Review articleMolecular methods for the assessment of bacterial viability
Introduction
Classical methods for the determination of bacterial viability rely on the ability of cells to actively grow and form visible colonies on solid media. Under some circumstances, the number of viable organisms may be severely underrepresented by such methods as sublethally damaged organisms (Blackburn and McCarthy, 2000), fastidious uncultivable bacteria (Ward et al., 1990) and viable cells that have lost the ability to form colonies under the test conditions will not be detected. Consequently, alternative methods for determining viability have been developed, based variously on demonstration of cellular integrity or activity. Alternatives to colony counting include the use of flow cytometry and fluorescent staining techniques Caron et al., 1998, Diaper and Edwards, 1994, Turner et al., 2000, the exploitation of physiological responsiveness (Kogure et al., 1979) or metabolic activity and nucleic acid-based analyses del Mar Lleó et al., 2000, McCarty and Atlas, 1993, Sheridan et al., 1998. Fig. 1 shows a schematic diagram illustrating the range of techniques used in assessment of bacterial viability status. For the purposes of this review, cells maintaining membrane integrity and retaining some metabolic activity or responsiveness are considered viable.
The analysis of environmental bacteria is performed for a variety of applications, ranging from the assessment of microbial diversity to monitoring pathogen presence for public health protection. A number of significant bacterial pathogens may be present in environmental reservoirs, including Verotoxigenic Escherichia coli (VTEC), Vibrio vulnificus, Salmonella spp., Campylobacter spp. and a variety of mycobacteria. Analyses for the detection of these and other pathogens in water supplies, in soil and as airborne contaminants have been developed. To determine if the presence of these pathogens poses an actual threat to public health, their viability needs to be determined, although the consistent detection of nucleic acid sequences from pathogenic bacteria in an environment can be taken as indicative of a hazardous contamination (Moore et al., 2001). The development of rapid, sensitive and specific methods for the detection and identification of environmental bacteria with concomitant determination of their viability status would significantly improve current surveillance regimes.
Section snippets
Molecular targets for assessment of viability
A range of molecular targets have been utilised in microbiological assays. Experiments utilising the polymerase chain reaction (PCR) have been used to detect DNA sequences and identify and enumerate bacterial species. More recently, the use of DNA microarrays (Ye et al., 2001) and PNA probes (Stender et al., 2002) has been employed to increase the speed and specificity of detection of bacterial species present in a sample. However, these assays do not give any indication as to viability of the
Impact of analytical method
The majority of current molecular analyses for viability utilise target amplification to maximise analytical sensitivity. Most DNA detection is undertaken using PCR Hellyer et al., 1999, McKillip et al., 1999, although hybridisation-based detection methods have also been employed (Meijer et al., 2000). The most commonly used amplification techniques for detecting mRNA are reverse transcriptase PCR (RT-PCR) and nucleic acid sequence-based amplification (NASBA) Chan and Fox, 1999, Simpkins et
Detection and significance of viable but nonculturable bacterial populations
Methods for the determination of viability depend on examination of a variety of cell characteristics. However, the definition of what constitutes a viable cell remains contentious, with the significance and precise physiological status of nonculturable organisms that retain some sign of viability, so-called VBNC forms, still undetermined Barer and Harwood, 1999, Kell et al., 1998. A number of bacterial species have been observed to undergo metabolic change leading to the production of cells
Molecular methods for pathogen detection
The main applications where determination of viability is central to the analysis are associated with detection of pathogens, either in environmental samples or clinical situations. Analysis of environmental samples includes the investigation of air and water quality, and assays have been developed to detect many significant human pathogens. A recent review considering the use of integrated biodetection systems for investigation of uncultivated environmental microorganisms has stressed the need
Molecular methods for ecological studies
Molecular methods have been introduced for the assessment of microbial diversity, contributing specificity of bacterial identification and sensitivity to such analyses. Viability of environmental bacteria is mainly determined by direct viable count (DVC) (Kogure et al., 1979) and staining methods Bernard et al., 2001, Miskin et al., 1998. However, the presence of specific mRNA transcripts in RNA libraries produced from environmental samples has been used as an indicator of active microbial
Reliability of molecular analysis of bacterial viability
The detection of both DNA and RNA species has been used as indicators of bacterial viability. However, these are indirect methods, and as the persistence of nucleic acids is highly dependent on the environmental conditions encountered by the bacterium after cell death Romanowski et al., 1992, Sheridan et al., 1999, the correlation with actual cell viability may vary greatly. Similarly, many of the staining techniques used for the assessment of bacterial viability are indirect analyses,
Future prospects
Many currently used methods for the determination of bacterial viability actually monitor decreasing levels of signal during cellular degradation after cell death. Molecular methods detect decaying nucleic acids, and both DNA and RNA persistence have been shown to correlate well with viability in certain well-characterised situations. Similarly, analysis of membrane integrity is used as a secondary indicator of cell death. All such indirect analyses rely on rapid clearance of dead cells to
Acknowledgments
This work was funded by the Department of Trade and Industry under the Valid Analytical Measurement (VAM) 2000–2003 programme. We thank Dr. Gavin Nixon and Carol Dawson for useful discussions during preparation of this manuscript, and the referees for their helpful suggestions.
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