Abstract
The coliform group has been used extensively as an indicator of water quality and has historically led to the public health protection concept. The aim of this review is to examine methods currently in use or which can be proposed for the monitoring of coliforms in drinking water. Actually, the need for more rapid, sensitive and specific tests is essential in the water industry. Routine and widely accepted techniques are discussed, as are methods which have emerged from recent research developments.
Approved traditional methods for coliform detection include the multiple-tube fermentation (MTF) technique and the membrane filter (MF) technique using different specific media and incubation conditions. These methods have limitations, however, such as duration of incubation, antagonistic organism interference, lack of specificity and poor detection of slow-growing or viable but non-culturable (VBNC) microorganisms. Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for routine enumeration of coliforms in drinking water.
The detection of coliforms based on specific enzymatic activity has improved the sensitivity of these methods. The enzymes β-d galactosidase and β-d glucuronidase are widely used for the detection and enumeration of total coliforms and Escherichia coli, respectively. Many chromogenic and fluorogenic substrates exist for the specific detection of these enzymatic activities, and various commercial tests based on these substrates are available. Numerous comparisons have shown these tests may be a suitable alternative to the classical techniques. They are, however, more expensive, and the incubation time, even though reduced, remains too long for same-day results. More sophisticated analytical tools such as solid phase cytometry can be employed to decrease the time needed for the detection of bacterial enzymatic activities, with a low detection threshold.
Detection of coliforms by molecular methods is also proposed, as these methods allow for very specific and rapid detection without the need for a cultivation step. Three molecular-based methods are evaluated here: the immunological, polymerase chain reaction (PCR) and in-situ hybridization (ISH) techniques. In the immunological approach, various antibodies against coliform bacteria have been produced, but the application of this technique often showed low antibody specificity. PCR can be used to detect coliform bacteria by means of signal amplification: DNA sequence coding for the lacZ gene (β-galactosidase gene) and the uidA gene (β-d glucuronidase gene) has been used to detect total coliforms and E. coli, respectively. However, quantification with PCR is still lacking in precision and necessitates extensive laboratory work. The FISH technique involves the use of oligonucleotide probes to detect complementary sequences inside specific cells. Oligonucleotide probes designed specifically for regions of the 16S RNA molecules of Enterobacteriaceae can be used for microbiological quality control of drinking water samples. FISH should be an interesting viable alternative to the conventional culture methods for the detection of coliforms in drinking water, as it provides quantitative data in a fairly short period of time (6 to 8 h), but still requires research effort.
This review shows that even though many innovative bacterial detection methods have been developed, few have the potential for becoming a standardized method for the detection of coliforms in drinking water samples.
Introduction
Public and environmental health protection requires safe drinking water, which means that it must be free of pathogenic bacteria. Among the pathogens disseminated in water sources, enteric pathogens are the ones most frequently encountered. As a consequence, sources of fecal pollution in waters devoted to human activity must be strictly controlled. Entero-pathogens, such as Escherischia coli O157:H7, are generally present at very low concentrations in environmental waters within a diversified microflora. Complex methods are required to detect them, and these are extremely time-consuming.
Most coliforms are present in large numbers among the intestinal flora of humans and other warm-blooded animals, and are thus found in fecal wastes. As a consequence, coliforms, detected in higher concentrations than pathogenic bacteria, are used as an index of the potential presence of entero-pathogens in water environments. The use of the coliform group, and more specifically E. coli, as an indicator of microbiological water quality dates from their first isolation from feces at the end of the 19th century. Coliforms are also routinely found in diversified natural environments, as some of them are of telluric origin, but drinking water is not a natural environment for them. Their presence in drinking water must at least be considered as a possible threat or indicative of microbiological water quality deterioration. Positive total coliform samples in a treated water which is usually coliform-free may indicate treatment ineffectiveness, loss of disinfectant, breakthrough (McFeters et al., 1986), intrusion of contaminated water into the potable water supply Geldreich et al., 1992, Clark et al., 1996 or regrowth problems (LeChevallier, 1990) in the distribution system, and, as a consequence, should not be tolerated.
The use of the coliform group as an indicator of the possible presence of enteric pathogens in aquatic systems has been a subject of debate for many years. Many authors have reported waterborne disease outbreaks in water meeting the coliform regulations Payment et al., 1991, Moore et al., 1994, MacKenzie et al., 1994, Gofti et al., 1999. However, the purpose of this review is not to discuss the indicator concept, but rather to identify methods currently in use or which can be proposed for the monitoring of coliforms in drinking water. The need for more rapid and sensitive tests is constant in the water industry, with the ultimate goal being the continuous on-line monitoring of water leaving treatment plants.
The coliform group includes a broad diversity in terms of genus and species, whether or not they belong to the Enterobacteriaceae family. Most definitions of coliforms are essentially based on common biochemical characteristics. In Standard Methods for the Examination of Water and Wastewater (Part 9221 and 9222; APHA et al., 1998), coliform group members are described as:
1.
all aerobic and facultative anaerobic, Gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas and acid formation within 48 h at 35 °C (multiple-tube fermentation technique; Section 3.1) or
2.
all aerobic and many facultative anaerobic, Gram-negative, non-spore-forming, rod-shaped bacteria that develop a red colony with a metallic sheen within 24 h at 35 °C on an Endo-type medium containing lactose (membrane filter technique; Section 3.2).
The definition of members of the coliform group has recently been extended to include other characteristics, such as β-d-galactosidase-positive reactions (Part 9223; APHA et al., 1998) (enzyme substrate test, Section 4.2). The search for β-galactosidase-positive and β-galactoside-permease-positive organisms also permits a confirmation step for lactose fermentation, when the multiple-tube fermentation technique is used. The cytochrome-oxidase test is also used as a confirmation test to eliminate some bacteria of the Aeromonas or Pseudomonas genera that would ferment lactose.
The definition of coliform bacteria differs slightly depending on the country or on the organization in charge of the microbiological monitoring regulations. In Canada, the definition is the same as in the US, and differs in some European countries. For example, the French Standardization Association (NFT90-413 and NFT90-414; AFNOR, 1990), which can be considered as a representative model for European legislation, defines total coliforms (TC) as:
⋅rod-shaped, non-spore-forming, Gram-negative, oxidase-negative, aerobic or facultative anaerobic bacteria that are able to grow in the presence of bile salts or other replacement surface active agents having an analogous growth inhibitory effect and that ferment lactose with gas and acid (or aldehyde) production within 48 h at 37±1 °C.
AFNOR (1990) goes further by defining other coliform groups, including the thermotolerant coliforms (also called fecal coliforms, FC) and, more specifically, E. coli:
⋅thermotolerant coliforms have the same fermentation properties as total coliforms (TC) but at a temperature of 44±0.5 °C.
⋅E. coli is a thermotolerant coliform which, among other things, produces indole from tryptophane at a temperature of 44±0.5 °C, gives a positive methyl red test result, is unable to produce acetyl–methyl carbinol and does not use citrate as its sole carbon source.
The use of the coliform group as an indicator of fecal contamination is subject to strict governmental regulations (Table 1). E. coli is the most common coliform among the intestinal flora of warm-blooded animals and its presence might be principally associated with fecal contamination. No E. coli are therefore allowed in drinking water.
The US Environmental Protection Agency (EPA) has approved several methods for coliform detection: the multiple-tube fermentation technique, the membrane filter technique and the presence/absence test (including the ONPG-MUG test). AFNOR (1990) has approved the multiple-tube fermentation technique and the membrane filter technique.
These methods have limitations, such as duration of incubation, antagonistic organisms interference, lack of specificity to the coliform group and a weak level of detection of slow-growing or stressed coliforms. Indeed, depending on the environmental system, only a small portion (0.1–15%) of the total bacterial population can be enumerated by cultivation-based methods (Amann et al., 1990). The proportion of non-culturable bacteria may be affected by unfavorable conditions for bacterial growth during culturing or by their entry into viable or active but non-culturable states (VBNC or ABNC) Roszak and Colwell, 1987, Joux and Lebaron, 2000, Colwell and Grimes, 2000.
Section snippets
Objectives
Since drinking waters constitute oligotrophic systems, the lack of sensitivity of cultivation methods in the detection of stressed and starved bacterial cells can generate serious limitations due to contamination-level underestimation. There exist other methods which may be used for coliform detection, and these are in various states of development and application. This review describes the principles and the usual protocols of the classical methods, as well as some innovative methods and
Multiple-tube fermentation technique
The technique of enumerating coliforms by means of multiple-tube fermentation (MTF) has been used for over 80 years as a water quality monitoring method. The method consists of inoculating a series of tubes with appropriate decimal dilutions of the water sample. Production of gas, acid formation or abundant growth in the test tubes after 48 h of incubation at 35 °C constitutes a positive presumptive reaction. Both lactose and lauryl tryptose broths can be used as presumptive media, but Seidler
General principles
The biochemical tests used for bacterial identification and enumeration in classical culture methods are generally based on metabolic reactions. For this reason, they are not fully specific, and many additional tests are sometimes required to obtain precise confirmation. The use of microbial enzyme profiles to detect indicator bacteria is an attractive alternative to classical methods. Enzymatic reactions can be group-, genus- or species-specific, depending on the enzyme targeted. Moreover,
Molecular methods
Molecular methods have been developed to increase the rapidity of analysis. They are able to achieve a high degree of sensitivity and specificity without the need for a complex cultivation and additional confirmation steps. Consequently, some of these methods permit the detection of specific culturable and/or non-culturable bacteria within hours, instead of the days required with the traditional methods. Several molecular methods applied to the specific detection of coliforms in waters and
Conclusions
The purposes of this work were as follows:
•
to compile an inventory of traditional and more recent coliform group detection methods, mainly those applied to the analysis of the microbiological quality of drinking water, and
•
to evaluate the limitations of these methods in terms of sensitivity and application.
Today, the MF technique is the method most widely used for the enumeration of coliforms in drinking water. This technique, simple to perform and inexpensive, requires at least an overnight
Acknowledgements
This review forms part of the research project “A comprehensive approach for controlling coliforms in drinking water distribution systems” supported by Vivendi Water-US Filter. The authors thank Dr. A. Camper and Dr. V. Gauthier for their helpful reviewing of the manuscript.