Results are a matter of time
Timothy Whitmore of WRc looks at rapid microbiological monitoring
The bacteriological quality of the public drinking water supply is required
to meet strict standards which are based on the presence of certain indicator
organisms, namely E. coli and enterococci. Their presence can indicate
potential faecal contamination and the presence of pathogenic organisms which
could pose a risk to public health. The current standard techniques for the
detection of E. coli and coliforms in potable water in the UK require
18-24h to produce a presumptive result with up to a further 24h for confirmation.
The advantages of current methods for the enumeration of water quality indicator
bacteria have been summarised by Sartory and Watkins (1999) as being relatively
simple to perform and requiring unsophisticated laboratory equipment. In addition,
a large number of samples (typically 200-500) can be analysed daily using junior
grade analysts. However, the methods are labour intensive and require at least
an overnight incubation.
Despite the water industry’s demand and identified need for more rapid microbiological
methods it has proved difficult to apply new technologies to the bacteriological
analysis of water (Sidorowicz and Whitmore 1995; Sartory and Watkins 1999).
The criteria for a new technique may be summarised as follows:
- elimination of the requirement for overnight incubation, to provide a same
day test of six-hour duration or less,
- quantitative result,
- close comparability with existing methods and legislation,
- cost comparable with existing methods,
- sensitive and specific,
- capable of a high throughput,
- low operator skill (although this issue would be negated if the assay platform
were highly automated),
An increasing need has been identified for the monitoring for specific pathogens
in water and other environments. For example, the 1999 revision of the Drinking
Water Supply Regulations requires that certain drinking water supplies are continuously
monitored for the presence of the protozoan parasite Cryptosporidium.
Concerns over other pathogens such as E. coli O157 could lead to similar
Nucleic acid-based techniques
1. Polymerase chain reaction
Nucleic acid-based methods are generally based on the detection of specific
nucleic acid sequences, through the use of complementary hybridisation probes,
or by their amplification using the polymerase chain reaction (PCR).
Conventional PCR does suffer from significant drawbacks including the inability
to provide quantitative results and the cumbersome detection procedure using
gel electrophoresis. Quantitative PCR (QPCR) is a comparatively new variation
which uses a sequence specific fluorescent probe and primers to quantify the
DNA template present in the sample in real-time (Jung et al 2000). Test
kits are already available for the QPCR detection of several pathogens including
E. coli O157:H7 and Cryptosporidium. However, a number of different chemistries
are available to undertake QPCR and the relative merits of each require investigation.
DNA-based detection methodology is essentially unable to differentiate between
viable and dead microbial cells. The use of alternative nucleic acid targets
which correlate with cellular viability have been investigated. Messenger RNA
(mRNA) and precursor RNA (pre-RNA) are potential candidate species. However,
the adoption of any RNA species as the target molecule would introduce further
complexity into a PCR-based assay since a reverse transcription step to generate
a DNA template for the PCR would be required. The comparative lability of RNA
species dictate the use of rapid extraction techniques coupled with careful
sample handling procedures.
2. Hybridisation probes
Hybridisation probes are often designed to target the naturally amplified ribosomal
RNA (rRNA) which is a universal constituent of bacterial ribosomes present in
high copy numbers (Scheu et al 1998). Peptide nucleic acid (PNA) is a
synthetic DNA analogue which appears to have considerable potential in hybridisation
assays (Nielsen et al 1994).
The simplicity and rapidity of the sample preparation (two-three hours) has
been exploited for the microscopic detection of individual cells using fluorescently-labelled
probes. Flow cytometry and laser scanning cytometry are alternative automated
Prescott and Fricker (1999) described the use of rRNA-targeted PNA probes to
detect E. coli in water within an assay time of three hours. However,
non-viable chlorinated cells were also detected, but the authors suggested the
assay could be combined with a viability marker, for example a redox dye such
as Cyanoditolyltetrazolium chloride (CTC).
The PCR requires long cycling times because of the large thermal mass of current
systems. Capillary electrophoresis (CE) can increase the throughput of the assay
but the preparation and manipulation of large numbers of capillaries can be
difficult and sample introduction inefficient (Woolley 1996).
Questions remain over the specificity of PCR detection and problems arising
through inhibition effects using environmental source material. However, QPCR
in which the kinetics of the reaction are monitored in real time should assist
in the development of more robust assays.
3. Microfabricated chips
Current developments in nucleic acid-based detection technologies are centred
around the miniaturisation of the assay system such that it can be performed
more rapidly in microfabricated bioelectronic silicon reactors. The separation
of E. coli cells from amended blood samples using an electrical separation
technique (dielectrophoresis) followed by electronic lysis, proteolytic digestion
and examination of the lysates by electronically enhanced hybridisation of 16S
rRNA probes has been demonstrated (Cheng et al 1998). Woolley et al
(1996) described the integration of a microfabricated silicon PCR reactor and
glass capillary electrophoresis chips to form an integrated DNA analysis system,
in which a rapid assay for Salmonella DNA was performed in under 45 minutes.
Microfabricated devices have the potential to revolutionise microbiological
analysis. The challenges which remain for the introduction of this technology
are the production of cheap disposable chips and the development of compatible
sample preparation techniques.
The past decade has seen the development of many enzyme and immunosensor-based
systems designed for the detection of pathogenic bacteria (Ivnitski et al
1999). The sensitivity and specificity of many is inadequate for the bacteriological
monitoring of water and hence only those which show immediate potential for
this application are described.
The detection of micro-organisms using antibody-conjugated fluorescent labels
is well established. Methods which may be more applicable to the routine monitoring
of water have been developed using fluorescently-labelled enzyme substrates
(fluorescein-di-ß-D-glucuronide for E. coli and fluorescein-di-ß-D-galactopyranoside
for total coliforms) (Pyle et al 2001). Fluorescence microcoscopy, flow
cytometry or laser scanning cytometry can be employed to detect the labelled
Flow cytometers can also be used to detect fluorescently-labelled cells. However,
it is doubtful whether commercial instruments could achieve the required sensitivity
without very careful optimisation. (Johnson et al 2001) has described
the construction of a flow cytometer which employed some innovative features
intended specifically for the detection of bacterial cells.
The introduction of matrix assisted laser desorption/ionisation time-of-flight
(MALDI-TOF) mass spectrometry has revolutionised the analysis of macromolecules
and the technique is being increasingly applied to the identification of bacteria
through their mass spectral fingerprints (Welham et al 1998). Spectra
from bacterial colonies can be obtained within minutes. However, the sensitivity,
discrimination, reproducibility and capacity of the technique require further
Infra-red Raman spectroscopy is based on the measurement of the inelastic scattering
of monochromatic light from molecules. It has been employed to identify microbial
colonies, including E. coli, to the genus and species level (Maquelin
et al 2000). Confocal microscopy has been employed to obtain spectra
from single microbial cells (Schuster et al 2000). This spectroscopic
technique is attractive since, in contrast to mass spectrometric methods, it
is non-destructive. Whether developments in the on-line detection of compounds
using Raman waveguide detectors could be adapted for microbial detection is
an intriguing possibility (Marquardt et al 1999).
The detection of bacterial microcolonies following a short period (5.5h) of
growth has been demonstrated for E. coli (Sartory et al 2001).
The microcolonies were enumerated under epifluorescence microscopy which was
used to visualise the fluorescent product of an enzyme specific substrate for
b-glucuronidase (Sartory et al 2001).
A chemiluminescent in situ hybridization method has provided the simultaneous
detection, identification and enumeration of culturable E. coli cells
in water within a working day (Stender et al 2001). Following filtration
and five hours of growth microcolonies were detected directly on membrane filters
using peroxidase-labelled PNA probes targeting a species-specific sequence of
E. coli 16S rRNA.
Although rapid techniques, for example laser scanning cytometry, are available
for the detection of single microbial cells, the cost of the instrumentation
is high and the throughput is currently inadequate for a busy analytical laboratory.
Flow cytometry probably has greater potential in terms of sample throughput,
but the reliable detection of single microbial cells is too demanding an application
for current instruments. Further development is hence required both in instrument
design and sensitive fluorescent labelling technologies.
The application of nucleic acid-based techniques is hampered by the requirement
to distinguish viable from non-viable cells, although PCR based methods could
find an application in the monitoring of specific pathogens when an incident
dictates that a rapid result is desirable.
An alternative technique which may have application to the identification of
microcolonies or even single cells is infra-red Raman spectroscopy.
The detection of bacterial microcolonies following a short period of growth
is a method which would appear to offer the greatest likelihood, in the short-term,
of being acceptable to the water industry regulators.
As indicated, some technologies show promise and warrant further research.
However, the concomitant development of compatible sample concentration and
separation techniques is required if the benefits of rapid detection technologies
are to be fully realised (Whitmore and Sidorowicz 1995).
The author wishes to thank Severn Trent Water, South East Water, West of Scotland
Water and Portsmouth Water for funding the contract on which this article is
based. The views expressed in the article are those of the author alone.
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