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I N V E S T I N G I N A U S T R A L I A ’ S H E A L T H

Review of ColiformsAs Microbial Indicators of Drinking Water Quality

Review of C

oliforms

As M

icrobial Indicators of Drinking W

ater Quality

RECOMMENDATIONS TO CHANGE THE USE OF COLIFORMS AS MICROBIAL INDICATORS OF DRINKING WATER QUALITY

Dr Melita Stevens Melbourne Water Corporation

Dr Nicholas Ashbolt

University of New South Wales

Dr David Cunliffe Department of Human Services, South Australia

Endorsed 10–11 April 2003

ISBN: 1864961651, Online ISBN: 1864961597

Material included in this document may be freely reproduced provided that it is accompanied by an acknowledgment stating the full title of the document, the National Health and Medical Research Council and the date of release.

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Front Cover: Image of Coliforms courtesy of Centre for Microscopy and Microanalysis, The University of Queensland.

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MICROBIAL INDICATORS OF DRINKING WATER QUALITY

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TABLE OF CONTENTS

EXECUTIVE SUMMARY VII

BACKGROUND VIII

1. INTRODUCTION 1

2. MICROBIAL INDICATORS OF WATER QUALITY 3

2.1 What are Microbial Indicators? 3

2.2 Total Coliforms 4

2.3 Escherichia coli (E. coli) 5

3. USE OF BACTERIAL INDICATORS OF WATER QUALITY 7

4. THE CHANGING FACE OF COLIFORMS AND INDICATORS 11

4.1 Changes in Coliform Definition 11

4.2 Molecular Methods for Detection of Microbial Indicators 12

5. TOTAL COLIFORMS AS INDICATORS 15

5.1 Growth in Distribution Systems 15

5.2 Normal Soil and Water Inhabitants 16

5.3 Waterborne Disease Outbreaks 16

6. . ALTERNATIVES TO TOTAL COLIFORMS 19

6.1 Current and Future Use of Coliforms 19

6.2 Water Quality Risk Management Approach 21

6.3 Escherichia coli and Enterococci – Key Faecal Indicators 23

6.4 Clostridium perfringens 24

6.5 Bacteriophages 24

6.6 Summary 25

7. CONCLUSIONS 27

8. RECOMMENDATIONS 29

9. REFERENCES 31


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APPENDIX A 37

Enzyme-based Methods for The Detection of Microbial Indicators 37

APPENDIX B 38

Molecular Methods for the Detection of Microbial Indicators 38

PROCESS REPORT 41


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EXECUTIVE SUMMARY

Assurance that water is microbially safe for drinking has traditionally been determined by measuring bacterial indicators of water quality, most commonly total coliforms and Escherichia coli (E. coli). The international water industry is questioning whether continued reliance on these indicators is sufficient to ensure microbial water quality and it has begun to adopt a more holistic approach to delivering safe water, through the development and adoption of risk management plans for drinking water quality.

In Australia, the ongoing revision of the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996), includes the development and trialing of a risk management framework for drinking water quality, and the development of the Third Edition of the WHO Guidelines for Drinking-water Quality is similarly focussed on the use of risk management with less reliance on end-point testing.

As a component of a risk-based approach to water quality management, measures used to verify water quality must support the risk management system, and provide useful information to water suppliers. Total coliforms have been shown to be a poor parameter for measuring the potential for faecal contamination of drinking water due to their presence as normal inhabitants of soil and water environments, their ability to grow in drinking water distribution systems and their inconsistent presence in water supplies during outbreaks of waterborne disease. These factors mean that it is difficult to interpret the sanitary significance of their presence (in the absence of E. coli) or have confidence in water quality in their absence.

The presence of E. coli in drinking water is still considered to indicate that faecal contamination of water has occurred. E. coli monitoring of drinking water as a verification measure is a useful tool within a risk management approach to water quality. There are a number of other useful indicators, both microbial and physical, which can be used to monitor both drinking water system operation and performance, and which provide better support for system management than total coliforms.

The key recommendations of this report are that:

1. total coliforms be removed as an indicator of faecal contamination in the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996); and,

2. E. coli be the primary indicator of faecal contamination in the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996).


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BACKGROUND

Almost 150 years ago, at a time when cholera and typhoid were common, the fact that water was a vehicle for disease was first proved (Snow, 1855). During the late 1800s and early 1900s, scientific knowledge about the nature and causes of disease increased rapidly and there was major focus on public health reform. During this period, techniques to identify and enumerate causative agents of disease were developed.

For more than 100 years, the microbial safety of drinking water has primarily been determined by testing for bacterial ‘indicators’ of faecal pollution, mainly Escherichia coli (E coli) (or alternatively thermotolerant (faecal) coliforms) and total coliforms. These indicators are used to assess the potential public health risk of drinking water, and their presence or absence are key elements of most drinking water quality guidelines, water supply operating licences and agreements between bulk water suppliers and retail water companies.

The rationale presented for testing water for total coliforms is that this functional group of bacteria is present in large numbers in the gut of humans and other warm-blooded animals. This means that if water is polluted by faeces, coliforms can be detected even after extensive dilution. However, the total coliform group lacks specificity as many of them can exist and proliferate in both soil and water environments, as well as drinking water distribution systems. The presence of total coliforms in water may be a result of natural processes and not of faecal pollution and the health significance of their presence in drinking water is not clear. Changes in methods for detecting total coliforms over the last 10 years have made the interpretation of their significance more difficult by broadening the functional definition of the group and incorporating more environmental species.

Of the total coliform group, E. coli is the most numerous in mammalian faeces and is considered the most specific indicator of faecal pollution. The presence of E. coli in water is still considered to represent the presence of faecal pollution and is used to indicate that pathogenic bacteria, viruses and protozoa may also be present. The drawback of relying on E. coli is that it is a poor indicator for the presence of viruses and parasitic protozoa that can survive for much longer periods than the bacterial indicator.

The relevance of testing for total coliforms and E. coli has been questioned since its introduction and it is again under challenge. In addition to changes in methodology, a new challenge has been initiated by a move to a more holistic and preventive approach to managing water quality. This new approach includes identifying appropriate monitoring strategies to measure the effectiveness of water treatment processes and the safety of drinking water. With increased attention being placed on non-bacterial pathogens including Cryptosporidium and viruses, effective testing of microbial water quality clearly requires more than simple testing for total coliforms and E. coli. Turbidity of filtered drinking water and measures of disinfection (such as C.t values) are being increasingly used as indicators of microbial quality, and confidence in the safety of water supply is being underpinned by comprehensive risk management plans. After so many years, is it time for a complete change or do the old indicators still have some merit?


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1. INTRODUCTION

One of the primary concerns of water authorities is to ensure that the drinking water they supply does not pose an unacceptable health risk to consumers. The safety of drinking water is generally monitored in a number of ways:

1. constituents of drinking water (such as chemicals and microbes) which can compromise human health can be measured directly;

2. barriers designed to protect water quality (such as catchment activities, filtration and disinfection) can be monitored; and

3. indicators of water quality (such as turbidity) can be measured to assess the potential presence of broad groups of parameters.

There are two major reasons for monitoring drinking water quality:

• to determine if the water supply system is being operated correctly, implying that the water is safe for consumers (Primary Assessment); and

• proof that the water was safe after it was supplied. This includes monitoring for compliance (Verification).

To address microbial health risk, primary assessment can only be achieved by monitoring source water and barriers. Monitoring treated water in distribution systems for microorganisms is a means of verification only.

Of the three methods used to assess drinking water listed above, indicators are most often used to monitor microbial water quality, as direct measurement of all pathogenic microorganisms is difficult, expensive and time consuming. In most cases risks from chemicals in drinking water are due to chronic exposure meaning that there is no urgency between sampling, testing and acting on results. This is not the case with the health risk from incidents of microbial pollution, which are generally short-lived with disease becoming apparent within a short period of time.

Focus on the use of barrier monitoring has increased as the water industry adopts complete system risk management, including identification of key elements which can be monitored to give useful information on potential health risk. Until these methodologies have become better established, the use of bacterial indicators for assessing water quality will remain an integral component of drinking water management. Indicators of water quality will be an important component of the verification step in risk management systems for drinking water.

The way in which bacterial indicators are used as a measure of microbial water quality has been questioned for some time, but this has increased with the development of more sensitive microbial techniques and increased understanding about the nature of environmental microorganisms, human pathogens and disease.

This paper describes the concept of indicator microorganisms and details the rationale behind their use. Current indicators are reviewed and their advantages and disadvantages discussed and alternative indicators are evaluated. The major purpose of this paper is to generate discussion about the relevance of current bacterial indicators of water quality as recommended in the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996) and to recommend changes to the current Guidelines. This paper was developed for consideration by operational and technical staff in State and Territory health authorities and water supply agencies.


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The alternative indicators discussed in this paper have not been used or studied as extensively as total coliforms and E. coli. There is limited information on their environmental significance, their presence in drinking water systems and relationship to waterborne disease outbreaks. Public consultation demonstrated that there was general agreement for removal of total coliforms as an indicator of faecal contamination, with E. coli supported as the primary indicator.

A regulatory impact statement (RIS) including a cost-benefit evaluation of regulatory alternatives, was not undertaken as part of this review. This document was developed to support consideration of microbial indicator organisms in the Australian Drinking Water Guidelines (ADWG). The Productivity Commission’s Office of Regulation Review has previously determined that the NHMRC is not required to undertake an RIS on the ADWG as the Guidelines do not have regulatory status (Productivity Commission, 2000). Implementation of the ADWG by the States and Territories is at the discretion of the State and Territory Health Departments, usually in consultation with water suppliers and should include an appropriate economic analysis prior to implementation.


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2. MICROBIAL INDICATORS OF WATER QUALITY

2.1 WHAT ARE MICROBIAL INDICATORS?

Microorganisms that can cause disease are called pathogens. Pathogens that can be spread through drinking water and cause waterborne disease include bacteria, viruses, and protozoa. The number of different types of pathogens that can be present in water as a result of pollution with human or animal faeces is very large and it is not possible to test water samples for each specific pathogen. For example, more than 100 types of enteric viruses have been isolated from human faeces and from sewage (Payment, 1993). Isolation and identification of some of these viruses is very difficult, or not currently possible. If these viruses or other pathogens are present in water as a result of faecal pollution, a measure is required which will alert water managers to their presence.

An indicator of microbial water quality is generally something (not necessarily bacteria), which has entered the water at the same time as faeces, but is easier to measure than the full range of microorganisms which pose the health risk. There are several qualities that are desirable for a useful water quality indicator (NHMRC-ARMCANZ, 1996; WHO, 1996):

• universally present in the faeces of humans and warm-blooded animals in large numbers

• readily detected by simple methods

• does not grow in natural waters, the general environment or water distribution systems

• persistence in water and the extent to which it is removed by water treatment is similar to those of waterborne pathogens.

The concept of coliforms as bacterial indicators of microbial water quality is based on the premise that because coliforms are present in high numbers in the faeces of humans and other warm-blooded animals, if faecal pollution has entered drinking water, it is likely that these bacteria will be present, even after significant dilution. With few exceptions, coliforms themselves are not considered to be a health risk, but their presence indicates that faecal pollution may have occurred and pathogens might be present as a result.

‘Coliform’ was the term first used in the 1880s to describe rod-shaped bacteria isolated from human faeces. The coliform group of bacteria, is a functionally-related group which all belong to a single taxonomic family (Enterobacteriaceae) and comprises many genera and species. Box 1 contains an example of the relationship between family, genera and species for coliforms. There are other genera in the Enterobacteriaceae family, such as Salmonella and Shigella, that are not considered coliforms.


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Box 1 Family, Genera and Species of Some Common Coliforms

Family Genera Species

Enterobacteriaceae Escherichia Escherichia coli

(E. coli)

Klebsiella Klebsiella pneumoniae

(K. pneumoniae)

Enterobacter Enterobacter amnigenus

(E. amnigenus)

Citrobacter Citrobacter freundii

(C. freundii)

Of the coliforms normally present in the gut of warm-blooded animals, E. coli is the most numerous and is also the only coliform which rarely grows in the environment. Box 2 shows the distribution of coliforms present in human and animal faeces.

Box 2 Distribution of Coliform Genera in Human and Animal Faeces(1)

Sample Type % of Total Coliforms

E. coli Klebsiella spp. Enterobacter/ Reference Citrobacter spp.

Human faeces 96.8 1.5 1.7 Dufour (1977)

94.1 5.9 Allen and Edberg (1995)

Animal faeces 94 2 4 Dufour (1977)

92.6 7.4 Allen and Edberg (1995)

Notes : (1) Once faeces leaves the body and makes its way down the sewer, the proportions of coliforms that are E. coli drops to about 30% as the other coliforms start to grow (Geldreich, 1978).

2.2 TOTAL COLIFORMS

The total coliform group of bacteria was originally used as a surrogate for E. coli (the name coming from ‘coli-form’ or like) which, in turn, was considered to show faecal pollution. This was due to three reasons:

1. coliform bacteria were readily isolated from human faecal material and water that had been impacted by pollution (these included E. coli and other coliforms, some of which also live naturally in soil and water environments);

2. most of the coliforms recovered from human faeces were E. coli, and it was assumed that the presence of total coliforms reflected the presence of E. coli; and

3. the technology available to easily distinguish E. coli from other coliforms in the early 1900s was not suitable for routine analysis.

As a result, total coliforms were adopted and considered to be equivalent to E. coli until more specific and rapid methods became available. It was not until 1948 that the more specific and well-known 48 hour test for thermotolerant (faecal) coliforms was accepted. Despite the development of this and other specific methods, the use of total coliforms was so commonplace that they were not dropped in favour of E. coli or thermotolerant coliforms, but rather have remained co-indicators.


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Originally total coliform bacteria were considered to be from four genera of the family Enterobacteriaceae that could all ferment lactose. These genera were Escherichia, Klebsiella, Enterobacter and Citrobacter. Of the total coliforms present in the human gut, Escherichia coli (E. coli) represents the majority of the population (see Box 2). Total coliforms represent only about 1% of the total population of bacteria in human faeces in concentrations of about 109 bacteria per gram (Brenner et al., 1982).

It is widely accepted that the total coliform group of bacteria is diverse and they can be considered normal inhabitants of many soil and water environments which have not been impacted by faecal pollution. Even though the presence of E. coli is considered an appropriate and specific indicator of faecal pollution, uncertainty surrounds the use of total coliforms as a health indicator.

As microbiological understanding about the nature of disease and the pathogens responsible increases, techniques have been developed to isolate and enumerate pathogenic viruses and protozoa from water. These techniques, however, are not sensible, specific, reliable, reproducible or inexpensive enough to replace the use of bacterial indicators.

2.3 ESCHERICHIA COLI (E. COLI)

More than 100 years ago scientists discovered that human faeces contained bacteria which if present in water, indicated that the water was not safe to drink. Escherich in 1885 observed 2 types of organisms present in faeces, one of which he named Bacterium coli (B. coli, which is now called Escherichia coli) and the concept that the presence of B. coli implied pollution of water was readily adopted. It is recorded that the concept of “indicators” had already been suggested in 1880 by van Fritsch based on his observations of Klebsiellae in human faeces that were also present in water (Hendricks, 1978).

Initially it was very difficult to distinguish B. coli from other coliform bacteria in water and faeces, so methods were developed to recover all coliform bacteria, and more detailed and lengthy analyses were carried out to confirm if any of the recovered coliforms were B. coli. Water bacteriologists for the next 50 years concentrated on developing these techniques to confirm the presence of B. coli in water and tell it apart from other gut bacteria. By the turn of the 20th century, methods were available that could distinguish B. coli from the bacteria that caused typhoid (Salmonella typhi), and it was known that B. coli produced acid and gas from lactose, whereas Salmonella typhi did not. The techniques founded in the late 1800s and early 1900s are still widely used to determine if faecal pollution of drinking water has occurred.


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3. USE OF BACTERIAL INDICATORS OF WATER QUALITY

Most international drinking water quality guidelines and standards include bacterial indicators as a measure of microbial water quality, and for compliance reporting. The two major international bodies, the United States Environmental Protection Agency (USEPA), and the European Union (EU) both include E. coli as a mandatory microbial indicator, and the USEPA regulates for total coliforms, via the Total Coliform Rule.

Most drinking water guidelines also refer to the use of total estimates of bacterial numbers in water. This measure is generally called ‘total heterotrophic plate count’ (HPC) or ‘standard plate count bacteria’, and is considered to represent the general cleanliness of a drinking water. As the HPC is not considered indicative of a potential health risk, these bacteria are not generally considered as a compliance measure, rather their numbers are monitored to understand changes in a drinking water system over time and to alert operators to increases in general bacterial numbers.

In response to a growing understanding and acceptance of the limitations of total coliforms, there has been a change of focus in Europe. In 1998, the EU, removed total coliforms as a mandatory primary indicator and added enterococci. In the EU standards, total coliforms are included with criteria whose presence can be negotiated with relevant Member State health departments. The relevant EU Legislation sections are shown in Box 3 and the USEPA Total Coliform Rule is described in Box 4, with the Australian Drinking Water Guidelines recommendations in Box 5.

The World Health Organization (WHO) provides extensive guidance for countries to develop local drinking water guidelines and standards. The Second Edition of the WHO Guidelines for Drinking-water Quality (WHO, 1993) include recommendations for assessment of microbial water quality based on the detection of E. coli and total coliforms. Volume 2 of the Second Edition (WHO, 1996) however, discusses in detail the inadequacies of total coliforms as an indicator of faecal pollution and debates the merits of alternative indicators such as enterococci and sulfite-reducing clostridia. The WHO Guidelines for Drinking-water Quality and the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996) are currently being updated to emphasise total system risk management with less focus on parametric values for acceptable water quality. The WHO is considering removing total coliforms as a primary compliance parameter in the revision for the Third Edition of the Guidelines for Drinking-water Quality.

The New Zealand Ministry of Health released their Drinking Water Standards for New Zealand 2000 (NZMoH, 2000) in August 2000. These revised drinking water standards contain only E. coli as a bacterial indicator of faecal pollution, and no longer rely on faecal coliforms or total coliforms. The rationale for the move to E. coli is based on the acknowledgement that both total coliforms and faecal coliforms can be found in natural waters and their presence in drinking water does not necessarily indicate a health risk.

Box 3 Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption

Relevant Tables:

Annex I: PARAMETERS AND PARAMETRIC VALUES

Part A Microbial parameters

Parameter Parametric Value (number/100 mL)

Escherichia coli (E. coli) 0

Enterococci 0

Part C Indicator parameters*

Parameter Parametric value Unit

Colony Count no abnormal change colonies/mL

Coliform Bacteria 0 numbers/100 mL

*Indicator parameters also include aesthetic parameters such as colour, conductivity, chloride and taste and odour.

Relevant Articles:

(189C, 14) Whereas a balance should be struck to prevent both microbial and chemical risks; whereas to that end, and in the light of a future review of the parametric values, the establishment of parametric values applicable to water intended for human consumption should be based on public-health considerations and on a method of assessing risk.

(189C, 27) Whereas, in the event of non-compliance with a parameter which has an indicator function, the Member State concerned must consider whether that non-compliance poses any risk to human health; whereas it should take remedial action to restore the quality of the water where that is necessary to protect human health.

Article 5 (2) The values set in accordance with paragraph 1 shall not be less stringent than those set out in Annex I. As regards the parameters set out in Annex I, Part C, the values need to be fixed only for monitoring purposes and for the fulfilment of the obligations imposed in Article 8.

Article 8 (6) In the event of non-compliance with the parametric values or with the specifications set out in Annex I, Part C, Member States will consider whether that non-compliance poses any risk to human health. They shall take remedial action to restore the quality of the water where that is necessary to protect human health.

Box 4 USEPA Total Coliform Rule

The Total Coliform Rule (TCR) is part of the USEPA Safe Drinking Water Act (SDWA) and was effective on 31 December 1990. The TCR sets both health goals (MCLGs) and legal limits (MCLs) for total coliforms. The Rule states that:

Systems must not find coliforms in more than 5% of samples. When a system finds coliforms, the system must collect a set of repeat samples within 24 hours. When a repeat sample tests positive for total coliforms, it must also be analysed for faecal coliforms and E. coli. A positive result to this last test signifies an acute MCL violation, which necessitates rapid state and public notification.


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Box 5 Australian Drinking Water Guidelines (1996)

Guidelines for Microbial Quality

Thermotolerant coliforms (or alternatively E. coli). 0/100 mL

Total coliforms 0/100 mL

Chapter 2 Microbial Quality of Drinking Water

Section 2.8 System Performance (Paragraph 4)

The performance of a system is judged by the number of times over a 12 month period that thermotolerant coliforms (or alternatively E. coli) and coliforms are detected in routine samples representative of water supplied to consumers.

For samples representative of the quality of water supplied to consumers, performance can be regarded as satisfactory if over the preceding 12 months:

• at least the minimum number of routine samples has been tested for indicator microorganisms;

and

• at least 98% of the scheduled samples (as distinct from repeat or special purpose samples) contain no thermotolerant coliforms (or alternatively E. coli);

and

• at least 95% of scheduled samples (as distinct from repeat or special purpose samples) contain no coliforms;

EXCEPT THAT

A higher level of coliform contamination might be tolerated in a particular area under certain conditions. These conditions should include –

– the system meets the guideline for thermotolerant coliforms; and

– that the water authority can satisfy the appropriate health authority that the coliforms are unlikely to be of faecal origin (based on careful evaluation of their species identity); and

– that there is a level of monitoring sufficient to detect any change in the pattern of coliform occurrence (species composition and density); and

– that there is a direct monitoring of the occurrence of pathogenic microorganisms as the health authority may select to ensure that the coliform level does not represent a risk to public health; and

– that agreed levels of service for total coliforms are negotiated with the appropriate health authority and the consumers.


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4. THE CHANGING FACE OF COLIFORMS AND INDICATORS

4.1 CHANGES IN COLIFORM DEFINITION

The last two decades in microbiology have seen a move away from selective growth media-based recovery methods for faecal bacteria to enzymatic and molecular methods. With these advances, the definition of what is considered a coliform has expanded, leading to increased scrutiny of the role that total coliforms play in water quality assessment and the validity of the information assumed by their presence or absence in drinking water systems.

Pre 1994 – acid and gas from lactose

Until the 1990s it was accepted that a coliform was a member of the Enterobacteriaceae, which displayed the biochemical characteristics of acid and gas production from lactose within 24–48 hours at 36±2°C. Thermotolerant or faecal coliforms were those that fitted the basic definition, but were able to grow and ferment lactose at 44.5±0.2°C. The vast majority of thermotolerant coliforms are E. coli and to a lesser extent Klebsiella, Enterobacter and Citrobacter. E coli are differentiated by their thermotolerance plus the ability to produce indole from tryptophan.

Report 71 (1994) – acid only from lactose

In 1994, the sixth edition of the UK, ‘Bacteriological Examination of Drinking Water Supplies 1982’ was published (HMSO, 1994). This report is referred to as Report 71. Report 71 altered a component of the biochemical definition of a coliform from:

acid and gas production from lactose

to

acid-only production from lactose.

This change in definition resulted in an increase in the number of bacterial species considered to be coliforms. Species of Enterobacter and Citrobacter, which do not produce gas from lactose were also included in the new definition. The ongoing review of the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996) has proposed adoption of this coliform definition.

Current and Future - presence of specific enzymes

With the advent of new technologies for bacterial analyses, the working definition of a coliform has again changed. Lactose fermentation is one of the key criteria in the coliform definition and fermentation of lactose is determined, in part, by the presence of a specific enzyme, ß-galactosidase. The presence of ß-galactosidase in a member of the Enterobacteriaceae is considered specific to coliforms. Many water companies in the UK, USA, Europe and Australasia use commercial kits for total coliform analyses based on specific enzymes. The USEPA has adopted this technology and the associated coliform definition. It is understood that the next Report 71 will also include this more specific coliform definition and it has been proposed that it will be included in the revised Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996).


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These enzyme-based methods appear to pick up coliforms that are traditionally not identified by selective media (George et al., 2000), so again the change in definition has expanded the range of bacteria recognised as coliforms as shown in Box 6. Appendix A contains a listing of currently available enzyme-based methods for total coliforms, E. coli and enterococci detection.

Box 6 Coliform Members by Evolving Definition

Pre 1994 Report 71, 1994 Enzyme-based Acid and Gas from Lactose Acid from Lactose ß-Galactosidase

Escherichia Escherichia Escherichia

Klebsiella Klebsiella Klebsiella

Enterobacter Enterobacter Enterobacter

Citrobacter Citrobacter Citrobacter

Yersinia Yersinia

Serratia Serratia

Hafnia Hafnia

Pantoea Pantoea

Kluyvera Kluyvera

Cedecea

Ewingella

Moellerella

Leclercia

Rahnella

Yokenella

bold type = coliforms which can be present in the environment as well as in human faeces.

bold and underline = coliforms which are considered to be primarily environmental.

Source: Kreig, 1984; Topley, 1997; Ewing, 1986; Ballows, 1992.


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4.2 MOLECULAR METHODS FOR DETECTION OF MICROBIAL INDICATORS

The future holds endless possibilities for methods to detect and identify indicator microorganisms and pathogens. On the horizon are methods based on sophisticated gene technology, which have only been used in medical research until now. A brief description of emerging technologies for microorganism detection is shown below, with detailed descriptions contained in Appendix B.

DNA Microarray Technology

This technique is based on testing water samples for the actual genetic material of a microorganism, rather than relying on growing the microbe, or using a microscope. Large amounts of known genetic information (DNA or RNA) can be stored on a very small surface and used to detect microbes in a sample by reacting with complementary DNA or RNA from the microbial population.

The microarray method was first developed by Stanford University (Elkins and Chu, 1999) and was called “DNA Microarray”. It is envisaged that these methods can reduce the time of analyses for faecal indicators to 4 hours and reduce the cost significantly.

Fluorescent in situ Hybridisation (FISH)

FISH is another genetic method for detecting microorganisms. The method uses a fluorescent marker attached to the DNA of the microorganism that is being investigated. The sample can be processed on a fixed surface, generally a microscope slide, and if the target microorganism is present, the reaction results in the microorganism “glowing”. This is then viewed using a fluorescence microscope.

A number of FISH methods have been developed for the detection of total coliforms and enterococci (Fuchs et al., 1998; Meier et al., 1997; Patel et al., 1998).


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5. TOTAL COLIFORMS AS INDICATORS

The information gained from the measurement of total coliforms can be confusing and the usefulness of coliforms (other than E. coli) as indicators of microbial water quality has been questioned for many years. This questioning has increased as research results have shown that total coliforms may not be an appropriate bacterial indicator of faecal pollution. The changing definition of total coliforms (Section 4.1) resulting in increased numbers of environmental bacteria is addressed in a draft revision of the Fact Sheet on coliforms in the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996, revised 2001), as follows:

‘Detection of coliform bacteria in the absence of thermotolerant coliforms (or E. coli) may be tolerated providing it can be shown that the organisms do not indicate faecal contamination ‘

and

‘Most coliforms including the thermotolerant coliforms (or alternatively E. coli) are not pathogenic but are used as indicators of the possible presence of faecal contamination and enteric pathogens. However, there are many environmental coliforms that are not of faecal origin and are of lesser significance (Fact Sheet 4 – Coliforms)’

The following three points support why total coliforms are not a reliable indicator of potential health risk in water, and each reason is discussed in the following sections. Coliforms have been found to:

1. grow in drinking water distribution systems;

2. be normal inhabitants of soil, water and plants; and

3. not always be present during waterborne disease outbreaks.

5.1 GROWTH IN DISTRIBUTION SYSTEMS

One of the requirements of a robust indicator of faecal pollution is that it enters the drinking water system with the pollutant and survives for a time that is consistent with the survival of pathogenic microorganisms. If a water quality indicator can multiply in the environment or in drinking water distribution systems, then detection does not necessarily imply that the system has been compromised by a pollution event, or that the water represents a potential public health risk.

Biofilms are microbial populations that grow on the inside of pipes and other surfaces. A number of research studies have shown that coliform bacteria can grow within drinking water distribution systems and can be a significant contributor to biofilm populations (Power and Nagy, 1989; LeChevallier, 1990). It has been shown that the presence of significant concentrations of coliforms within distribution systems, in themselves, do not represent a health risk to water consumers. For example, elevated concentrations of the coliform, Enterobacter cloacae isolated from within a water supply system were compared to Enterobacter cloacae isolated from the source water and from within several hospital environments by DNA analysis. Bacteria isolated from the three different environments were all different indicating the high numbers present within the system were not due to a failure in treatment processes but regrowth and those within the hospital were not from the water supply system. None of the isolates from the hospital environments were similar to those in the source water or treated water. In addition Enterobacter cloacae isolates from patients were different from those in the distribution system , indicating that the distribution system bacteria were not causing a public health risk (Edberg et al., 1994).


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Studies on the growth of coliforms under low nutrient conditions such as those in drinking water distribution systems showed they could grow on surfaces and remain within biofilms successfully competing with other bacteria (Camper et al., 1996).

To support the theory that bacteria growing in drinking water distribution systems do not represent a direct health risk to consumers, a review on the health significance of such bacteria found no evidence of any reported outbreaks of waterborne infection caused by typical bacteria growing within the system (PHLS, 1994). The review and application of risk ranking to bacteria in water supplies indicated that, while many are implicated in hospital infections they are not high risk and require specialised environments to grow and, frequently, only cause infections in already debilitated or immunocompromised people. Most hospitals are aware that drinking water is not sterile and adequate procedures for cleaning should be in place for potential niche sites as well as ensuring cleaning of wounds is not carried out with non-sterile solutions.

5.2 NORMAL SOIL AND WATER INHABITANTS

Many coliform bacteria, other than E. coli, form a small component of the normal intestinal population in humans and animals. It is well recognised and reported that E. coli is the only coliform that is an exclusive inhabitant of the gastrointestinal tract (Edberg et al., 2000). Most coliforms have an environmental origin and include plant pathogens and normal inhabitants of soil and water environments.

For example, coliforms of the genus Serratia are soil, water and plant microorganisms and play a role in insect disease (Villalobos et al., 1997). Another common coliform genera, Enterobacter, is widely distributed in nature, and is a common member of the community of bacteria which live in and around the roots of plants (Hinton and Watson, 1995). Most genera of coliforms have members that are found in natural environments more often than they are found in the intestines of humans and animals. It is interaction with the environment which results in the initial colonisation of the human intestine with coliform bacteria.

5.3 WATERBORNE DISEASE OUTBREAKS

Total coliforms have been shown not to be a sensitive indicator of the risk of waterborne disease. In some reported waterborne disease outbreaks, coliforms and E. coli have been detected in drinking water, while in others they are not present. The presence of E. coli is more representative of faecal pollution than other coliforms, because it occurs in higher numbers in faecal material, and generally does not occur elsewhere in the environment.

Waterborne disease outbreaks have been reported where drinking water did not contain detectable total coliform bacteria. In the USA between 1978–1986 there were 502 reported outbreaks of waterborne disease involving more than 110 000 cases of gastrointestinal illness. Many of the implicated water supplies in these outbreaks met the coliform compliance requirements of the USEPA (Sobsey, 1989). An additional study of outbreaks of waterborne disease in the USA found that one third of water supplies responsible for disease outbreaks did not have any total coliforms isolated from within the system (Craun, et al., 1997). Craun concluded that the presence of coliforms (including E. coli) is sometimes a useful indicator for viruses and bacteria, but not for protozoan parasites.


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The study of waterborne disease outbreaks increasingly show that outbreaks are being attributed to non-bacterial pathogens (viruses, protozoa) (Rose, 1990). This is most likely due to an increased understanding of the role of these pathogens in waterborne disease and the availability of more sophisticated and reproducible laboratory methods to recover and identify them from source and treated water.

Waterborne disease outbreaks where total coliforms are detected are generally attributed to bacteria, viruses or unknown agents, as shown by Moore, et al., 1994, who reported that 88% of such outbreaks had coliforms present in water samples. For outbreaks attributed to protozoan parasites, only 33% of waters were positive for coliforms.

Much of the difficulty in correlating the presence of bacterial indicators to the presence of protozoa is due to different susceptibility of protozoa to treatment processes, particularly chlorine. Chlorine as a disinfectant is more effective against bacteria and viruses than protozoa making coliforms limited in their use as a measure of the effectiveness of treatment processes for the removal of these organisms (Sobsey, 1989).


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6. ALTERNATIVES TO TOTAL COLIFORMS

6.1 CURRENT AND FUTURE USE OF COLIFORMS

There is now sufficient evidence that the presence of coliform bacteria (other than E. coli) within drinking water does not clearly indicate the presence of a health risk nor their absence, an absence of health risk. The appropriate role of coliforms needs to be addressed along with the suitability of the emphasis placed on their presence or absence for compliance. Coliforms can indicate a range of different things within a drinking water system, but indicate no single thing with confidence.

The interpretation of the presence or absence of total coliforms in drinking water, which is driven by the need to meet compliance targets, does not enable water quality managers or health officials to make confident decisions about the microbial safety of the water. This end-point, compliance-driven system needs to be replaced with a complete management system, which incorporates an understanding of risks posed, the best ways to manage them and specific testing to validate the effectiveness of its implementation.

Drinking water supply and system monitoring for bacterial indicators has four major purposes:

1. to identify general faecal contamination of source waters;

2. to demonstrate that treatment and/or disinfection processes are working effectively;

3. to alert for possible in-system contamination through cross connections, ingress, pipe break contamination and contamination from open storages; and

4. to monitor biofilm growth, general system cleanliness and the potential presence of opportunistic pathogens.

The current use of total coliforms for each purpose gives limited useful information, and generally better indicators are available. This is summarised in Box 7.

Many alternative indicators to total coliforms have been proposed including enterococci, sulfite-reducing clostridia, Bacteroides fragilis, Bifidobacteria, bacteriophages, and non-microbial indicators such as faecal sterols. Of these proposed indicators, enterococci has gained the most acceptance, particularly when used in conjunction with E. coli (Edberg et al., 2000; Pinto et al., 1999; Sinton et al., 1993; WHO, 1996).


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Box 7 Current use of Coliforms and Alternatives

Information To identify general faecal contamination of source waters Need

Currently Use Presence of total coliforms/thermotolerant coliforms.

Issue Short-term changes missed, aged faecal material may not contain coliforms, yet persistent pathogens could be present. Unknown source of faecal contamination and what to manage.

Future Use E. coli and other more specific faecal indicators such as enterococci, sulfite-reducing clostridia and faecal sterols. On-line turbidity measurement.

Information To demonstrate that treatment and/or disinfection processes are working effectively Need

Currently Use Absence of total coliforms and E. coli after treatment.

Issue Sampling frequency generally too low to detect chlorination upset or treatment failure. Coliforms more sensitive to chlorination than viral or protozoan pathogens.

Future Use Barrier measurement such as on-line particle sizing and chlorine analysers, ensure adequate disinfection.

Information To alert water managers and operators to in-system contamination through Need pipebreaks,ingress, cross connections and contamination from open storages

Currently Use Presence of coliforms or E. coli in distribution system samples.

Issue Cross connection contamination will result in high numbers of faecal organisms entering a system. In this case, E. coli is a suitable indicator and will be present in high numbers. Coliforms can already be present in the system from biofilms or regrowth, so their detection confuses the issue. The use of a chlorine residual has been shown to effectively reduce numbers of coliforms and indicator bacteria, while leaving other ingress pathogens unaffected.

Future Use E. coli and other more specific faecal indicators such as enterococci and faecal sterols. On-line chlorine analysers and pressure measurement.

Information To monitor growth of biofilms and general system cleanliness Need

Currently Use Coliforms

Issue Although coliforms can become part of a biofilm community, they do not represent the majority of bacteria within the biofilm. Some biofilms have very few, or no coliforms associated with them, yet may contain opportunistic pathogens.

Future Use Plate count (heterotrophic) bacteria comprise the vast majority of bacteria found in biofilms. Constant erosion or random sloughing-off of biofilm into the water flow may result in high numbers of plate count bacteria in the absence of coliforms.


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6.2 WATER QUALITY RISK MANAGEMENT APPROACH

Reliance on total coliforms for measurement of the microbial safety of a drinking water can result in a false sense of assurance from negative results. In many instances, E. coli and total coliforms are the sole indicators analysed to determine microbial water quality. The retrospective study of waterborne disease outbreaks and advances in the understanding of the behaviour of pathogens in water, has shown that continued reliance on bacterial indicators alone, and assumptions surrounding the absence or presence of total coliforms does not ensure that informed decisions are made regarding water quality.

A risk management approach to drinking water supply is being adopted across Australia to increase confidence in the safety of drinking water and reduce reliance on end-point testing. Several major Australian water suppliers have developed risk management plans that are a holistic approach to water management. These plans systematically assess risks throughout a drinking water supply, from the catchment and source water, through to the customer tap, and identify the ways that these risks can be managed and methods to ensure that barriers and control measures are working effectively. A risk management plan assesses the integrity of the entire water supply system and is able to incorporate strategies to deal with day-to-day management of water quality as well as upsets and failures.

The ongoing review of the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996), is resulting in the development of a comprehensive drinking water quality management framework. The framework supplements system management information currently included in the Guidelines with principles from existing management systems such as the International Organisation for Standardisation (ISO) series and the Hazard Analysis and Critical Control Point (HACCP) system. HACCP is a risk prevention/risk management system that has been used extensively in the food industry and is now being adopted for risk management of water production and supply. The draft framework, which has been trialed in a number of water supplies, will enable water managers to identify and rank risks within the water supply and establish critical control points where these risks can be managed. The framework will focus on total system management, measurement of barriers and verification using end-point testing.

Internationally, the World Health Organization (WHO, 1999) has developed a risk management approach to water quality as a model for assessing the safety of recreational waters (the Annapolis Protocol). This approach is being proposed as part of a harmonised framework for managing risks from drinking water and food production. The WHO is further developing this risk management approach in the current development of the Third Edition of the Guidelines for Drinking-water Quality.

A risk management approach for drinking water includes (1) end-point monitoring to verify that the water supplied to consumers was safe; and (2) operational monitoring to show that treatment processes are functioning properly and that distribution system integrity is maintained. End-point monitoring cannot be used as a system control measure, only as a final verification step in a complete risk management plan. Operational monitoring is a means of assessing system performance and results are used to modify system controls to ensure that processes are working within specification. For this reason, on-line and continuous monitoring for operational purposes is better able to support system management.


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Parameters within a risk management approach to monitor and verify water quality should be simple and include a range of parameters, which can indicate:

• Faecal contamination of source waters;

• Treatment effectiveness;

• Faecal contamination from in-system ingress; and

• Water stagnation, biofilm growth, system cleanliness and the potential presence of opportunistic pathogens.

Box 8 shows a number of indicators, both physical and microbial, which can be incorporated as part of a risk management framework.

Box 8 Water Quality Matrix Indicators

Hazard Indicator

Faecal contamination of source water • sanitary survey

• turbidity

• E. coli

Treatment effectiveness • total chlorine

• HPC(1)

• E. coli

Faecal contamination from in-system ingress • ammonia

• enterococci

• E. col

• dissolved oxygen (sudden change)

• free chlorine (sudden change)

• pressure (sudden change)

Water stagnation • loss of disinfectant residual

• dissolved oxygen

• HPC(1)

Potential presence of opportunistic pathogens • HPC(1)

• free chlorine

Notes: (1) HPC = Heterotrophic Plate Count


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6.3 ESCHERICHIA COLI AND ENTEROCOCCI – KEY FAECAL INDICATORS

It is widely acknowledged that the major threat to public health from drinking water is from microbial contamination with human, and to a lesser degree, animal faeces. One detailed risk assessment of pathogens and chemicals in drinking water concluded (Regli et al., 1993):

“risk of death from known pathogens in untreated water is 100 to 1000 times greater than risk of cancer from known disinfection by-products in chlorinated drinking water

and

the risk of illness from pathogens in untreated surface water is 10 000 to 1 000 000 times greater than risk of cancer from disinfection by-products in chlorinated drinking water”

As a component of the assessment of public health risk through monitoring of water quality at consumer’s taps, E. coli is regarded as the most sensitive indicator of faecal pollution. The large numbers of E. coli present in the gut of humans and other warm-blooded animals and the fact that they are not generally present in other environments support their continued use as the most sensitive indicator of faecal pollution available (Edberg et al., 2000).

To increase the confidence of water quality results, especially when monitoring for faecal pollution, analysis for enterococci has been used (eg. EU guidelines, Section 3, Box 3). The enterococci are the group of bacteria most often suggested as alternatives to coliforms, and interest in their use as a water quality indicator date back to 1900 when they were found to be common commensal bacteria in the gut of warm-blooded animals (Gleeson and Gray, 1997).

The enterococci were included in the functional group of bacteria known as “faecal streptococci” and now largely belong in the genus Enterococcus which was formed by the splitting of Streptococcus faecalis and Streptococcus faecium, along with less important streptococci, from the genus Streptococcus (Schleifer and Klipper-Balz, 1984). There are now 19 species that are included as enterococci (Topley, 1997). The predominant intestinal enterococci are Enterococcus faecalis, E. faecium, E. durans and E. hirae. In addition, other Enterococcus species and some species of Streptococcus (namely S. bovis, and S. equinus) may occasionally be detected. Generally, for water examination purposes enterococci can be regarded as indicators of faecal pollution, although some can occasionally originate from other habitats.

Enterococci have a number of advantages as indicators over total coliforms and even E. coli, including that they generally do not grow in the environment (WHO, 1993) and they have been shown to survive longer (McFeters et al., 1974). Despite being approximately an order of magnitude less numerous than faecal coliforms and E. coli in human faeces (Feacham et al., 1983), they are still numerous enough to be detected after significant dilution. Rapid and simple methods, based on defined substrate technology, are available for the detection and enumeration of enterococci and routinely employed in many laboratories (see Appendix A for description of methods).

There is some concern that enterococci are a diverse group of bacteria, and that the group contains species that are environmental and their presence in water is not necessarily indicative of faecal pollution. This concern is driven by the problems associated with the use of total coliforms as an indicator of faecal pollution. An early research report showed that Enterococcus faecalis var liquefaciens was common in good quality water and its relevance was not considered clear if recovered in waters in concentrations of less than 100 organisms/100 mL (Geldreich, 1970). More recent research on the relevance of faecal streptococci as indicators of pollution, showed that the majority of enterococci (84%) isolated from a variety of polluted water sources were “true faecal species” (Pinto et al., 1999).


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Measurement for enterococci in water is used in South Australia as an additional indicator when total coliforms are present in the absence of E. coli (Cunliffe, 2000), while in Sydney faecal streptococci are used to confirm faecal contamination if either total coliforms or E. coli are detected (Ashbolt pers. comm.). The WHO (1996) also recommends the use of faecal streptococci (of which enterococci are a sub-group) as an additional indicator of faecal pollution. When combined with the measurement of E. coli, the result is increased confidence in the absence or presence of faecal pollution.

6.4 CLOSTRIDIUM PERFRINGENS

Clostridium perfringens (C. perfringens) are sulfite-reducing, spore-forming, clostridia, which are hardy rod-shaped anaerobic bacteria. They are widely spread through nature and have been isolated from the intestines of many animals (Cato et al., 1986). It is reported that the use of C. perfringens as an indicator organism was first proposed in 1899 (cited in Gleeson and Gray, 1997). The spores produced by C. perfringens are very resistant to disinfection and the WHO (1996) suggests that their presence in filtered supplies may not be an indication of treatment inefficiencies. In disinfected supplies, their presence may not be an indication of poor inactivation performance for this same reason (Fujioka and Shizumura, 1985).

Spores of C. perfringens are largely of faecal origin (Sorensen et al., 1989) and are always present in sewage. Their spores are highly resistant in the environment, and vegetative cells appear not to reproduce in aquatic sediments, unlike many traditional indicator bacteria (Davies et al., 1995). There is evidence to show that C. perfringens may be a suitable indicator for viruses and parasitic protozoa when sewage is the suspected cause of contamination (Payment and Franco, 1993).

Nonetheless, C. perfringens is not generally considered a robust indicator of microbial water quality because they can survive and accumulate in drinking water systems and may be detected long after a pollution event has occurred and far from the source (WHO, 1996). Their preferred role is to aid identification of faecal contamination in sanitary surveys.

6.5 BACTERIOPHAGES

Bacteriophages are viruses that infect bacteria and those that infect coliforms are known as coliphages, or more generally, phages. Phages have been proposed as microbial indicators as they behave more like the human enteric viruses which pose a health risk to water consumers if water has been contaminated with human faeces.

The use of phages as water quality indicators has been extensively researched and the limitations of their use widely debated. Box 9 shows a summary of the limitations of phages as reliable water quality indicators. Research results show that phages cannot be considered as reliable indicators, models or surrogates for enteric viruses in water. This is underlined by the detection of enteric viruses in treated drinking water supplies which were negative for phages (Grabow et al., 2000). Phages are probably best applied as models/surrogates in laboratory experiments where the survival or behaviour of selected phages and viruses are directly compared under controlled conditions (Grabow et al., 1983, 1999b; Naranjo et al., 1997).


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Box 9 Limitations of Phages Reference

Phages are excreted by a certain percentage of humans and animals all the time Vaughn and Metcalf, 1975 while viruses are excreted only by infected individuals for a short period of time. Borrego et al., 1990 There is no direct correlation in numbers of phages and viruses in human faeces. Grabow et al., 1993 Grabow et al., 1999a

Enteric viruses have been detected in water environments in the absence of coliphages Montgomery, 1982 Morinigo et al., 1992

Human enteric viruses associated with waterborne diseases are excreted almost exclusively Osawa et al., 1981 by humans, whereas phages used as models/surrogates in water quality assessment are Furuse et al., 1983 excreted by humans and animals. The faeces of animals such as cows and pigs generally Grabow et al., 1993 contains higher densities of coliphages than that of humans, and the percentage of many Grabow et al.,1995 animals which excrete phages tends to be higher than for humans. Grabow, 1996

Some coliphages may replicate in water environments Seeley and Primrose, 1982; Grabow et al., 1984; Borrego et al., 1990

6.6 SUMMARY

There is a substantial amount of information currently available on the advantages and disadvantages of total coliforms and other indicators of water quality (Gleeson and Gray, 1997; Ashbolt et al., 2001). Most of the scientific literature and water quality guideline development supports a more scientifically defensible and risk-based approach for public health protection. There is widespread agreement that the presence of total coliforms, other than E. coli, does not assist water managers determine if there is an associated public health risk. The presence of E. coli can be relied upon to indicate faecal pollution has occurred, as can generally the presence of enterococci.

Other faecal indicators superior to E. coli and enterococci have not been developed to a point where there are methods readily available that are inexpensive and simple for routine use.

The monitoring of water filtration plants requires a continuous type of assessment using criteria such as turbidity and particle size distribution. Monitoring of these parameters in source and treated water allows early warning and treatment efficacy assessment. Disinfection effectiveness and system cleanliness assessment is better achieved using disinfectant residual (C.t), which is a measure of the concentration of disinfectant and contact time, combined with measurement of total heterotrophic bacteria (of which coliforms are a sub-set). Water operators should be alerted by sudden changes in these parameters.


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7. CONCLUSIONS

Water suppliers have been aware of the role of water in disease transmission for more than 150 years, during which time the primary focus of managing drinking water has been the protection of public health. The fundamental issues associated with public health impacts and the need for safe drinking water are currently well understood.

The Australian and international water industry is making a positive move towards understanding and managing risks to public health from drinking water. This has been driven by advances in methods for detecting pathogens, epidemiological studies to measure background levels of waterborne disease, and a realisation that over reliance on treatment processes to protect public health is not a sustainable management approach.

Detailed assessment of the factors that influence water quality necessarily includes a review of the way in which the microbial safety of water delivered is measured. The current focus on the absence of total coliforms and E. coli to ensure water quality has been shown to be flawed and the water industry and regulators internationally are evaluating alternative ways to protect public health and the usefulness of these indicators.

The collection of information on water quality is costly, therefore data needs to be unambiguous, and able to assist risk management decisions. The presence of E. coli in treated water is still considered to indicate faecal pollution and remains a key verification tool. The uncertainty surrounding the relevance of other coliforms in drinking water means that their value as an indicator is limited. A set of indicators, used in conjunction with total-system risk management, is required that increases confidence in the safety of water delivered to consumers.

The move by the EU to downgrade total coliforms and include mandatory measurement of enterococci as well as E. coli is one that recognises the worth of using specific indicators of faecal pollution, and assigns a minor role to total coliforms.

The limitation of total coliforms in Australia has been reaffirmed in the ongoing review of the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996). The revised Fact Sheets for thermotolerant coliforms/E. coli and total coliforms acknowledge that coliforms can be of environmental origin and that their presence in drinking water is not necessarily indicative of a health risk.

The importance of risk management is also addressed in the ongoing review of the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996) with the development and trialing of a framework for the risk management of drinking water systems. The development of a set of criteria to verify that risk management plans are effective is a necessary part of this approach to water quality. In the long-term review of appropriate water quality monitoring, it is envisaged that a matrix approach will be adopted, with less significance on single parameters such as total coliforms.

The next step in the current direction of water quality management and compliance monitoring for Australia is to reduce reliance on total coliforms and increase emphasis on implementation of risk management systems, measurement of specific faecal indicators, and assessment of the effectiveness of source water protection, treatment and disinfection barriers.


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Information currently available supports the use of Escherichia coli as the primary indicator of faecal pollution supported by other measurements such as heterotrophic bacteria, C.t, chlorine residual and turbidity to verify treatment and disinfection effectiveness and assess system cleanliness.

Above all, a move away from reliance on total coliform bacteria and adoption of a risk management approach to drinking water quality for Australia will ensure that suppliers and consumers can have increased confidence in the safety of their drinking water.


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8. RECOMMENDATIONS

It is recommended that:

1. Total coliforms be removed as an indicator of faecal contamination in the Australian Drinking Water Guidelines (NHMRC–ARMCANZ, 1996); and

2. E. coli be the primary indicator of faecal contamination in the Australian Drinking Water Guidelines (NHMRC–ARMCANZ, 1996).


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APPENDIX A

ENZYME-BASED METHODS FOR THE DETECTION OF MICROBIAL INDICATORS

A number of colourimetric media enabling quantification of total coliforms and E. coli within 24h are now available, as well as for the enterococci as follows and shown in Box A1:

• Enteroler®, manufactured by IDEXX (Hernandez et al., 1991; Manafi, 1996);

• Colisure® manufactured by IDEXX (McFeters et al., 1995);

• Colilert®, manufactured by IDEXX (Edberg et al., 1988; Edberg et al., 1991);

• m-ColiBlue®, manufactured by Hach:

• ColiComplete®, manufactured by BioControl;

• Chromocult®, manufactured by Merck; and

• MicroSure®, manufactured by Gelman.

The Colilert® method is based upon the water sample turning yellow, indicating coliforms with b-galactosidase activity on the substrate ONPG (O-nitrophenyl-ß-D-galactopyranoside), and fluorescence under long-wavelength UV light when the substrate MUG (5-methylumbelliferyl-ß ß D-glucuronide) is metabolised by E. coli containing ß-glucuronidase. The analytical method involves adding commercial dried indicator nutrients containing the two defined substrates to a 100 mL volume of water and incubation at 35-37°C as described in Standard Methods (1998). The result is either a presence/absence testing in the 100 mL volume or quantification in a propriety tray (QuantiTray™) which separates the sample into a series of test wells and provides a most probable number (MPN) per 100 mL of water.

Box A1 Chromogenic substances available for the detection of indicator bacteria Bacteria Enzyme tested Chromogenic substance

Coliform bacteria • o-nitrophenyl-ß-D-galactopyranoside (ONPG) ß-D-galactosidase • 6-bromo-2-naphtyl-ß-D-galactopyranoside (E.C.3.2.1.23.) • 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (XGAL)

E. coli • 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (XGLUC) ß-D-glucuronidase • 4-methylumbelliferyl-ß-D-glucuronide (MUG) (GUD, E.C3.2.1.31) • p-nitrophenol-ß-D-glucuronide (PNPG)

Enterococci • 4-methylumbelliferyl-ß-D-glucoside (MUD) ß-D-glucosidase • indoxyl-ß-D-glucoside (ß-GLU, EC.3.2.21)

Source: Adapted from Manafi (1996)


38

APPENDIX B

MOLECULAR METHODS FOR THE DETECTION OF MICROBIAL INDICATORS

This section contains detailed description of novel methods for the detection of indicator microorganisms and pathogens, based on molecular techniques.

Microarray Technology

There are two variants of the DNA microarray technology, in terms of the property of arrayed DNA sequence with known identity:

• probe cDNA (500~5000 bases long) is immobilised to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, ‘traditionally’ called DNA microarray, is widely considered as developed at Stanford University (Ekins and Chu, 1999).

• an array of oligonucleotide (20~25-mer oligos) or peptide nucleic acid (PNA) probes is synthesised either in situ (on-chip) or by conventional synthesis followed by on-chip immobilisation. The array is exposed to labelled sample DNA, hybridised, and the identity/abundance of complementary sequences are determined. This method, “historically” called GeneChip® arrays or DNA chips, was first developed at Affymetrix Inc. (Lemieux et al., 1998; Lipshutz et al., 1999).

Microarrays using DNA/RNA probe-based rRNA targets may be coupled to adjacent CCD detectors (Guschin et al., 1997). Eggers et al., (1997) have demonstrated the detection of E. coli and Vibrio proteolyticus using a microarray containing hundreds of probes within a single well (1cm2) of a conventional microtitre plate (96 well). The complete assay with quantification took less than 1 minute. The basic steps in a microarray assay are given in Box B1.

DNA sensing protocols, based on different modes of nucleic acid interaction, possess an enormous potential for environmental monitoring. Carbon strip or paste electrode transducers, supporting the DNA recognition layer, are used with a highly sensitive chronopotentiometric transduction of the DNA analyte recognition event. Pathogens targeted to date include Mycobacterium tuberculosis, Cryptosporidium parvum and Human Immunodeficiency Virus HIV-1 (Wang et al., 1997; Vahey et al., 1999).

The microarray under development by bioMerieux (using Affymetrix Inc. GeneChip® technology) for an international water company (Lyonnaise des Eaux, Paris, France) is expected to reduce test time for faecal indicators from the current average of 48 hours to 4 hours. In addition, the cost for the standard water microbiology test is expected to be 10 times less than present methods. The high resolution DNA chip technology is expected to target a range of key microorganisms in water. The prototype GeneChip® measures about 1 cm2, on which hybridisation occurs with up to 400 000 oligonucleotide probes. Nonetheless, such technology may be limited by effective means of concentrating the target indicator to the small volumes used in these assays.

Of the better-evaluated molecular-based methods, the polymerase chain reaction (PCR) amplification of nucleic acids also suffers from the need to use small reaction volumes. Consequently, and due to problems of environmental inhibitors, its use in the water industry is likely to be limited to confirmation testing of cultures or presence/absent testing of specific pathogens in concentrates (Stinear et al., 1996; Dombek et al., 2000).


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Fluorescence In-situ Hybridisation (FISH)

Solid-state cytometry is a methodology that is rapidly taking off in water microbiology, utilising either chromogenic substrates (see Appendix A) or the molecular labelling method called fluorescence in situ hybridisation (FISH).

FISH detection makes use of gene probes with a fluorescent marker, typically targeting the 16S ribosomal RNA (16S rRNA) (Amann et al., 1995). Concentrated and fixed cells are permeabilised and mixed with the probe. Incubation temperature and addition of chemicals can influence the stringency of the match between the gene probe and the target sequence. Since the signal of a single fluorescent molecule within a cell does not allow detection, target sequences with multiple copies in a cell have to be selected (eg. there are 102-104 copies of 16S rRNA in active cells). A number of FISH methods for the detection of coliforms and enterococci have been developed (Fuchs et al., 1998; Meier et al., 1997; Patel et al., 1998). A number of studies indicate that FISH detection-based methods may better report the presence of infective pathogens and viable, but not necessarily culturable indicator bacteria. As a further extension of the FISH approach, peptide nucleic acid probes targeted against the 16S rRNA molecule were designed and used to detect E. coli from water (Prescott et al., 1998). The probe was labelled with biotin, which was subsequently detected with streptavidin horseradish-peroxidase and the tyramide signal amplification system. E. coli cells were concentrated by membrane filtration prior to hybridisation and the labelled cells detected by a commercial laser-scanning solid-state cytometer (eg. ChemScanTM) within 3h. Detection and enumeration is also possible by the use of a flow cytometer (Fuchs et al., 1998). These cytometers, however, are expensive, and high sample throughput is necessary to justify their purchase.


40

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41

PROCESS REPORT

In 2000, the NHMRC Drinking Water Review Coordinating Group recognised increasing uncertainty in relation to the use of traditional indicator organisms, including thermotolerant coliforms and total coliforms, as a measure of the microbial quality of drinking water. In response, a discussion paper was commissioned to consider the concepts of indicator microorganisms, the rationale behind their use, and to provide an evaluation of alternative indicators of microbial water quality.

The Coordinating Group requested Dr Melita Stevens (Melbourne Water), Dr Nick Ashbolt (University of New South Wales) and Dr David Cunliffe (SA Department of Human Services) to undertake a review on microbial indicators of water quality that addressed the:

• status of bacterial indicators of drinking water quality in Australia and internationally;

• impact of emerging technologies for indicator enumeration and identification;

• usefulness of current bacterial indicators in supporting the risk-based water quality management systems;

• usefulness of emerging indicators such as faecal sterols and bacteriophage; and

• possible alternative approaches for microbial indicators in any future revision to the Australian Drinking Water Guidelines.

The review has highlighted a number of potential activities, including the need to:

• revise guidance on heterotrophic plate counts in operational management;

• revise guidance on coliforms in operational management; and

• remove total coliforms to be removed as public health indicators

Consultation on the draft report, Review of Coliforms as Microbial Indicators of Drinking Water Quality took place from September to November 2001 and involved a call for submissions on the draft document publicised in the Commonwealth Notices Gazette and The Weekend Australia. Invitations were also forwarded to known interested parties through enHealth Council, the Australian Water Association and Water Services Association of Australia.

All submissions received were taken into consideration by the Coordinating Group in finalising this review. Submissions were received from the following individuals/organisations:

Sam Austin Yarra Valley Water

Harry Ferguson Brisbane Water

Dr Philip Berger United States Environment Protection Authority

Brian Bailey Melbourne Water

Ian Tanner Sydney Catchment Authority

Keith Neaves Lower Murray ater Authority

Dr Chris Saint, Australian Water Quality Centre Phil Adcock

Les Mathieson East Gippsland Water

Jacqui Goonrey ActewAGL

Mark Harvey Victorian Water Industry Association Inc

David Heap City West Water


42

Greg Ryan South East Water Limited

Martha Sinclair, Monash University Samantha Rizak

Jim Martin North East Water

Christine Cowie NSW Department of Health

Dr Paul Van Buynder Department of Human Services, Victoria

David Sheehan Queensland

Membership of the NHMRC Drinking Water Review Coordinating Group

Professor Don Bursill (Chair) Cooperative Research Centre for Water Quality and Treatment

Dr David Cunliffe Department of Human Services, South Australia

Peter Scott Melbourne Water Corporation

Dr Anne Neller University of the Sunshine Coast

Alec Percival Consumer’s Health Forum

Dr John Langford Water Services Association of Australia

Brian McRae Australian Water Association

Secretariat

Phil Callan National Health and Medical Research Council Peer Reviewer

Emeritus Professor Nancy Millis University of Melbourne Technical Editor

Dr Andrew Langley Department of Health, Queensland

Prior to approval by the NHMRC, Review of Coliforms as Microbial Indicators of Drinking Water Quality was subjected to an independent review against the NHMRC key criteria for assessing information reports

by Hawkless Consulting Pty Ltd.

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