 1989; Lucena et al., 1994). Interestingly somatic

coliphages were the most abundant organisms tested for in

river samples impacted by STW and faecal coliforms were the

most abundant of the organisms tested for in river samples

impacted by animal sources. Under current EU legislation

only those river waters designated as ‘inland bathing waters’

are required to meet microbiological water quality standards

(CEC, 1976). However, all river water monitoring sites recorded

ARTICLE IN PRESS

1

10

100

1000

10000

100000

River < 0.5 km

d/sSTW

River < 2.0 km

d/s STW

River > 2.0km

d/s STW

and d/s farms

River directly

d/s farm

Site

Mean

PF

U/1

00M

L

High rainfallLow rainfall

Fig. 2 – Mean numbers of GB-124 phages during both ‘high’ and ‘low’ rainfall in river water samples (n ¼ 166).

1

10

100

1000

10000

100000

River < 0.5 km d/s

STW

River < 2.0 km d/s

STW

River > 2.0km d/s

STW and d/s farms

River directly d/s farm River close to origin

Site

Mean

CF

U a

nd

PF

U/1

00M

LFC

FE

WG5

GB124

Fig. 1 – Mean faecal organism numbers in river water samples (n ¼ 166). FC ¼ faecal coliforms; FE ¼ faecal enterococci; WG-

5 ¼ somatic coliphage; GB-124 ¼ Bacteroides.

WAT E R R E S E A R C H 41 (2007) 3683– 3690 3687

numbers of faecal coliforms and intestinal enterococci, well

in excess of these standards, particularly the sites down-

stream of dairy farm and STW.

The numbers of GB-124 phages in samples ‘o 0.5 km

downstream of STW’ were similar to those of B. fragilis

phages found in Spanish rivers contaminated with human

ARTICLE IN PRESS

1

10

100

1000

10000

100000

River < 0.5 km

d/s STW

River < 2.0 km

d/s STW

River > 2.0km

d/s STW

and d/s farms

River directly

d/s farm

River close

to origin

Site

Mean

PF

U/1

00M

L

High rainfall

Low rainfall

Fig. 3 – Mean numbers of somatic coliphages during both ‘high’ and ‘low’ rainfall in river water samples (n ¼ 166).

WAT E R R E S E A R C H 4 1 ( 2 0 0 7 ) 3 6 8 3 – 3 6 9 03688

sewage (Lucena et al., 1996). Figs. 1 and 2 show a reduction in

the mean numbers of GB-124 phages and somatic coliphages

(with respect to distance downstream of STW), suggesting

that MW is a major input of faecal organisms at these points

on the river, under the flow conditions investigated.

3.3. ‘High’ and ‘low’ rainfall

In order to evaluate the effect of rainfall on the numbers of

GB-124 phages present in river waters, the sampling occa-

sions were retrospectively divided into ‘high’ and ‘low’

rainfall. An arbitrary ‘high/low’ rainfall level was then

established based on the combined 12 and 24 h antecedent

rainfall values (measured at four weather stations within the

catchment). According to this method, approximately a third

of the sampling occasions could be regarded as ‘high’ rainfall

events (410 mm per day), and two-thirds as ‘low’ rainfall

events (p10 mm per day). Rainfall data (not shown) was

supplied by the UK Environment Agency (Pers. Comm., 2006).

As expected, mean levels of all the target organisms

increased during ‘high’ rainfall events, typically by a factor

of approximately 1.0 Log10 (for FC and ENT) and 0.5 Log10 (for

somatic coliphages and GB-124 phages), compared with the

mean ‘low’ rainfall values. The highest levels of FC and ENT

(5.24 and 5.08 Log10 CFU/100 ml, respectively) and somatic

coliphages and GB-124 phages (4.81 and 4.30 Log10 PFU/100 ml,

respectively) were recorded on the 25 and 27 July, 2005 and

coincided with the highest antecedent rainfall figs

(13.9–23.7 mm) (Tables 2 and 3). The mean number of phages

infecting GB-124 present in river samples ‘o0.5 km down-

stream of STW’ varied from 3.37 (Log10)PFU/100 ml during

‘low’ rainfall, to 3.89 (Log 10) PFU/100 ml during ‘high’ rainfall

conditions (Fig. 2). The levels of GB-124 phages present in

river samples ‘o0.5 km downstream from STW’ were greater

during ‘high’ rainfall conditions than those found at the same

site during ‘low’ rainfall conditions (Student’s t-test; Po0.05).

The number of phages infecting strain GB-124 at each of the

sites ‘40.5 km downstream of the STW’ did not vary

significantly between ‘low’ and ‘high’ rainfall events (Fig. 2).

Fig. 2 shows that GB-124 phages were not detected at the site

‘directly downstream of the dairy farm’ during ‘low’ rainfall

conditions and were only detected in very low numbers at

this site (Po0.001) during ‘high’ rainfall conditions.

Fig. 3 shows that the number of somatic coliphages

detected in samples ‘directly downstream of the dairy farm’

and samples ‘o2.0 km downstream of STW’ both differ

significantly between ‘high’ and ‘low’ rainfall conditions.

The highest numbers of somatic coliphages (4.81 and

4.72 Log10 PFU/100 ml) were observed in samples ‘directly

downstream of the dairy farm’ and samples ‘o 0.5 km

downstream of STW’, respectively, during ‘high’ rainfall

conditions (Table 2). Most importantly, phages infecting strain

GB-124 appeared to be present in sufficiently high numbers

during both ‘high’ and ‘low’ rainfall conditions, to allow their

direct detection in 1 ml of river water.

4. Discussion

Our data shows that the relative numbers of faecal indicator

organisms appear to differ between municipal wastewaters,

runoff from animals and river waters impacted by both these

sources. For instance, the number of FC exceeded the number

of somatic coliphages in MW, animal faeces and at sites

directly downstream of farms but was lower than the number

of somatic coliphages in river waters downstream of STW.

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WAT E R R E S E A R C H 41 (2007) 3683– 3690 3689

A similar pattern was observed by Bell (1976), Borrego et al.

(1987) and Skraber et al. (2002). It has been demonstrated that

differences in water temperature, levels of predation, adsorp-

tion to sediments and the effects of UV irradiation can

all influence the survival of faecal organisms in aquatic

environments (Mancini, 1978; Fujioka et al., 1981; Wilkinson

et al., 1995).

Whilst numbers of faecal indicator bacteria were signifi-

cantly lower in 100 ml of river water than those found in MW

and animal faeces (per 100 ml or 100 g), phages were present

in contaminated river waters at similar numbers to those

found in untreated MW and animal faeces. This would

suggest that phages are more resilient to inactivation

processes than traditional bacterial indicators and the

observation is in agreement with the data of Duran et al.

(2002). Phages may also be released during destruction of the

host cells during wastewater treatment. Ewart and Paynter

(1980) found that phage numbers were consistently higher in

reactor effluent and mixed liquor compared with inflowing

sewage. Duran et al. (2002) showed that somatic coliphages

and phages infecting B. fragilis are capable of surviving for

significantly longer periods in river waters than are faecal

coliforms and intestinal enterococci. However, research by

Bradley et al. (1999) also suggests that it is possible that

somatic coliphages are able to attack E. coli host strains and be

subsequently released outside of the gastrointestinal tract.

Conversely, Tartera et al. (1989), demonstrated that B. fragilis

phages can only replicate in metabolically active host cells

and therefore replication of the phages in the environment is

highly unlikely. Bacteriophages of Bacteroides spp. may there-

fore provide useful information on longer-term pollution.

The host specificity of Bacteroides GB-124 phages and the

high numbers of somatic coliphages present in river waters

impacted by MW also support previous research findings

(Tartera et al., 1989; Payment et al., 1991; Skraber et al., 2002),

which suggest that phages may offer a better indication of the

presence of enteric viruses than current bacterial indicators.

However, further research is necessary in order to determine

both the relationship between GB-124 phages and human

enteric viruses and the numbers of GB-124 excreted by

individual humans. The presence of GB-124 phages in the

final effluent from all the STW (including those with

population equivalents as low as 28) suggests that a

significant proportion of the human population in the study

area is excreting these phages. A previous study of 600

healthy adults found that 34% of them were positive for

somatic coliphages (Furuse, 1987).

This study has provided useful information on the numbers

and distribution of phages infecting strains GB-124 and

somatic coliphages found in UK river waters, MW and animal

faeces. It should be noted that Bacteroides strain GB-124 used

in this study also produced less turbid plaques than other

Bacteroides host strains used in previous UK FST investigations

(Blanch et al., 2006), making it quicker and easier to interpret

the results. Interestingly, phages infecting GB-124 were

detected in two MW samples from the north of England and

from Denmark, and more recently the authors have also

detected phages to GB-124 in MW from Spain, Sicily and

Uganda (unpublished data). Further work is currently under-

way to determine whether GB-124 can distinguish human and

non-human sources of faecal pollution in other parts of the

world as successfully as it has in this study.

The Bacteroides (GB-124) phage assay is also being used in an

extended study of the same catchment in which additional

water quality parameters, and meteorological and geographi-

cal data will be included in a GIS. The aim of the extended

project is to produce a model to predict the impact of human

and animal faeces on a river catchment under a range of

meteorological conditions. It is anticipated that this informa-

tion will help to predict faecal inputs thereby allowing more

effective sampling of river catchments and coastal waters in

the future.

5. Conclusions

1.

High levels of GB-124 phages within MW and river samples

impacted by partially treated MW, and their absence in

animal faeces suggests that GB-124 may be used as a

marker of human pollution.

2.

The technique is low-cost, rapid (requiring no sample

concentration) and is database (or ‘library’) independent.

3.

Initial results suggest that it may also be possible to use

GB-124 to determine successfully sources of faecal pollu-

tion in other parts of the world, although further research

is necessary.

4.

In addition to surface waters, GB-124 could also be used to

identify and assess sources of faecal pollution present in

groundwater, shellfish, soil, and sediments.

5.

As such GB-124 could assist in the management of micro-

bial water quality through the identification and remedia-

tion of point and diffuse sources of faecal pollution.

Acknowledgements

This study was supported by the EU INTERREG IIIA Regional

Development Fund (162/025/263) ‘‘Advanced Monitoring and

Control of Microbial Water Quality (AMACOM)’’. The authors

would like to acknowledge the cooperation of Southern Water

Scientific Services, the Sussex Ouse Conservation Society

(SOCS) and the UK Environment Agency (Southern Region).

Thanks are also expressed to Professor Joan Jofre of the

University of Barcelona for his support and advice and to

Søren Bastholm of Aalborg University (Denmark), for the

provision of municipal wastewater samples.

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