Review of Coliforms - National Health and Medical Research Council
Review of Coliforms - National Health and Medical Research Council
Review of Coliforms - National Health and Medical Research Council
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<strong>Review</strong> <strong>of</strong> <strong>Coliforms</strong><br />
As Microbial Indicators <strong>of</strong> Drinking Water Quality<br />
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
RECOMMENDATIONS TO CHANGE THE USE OF COLIFORMS AS<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Dr Melita Stevens<br />
Melbourne Water Corporation<br />
Dr Nicholas Ashbolt<br />
University <strong>of</strong> New South Wales<br />
Dr David Cunliffe<br />
Department <strong>of</strong> Human Services, South Australia<br />
Endorsed 10–11 April 2003
ISBN: 1864961651, Online ISBN: 1864961597<br />
Material included in this document may be freely reproduced provided that it is accompanied<br />
by an acknowledgment stating the full title <strong>of</strong> the document, the <strong>National</strong> <strong>Health</strong> <strong>and</strong> <strong>Medical</strong><br />
<strong>Research</strong> <strong>Council</strong> <strong>and</strong> the date <strong>of</strong> release.<br />
Disclaimer<br />
The contents <strong>of</strong> this document have been compiled using a range <strong>of</strong> source material <strong>and</strong> while<br />
due care has been taken in its compilation, the Commonwealth, member governments <strong>of</strong><br />
NHMRC <strong>and</strong> the organisations <strong>and</strong> individuals involved with the compilation <strong>of</strong> this document<br />
shall not be liable for any consequences which may result from using the contents <strong>of</strong> this<br />
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before relying on the information in any important matter.<br />
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Front Cover: Image <strong>of</strong> <strong>Coliforms</strong> courtesy <strong>of</strong> Centre for Microscopy <strong>and</strong> Microanalysis,<br />
The University <strong>of</strong> Queensl<strong>and</strong>.<br />
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Interent: http://www.nhmrc.gov.au
TABLE OF CONTENTS<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
EXECUTIVE SUMMARY VII<br />
BACKGROUND VIII<br />
1. INTRODUCTION 1<br />
2. MICROBIAL INDICATORS OF WATER QUALITY 3<br />
2.1 What are Microbial Indicators? 3<br />
2.2 Total <strong>Coliforms</strong> 4<br />
2.3 Escherichia coli (E. coli) 5<br />
3. USE OF BACTERIAL INDICATORS OF WATER QUALITY 7<br />
4. THE CHANGING FACE OF COLIFORMS AND INDICATORS 11<br />
4.1 Changes in Coliform Definition 11<br />
4.2 Molecular Methods for Detection <strong>of</strong> Microbial Indicators 12<br />
5. TOTAL COLIFORMS AS INDICATORS 15<br />
5.1 Growth in Distribution Systems 15<br />
5.2 Normal Soil <strong>and</strong> Water Inhabitants 16<br />
5.3 Waterborne Disease Outbreaks 16<br />
6. . ALTERNATIVES TO TOTAL COLIFORMS 19<br />
6.1 Current <strong>and</strong> Future Use <strong>of</strong> <strong>Coliforms</strong> 19<br />
6.2 Water Quality Risk Management Approach 21<br />
6.3 Escherichia coli <strong>and</strong> Enterococci – Key Faecal Indicators 23<br />
6.4 Clostridium perfringens 24<br />
6.5 Bacteriophages 24<br />
6.6 Summary 25<br />
7. CONCLUSIONS 27<br />
8. RECOMMENDATIONS 29<br />
9. REFERENCES 31<br />
iii
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
APPENDIX A 37<br />
Enzyme-based Methods for The Detection <strong>of</strong> Microbial Indicators 37<br />
APPENDIX B 38<br />
Molecular Methods for the Detection <strong>of</strong> Microbial Indicators 38<br />
PROCESS REPORT 41<br />
iv
EXECUTIVE SUMMARY<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Assurance that water is microbially safe for drinking has traditionally been determined by<br />
measuring bacterial indicators <strong>of</strong> water quality, most commonly total coliforms <strong>and</strong> Escherichia<br />
coli (E. coli). The international water industry is questioning whether continued reliance on<br />
these indicators is sufficient to ensure microbial water quality <strong>and</strong> it has begun to adopt a<br />
more holistic approach to delivering safe water, through the development <strong>and</strong> adoption <strong>of</strong> risk<br />
management plans for drinking water quality.<br />
In Australia, the ongoing revision <strong>of</strong> the Australian Drinking Water Guidelines (NHMRC-<br />
ARMCANZ, 1996), includes the development <strong>and</strong> trialing <strong>of</strong> a risk management framework<br />
for drinking water quality, <strong>and</strong> the development <strong>of</strong> the Third Edition <strong>of</strong> the WHO Guidelines<br />
for Drinking-water Quality is similarly focussed on the use <strong>of</strong> risk management with less<br />
reliance on end-point testing.<br />
As a component <strong>of</strong> a risk-based approach to water quality management, measures used to<br />
verify water quality must support the risk management system, <strong>and</strong> provide useful information<br />
to water suppliers. Total coliforms have been shown to be a poor parameter for measuring<br />
the potential for faecal contamination <strong>of</strong> drinking water due to their presence as normal<br />
inhabitants <strong>of</strong> soil <strong>and</strong> water environments, their ability to grow in drinking water distribution<br />
systems <strong>and</strong> their inconsistent presence in water supplies during outbreaks <strong>of</strong> waterborne<br />
disease. These factors mean that it is difficult to interpret the sanitary significance <strong>of</strong> their<br />
presence (in the absence <strong>of</strong> E. coli) or have confidence in water quality in their absence.<br />
The presence <strong>of</strong> E. coli in drinking water is still considered to indicate that faecal<br />
contamination <strong>of</strong> water has occurred. E. coli monitoring <strong>of</strong> drinking water as a verification<br />
measure is a useful tool within a risk management approach to water quality. There are a<br />
number <strong>of</strong> other useful indicators, both microbial <strong>and</strong> physical, which can be used to monitor<br />
both drinking water system operation <strong>and</strong> performance, <strong>and</strong> which provide better support<br />
for system management than total coliforms.<br />
The key recommendations <strong>of</strong> this report are that:<br />
1. total coliforms be removed as an indicator <strong>of</strong> faecal contamination in the Australian<br />
Drinking Water Guidelines (NHMRC-ARMCANZ, 1996); <strong>and</strong>,<br />
2. E. coli be the primary indicator <strong>of</strong> faecal contamination in the Australian Drinking<br />
Water Guidelines (NHMRC-ARMCANZ, 1996).<br />
v
BACKGROUND<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Almost 150 years ago, at a time when cholera <strong>and</strong> typhoid were common, the fact that water<br />
was a vehicle for disease was first proved (Snow, 1855). During the late 1800s <strong>and</strong> early 1900s,<br />
scientific knowledge about the nature <strong>and</strong> causes <strong>of</strong> disease increased rapidly <strong>and</strong> there was<br />
major focus on public health reform. During this period, techniques to identify <strong>and</strong> enumerate<br />
causative agents <strong>of</strong> disease were developed.<br />
For more than 100 years, the microbial safety <strong>of</strong> drinking water has primarily been determined<br />
by testing for bacterial ‘indicators’ <strong>of</strong> faecal pollution, mainly Escherichia coli (E coli)<br />
(or alternatively thermotolerant (faecal) coliforms) <strong>and</strong> total coliforms. These indicators are<br />
used to assess the potential public health risk <strong>of</strong> drinking water, <strong>and</strong> their presence or absence<br />
are key elements <strong>of</strong> most drinking water quality guidelines, water supply operating licences<br />
<strong>and</strong> agreements between bulk water suppliers <strong>and</strong> retail water companies.<br />
The rationale presented for testing water for total coliforms is that this functional group <strong>of</strong><br />
bacteria is present in large numbers in the gut <strong>of</strong> humans <strong>and</strong> other warm-blooded animals.<br />
This means that if water is polluted by faeces, coliforms can be detected even after extensive<br />
dilution. However, the total coliform group lacks specificity as many <strong>of</strong> them can exist <strong>and</strong><br />
proliferate in both soil <strong>and</strong> water environments, as well as drinking water distribution systems.<br />
The presence <strong>of</strong> total coliforms in water may be a result <strong>of</strong> natural processes <strong>and</strong> not <strong>of</strong> faecal<br />
pollution <strong>and</strong> the health significance <strong>of</strong> their presence in drinking water is not clear. Changes<br />
in methods for detecting total coliforms over the last 10 years have made the interpretation<br />
<strong>of</strong> their significance more difficult by broadening the functional definition <strong>of</strong> the group <strong>and</strong><br />
incorporating more environmental species.<br />
Of the total coliform group, E. coli is the most numerous in mammalian faeces <strong>and</strong> is<br />
considered the most specific indicator <strong>of</strong> faecal pollution. The presence <strong>of</strong> E. coli in water<br />
is still considered to represent the presence <strong>of</strong> faecal pollution <strong>and</strong> is used to indicate that<br />
pathogenic bacteria, viruses <strong>and</strong> protozoa may also be present. The drawback <strong>of</strong> relying on<br />
E. coli is that it is a poor indicator for the presence <strong>of</strong> viruses <strong>and</strong> parasitic protozoa that can<br />
survive for much longer periods than the bacterial indicator.<br />
The relevance <strong>of</strong> testing for total coliforms <strong>and</strong> E. coli has been questioned since its<br />
introduction <strong>and</strong> it is again under challenge. In addition to changes in methodology, a<br />
new challenge has been initiated by a move to a more holistic <strong>and</strong> preventive approach to<br />
managing water quality. This new approach includes identifying appropriate monitoring<br />
strategies to measure the effectiveness <strong>of</strong> water treatment processes <strong>and</strong> the safety <strong>of</strong><br />
drinking water. With increased attention being placed on non-bacterial pathogens including<br />
Cryptosporidium <strong>and</strong> viruses, effective testing <strong>of</strong> microbial water quality clearly requires more<br />
than simple testing for total coliforms <strong>and</strong> E. coli. Turbidity <strong>of</strong> filtered drinking water <strong>and</strong><br />
measures <strong>of</strong> disinfection (such as C.t values) are being increasingly used as indicators<br />
<strong>of</strong> microbial quality, <strong>and</strong> confidence in the safety <strong>of</strong> water supply is being underpinned by<br />
comprehensive risk management plans. After so many years, is it time for a complete change<br />
or do the old indicators still have some merit?<br />
vii
1. INTRODUCTION<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
One <strong>of</strong> the primary concerns <strong>of</strong> water authorities is to ensure that the drinking water they<br />
supply does not pose an unacceptable health risk to consumers. The safety <strong>of</strong> drinking water<br />
is generally monitored in a number <strong>of</strong> ways:<br />
1. constituents <strong>of</strong> drinking water (such as chemicals <strong>and</strong> microbes) which can compromise<br />
human health can be measured directly;<br />
2. barriers designed to protect water quality (such as catchment activities, filtration<br />
<strong>and</strong> disinfection) can be monitored; <strong>and</strong><br />
3. indicators <strong>of</strong> water quality (such as turbidity) can be measured to assess the potential<br />
presence <strong>of</strong> broad groups <strong>of</strong> parameters.<br />
There are two major reasons for monitoring drinking water quality:<br />
• to determine if the water supply system is being operated correctly, implying that the<br />
water is safe for consumers (Primary Assessment); <strong>and</strong><br />
• pro<strong>of</strong> that the water was safe after it was supplied. This includes monitoring for<br />
compliance (Verification).<br />
To address microbial health risk, primary assessment can only be achieved by monitoring<br />
source water <strong>and</strong> barriers. Monitoring treated water in distribution systems for microorganisms<br />
is a means <strong>of</strong> verification only.<br />
Of the three methods used to assess drinking water listed above, indicators are most<br />
<strong>of</strong>ten used to monitor microbial water quality, as direct measurement <strong>of</strong> all pathogenic<br />
microorganisms is difficult, expensive <strong>and</strong> time consuming. In most cases risks from chemicals<br />
in drinking water are due to chronic exposure meaning that there is no urgency between<br />
sampling, testing <strong>and</strong> acting on results. This is not the case with the health risk from incidents<br />
<strong>of</strong> microbial pollution, which are generally short-lived with disease becoming apparent within<br />
a short period <strong>of</strong> time.<br />
Focus on the use <strong>of</strong> barrier monitoring has increased as the water industry adopts complete<br />
system risk management, including identification <strong>of</strong> key elements which can be monitored to<br />
give useful information on potential health risk. Until these methodologies have become better<br />
established, the use <strong>of</strong> bacterial indicators for assessing water quality will remain an integral<br />
component <strong>of</strong> drinking water management. Indicators <strong>of</strong> water quality will be an important<br />
component <strong>of</strong> the verification step in risk management systems for drinking water.<br />
The way in which bacterial indicators are used as a measure <strong>of</strong> microbial water quality<br />
has been questioned for some time, but this has increased with the development <strong>of</strong> more<br />
sensitive microbial techniques <strong>and</strong> increased underst<strong>and</strong>ing about the nature <strong>of</strong> environmental<br />
microorganisms, human pathogens <strong>and</strong> disease.<br />
This paper describes the concept <strong>of</strong> indicator microorganisms <strong>and</strong> details the rationale<br />
behind their use. Current indicators are reviewed <strong>and</strong> their advantages <strong>and</strong> disadvantages<br />
discussed <strong>and</strong> alternative indicators are evaluated. The major purpose <strong>of</strong> this paper is<br />
to generate discussion about the relevance <strong>of</strong> current bacterial indicators <strong>of</strong> water quality<br />
as recommended in the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996)<br />
<strong>and</strong> to recommend changes to the current Guidelines. This paper was developed for<br />
consideration by operational <strong>and</strong> technical staff in State <strong>and</strong> Territory health authorities<br />
<strong>and</strong> water supply agencies.<br />
1
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
The alternative indicators discussed in this paper have not been used or studied as extensively as<br />
total coliforms <strong>and</strong> E. coli. There is limited information on their environmental significance, their<br />
presence in drinking water systems <strong>and</strong> relationship to waterborne disease outbreaks. Public<br />
consultation demonstrated that there was general agreement for removal <strong>of</strong> total coliforms as<br />
an indicator <strong>of</strong> faecal contamination, with E. coli supported as the primary indicator.<br />
A regulatory impact statement (RIS) including a cost-benefit evaluation <strong>of</strong> regulatory<br />
alternatives, was not undertaken as part <strong>of</strong> this review. This document was developed to<br />
support consideration <strong>of</strong> microbial indicator organisms in the Australian Drinking Water<br />
Guidelines (ADWG). The Productivity Commission’s Office <strong>of</strong> Regulation <strong>Review</strong> has<br />
previously determined that the NHMRC is not required to undertake an RIS on the ADWG as<br />
the Guidelines do not have regulatory status (Productivity Commission, 2000). Implementation<br />
<strong>of</strong> the ADWG by the States <strong>and</strong> Territories is at the discretion <strong>of</strong> the State <strong>and</strong> Territory <strong>Health</strong><br />
Departments, usually in consultation with water suppliers <strong>and</strong> should include an appropriate<br />
economic analysis prior to implementation.<br />
2
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
2. MICROBIAL INDICATORS OF WATER QUALITY<br />
2.1 WHAT ARE MICROBIAL INDICATORS?<br />
Microorganisms that can cause disease are called pathogens. Pathogens that can be spread<br />
through drinking water <strong>and</strong> cause waterborne disease include bacteria, viruses, <strong>and</strong> protozoa.<br />
The number <strong>of</strong> different types <strong>of</strong> pathogens that can be present in water as a result <strong>of</strong><br />
pollution with human or animal faeces is very large <strong>and</strong> it is not possible to test water samples<br />
for each specific pathogen. For example, more than 100 types <strong>of</strong> enteric viruses have been<br />
isolated from human faeces <strong>and</strong> from sewage (Payment, 1993). Isolation <strong>and</strong> identification<br />
<strong>of</strong> some <strong>of</strong> these viruses is very difficult, or not currently possible. If these viruses or other<br />
pathogens are present in water as a result <strong>of</strong> faecal pollution, a measure is required which<br />
will alert water managers to their presence.<br />
An indicator <strong>of</strong> microbial water quality is generally something (not necessarily bacteria), which<br />
has entered the water at the same time as faeces, but is easier to measure than the full range<br />
<strong>of</strong> microorganisms which pose the health risk. There are several qualities that are desirable for<br />
a useful water quality indicator (NHMRC-ARMCANZ, 1996; WHO, 1996):<br />
• universally present in the faeces <strong>of</strong> humans <strong>and</strong> warm-blooded animals<br />
in large numbers<br />
• readily detected by simple methods<br />
• does not grow in natural waters, the general environment or water distribution<br />
systems<br />
• persistence in water <strong>and</strong> the extent to which it is removed by water treatment<br />
is similar to those <strong>of</strong> waterborne pathogens.<br />
The concept <strong>of</strong> coliforms as bacterial indicators <strong>of</strong> microbial water quality is based on the<br />
premise that because coliforms are present in high numbers in the faeces <strong>of</strong> humans <strong>and</strong><br />
other warm-blooded animals, if faecal pollution has entered drinking water, it is likely that<br />
these bacteria will be present, even after significant dilution. With few exceptions, coliforms<br />
themselves are not considered to be a health risk, but their presence indicates that faecal<br />
pollution may have occurred <strong>and</strong> pathogens might be present as a result.<br />
‘Coliform’ was the term first used in the 1880s to describe rod-shaped bacteria isolated from<br />
human faeces. The coliform group <strong>of</strong> bacteria, is a functionally-related group which all belong to<br />
a single taxonomic family (Enterobacteriaceae) <strong>and</strong> comprises many genera <strong>and</strong> species. Box 1<br />
contains an example <strong>of</strong> the relationship between family, genera <strong>and</strong> species for coliforms. There<br />
are other genera in the Enterobacteriaceae family, such as Salmonella <strong>and</strong> Shigella, that are not<br />
considered coliforms.<br />
3
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
4<br />
Box 1 Family, Genera <strong>and</strong> Species <strong>of</strong> Some Common <strong>Coliforms</strong><br />
Family Genera Species<br />
Enterobacteriaceae Escherichia Escherichia coli<br />
(E. coli)<br />
Klebsiella Klebsiella pneumoniae<br />
(K. pneumoniae)<br />
Enterobacter Enterobacter amnigenus<br />
(E. amnigenus)<br />
Citrobacter Citrobacter freundii<br />
(C. freundii)<br />
Of the coliforms normally present in the gut <strong>of</strong> warm-blooded animals, E. coli is the most<br />
numerous <strong>and</strong> is also the only coliform which rarely grows in the environment. Box 2 shows<br />
the distribution <strong>of</strong> coliforms present in human <strong>and</strong> animal faeces.<br />
Box 2 Distribution <strong>of</strong> Coliform Genera in Human <strong>and</strong> Animal Faeces (1)<br />
Sample Type % <strong>of</strong> Total <strong>Coliforms</strong><br />
E. coli Klebsiella spp. Enterobacter/<br />
Citrobacter spp.<br />
Reference<br />
Human faeces 96.8 1.5 1.7 Dufour (1977)<br />
94.1 5.9 Allen <strong>and</strong> Edberg (1995)<br />
Animal faeces 94 2 4 Dufour (1977)<br />
92.6 7.4 Allen <strong>and</strong> Edberg (1995)<br />
Notes : (1) Once faeces leaves the body <strong>and</strong> makes its way down the sewer, the proportions <strong>of</strong> coliforms that are<br />
E. coli drops to about 30% as the other coliforms start to grow (Geldreich, 1978).<br />
2.2 TOTAL COLIFORMS<br />
The total coliform group <strong>of</strong> bacteria was originally used as a surrogate for E. coli (the name<br />
coming from ‘coli-form’ or like) which, in turn, was considered to show faecal pollution.<br />
This was due to three reasons:<br />
1. coliform bacteria were readily isolated from human faecal material <strong>and</strong> water that had<br />
been impacted by pollution (these included E. coli <strong>and</strong> other coliforms, some <strong>of</strong> which<br />
also live naturally in soil <strong>and</strong> water environments);<br />
2. most <strong>of</strong> the coliforms recovered from human faeces were E. coli, <strong>and</strong> it was assumed<br />
that the presence <strong>of</strong> total coliforms reflected the presence <strong>of</strong> E. coli; <strong>and</strong><br />
3. the technology available to easily distinguish E. coli from other coliforms in the early<br />
1900s was not suitable for routine analysis.<br />
As a result, total coliforms were adopted <strong>and</strong> considered to be equivalent to E. coli until<br />
more specific <strong>and</strong> rapid methods became available. It was not until 1948 that the more<br />
specific <strong>and</strong> well-known 48 hour test for thermotolerant (faecal) coliforms was accepted.<br />
Despite the development <strong>of</strong> this <strong>and</strong> other specific methods, the use <strong>of</strong> total coliforms was so<br />
commonplace that they were not dropped in favour <strong>of</strong> E. coli or thermotolerant coliforms, but<br />
rather have remained co-indicators.
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Originally total coliform bacteria were considered to be from four genera <strong>of</strong> the family<br />
Enterobacteriaceae that could all ferment lactose. These genera were Escherichia, Klebsiella,<br />
Enterobacter <strong>and</strong> Citrobacter. Of the total coliforms present in the human gut, Escherichia coli<br />
(E. coli) represents the majority <strong>of</strong> the population (see Box 2). Total coliforms represent only<br />
about 1% <strong>of</strong> the total population <strong>of</strong> bacteria in human faeces in concentrations <strong>of</strong> about 10 9<br />
bacteria per gram (Brenner et al., 1982).<br />
It is widely accepted that the total coliform group <strong>of</strong> bacteria is diverse <strong>and</strong> they can be<br />
considered normal inhabitants <strong>of</strong> many soil <strong>and</strong> water environments which have not been<br />
impacted by faecal pollution. Even though the presence <strong>of</strong> E. coli is considered an appropriate<br />
<strong>and</strong> specific indicator <strong>of</strong> faecal pollution, uncertainty surrounds the use <strong>of</strong> total coliforms as a<br />
health indicator.<br />
As microbiological underst<strong>and</strong>ing about the nature <strong>of</strong> disease <strong>and</strong> the pathogens responsible<br />
increases, techniques have been developed to isolate <strong>and</strong> enumerate pathogenic viruses<br />
<strong>and</strong> protozoa from water. These techniques, however, are not sensible, specific, reliable,<br />
reproducible or inexpensive enough to replace the use <strong>of</strong> bacterial indicators.<br />
2.3 ESCHERICHIA COLI (E. COLI)<br />
More than 100 years ago scientists discovered that human faeces contained bacteria which if<br />
present in water, indicated that the water was not safe to drink. Escherich in 1885 observed<br />
2 types <strong>of</strong> organisms present in faeces, one <strong>of</strong> which he named Bacterium coli (B. coli, which<br />
is now called Escherichia coli) <strong>and</strong> the concept that the presence <strong>of</strong> B. coli implied pollution<br />
<strong>of</strong> water was readily adopted. It is recorded that the concept <strong>of</strong> “indicators” had already been<br />
suggested in 1880 by van Fritsch based on his observations <strong>of</strong> Klebsiellae in human faeces that<br />
were also present in water (Hendricks, 1978).<br />
Initially it was very difficult to distinguish B. coli from other coliform bacteria in water <strong>and</strong><br />
faeces, so methods were developed to recover all coliform bacteria, <strong>and</strong> more detailed <strong>and</strong><br />
lengthy analyses were carried out to confirm if any <strong>of</strong> the recovered coliforms were B. coli.<br />
Water bacteriologists for the next 50 years concentrated on developing these techniques to<br />
confirm the presence <strong>of</strong> B. coli in water <strong>and</strong> tell it apart from other gut bacteria. By the turn<br />
<strong>of</strong> the 20 th century, methods were available that could distinguish B. coli from the bacteria that<br />
caused typhoid (Salmonella typhi), <strong>and</strong> it was known that B. coli produced acid <strong>and</strong> gas from<br />
lactose, whereas Salmonella typhi did not. The techniques founded in the late 1800s <strong>and</strong> early<br />
1900s are still widely used to determine if faecal pollution <strong>of</strong> drinking water has occurred.<br />
5
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
3. USE OF BACTERIAL INDICATORS OF WATER QUALITY<br />
Most international drinking water quality guidelines <strong>and</strong> st<strong>and</strong>ards include bacterial indicators<br />
as a measure <strong>of</strong> microbial water quality, <strong>and</strong> for compliance reporting. The two major<br />
international bodies, the United States Environmental Protection Agency (USEPA), <strong>and</strong> the<br />
European Union (EU) both include E. coli as a m<strong>and</strong>atory microbial indicator, <strong>and</strong> the USEPA<br />
regulates for total coliforms, via the Total Coliform Rule.<br />
Most drinking water guidelines also refer to the use <strong>of</strong> total estimates <strong>of</strong> bacterial numbers<br />
in water. This measure is generally called ‘total heterotrophic plate count’ (HPC) or ‘st<strong>and</strong>ard<br />
plate count bacteria’, <strong>and</strong> is considered to represent the general cleanliness <strong>of</strong> a drinking<br />
water. As the HPC is not considered indicative <strong>of</strong> a potential health risk, these bacteria are<br />
not generally considered as a compliance measure, rather their numbers are monitored to<br />
underst<strong>and</strong> changes in a drinking water system over time <strong>and</strong> to alert operators to increases<br />
in general bacterial numbers.<br />
In response to a growing underst<strong>and</strong>ing <strong>and</strong> acceptance <strong>of</strong> the limitations <strong>of</strong> total coliforms,<br />
there has been a change <strong>of</strong> focus in Europe. In 1998, the EU, removed total coliforms as<br />
a m<strong>and</strong>atory primary indicator <strong>and</strong> added enterococci. In the EU st<strong>and</strong>ards, total coliforms<br />
are included with criteria whose presence can be negotiated with relevant Member State<br />
health departments. The relevant EU Legislation sections are shown in Box 3 <strong>and</strong> the USEPA<br />
Total Coliform Rule is described in Box 4, with the Australian Drinking Water Guidelines<br />
recommendations in Box 5.<br />
The World <strong>Health</strong> Organization (WHO) provides extensive guidance for countries to develop<br />
local drinking water guidelines <strong>and</strong> st<strong>and</strong>ards. The Second Edition <strong>of</strong> the WHO Guidelines<br />
for Drinking-water Quality (WHO, 1993) include recommendations for assessment <strong>of</strong><br />
microbial water quality based on the detection <strong>of</strong> E. coli <strong>and</strong> total coliforms. Volume 2 <strong>of</strong> the<br />
Second Edition (WHO, 1996) however, discusses in detail the inadequacies <strong>of</strong> total coliforms<br />
as an indicator <strong>of</strong> faecal pollution <strong>and</strong> debates the merits <strong>of</strong> alternative indicators such as<br />
enterococci <strong>and</strong> sulfite-reducing clostridia. The WHO Guidelines for Drinking-water Quality<br />
<strong>and</strong> the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996) are currently being<br />
updated to emphasise total system risk management with less focus on parametric values<br />
for acceptable water quality. The WHO is considering removing total coliforms as a primary<br />
compliance parameter in the revision for the Third Edition <strong>of</strong> the Guidelines for Drinkingwater<br />
Quality.<br />
The New Zeal<strong>and</strong> Ministry <strong>of</strong> <strong>Health</strong> released their Drinking Water St<strong>and</strong>ards for New Zeal<strong>and</strong><br />
2000 (NZMoH, 2000) in August 2000. These revised drinking water st<strong>and</strong>ards contain only<br />
E. coli as a bacterial indicator <strong>of</strong> faecal pollution, <strong>and</strong> no longer rely on faecal coliforms or<br />
total coliforms. The rationale for the move to E. coli is based on the acknowledgement that<br />
both total coliforms <strong>and</strong> faecal coliforms can be found in natural waters <strong>and</strong> their presence<br />
in drinking water does not necessarily indicate a health risk.<br />
7
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Box 3 <strong>Council</strong> Directive 98/83/EC <strong>of</strong> 3 November 1998 on the quality <strong>of</strong> water intended<br />
for human consumption<br />
Relevant Tables:<br />
Annex I: PARAMETERS AND PARAMETRIC VALUES<br />
Part A Microbial parameters<br />
Parameter Parametric Value (number/100 mL)<br />
Escherichia coli (E. coli) 0<br />
Enterococci 0<br />
Part C Indicator parameters*<br />
Parameter Parametric value Unit<br />
Colony Count no abnormal change colonies/mL<br />
Coliform Bacteria 0 numbers/100 mL<br />
*Indicator parameters also include aesthetic parameters such as colour, conductivity, chloride <strong>and</strong> taste <strong>and</strong> odour.<br />
Relevant Articles:<br />
(189C, 14) Whereas a balance should be struck to prevent both microbial <strong>and</strong> chemical risks; whereas to that<br />
end, <strong>and</strong> in the light <strong>of</strong> a future review <strong>of</strong> the parametric values, the establishment <strong>of</strong> parametric values<br />
applicable to water intended for human consumption should be based on public-health considerations <strong>and</strong><br />
on a method <strong>of</strong> assessing risk.<br />
(189C, 27) Whereas, in the event <strong>of</strong> non-compliance with a parameter which has an indicator function, the Member<br />
State concerned must consider whether that non-compliance poses any risk to human health; whereas<br />
it should take remedial action to restore the quality <strong>of</strong> the water where that is necessary to protect<br />
human health.<br />
Article 5 (2) The values set in accordance with paragraph 1 shall not be less stringent than those set out in Annex I.<br />
As regards the parameters set out in Annex I, Part C, the values need to be fixed only for monitoring<br />
purposes <strong>and</strong> for the fulfilment <strong>of</strong> the obligations imposed in Article 8.<br />
Article 8 (6) In the event <strong>of</strong> non-compliance with the parametric values or with the specifications set out in Annex I,<br />
Part C, Member States will consider whether that non-compliance poses any risk to human health. They shall<br />
take remedial action to restore the quality <strong>of</strong> the water where that is necessary to protect human health.<br />
Box 4 USEPA Total Coliform Rule<br />
The Total Coliform Rule (TCR) is part <strong>of</strong> the USEPA Safe Drinking Water Act (SDWA) <strong>and</strong> was effective on 31 December<br />
1990. The TCR sets both health goals (MCLGs) <strong>and</strong> legal limits (MCLs) for total coliforms. The Rule states that:<br />
Systems must not find coliforms in more than 5% <strong>of</strong> samples. When a system finds coliforms, the system must collect a set <strong>of</strong> repeat<br />
samples within 24 hours. When a repeat sample tests positive for total coliforms, it must also be analysed for faecal coliforms <strong>and</strong><br />
E. coli. A positive result to this last test signifies an acute MCL violation, which necessitates rapid state <strong>and</strong> public notification.<br />
8
Box 5 Australian Drinking Water Guidelines (1996)<br />
Guidelines for Microbial Quality<br />
Thermotolerant coliforms (or alternatively E. coli). 0/100 mL<br />
Total coliforms 0/100 mL<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Chapter 2 Microbial Quality <strong>of</strong> Drinking Water<br />
Section 2.8 System Performance (Paragraph 4)<br />
The performance <strong>of</strong> a system is judged by the number <strong>of</strong> times over a 12 month period that thermotolerant coliforms<br />
(or alternatively E. coli) <strong>and</strong> coliforms are detected in routine samples representative <strong>of</strong> water supplied to consumers.<br />
For samples representative <strong>of</strong> the quality <strong>of</strong> water supplied to consumers, performance can be regarded as satisfactory<br />
if over the preceding 12 months:<br />
• at least the minimum number <strong>of</strong> routine samples has been tested for indicator microorganisms;<br />
<strong>and</strong><br />
• at least 98% <strong>of</strong> the scheduled samples (as distinct from repeat or special purpose samples) contain<br />
no thermotolerant coliforms (or alternatively E. coli);<br />
<strong>and</strong><br />
• at least 95% <strong>of</strong> scheduled samples (as distinct from repeat or special purpose samples) contain no coliforms;<br />
EXCEPT THAT<br />
A higher level <strong>of</strong> coliform contamination might be tolerated in a particular area under<br />
certain conditions. These conditions should include –<br />
– the system meets the guideline for thermotolerant coliforms; <strong>and</strong><br />
– that the water authority can satisfy the appropriate health authority that the coliforms are unlikely<br />
to be <strong>of</strong> faecal origin (based on careful evaluation <strong>of</strong> their species identity); <strong>and</strong><br />
– that there is a level <strong>of</strong> monitoring sufficient to detect any change in the pattern <strong>of</strong> coliform occurrence<br />
(species composition <strong>and</strong> density); <strong>and</strong><br />
– that there is a direct monitoring <strong>of</strong> the occurrence <strong>of</strong> pathogenic microorganisms as the health authority<br />
may select to ensure that the coliform level does not represent a risk to public health; <strong>and</strong><br />
– that agreed levels <strong>of</strong> service for total coliforms are negotiated with the appropriate health authority <strong>and</strong> the<br />
consumers.<br />
9
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
4. THE CHANGING FACE OF COLIFORMS AND INDICATORS<br />
4.1 CHANGES IN COLIFORM DEFINITION<br />
The last two decades in microbiology have seen a move away from selective growth mediabased<br />
recovery methods for faecal bacteria to enzymatic <strong>and</strong> molecular methods. With these<br />
advances, the definition <strong>of</strong> what is considered a coliform has exp<strong>and</strong>ed, leading to increased<br />
scrutiny <strong>of</strong> the role that total coliforms play in water quality assessment <strong>and</strong> the validity <strong>of</strong> the<br />
information assumed by their presence or absence in drinking water systems.<br />
Pre 1994 – acid <strong>and</strong> gas from lactose<br />
Until the 1990s it was accepted that a coliform was a member <strong>of</strong> the Enterobacteriaceae,<br />
which displayed the biochemical characteristics <strong>of</strong> acid <strong>and</strong> gas production from lactose<br />
within 24–48 hours at 36±2°C. Thermotolerant or faecal coliforms were those that fitted<br />
the basic definition, but were able to grow <strong>and</strong> ferment lactose at 44.5±0.2°C. The<br />
vast majority <strong>of</strong> thermotolerant coliforms are E. coli <strong>and</strong> to a lesser extent Klebsiella,<br />
Enterobacter <strong>and</strong> Citrobacter. E coli are differentiated by their thermotolerance plus<br />
the ability to produce indole from tryptophan.<br />
Report 71 (1994) – acid only from lactose<br />
In 1994, the sixth edition <strong>of</strong> the UK, ‘Bacteriological Examination <strong>of</strong> Drinking Water<br />
Supplies 1982’ was published (HMSO, 1994). This report is referred to as Report 71.<br />
Report 71 altered a component <strong>of</strong> the biochemical definition <strong>of</strong> a coliform from:<br />
acid <strong>and</strong> gas production from lactose<br />
to<br />
acid-only production from lactose.<br />
This change in definition resulted in an increase in the number <strong>of</strong> bacterial species<br />
considered to be coliforms. Species <strong>of</strong> Enterobacter <strong>and</strong> Citrobacter, which do not<br />
produce gas from lactose were also included in the new definition. The ongoing review<br />
<strong>of</strong> the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996) has proposed<br />
adoption <strong>of</strong> this coliform definition.<br />
Current <strong>and</strong> Future - presence <strong>of</strong> specific enzymes<br />
With the advent <strong>of</strong> new technologies for bacterial analyses, the working definition <strong>of</strong><br />
a coliform has again changed. Lactose fermentation is one <strong>of</strong> the key criteria in the<br />
coliform definition <strong>and</strong> fermentation <strong>of</strong> lactose is determined, in part, by the presence<br />
<strong>of</strong> a specific enzyme, ß-galactosidase. The presence <strong>of</strong> ß-galactosidase in a member<br />
<strong>of</strong> the Enterobacteriaceae is considered specific to coliforms. Many water companies<br />
in the UK, USA, Europe <strong>and</strong> Australasia use commercial kits for total coliform analyses<br />
based on specific enzymes. The USEPA has adopted this technology <strong>and</strong> the associated<br />
coliform definition. It is understood that the next Report 71 will also include this more<br />
specific coliform definition <strong>and</strong> it has been proposed that it will be included in the<br />
revised Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996).<br />
11
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
12<br />
These enzyme-based methods appear to pick up coliforms that are traditionally not<br />
identified by selective media (George et al., 2000), so again the change in definition has<br />
exp<strong>and</strong>ed the range <strong>of</strong> bacteria recognised as coliforms as shown in Box 6. Appendix A<br />
contains a listing <strong>of</strong> currently available enzyme-based methods for total coliforms,<br />
E. coli <strong>and</strong> enterococci detection.<br />
Box 6 Coliform Members by Evolving Definition<br />
Pre 1994 Report 71, 1994 Enzyme-based<br />
Acid <strong>and</strong> Gas from Lactose Acid from Lactose ß-Galactosidase<br />
Escherichia Escherichia Escherichia<br />
Klebsiella Klebsiella Klebsiella<br />
Enterobacter Enterobacter Enterobacter<br />
Citrobacter Citrobacter Citrobacter<br />
Yersinia Yersinia<br />
Serratia Serratia<br />
Hafnia Hafnia<br />
Pantoea Pantoea<br />
Kluyvera Kluyvera<br />
Cedecea<br />
Ewingella<br />
Moellerella<br />
Leclercia<br />
Rahnella<br />
Yokenella<br />
bold type = coliforms which can be present in the environment as well as in human faeces.<br />
bold <strong>and</strong> underline = coliforms which are considered to be primarily environmental.<br />
Source: Kreig, 1984; Topley, 1997; Ewing, 1986; Ballows, 1992.
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
4.2 MOLECULAR METHODS FOR DETECTION OF MICROBIAL INDICATORS<br />
The future holds endless possibilities for methods to detect <strong>and</strong> identify indicator<br />
microorganisms <strong>and</strong> pathogens. On the horizon are methods based on sophisticated gene<br />
technology, which have only been used in medical research until now. A brief description<br />
<strong>of</strong> emerging technologies for microorganism detection is shown below, with detailed<br />
descriptions contained in Appendix B.<br />
DNA Microarray Technology<br />
This technique is based on testing water samples for the actual genetic material <strong>of</strong><br />
a microorganism, rather than relying on growing the microbe, or using a microscope.<br />
Large amounts <strong>of</strong> known genetic information (DNA or RNA) can be stored on a very<br />
small surface <strong>and</strong> used to detect microbes in a sample by reacting with complementary<br />
DNA or RNA from the microbial population.<br />
The microarray method was first developed by Stanford University (Elkins <strong>and</strong> Chu,<br />
1999) <strong>and</strong> was called “DNA Microarray”. It is envisaged that these methods can reduce<br />
the time <strong>of</strong> analyses for faecal indicators to 4 hours <strong>and</strong> reduce the cost significantly.<br />
Fluorescent in situ Hybridisation (FISH)<br />
FISH is another genetic method for detecting microorganisms. The method uses a<br />
fluorescent marker attached to the DNA <strong>of</strong> the microorganism that is being investigated.<br />
The sample can be processed on a fixed surface, generally a microscope slide, <strong>and</strong> if<br />
the target microorganism is present, the reaction results in the microorganism “glowing”.<br />
This is then viewed using a fluorescence microscope.<br />
A number <strong>of</strong> FISH methods have been developed for the detection <strong>of</strong> total coliforms<br />
<strong>and</strong> enterococci (Fuchs et al., 1998; Meier et al., 1997; Patel et al., 1998).<br />
13
5. TOTAL COLIFORMS AS INDICATORS<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
The information gained from the measurement <strong>of</strong> total coliforms can be confusing <strong>and</strong> the<br />
usefulness <strong>of</strong> coliforms (other than E. coli) as indicators <strong>of</strong> microbial water quality has been<br />
questioned for many years. This questioning has increased as research results have shown that<br />
total coliforms may not be an appropriate bacterial indicator <strong>of</strong> faecal pollution. The changing<br />
definition <strong>of</strong> total coliforms (Section 4.1) resulting in increased numbers <strong>of</strong> environmental<br />
bacteria is addressed in a draft revision <strong>of</strong> the Fact Sheet on coliforms in the Australian<br />
Drinking Water Guidelines (NHMRC-ARMCANZ, 1996, revised 2001), as follows:<br />
‘Detection <strong>of</strong> coliform bacteria in the absence <strong>of</strong> thermotolerant coliforms (or E. coli)<br />
may be tolerated providing it can be shown that the organisms do not indicate faecal<br />
contamination ‘<br />
<strong>and</strong><br />
‘Most coliforms including the thermotolerant coliforms (or alternatively E. coli) are not<br />
pathogenic but are used as indicators <strong>of</strong> the possible presence <strong>of</strong> faecal contamination<br />
<strong>and</strong> enteric pathogens. However, there are many environmental coliforms that are not<br />
<strong>of</strong> faecal origin <strong>and</strong> are <strong>of</strong> lesser significance (Fact Sheet 4 – <strong>Coliforms</strong>)’<br />
The following three points support why total coliforms are not a reliable indicator <strong>of</strong> potential<br />
health risk in water, <strong>and</strong> each reason is discussed in the following sections. <strong>Coliforms</strong> have<br />
been found to:<br />
1. grow in drinking water distribution systems;<br />
2. be normal inhabitants <strong>of</strong> soil, water <strong>and</strong> plants; <strong>and</strong><br />
3. not always be present during waterborne disease outbreaks.<br />
5.1 GROWTH IN DISTRIBUTION SYSTEMS<br />
One <strong>of</strong> the requirements <strong>of</strong> a robust indicator <strong>of</strong> faecal pollution is that it enters the drinking<br />
water system with the pollutant <strong>and</strong> survives for a time that is consistent with the survival <strong>of</strong><br />
pathogenic microorganisms. If a water quality indicator can multiply in the environment or in<br />
drinking water distribution systems, then detection does not necessarily imply that the system<br />
has been compromised by a pollution event, or that the water represents a potential public<br />
health risk.<br />
Bi<strong>of</strong>ilms are microbial populations that grow on the inside <strong>of</strong> pipes <strong>and</strong> other surfaces.<br />
A number <strong>of</strong> research studies have shown that coliform bacteria can grow within drinking<br />
water distribution systems <strong>and</strong> can be a significant contributor to bi<strong>of</strong>ilm populations<br />
(Power <strong>and</strong> Nagy, 1989; LeChevallier, 1990). It has been shown that the presence <strong>of</strong><br />
significant concentrations <strong>of</strong> coliforms within distribution systems, in themselves, do not<br />
represent a health risk to water consumers. For example, elevated concentrations <strong>of</strong> the<br />
coliform, Enterobacter cloacae isolated from within a water supply system were compared<br />
to Enterobacter cloacae isolated from the source water <strong>and</strong> from within several hospital<br />
environments by DNA analysis. Bacteria isolated from the three different environments were<br />
all different indicating the high numbers present within the system were not due to a failure<br />
in treatment processes but regrowth <strong>and</strong> those within the hospital were not from the water<br />
supply system. None <strong>of</strong> the isolates from the hospital environments were similar to those in<br />
the source water or treated water. In addition Enterobacter cloacae isolates from patients were<br />
different from those in the distribution system , indicating that the distribution system bacteria<br />
were not causing a public health risk (Edberg et al., 1994).<br />
15
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Studies on the growth <strong>of</strong> coliforms under low nutrient conditions such as those in drinking<br />
water distribution systems showed they could grow on surfaces <strong>and</strong> remain within bi<strong>of</strong>ilms<br />
successfully competing with other bacteria (Camper et al., 1996).<br />
To support the theory that bacteria growing in drinking water distribution systems do not<br />
represent a direct health risk to consumers, a review on the health significance <strong>of</strong> such<br />
bacteria found no evidence <strong>of</strong> any reported outbreaks <strong>of</strong> waterborne infection caused by<br />
typical bacteria growing within the system (PHLS, 1994). The review <strong>and</strong> application <strong>of</strong> risk<br />
ranking to bacteria in water supplies indicated that, while many are implicated in hospital<br />
infections they are not high risk <strong>and</strong> require specialised environments to grow <strong>and</strong>, frequently,<br />
only cause infections in already debilitated or immunocompromised people. Most hospitals<br />
are aware that drinking water is not sterile <strong>and</strong> adequate procedures for cleaning should be<br />
in place for potential niche sites as well as ensuring cleaning <strong>of</strong> wounds is not carried out<br />
with non-sterile solutions.<br />
5.2 NORMAL SOIL AND WATER INHABITANTS<br />
Many coliform bacteria, other than E. coli, form a small component <strong>of</strong> the normal intestinal<br />
population in humans <strong>and</strong> animals. It is well recognised <strong>and</strong> reported that E. coli is the only<br />
coliform that is an exclusive inhabitant <strong>of</strong> the gastrointestinal tract (Edberg et al., 2000). Most<br />
coliforms have an environmental origin <strong>and</strong> include plant pathogens <strong>and</strong> normal inhabitants<br />
<strong>of</strong> soil <strong>and</strong> water environments.<br />
For example, coliforms <strong>of</strong> the genus Serratia are soil, water <strong>and</strong> plant microorganisms <strong>and</strong><br />
play a role in insect disease (Villalobos et al., 1997). Another common coliform genera,<br />
Enterobacter, is widely distributed in nature, <strong>and</strong> is a common member <strong>of</strong> the community <strong>of</strong><br />
bacteria which live in <strong>and</strong> around the roots <strong>of</strong> plants (Hinton <strong>and</strong> Watson, 1995). Most genera<br />
<strong>of</strong> coliforms have members that are found in natural environments more <strong>of</strong>ten than they are<br />
found in the intestines <strong>of</strong> humans <strong>and</strong> animals. It is interaction with the environment which<br />
results in the initial colonisation <strong>of</strong> the human intestine with coliform bacteria.<br />
5.3 WATERBORNE DISEASE OUTBREAKS<br />
Total coliforms have been shown not to be a sensitive indicator <strong>of</strong> the risk <strong>of</strong> waterborne<br />
disease. In some reported waterborne disease outbreaks, coliforms <strong>and</strong> E. coli have been<br />
detected in drinking water, while in others they are not present. The presence <strong>of</strong> E. coli<br />
is more representative <strong>of</strong> faecal pollution than other coliforms, because it occurs in higher<br />
numbers in faecal material, <strong>and</strong> generally does not occur elsewhere in the environment.<br />
Waterborne disease outbreaks have been reported where drinking water did not contain<br />
detectable total coliform bacteria. In the USA between 1978–1986 there were 502 reported<br />
outbreaks <strong>of</strong> waterborne disease involving more than 110 000 cases <strong>of</strong> gastrointestinal illness.<br />
Many <strong>of</strong> the implicated water supplies in these outbreaks met the coliform compliance<br />
requirements <strong>of</strong> the USEPA (Sobsey, 1989). An additional study <strong>of</strong> outbreaks <strong>of</strong> waterborne<br />
disease in the USA found that one third <strong>of</strong> water supplies responsible for disease outbreaks<br />
did not have any total coliforms isolated from within the system (Craun, et al., 1997). Craun<br />
concluded that the presence <strong>of</strong> coliforms (including E. coli) is sometimes a useful indicator<br />
for viruses <strong>and</strong> bacteria, but not for protozoan parasites.<br />
16
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
The study <strong>of</strong> waterborne disease outbreaks increasingly show that outbreaks are being<br />
attributed to non-bacterial pathogens (viruses, protozoa) (Rose, 1990). This is most likely due<br />
to an increased underst<strong>and</strong>ing <strong>of</strong> the role <strong>of</strong> these pathogens in waterborne disease <strong>and</strong> the<br />
availability <strong>of</strong> more sophisticated <strong>and</strong> reproducible laboratory methods to recover <strong>and</strong> identify<br />
them from source <strong>and</strong> treated water.<br />
Waterborne disease outbreaks where total coliforms are detected are generally attributed<br />
to bacteria, viruses or unknown agents, as shown by Moore, et al., 1994, who reported that<br />
88% <strong>of</strong> such outbreaks had coliforms present in water samples. For outbreaks attributed to<br />
protozoan parasites, only 33% <strong>of</strong> waters were positive for coliforms.<br />
Much <strong>of</strong> the difficulty in correlating the presence <strong>of</strong> bacterial indicators to the presence<br />
<strong>of</strong> protozoa is due to different susceptibility <strong>of</strong> protozoa to treatment processes, particularly<br />
chlorine. Chlorine as a disinfectant is more effective against bacteria <strong>and</strong> viruses than protozoa<br />
making coliforms limited in their use as a measure <strong>of</strong> the effectiveness <strong>of</strong> treatment processes<br />
for the removal <strong>of</strong> these organisms (Sobsey, 1989).<br />
17
6. ALTERNATIVES TO TOTAL COLIFORMS<br />
6.1 CURRENT AND FUTURE USE OF COLIFORMS<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
There is now sufficient evidence that the presence <strong>of</strong> coliform bacteria (other than E. coli)<br />
within drinking water does not clearly indicate the presence <strong>of</strong> a health risk nor their absence,<br />
an absence <strong>of</strong> health risk. The appropriate role <strong>of</strong> coliforms needs to be addressed along with<br />
the suitability <strong>of</strong> the emphasis placed on their presence or absence for compliance. <strong>Coliforms</strong><br />
can indicate a range <strong>of</strong> different things within a drinking water system, but indicate no single<br />
thing with confidence.<br />
The interpretation <strong>of</strong> the presence or absence <strong>of</strong> total coliforms in drinking water, which is<br />
driven by the need to meet compliance targets, does not enable water quality managers or<br />
health <strong>of</strong>ficials to make confident decisions about the microbial safety <strong>of</strong> the water. This endpoint,<br />
compliance-driven system needs to be replaced with a complete management system,<br />
which incorporates an underst<strong>and</strong>ing <strong>of</strong> risks posed, the best ways to manage them <strong>and</strong><br />
specific testing to validate the effectiveness <strong>of</strong> its implementation.<br />
Drinking water supply <strong>and</strong> system monitoring for bacterial indicators has four major purposes:<br />
1. to identify general faecal contamination <strong>of</strong> source waters;<br />
2. to demonstrate that treatment <strong>and</strong>/or disinfection processes are working effectively;<br />
3. to alert for possible in-system contamination through cross connections, ingress,<br />
pipe break contamination <strong>and</strong> contamination from open storages; <strong>and</strong><br />
4. to monitor bi<strong>of</strong>ilm growth, general system cleanliness <strong>and</strong> the potential presence<br />
<strong>of</strong> opportunistic pathogens.<br />
The current use <strong>of</strong> total coliforms for each purpose gives limited useful information, <strong>and</strong><br />
generally better indicators are available. This is summarised in Box 7.<br />
Many alternative indicators to total coliforms have been proposed including enterococci,<br />
sulfite-reducing clostridia, Bacteroides fragilis, Bifidobacteria, bacteriophages, <strong>and</strong> nonmicrobial<br />
indicators such as faecal sterols. Of these proposed indicators, enterococci has<br />
gained the most acceptance, particularly when used in conjunction with E. coli (Edberg et al.,<br />
2000; Pinto et al., 1999; Sinton et al., 1993; WHO, 1996).<br />
19
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Box 7 Current use <strong>of</strong> <strong>Coliforms</strong> <strong>and</strong> Alternatives<br />
Information To identify general faecal contamination <strong>of</strong> source waters<br />
Need<br />
Currently Use Presence <strong>of</strong> total coliforms/thermotolerant coliforms.<br />
Issue Short-term changes missed, aged faecal material may not contain coliforms, yet persistent pathogens could be<br />
present. Unknown source <strong>of</strong> faecal contamination <strong>and</strong> what to manage.<br />
Future Use E. coli <strong>and</strong> other more specific faecal indicators such as enterococci, sulfite-reducing clostridia <strong>and</strong> faecal<br />
sterols. On-line turbidity measurement.<br />
Information To demonstrate that treatment <strong>and</strong>/or disinfection processes are working effectively<br />
Need<br />
Currently Use Absence <strong>of</strong> total coliforms <strong>and</strong> E. coli after treatment.<br />
Issue Sampling frequency generally too low to detect chlorination upset or treatment failure. <strong>Coliforms</strong> more<br />
sensitive to chlorination than viral or protozoan pathogens.<br />
Future Use Barrier measurement such as on-line particle sizing <strong>and</strong> chlorine analysers, ensure adequate disinfection.<br />
Information To alert water managers <strong>and</strong> operators to in-system contamination through<br />
Need pipebreaks,ingress, cross connections <strong>and</strong> contamination from open storages<br />
Currently Use Presence <strong>of</strong> coliforms or E. coli in distribution system samples.<br />
Issue Cross connection contamination will result in high numbers <strong>of</strong> faecal organisms entering a system. In this<br />
case, E. coli is a suitable indicator <strong>and</strong> will be present in high numbers. <strong>Coliforms</strong> can already be present in<br />
the system from bi<strong>of</strong>ilms or regrowth, so their detection confuses the issue. The use <strong>of</strong> a chlorine residual<br />
has been shown to effectively reduce numbers <strong>of</strong> coliforms <strong>and</strong> indicator bacteria, while leaving other ingress<br />
pathogens unaffected.<br />
Future Use E. coli <strong>and</strong> other more specific faecal indicators such as enterococci <strong>and</strong> faecal sterols. On-line chlorine<br />
analysers <strong>and</strong> pressure measurement.<br />
Information To monitor growth <strong>of</strong> bi<strong>of</strong>ilms <strong>and</strong> general system cleanliness<br />
Need<br />
Currently Use <strong>Coliforms</strong><br />
Issue Although coliforms can become part <strong>of</strong> a bi<strong>of</strong>ilm community, they do not represent the majority <strong>of</strong> bacteria<br />
within the bi<strong>of</strong>ilm. Some bi<strong>of</strong>ilms have very few, or no coliforms associated with them, yet may contain<br />
opportunistic pathogens.<br />
Future Use Plate count (heterotrophic) bacteria comprise the vast majority <strong>of</strong> bacteria found in bi<strong>of</strong>ilms. Constant<br />
erosion or r<strong>and</strong>om sloughing-<strong>of</strong>f <strong>of</strong> bi<strong>of</strong>ilm into the water flow may result in high numbers <strong>of</strong> plate count<br />
bacteria in the absence <strong>of</strong> coliforms.<br />
20
6.2 WATER QUALITY RISK MANAGEMENT APPROACH<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Reliance on total coliforms for measurement <strong>of</strong> the microbial safety <strong>of</strong> a drinking water can<br />
result in a false sense <strong>of</strong> assurance from negative results. In many instances, E. coli <strong>and</strong> total<br />
coliforms are the sole indicators analysed to determine microbial water quality. The retrospective<br />
study <strong>of</strong> waterborne disease outbreaks <strong>and</strong> advances in the underst<strong>and</strong>ing <strong>of</strong> the behaviour<br />
<strong>of</strong> pathogens in water, has shown that continued reliance on bacterial indicators alone, <strong>and</strong><br />
assumptions surrounding the absence or presence <strong>of</strong> total coliforms does not ensure that<br />
informed decisions are made regarding water quality.<br />
A risk management approach to drinking water supply is being adopted across Australia to<br />
increase confidence in the safety <strong>of</strong> drinking water <strong>and</strong> reduce reliance on end-point testing.<br />
Several major Australian water suppliers have developed risk management plans that are a<br />
holistic approach to water management. These plans systematically assess risks throughout<br />
a drinking water supply, from the catchment <strong>and</strong> source water, through to the customer tap,<br />
<strong>and</strong> identify the ways that these risks can be managed <strong>and</strong> methods to ensure that barriers<br />
<strong>and</strong> control measures are working effectively. A risk management plan assesses the integrity<br />
<strong>of</strong> the entire water supply system <strong>and</strong> is able to incorporate strategies to deal with day-to-day<br />
management <strong>of</strong> water quality as well as upsets <strong>and</strong> failures.<br />
The ongoing review <strong>of</strong> the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996),<br />
is resulting in the development <strong>of</strong> a comprehensive drinking water quality management<br />
framework. The framework supplements system management information currently included<br />
in the Guidelines with principles from existing management systems such as the International<br />
Organisation for St<strong>and</strong>ardisation (ISO) series <strong>and</strong> the Hazard Analysis <strong>and</strong> Critical Control<br />
Point (HACCP) system. HACCP is a risk prevention/risk management system that has been<br />
used extensively in the food industry <strong>and</strong> is now being adopted for risk management <strong>of</strong> water<br />
production <strong>and</strong> supply. The draft framework, which has been trialed in a number <strong>of</strong> water<br />
supplies, will enable water managers to identify <strong>and</strong> rank risks within the water supply <strong>and</strong><br />
establish critical control points where these risks can be managed. The framework will focus<br />
on total system management, measurement <strong>of</strong> barriers <strong>and</strong> verification using end-point testing.<br />
Internationally, the World <strong>Health</strong> Organization (WHO, 1999) has developed a risk management<br />
approach to water quality as a model for assessing the safety <strong>of</strong> recreational waters (the<br />
Annapolis Protocol). This approach is being proposed as part <strong>of</strong> a harmonised framework<br />
for managing risks from drinking water <strong>and</strong> food production. The WHO is further developing<br />
this risk management approach in the current development <strong>of</strong> the Third Edition <strong>of</strong> the<br />
Guidelines for Drinking-water Quality.<br />
A risk management approach for drinking water includes (1) end-point monitoring to verify<br />
that the water supplied to consumers was safe; <strong>and</strong> (2) operational monitoring to show<br />
that treatment processes are functioning properly <strong>and</strong> that distribution system integrity is<br />
maintained. End-point monitoring cannot be used as a system control measure, only as a final<br />
verification step in a complete risk management plan. Operational monitoring is a means <strong>of</strong><br />
assessing system performance <strong>and</strong> results are used to modify system controls to ensure that<br />
processes are working within specification. For this reason, on-line <strong>and</strong> continuous monitoring<br />
for operational purposes is better able to support system management.<br />
21
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Parameters within a risk management approach to monitor <strong>and</strong> verify water quality should be<br />
simple <strong>and</strong> include a range <strong>of</strong> parameters, which can indicate:<br />
• Faecal contamination <strong>of</strong> source waters;<br />
• Treatment effectiveness;<br />
• Faecal contamination from in-system ingress; <strong>and</strong><br />
• Water stagnation, bi<strong>of</strong>ilm growth, system cleanliness <strong>and</strong> the potential presence <strong>of</strong><br />
opportunistic pathogens.<br />
Box 8 shows a number <strong>of</strong> indicators, both physical <strong>and</strong> microbial, which can be incorporated<br />
as part <strong>of</strong> a risk management framework.<br />
Box 8 Water Quality Matrix Indicators<br />
Hazard Indicator<br />
Faecal contamination <strong>of</strong> source water • sanitary survey<br />
• turbidity<br />
• E. coli<br />
Treatment effectiveness • total chlorine<br />
• HPC (1)<br />
• E. coli<br />
Faecal contamination from in-system ingress • ammonia<br />
• enterococci<br />
• E. col<br />
• dissolved oxygen (sudden change)<br />
• free chlorine (sudden change)<br />
• pressure (sudden change)<br />
Water stagnation • loss <strong>of</strong> disinfectant residual<br />
• dissolved oxygen<br />
• HPC (1)<br />
Potential presence <strong>of</strong> opportunistic pathogens • HPC (1)<br />
Notes: (1) HPC = Heterotrophic Plate Count<br />
22<br />
• free chlorine
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
6.3 ESCHERICHIA COLI AND ENTEROCOCCI – KEY FAECAL INDICATORS<br />
It is widely acknowledged that the major threat to public health from drinking water is from<br />
microbial contamination with human, <strong>and</strong> to a lesser degree, animal faeces. One detailed risk<br />
assessment <strong>of</strong> pathogens <strong>and</strong> chemicals in drinking water concluded (Regli et al., 1993):<br />
“risk <strong>of</strong> death from known pathogens in untreated water is 100 to 1000 times greater<br />
than risk <strong>of</strong> cancer from known disinfection by-products in chlorinated drinking water<br />
<strong>and</strong><br />
the risk <strong>of</strong> illness from pathogens in untreated surface water is 10 000 to 1 000 000 times<br />
greater than risk <strong>of</strong> cancer from disinfection by-products in chlorinated drinking water”<br />
As a component <strong>of</strong> the assessment <strong>of</strong> public health risk through monitoring <strong>of</strong> water quality<br />
at consumer’s taps, E. coli is regarded as the most sensitive indicator <strong>of</strong> faecal pollution. The<br />
large numbers <strong>of</strong> E. coli present in the gut <strong>of</strong> humans <strong>and</strong> other warm-blooded animals <strong>and</strong><br />
the fact that they are not generally present in other environments support their continued use<br />
as the most sensitive indicator <strong>of</strong> faecal pollution available (Edberg et al., 2000).<br />
To increase the confidence <strong>of</strong> water quality results, especially when monitoring for faecal<br />
pollution, analysis for enterococci has been used (eg. EU guidelines, Section 3, Box 3). The<br />
enterococci are the group <strong>of</strong> bacteria most <strong>of</strong>ten suggested as alternatives to coliforms, <strong>and</strong><br />
interest in their use as a water quality indicator date back to 1900 when they were found to be<br />
common commensal bacteria in the gut <strong>of</strong> warm-blooded animals (Gleeson <strong>and</strong> Gray, 1997).<br />
The enterococci were included in the functional group <strong>of</strong> bacteria known as “faecal<br />
streptococci” <strong>and</strong> now largely belong in the genus Enterococcus which was formed by<br />
the splitting <strong>of</strong> Streptococcus faecalis <strong>and</strong> Streptococcus faecium, along with less important<br />
streptococci, from the genus Streptococcus (Schleifer <strong>and</strong> Klipper-Balz, 1984). There are<br />
now 19 species that are included as enterococci (Topley, 1997). The predominant intestinal<br />
enterococci are Enterococcus faecalis, E. faecium, E. durans <strong>and</strong> E. hirae. In addition, other<br />
Enterococcus species <strong>and</strong> some species <strong>of</strong> Streptococcus (namely S. bovis, <strong>and</strong> S. equinus)<br />
may occasionally be detected. Generally, for water examination purposes enterococci can<br />
be regarded as indicators <strong>of</strong> faecal pollution, although some can occasionally originate<br />
from other habitats.<br />
Enterococci have a number <strong>of</strong> advantages as indicators over total coliforms <strong>and</strong> even E. coli,<br />
including that they generally do not grow in the environment (WHO, 1993) <strong>and</strong> they have<br />
been shown to survive longer (McFeters et al., 1974). Despite being approximately an order<br />
<strong>of</strong> magnitude less numerous than faecal coliforms <strong>and</strong> E. coli in human faeces (Feacham et<br />
al., 1983), they are still numerous enough to be detected after significant dilution. Rapid <strong>and</strong><br />
simple methods, based on defined substrate technology, are available for the detection <strong>and</strong><br />
enumeration <strong>of</strong> enterococci <strong>and</strong> routinely employed in many laboratories (see Appendix A<br />
for description <strong>of</strong> methods).<br />
There is some concern that enterococci are a diverse group <strong>of</strong> bacteria, <strong>and</strong> that the group<br />
contains species that are environmental <strong>and</strong> their presence in water is not necessarily<br />
indicative <strong>of</strong> faecal pollution. This concern is driven by the problems associated with the use<br />
<strong>of</strong> total coliforms as an indicator <strong>of</strong> faecal pollution. An early research report showed that<br />
Enterococcus faecalis var liquefaciens was common in good quality water <strong>and</strong> its relevance<br />
was not considered clear if recovered in waters in concentrations <strong>of</strong> less than 100 organisms/<br />
100 mL (Geldreich, 1970). More recent research on the relevance <strong>of</strong> faecal streptococci as<br />
indicators <strong>of</strong> pollution, showed that the majority <strong>of</strong> enterococci (84%) isolated from a variety<br />
<strong>of</strong> polluted water sources were “true faecal species” (Pinto et al., 1999).<br />
23
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Measurement for enterococci in water is used in South Australia as an additional indicator<br />
when total coliforms are present in the absence <strong>of</strong> E. coli (Cunliffe, 2000), while in Sydney<br />
faecal streptococci are used to confirm faecal contamination if either total coliforms or<br />
E. coli are detected (Ashbolt pers. comm.). The WHO (1996) also recommends the use <strong>of</strong><br />
faecal streptococci (<strong>of</strong> which enterococci are a sub-group) as an additional indicator <strong>of</strong> faecal<br />
pollution. When combined with the measurement <strong>of</strong> E. coli, the result is increased confidence<br />
in the absence or presence <strong>of</strong> faecal pollution.<br />
6.4 CLOSTRIDIUM PERFRINGENS<br />
Clostridium perfringens (C. perfringens) are sulfite-reducing, spore-forming, clostridia, which<br />
are hardy rod-shaped anaerobic bacteria. They are widely spread through nature <strong>and</strong> have<br />
been isolated from the intestines <strong>of</strong> many animals (Cato et al., 1986). It is reported that the<br />
use <strong>of</strong> C. perfringens as an indicator organism was first proposed in 1899 (cited in Gleeson<br />
<strong>and</strong> Gray, 1997). The spores produced by C. perfringens are very resistant to disinfection <strong>and</strong><br />
the WHO (1996) suggests that their presence in filtered supplies may not be an indication<br />
<strong>of</strong> treatment inefficiencies. In disinfected supplies, their presence may not be an indication<br />
<strong>of</strong> poor inactivation performance for this same reason (Fujioka <strong>and</strong> Shizumura, 1985).<br />
Spores <strong>of</strong> C. perfringens are largely <strong>of</strong> faecal origin (Sorensen et al., 1989) <strong>and</strong> are always<br />
present in sewage. Their spores are highly resistant in the environment, <strong>and</strong> vegetative cells<br />
appear not to reproduce in aquatic sediments, unlike many traditional indicator bacteria<br />
(Davies et al., 1995). There is evidence to show that C. perfringens may be a suitable indicator<br />
for viruses <strong>and</strong> parasitic protozoa when sewage is the suspected cause <strong>of</strong> contamination<br />
(Payment <strong>and</strong> Franco, 1993).<br />
Nonetheless, C. perfringens is not generally considered a robust indicator <strong>of</strong> microbial water<br />
quality because they can survive <strong>and</strong> accumulate in drinking water systems <strong>and</strong> may be<br />
detected long after a pollution event has occurred <strong>and</strong> far from the source (WHO, 1996).<br />
Their preferred role is to aid identification <strong>of</strong> faecal contamination in sanitary surveys.<br />
6.5 BACTERIOPHAGES<br />
Bacteriophages are viruses that infect bacteria <strong>and</strong> those that infect coliforms are known as<br />
coliphages, or more generally, phages. Phages have been proposed as microbial indicators as<br />
they behave more like the human enteric viruses which pose a health risk to water consumers<br />
if water has been contaminated with human faeces.<br />
The use <strong>of</strong> phages as water quality indicators has been extensively researched <strong>and</strong> the<br />
limitations <strong>of</strong> their use widely debated. Box 9 shows a summary <strong>of</strong> the limitations <strong>of</strong> phages<br />
as reliable water quality indicators. <strong>Research</strong> results show that phages cannot be considered as<br />
reliable indicators, models or surrogates for enteric viruses in water. This is underlined by the<br />
detection <strong>of</strong> enteric viruses in treated drinking water supplies which were negative for phages<br />
(Grabow et al., 2000). Phages are probably best applied as models/surrogates in laboratory<br />
experiments where the survival or behaviour <strong>of</strong> selected phages <strong>and</strong> viruses are directly<br />
compared under controlled conditions (Grabow et al., 1983, 1999b; Naranjo et al., 1997).<br />
24
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Box 9 Limitations <strong>of</strong> Phages Reference<br />
Phages are excreted by a certain percentage <strong>of</strong> humans <strong>and</strong> animals all the time Vaughn <strong>and</strong> Metcalf, 1975<br />
while viruses are excreted only by infected individuals for a short period <strong>of</strong> time. Borrego et al., 1990<br />
There is no direct correlation in numbers <strong>of</strong> phages <strong>and</strong> viruses in human faeces. Grabow et al., 1993<br />
Grabow et al., 1999a<br />
Enteric viruses have been detected in water environments in the absence <strong>of</strong> coliphages Montgomery, 1982<br />
Morinigo et al., 1992<br />
Human enteric viruses associated with waterborne diseases are excreted almost exclusively Osawa et al., 1981<br />
by humans, whereas phages used as models/surrogates in water quality assessment are Furuse et al., 1983<br />
excreted by humans <strong>and</strong> animals. The faeces <strong>of</strong> animals such as cows <strong>and</strong> pigs generally Grabow et al., 1993<br />
contains higher densities <strong>of</strong> coliphages than that <strong>of</strong> humans, <strong>and</strong> the percentage <strong>of</strong> many Grabow et al.,1995<br />
animals which excrete phages tends to be higher than for humans. Grabow, 1996<br />
Some coliphages may replicate in water environments Seeley <strong>and</strong> Primrose, 1982;<br />
Grabow et al., 1984;<br />
Borrego et al., 1990<br />
6.6 SUMMARY<br />
There is a substantial amount <strong>of</strong> information currently available on the advantages <strong>and</strong><br />
disadvantages <strong>of</strong> total coliforms <strong>and</strong> other indicators <strong>of</strong> water quality (Gleeson <strong>and</strong> Gray, 1997;<br />
Ashbolt et al., 2001). Most <strong>of</strong> the scientific literature <strong>and</strong> water quality guideline development<br />
supports a more scientifically defensible <strong>and</strong> risk-based approach for public health protection.<br />
There is widespread agreement that the presence <strong>of</strong> total coliforms, other than E. coli, does<br />
not assist water managers determine if there is an associated public health risk. The presence<br />
<strong>of</strong> E. coli can be relied upon to indicate faecal pollution has occurred, as can generally the<br />
presence <strong>of</strong> enterococci.<br />
Other faecal indicators superior to E. coli <strong>and</strong> enterococci have not been developed to a point<br />
where there are methods readily available that are inexpensive <strong>and</strong> simple for routine use.<br />
The monitoring <strong>of</strong> water filtration plants requires a continuous type <strong>of</strong> assessment using<br />
criteria such as turbidity <strong>and</strong> particle size distribution. Monitoring <strong>of</strong> these parameters in<br />
source <strong>and</strong> treated water allows early warning <strong>and</strong> treatment efficacy assessment. Disinfection<br />
effectiveness <strong>and</strong> system cleanliness assessment is better achieved using disinfectant residual<br />
(C.t), which is a measure <strong>of</strong> the concentration <strong>of</strong> disinfectant <strong>and</strong> contact time, combined with<br />
measurement <strong>of</strong> total heterotrophic bacteria (<strong>of</strong> which coliforms are a sub-set). Water operators<br />
should be alerted by sudden changes in these parameters.<br />
25
7. CONCLUSIONS<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Water suppliers have been aware <strong>of</strong> the role <strong>of</strong> water in disease transmission for more than<br />
150 years, during which time the primary focus <strong>of</strong> managing drinking water has been the<br />
protection <strong>of</strong> public health. The fundamental issues associated with public health impacts<br />
<strong>and</strong> the need for safe drinking water are currently well understood.<br />
The Australian <strong>and</strong> international water industry is making a positive move towards<br />
underst<strong>and</strong>ing <strong>and</strong> managing risks to public health from drinking water. This has been<br />
driven by advances in methods for detecting pathogens, epidemiological studies to measure<br />
background levels <strong>of</strong> waterborne disease, <strong>and</strong> a realisation that over reliance on treatment<br />
processes to protect public health is not a sustainable management approach.<br />
Detailed assessment <strong>of</strong> the factors that influence water quality necessarily includes a review<br />
<strong>of</strong> the way in which the microbial safety <strong>of</strong> water delivered is measured. The current focus<br />
on the absence <strong>of</strong> total coliforms <strong>and</strong> E. coli to ensure water quality has been shown to be<br />
flawed <strong>and</strong> the water industry <strong>and</strong> regulators internationally are evaluating alternative ways<br />
to protect public health <strong>and</strong> the usefulness <strong>of</strong> these indicators.<br />
The collection <strong>of</strong> information on water quality is costly, therefore data needs to be<br />
unambiguous, <strong>and</strong> able to assist risk management decisions. The presence <strong>of</strong> E. coli in<br />
treated water is still considered to indicate faecal pollution <strong>and</strong> remains a key verification<br />
tool. The uncertainty surrounding the relevance <strong>of</strong> other coliforms in drinking water means<br />
that their value as an indicator is limited. A set <strong>of</strong> indicators, used in conjunction with<br />
total-system risk management, is required that increases confidence in the safety <strong>of</strong> water<br />
delivered to consumers.<br />
The move by the EU to downgrade total coliforms <strong>and</strong> include m<strong>and</strong>atory measurement<br />
<strong>of</strong> enterococci as well as E. coli is one that recognises the worth <strong>of</strong> using specific indicators<br />
<strong>of</strong> faecal pollution, <strong>and</strong> assigns a minor role to total coliforms.<br />
The limitation <strong>of</strong> total coliforms in Australia has been reaffirmed in the ongoing review <strong>of</strong><br />
the Australian Drinking Water Guidelines (NHMRC-ARMCANZ, 1996). The revised Fact Sheets<br />
for thermotolerant coliforms/E. coli <strong>and</strong> total coliforms acknowledge that coliforms can be<br />
<strong>of</strong> environmental origin <strong>and</strong> that their presence in drinking water is not necessarily indicative<br />
<strong>of</strong> a health risk.<br />
The importance <strong>of</strong> risk management is also addressed in the ongoing review <strong>of</strong> the Australian<br />
Drinking Water Guidelines (NHMRC-ARMCANZ, 1996) with the development <strong>and</strong> trialing<br />
<strong>of</strong> a framework for the risk management <strong>of</strong> drinking water systems. The development <strong>of</strong><br />
a set <strong>of</strong> criteria to verify that risk management plans are effective is a necessary part <strong>of</strong> this<br />
approach to water quality. In the long-term review <strong>of</strong> appropriate water quality monitoring,<br />
it is envisaged that a matrix approach will be adopted, with less significance on single<br />
parameters such as total coliforms.<br />
The next step in the current direction <strong>of</strong> water quality management <strong>and</strong> compliance monitoring<br />
for Australia is to reduce reliance on total coliforms <strong>and</strong> increase emphasis on implementation<br />
<strong>of</strong> risk management systems, measurement <strong>of</strong> specific faecal indicators, <strong>and</strong> assessment <strong>of</strong> the<br />
effectiveness <strong>of</strong> source water protection, treatment <strong>and</strong> disinfection barriers.<br />
27
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Information currently available supports the use <strong>of</strong> Escherichia coli as the primary indicator<br />
<strong>of</strong> faecal pollution supported by other measurements such as heterotrophic bacteria, C.t,<br />
chlorine residual <strong>and</strong> turbidity to verify treatment <strong>and</strong> disinfection effectiveness <strong>and</strong> assess<br />
system cleanliness.<br />
Above all, a move away from reliance on total coliform bacteria <strong>and</strong> adoption <strong>of</strong> a risk<br />
management approach to drinking water quality for Australia will ensure that suppliers<br />
<strong>and</strong> consumers can have increased confidence in the safety <strong>of</strong> their drinking water.<br />
28
8. RECOMMENDATIONS<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
It is recommended that:<br />
1. Total coliforms be removed as an indicator <strong>of</strong> faecal contamination in the Australian<br />
Drinking Water Guidelines (NHMRC–ARMCANZ, 1996); <strong>and</strong><br />
2. E. coli be the primary indicator <strong>of</strong> faecal contamination in the Australian Drinking<br />
Water Guidelines (NHMRC–ARMCANZ, 1996).<br />
29
9. REFERENCES<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Allen, M.J. <strong>and</strong> Edberg, S.C. (1995) The public health significance <strong>of</strong> bacterial indicators<br />
in drinking water. The Royal Society <strong>of</strong> Chemistry 1999. Special Publication. Athenaeum Press,<br />
UK.<br />
Amann, R.I., Ludwig, W. <strong>and</strong> Schleifer, K-H. (1995) Phylogenetic identification <strong>and</strong> in situ<br />
detection <strong>of</strong> individual microbial cells without cultivation. Microbiology <strong>Review</strong>s. 59:143-169.<br />
Ashbolt, N. J., Grabow, W. O. K., <strong>and</strong> Snozzi, M. (2001) Indicators <strong>of</strong> microbial water quality.<br />
In: Water Quality: Guidelines, St<strong>and</strong>ards <strong>and</strong> <strong>Health</strong>. Risk assessment <strong>and</strong> management for<br />
water-related infectious disease. (Eds.: Fewtrell, L, <strong>and</strong> J. Bartram) IWA Press, London.<br />
Pp.289-316.<br />
Ballows, A. (1992) The Prokaryotes. 2 nd Edition. Springer Verlag, New York.<br />
Brenner, D.J., David, B.R., <strong>and</strong> Steigerwalt, A.G. (1982) Atypical biogroups <strong>of</strong> Escherichia<br />
coli found in clinical specimens <strong>and</strong> description <strong>of</strong> Escherichia hermanii sp. nov. Journal <strong>of</strong><br />
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36
APPENDIX A<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
ENZYME-BASED METHODS FOR THE DETECTION OF MICROBIAL INDICATORS<br />
A number <strong>of</strong> colourimetric media enabling quantification <strong>of</strong> total coliforms <strong>and</strong> E. coli within<br />
24h are now available, as well as for the enterococci as follows <strong>and</strong> shown in Box A1:<br />
• Enteroler®, manufactured by IDEXX (Hern<strong>and</strong>ez et al., 1991; Manafi, 1996);<br />
• Colisure® manufactured by IDEXX (McFeters et al., 1995);<br />
• Colilert®, manufactured by IDEXX (Edberg et al., 1988; Edberg et al., 1991);<br />
• m-ColiBlue®, manufactured by Hach:<br />
• ColiComplete®, manufactured by BioControl;<br />
• Chromocult®, manufactured by Merck; <strong>and</strong><br />
• MicroSure®, manufactured by Gelman.<br />
The Colilert® method is based upon the water sample turning yellow, indicating coliforms<br />
with b-galactosidase activity on the substrate ONPG (O-nitrophenyl-ß-D-galactopyranoside),<br />
<strong>and</strong> fluorescence under long-wavelength UV light when the substrate MUG<br />
(5-methylumbelliferyl-ß ß D-glucuronide) is metabolised by E. coli containing ß-glucuronidase.<br />
The analytical method involves adding commercial dried indicator nutrients containing the<br />
two defined substrates to a 100 mL volume <strong>of</strong> water <strong>and</strong> incubation at 35-37°C as described in<br />
St<strong>and</strong>ard Methods (1998). The result is either a presence/absence testing in the 100 mL volume<br />
or quantification in a propriety tray (QuantiTray) which separates the sample into<br />
a series <strong>of</strong> test wells <strong>and</strong> provides a most probable number (MPN) per 100 mL <strong>of</strong> water.<br />
Box A1 Chromogenic substances available for the detection <strong>of</strong> indicator bacteria<br />
Bacteria Enzyme tested Chromogenic substance<br />
Coliform bacteria • o-nitrophenyl-ß-D-galactopyranoside (ONPG) ß-D-galactosidase<br />
• 6-bromo-2-naphtyl-ß-D-galactopyranoside (E.C.3.2.1.23.)<br />
• 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (XGAL)<br />
E. coli • 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (XGLUC) ß-D-glucuronidase<br />
• 4-methylumbelliferyl-ß-D-glucuronide (MUG) (GUD, E.C3.2.1.31)<br />
• p-nitrophenol-ß-D-glucuronide (PNPG)<br />
Enterococci • 4-methylumbelliferyl-ß-D-glucoside (MUD) ß-D-glucosidase<br />
• indoxyl-ß-D-glucoside<br />
Source: Adapted from Manafi (1996)<br />
(ß-GLU, EC.3.2.21)<br />
37
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
APPENDIX B<br />
MOLECULAR METHODS FOR THE DETECTION OF MICROBIAL INDICATORS<br />
This section contains detailed description <strong>of</strong> novel methods for the detection <strong>of</strong> indicator<br />
microorganisms <strong>and</strong> pathogens, based on molecular techniques.<br />
Microarray Technology<br />
There are two variants <strong>of</strong> the DNA microarray technology, in terms <strong>of</strong> the property <strong>of</strong> arrayed<br />
DNA sequence with known identity:<br />
• probe cDNA (500~5000 bases long) is immobilised to a solid surface such as glass<br />
using robot spotting <strong>and</strong> exposed to a set <strong>of</strong> targets either separately or in a mixture.<br />
This method, ‘traditionally’ called DNA microarray, is widely considered as developed<br />
at Stanford University (Ekins <strong>and</strong> Chu, 1999).<br />
• an array <strong>of</strong> oligonucleotide (20~25-mer oligos) or peptide nucleic acid (PNA) probes<br />
is synthesised either in situ (on-chip) or by conventional synthesis followed by onchip<br />
immobilisation. The array is exposed to labelled sample DNA, hybridised, <strong>and</strong><br />
the identity/abundance <strong>of</strong> complementary sequences are determined. This method,<br />
“historically” called GeneChip® arrays or DNA chips, was first developed at Affymetrix<br />
Inc. (Lemieux et al., 1998; Lipshutz et al., 1999).<br />
Microarrays using DNA/RNA probe-based rRNA targets may be coupled to adjacent CCD<br />
detectors (Guschin et al., 1997). Eggers et al., (1997) have demonstrated the detection <strong>of</strong><br />
E. coli <strong>and</strong> Vibrio proteolyticus using a microarray containing hundreds <strong>of</strong> probes within<br />
a single well (1cm 2 ) <strong>of</strong> a conventional microtitre plate (96 well). The complete assay with<br />
quantification took less than 1 minute. The basic steps in a microarray assay are given<br />
in Box B1.<br />
DNA sensing protocols, based on different modes <strong>of</strong> nucleic acid interaction, possess an<br />
enormous potential for environmental monitoring. Carbon strip or paste electrode transducers,<br />
supporting the DNA recognition layer, are used with a highly sensitive chronopotentiometric<br />
transduction <strong>of</strong> the DNA analyte recognition event. Pathogens targeted to date include<br />
Mycobacterium tuberculosis, Cryptosporidium parvum <strong>and</strong> Human Immunodeficiency<br />
Virus HIV-1 (Wang et al., 1997; Vahey et al., 1999).<br />
The microarray under development by bioMerieux (using Affymetrix Inc. GeneChip®<br />
technology) for an international water company (Lyonnaise des Eaux, Paris, France) is<br />
expected to reduce test time for faecal indicators from the current average <strong>of</strong> 48 hours to<br />
4 hours. In addition, the cost for the st<strong>and</strong>ard water microbiology test is expected to be<br />
10 times less than present methods. The high resolution DNA chip technology is expected<br />
to target a range <strong>of</strong> key microorganisms in water. The prototype GeneChip® measures about<br />
1 cm 2 , on which hybridisation occurs with up to 400 000 oligonucleotide probes. Nonetheless,<br />
such technology may be limited by effective means <strong>of</strong> concentrating the target indicator to<br />
the small volumes used in these assays.<br />
Of the better-evaluated molecular-based methods, the polymerase chain reaction (PCR)<br />
amplification <strong>of</strong> nucleic acids also suffers from the need to use small reaction volumes.<br />
Consequently, <strong>and</strong> due to problems <strong>of</strong> environmental inhibitors, its use in the water industry<br />
is likely to be limited to confirmation testing <strong>of</strong> cultures or presence/absent testing <strong>of</strong> specific<br />
pathogens in concentrates (Stinear et al., 1996; Dombek et al., 2000).<br />
38
Fluorescence In-situ Hybridisation (FISH)<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Solid-state cytometry is a methodology that is rapidly taking <strong>of</strong>f in water microbiology,<br />
utilising either chromogenic substrates (see Appendix A) or the molecular labelling method<br />
called fluorescence in situ hybridisation (FISH).<br />
FISH detection makes use <strong>of</strong> gene probes with a fluorescent marker, typically targeting<br />
the 16S ribosomal RNA (16S rRNA) (Amann et al., 1995). Concentrated <strong>and</strong> fixed cells are<br />
permeabilised <strong>and</strong> mixed with the probe. Incubation temperature <strong>and</strong> addition <strong>of</strong> chemicals<br />
can influence the stringency <strong>of</strong> the match between the gene probe <strong>and</strong> the target sequence.<br />
Since the signal <strong>of</strong> a single fluorescent molecule within a cell does not allow detection, target<br />
sequences with multiple copies in a cell have to be selected (eg. there are 10 2 -10 4 copies<br />
<strong>of</strong> 16S rRNA in active cells). A number <strong>of</strong> FISH methods for the detection <strong>of</strong> coliforms <strong>and</strong><br />
enterococci have been developed (Fuchs et al., 1998; Meier et al., 1997; Patel et al., 1998).<br />
A number <strong>of</strong> studies indicate that FISH detection-based methods may better report the<br />
presence <strong>of</strong> infective pathogens <strong>and</strong> viable, but not necessarily culturable indicator bacteria.<br />
As a further extension <strong>of</strong> the FISH approach, peptide nucleic acid probes targeted against<br />
the 16S rRNA molecule were designed <strong>and</strong> used to detect E. coli from water (Prescott et al.,<br />
1998). The probe was labelled with biotin, which was subsequently detected with streptavidin<br />
horseradish-peroxidase <strong>and</strong> the tyramide signal amplification system. E. coli cells were<br />
concentrated by membrane filtration prior to hybridisation <strong>and</strong> the labelled cells detected<br />
by a commercial laser-scanning solid-state cytometer (eg. ChemScan TM ) within 3h. Detection<br />
<strong>and</strong> enumeration is also possible by the use <strong>of</strong> a flow cytometer (Fuchs et al., 1998). These<br />
cytometers, however, are expensive, <strong>and</strong> high sample throughput is necessary to justify<br />
their purchase.<br />
39
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Box B1 Six steps in the design <strong>and</strong> implementation <strong>of</strong> a DNA microarray assay<br />
40<br />
1 2 3 4 5 6<br />
Probe Chip fabrication Target Assay Readout Informatics<br />
(cDNA/ oligo with known idenity) (Putting probes on the chip) (fluorescently labelled sample)<br />
Small oligos, cDNAs, Photolithography, RNA, (mRNA) to cDNA Hybridisation, ligase, Fluorescence, probeless Robotics control,<br />
chromosome (whole organism pipette, drop-touch, ligase, base addition, (conductance, electrophoresis), Image processing,<br />
on a chip?) piezoelectric (ink-jet), electric, electrophoresis, electronic DBMS, WWW,<br />
electric flow cytometry PCR-DIRECT, bioinformatics<br />
TaqMan<br />
Source: http://www.Gene-Chips.com
PROCESS REPORT<br />
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
In 2000, the NHMRC Drinking Water <strong>Review</strong> Coordinating Group recognised increasing<br />
uncertainty in relation to the use <strong>of</strong> traditional indicator organisms, including thermotolerant<br />
coliforms <strong>and</strong> total coliforms, as a measure <strong>of</strong> the microbial quality <strong>of</strong> drinking water.<br />
In response, a discussion paper was commissioned to consider the concepts <strong>of</strong> indicator<br />
microorganisms, the rationale behind their use, <strong>and</strong> to provide an evaluation <strong>of</strong> alternative<br />
indicators <strong>of</strong> microbial water quality.<br />
The Coordinating Group requested Dr Melita Stevens (Melbourne Water), Dr Nick Ashbolt<br />
(University <strong>of</strong> New South Wales) <strong>and</strong> Dr David Cunliffe (SA Department <strong>of</strong> Human Services)<br />
to undertake a review on microbial indicators <strong>of</strong> water quality that addressed the:<br />
• status <strong>of</strong> bacterial indicators <strong>of</strong> drinking water quality in Australia <strong>and</strong> internationally;<br />
• impact <strong>of</strong> emerging technologies for indicator enumeration <strong>and</strong> identification;<br />
• usefulness <strong>of</strong> current bacterial indicators in supporting the risk-based water quality<br />
management systems;<br />
• usefulness <strong>of</strong> emerging indicators such as faecal sterols <strong>and</strong> bacteriophage; <strong>and</strong><br />
• possible alternative approaches for microbial indicators in any future revision<br />
to the Australian Drinking Water Guidelines.<br />
The review has highlighted a number <strong>of</strong> potential activities, including the need to:<br />
• revise guidance on heterotrophic plate counts in operational management;<br />
• revise guidance on coliforms in operational management; <strong>and</strong><br />
• remove total coliforms to be removed as public health indicators<br />
Consultation on the draft report, <strong>Review</strong> <strong>of</strong> <strong>Coliforms</strong> as Microbial Indicators <strong>of</strong> Drinking Water<br />
Quality took place from September to November 2001 <strong>and</strong> involved a call for submissions<br />
on the draft document publicised in the Commonwealth Notices Gazette <strong>and</strong> The Weekend<br />
Australia. Invitations were also forwarded to known interested parties through en<strong>Health</strong><br />
<strong>Council</strong>, the Australian Water Association <strong>and</strong> Water Services Association <strong>of</strong> Australia.<br />
All submissions received were taken into consideration by the Coordinating Group in finalising<br />
this review. Submissions were received from the following individuals/organisations:<br />
Sam Austin Yarra Valley Water<br />
Harry Ferguson Brisbane Water<br />
Dr Philip Berger United States Environment Protection Authority<br />
Brian Bailey Melbourne Water<br />
Ian Tanner Sydney Catchment Authority<br />
Keith Neaves Lower Murray ater Authority<br />
Dr Chris Saint,<br />
Phil Adcock<br />
Australian Water Quality Centre<br />
Les Mathieson East Gippsl<strong>and</strong> Water<br />
Jacqui Goonrey ActewAGL<br />
Mark Harvey Victorian Water Industry Association Inc<br />
David Heap City West Water<br />
41
MICROBIAL INDICATORS OF DRINKING WATER QUALITY<br />
Greg Ryan South East Water Limited<br />
Martha Sinclair,<br />
Samantha Rizak<br />
Monash University<br />
Jim Martin North East Water<br />
Christine Cowie NSW Department <strong>of</strong> <strong>Health</strong><br />
Dr Paul Van Buynder Department <strong>of</strong> Human Services, Victoria<br />
David Sheehan Queensl<strong>and</strong><br />
Membership <strong>of</strong> the NHMRC Drinking Water <strong>Review</strong> Coordinating Group<br />
Pr<strong>of</strong>essor Don Bursill (Chair)<br />
Treatment<br />
Cooperative <strong>Research</strong> Centre for Water Quality <strong>and</strong><br />
Dr David Cunliffe Department <strong>of</strong> Human Services, South Australia<br />
Peter Scott Melbourne Water Corporation<br />
Dr Anne Neller University <strong>of</strong> the Sunshine Coast<br />
Alec Percival Consumer’s <strong>Health</strong> Forum<br />
Dr John Langford Water Services Association <strong>of</strong> Australia<br />
Brian McRae Australian Water Association<br />
Secretariat<br />
Phil Callan <strong>National</strong> <strong>Health</strong> <strong>and</strong> <strong>Medical</strong> <strong>Research</strong> <strong>Council</strong><br />
Peer <strong>Review</strong>er<br />
Emeritus Pr<strong>of</strong>essor Nancy Millis University <strong>of</strong> Melbourne<br />
Technical Editor<br />
Dr Andrew Langley Department <strong>of</strong> <strong>Health</strong>, Queensl<strong>and</strong><br />
Prior to approval by the NHMRC, <strong>Review</strong> <strong>of</strong> <strong>Coliforms</strong> as Microbial Indicators <strong>of</strong> Drinking Water Quality<br />
was subjected to an independent review against the NHMRC key criteria for assessing information reports<br />
by Hawkless Consulting Pty Ltd.<br />
42