Results are a matter of time

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),

• reliable.

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

regulation.

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

detection techniques.

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.

Biosensors

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

micro-organism.

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.

Mass spectrometry

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

investigation.

Infra-red spectroscopy

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).

Microcolonies

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.

Conclusions

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).

Acknowledgments

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.

References

Cheng J, Sheldon EL, Wu L, Uribe A, Gerrue LO, Carrino J, Heller MJ and O’Connell

JP (1998) Nature Biotechnology 16, 541-546.

Johnson PE, Pienlazek NJ, Griffin DW, Misener L and Rose JB (2001) Biomedical

Sciences Instrumentation 37, 191-196.

Jung R, Soondrum K and Neumaier, M (2000) Clinical Laboratory Medicine 38,

833-836.

Maquelin K, Choo-Smith L-P, van Vreeswijk T, Endtz H Ph, Smith B, Bennett R,

Bruining HA and Puppels GJ (2000). Analytical Chemistry 72, 12-19.

Marquardt BJ, Vahey PG, Synovec RE and Burgess LW (1999) Analytical Chemistry

71, 4808-4014

Neilsen PE, Egholm M and Buchardt O (1994) Bioconjugate Chemistry 5, 3-7.

Prescott AM and Fricker CR (1999). Molecular and Cellular Probes 13, 261-268.

Pyle BH, McFeters GA, Poucke SO and Nelis HJ (2001). In Rapid Detection Assays

for Food and Water. Eds. Clark S, Thompson KC, Keevil CW and Smith M. Royal

Society of Chemistry, Cambridge. pp. 31-37.

Sartory DP and Watkins J. (1999) Journal of Applied Microbiology Symposium

Supplement 85, 225S-233S.

Sartory DP, Parton A and Rackstraw R (2001) In Rapid Detection Assays in

Food and Water. Eds. Clark S, Thompson KC, Keevil CW and Smith M Royal Society

of Chemistry, Cambridge. pp 31-37.

Scheu PM, Berghof K and Stahl U (1998). Food Microbiology 15, 13-31.

Schuster KC, Reese I, Urlaub E, Gapes JR and Lendl B (2000). Analytical Chemistry

72, 5529-5534.

Sidorowicz SV and Whitmore TN (1995) Journal of the Institute of Water and

Environment Management (IWEM) 9, 92-98.

Stender H, Broomer AJ, Oliveira K, Perry-O’Keefe H, Hyldig-Nielsen JJ, Sage

A and Coull J (2001). Applied and Environmental Microbiology 67, 142-147.

Welham KJ, Domin MA, Scannell DE, Cohen E and Ashton DS (1998) Rapid Communications

in Mass Spectrometry 12, 176-180

Whitmore TN and Sidorowicz SV (1995). Microbiology Europe 3(5), 16-22.

Woolley AT, Hadley D, Landre P, deMello AJ, Mathies RA and Northrup MA (1996)

Analytical Chemistry 68, 4081-4086.