Results are a matter of time

Timothy Whitmore of WRc looks at rapid microbiological monitoring

Fluorescent dyes detect individual microbial cells

Fluorescent dyes detect individual microbial cells

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
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