“We need a better understanding of how water and different types of pollution move and interact, while passing through

river basins towards the sea”.

Source: UK Environment Agency 2005

Introduction to MST

Outline

• Composition of faecal pollution • What is sewage? • Transmission of faecal pollution • Sources and detection of faecal contaminants •Why understanding sources of pollution is important •Microbial Source Tracking (MST) • Current research and recent developments • Where and what next?

What is sewage?

Organic particles

Micro-organisms (pathogens) Inorganic particles

Emulsions

Water

Gases (H2S, CO2, CH4 etc)

Toxins

Soluble organic material

Animals

Macrosolids

One gramme of faeces may contain: 10,000,000 viruses 1,000,000 bacteria 1,000 parasite cysts 100 parasite eggs

Micro-organisms (pathogens)

Water quality is currently monitored in EU using two groups of faecal indicator organisms

Isolated from the faeces of all warm-blooded mammals

Indicate presence of faecal contamination

• Faecal coliforms

• Intestinal enterococci

However, they do not indicate where the pollution is coming from and can take up to 48 hrs to get result!

Major Sources of Faecal Pollution

RSPB survey of five most common species of gull estimates 543,500 breeding pairs in UK.

Equivalent impact on water quality as two human adults (faecal bacterial load per day).

Seagulls (Larus spp.)

Equivalent to over 2 million humans (or entire population of Wales) defecating every day!

Understanding sources of contamination is important because:

• It helps target mitigation methods more effectively

• It helps establish responsibility/liability

• It improves understanding of the risks to human health

• It improves understanding of the risks to the environment (habitat loss, biodiversity etc)

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Microbial Source Tracking (MST)

Seagull

Pig

Cow

Deer

Human

Dog

Livestock

Wildlife

Human

Decr

easi

ng r

eso

lution

Theoretical scenarios describing levels of discriminatory resolution possible with source tracking technologies

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2

Non-human

Human

Non-human

Human

Decr

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lution

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4

Microbial Source Tracking (MST)

Long-chain alkylbenzenes (detergents)

Antibiotic resistance profiling

Target organisms (Bifidobacteria, Clostridium)

Molecular methods (PCR, PFGE, MLST, Ribotyping)

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Caffeine

Carbon source utilisation

Fluorescent whitening agents

Ratio Faecal coliforms

Faecal Strep

FC:FS

Bacteroides

• Anaerobic bacteria that constitute the most substantial portion of the human gut flora

• Bacteroides don’t survive for long outside the human body (e.g. in the environment)

• However viruses (phage) which infect Bacteroides do survive outside the human body

• Present at high numbers in the faeces of humans (1010-1011 cells/gram)

• Presence of these phages in wastewaters, and in surface waters, shellfish, and sediments suggests human faecal contamination

Bacteriophage

• Have very narrow host ranges

• More persistent in the environment than bacteria they infect

• Virus capable of infecting bacteria

• Bacteriophage comes from Greek “Bacteria-eater”

• Potential indicators source

• Capable of surviving sewage treatment processes

Bacteria

Bacteriophage

• Capable of bio-accumulating in shellfish

Method of action Bacteriophage Lunar landing craft

(b) Injection of phage DNA into cell

(d) Multiple phage progeny produced

(c) Phage synthesis begins

(e) Cell lysed (burst) and phage released

(a) Attachment to cell wall

Bacteroides strain GB-124 was isolated from influent at Scaynes Hill STW, East Sussex in early 2005.

X

Population Equivalent* = 37,327 *Courtesy of Southern Water

Scaynes Hill sewage treatment works

X = Haywards Heath

Why Haywards Heath?

Enumeration of bacteriophages*

Methodology:

Grow bacterial host in broth (Bacteroides GB-124)

Step 1

Results expressed as plaque forming units (PFU) per 100 ml

Mix host with sample in semi-solid agar and pour onto agar plate

Step 2

*In accordance with ISO 10705-4. Water Quality. Detection and Enumeration of Bacteriophages-Part 4.

Incubate anaerobically for 18-24hrs at 37 oC, count zones of lysis (plaques)

Step 3

Municipal wastewaters and faecal material from pooled animal sources were tested for the presence of a range of faecal organisms/indicators including Bacteroides GB-124

Spatham Lane (< 0.5km downstream of Ditchling STW)

Wales Farm (directly downstream of farm)

Log10 PFU/100ml

Description n Mean Range % Positive

STW (final effluent) 45 sites 152 4.20 4.70 - 2.00 100

River < 0.5 km d/s of STW 55 3.65 4.30 - 2.48 100

River < 2.0 km d/s of STW 55 3.12 3.90 - 2.00 100

River >2.0 km d/s STW 194 2.38 3.63 - <DT 56

River directly d/s of farm no

STW

55 1.15 2.48 - <DT 12

River close to origin 25 ND - 0

Animal samples 45 ND - 0

Table 1. Numbers of bacteriophages infecting GB-124 in samples from different sources (n = 581)

STW = Sewage treatment works; PFU= Plaque Forming Units; ND= Not Detected; <DT = Below Detection Threshold (1 PFU/ml)

Fig 1. Mean levels of each parameter during different stages of treatment at ‘Site A’ (n=30)

• Do GB-124 phage bio-accumulate in bivalve molluscs? • If so where? • Is there a seasonality to their presence? • Is their presence linked to that of human pathogens? • If so what does it all mean?

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18/11/10 26/2/11 6/6/11 14/9/11 23/12/11 1/4/12

PFU

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Date

GB124 phage and Nv concentrations in mussels from the R. Ouse (Feb 2011 to Jan 2012)

GB124 Mussel Flesh PFU/100 g

NOROVIRUS copies/g

R² = 0.4452

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14000

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Nv

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GB124 phage PFU/100g

GB124 phage (mussel flesh) vs. Norovirus (Nv) copies/g

R² = 0.8105

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GB124 phage PFU/100g

GB124 phage (mussel flesh) vs. Norovirus (Nv) copies/g

Extreme event!

• Strong human signal • Elevated risk to human health (presence of human viral pathogens)

Results

• GB-124 phages present in 100% of sewage samples from 10 countries (n = >200)

• Phages infecting GB124 were not detected in any animal samples and were seldom found downstream of farms

• Highest counts found in wastewater & river samples downstream of STW (>500 PFU/ml)

• All samples positive for pathogens (adenovirus and/or norovirus) were also positive for GB-124 phages

• GB-124 phages, pathogens (adenovirus and norovirus) absent from all the non-human faecal samples

A toolbox approach Different methods for different applications (e.g. Drinking water, shellfish waters)

No single method suitable for all situations

Different cost implications

Depends on the level of detail we require (resolution)

Different methods for different geographical regions

Conclusion

Library independent (potentially culture independent)

Quantitative (results expressed PFU/100ml)

Low-cost (compared to other MST methods)

Possible to analyse multiple samples per day (e.g. 100+)

Ease of detection (method can be carried out in the field)

Phage behaviour more similar to enteric viruses than

traditional bacterial indicators (better indicator of risk)

Thanks for your attention!