Molecular methods for the assessment of bacterial viability.

A significant number of pathogenic microorganisms can be found in environmental reservoirs (air, water, soil). It is important to assess the viability status of these organisms to determine whether they pose a threat to public health. Classical methods for determining viability are time consuming. Hence, molecular methods have been developed to address this problem. Molecular methods offer speed, sensitivity and specificity. Both DNA and RNA have been analysed using molecular amplification methods such as polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and nucleic acid sequence-based amplification (NASBA). However, due to the variable persistence of nucleic acids in cells post-death, the correlation between presence of DNA and RNA and viability is not clear-cut. Similarly, the choice of target and sensitivity of the method can significantly affect the validity of the viability assay. This review assesses the molecular methods currently available and evaluates their ability to assess cell viability with emphasis on environmental pathogens.

A comparison of nucleic acid amplification techniques for the assessment of bacterial viability.

AIMS: The ability to determine the presence and viability status of bacteria by molecular methods could offer significant advantages to the food, environmental and health sectors, in terms of improved speed and sensitivity of detection. METHODS AND RESULTS: In this study, we have assessed three amplification techniques, PCR, RT-PCR and NASBA, for their ability to detect nucleic acid persistence in an E. coli strain following heat-killing. NASBA offered the greatest sensitivity of the three methods tested. The presence of residual DNA and mRNA could be detected by PCR and NASBA, respectively, for up to 30 h postdeath, by which time cell death had been confirmed by culture methods. Thus a single quantitative measurement based on nucleic acid amplification did not permit unequivocal determination of cell viability. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation between cell viability and persistence of nucleic acids must be well characterized for a particular analytical situation before molecular techniques can be substituted for traditional culture methods.

Mycobacterial recA is cotranscribed with a potential regulatory gene called recX.

The recA gene of Mycobacterium smegmatis has been cloned and sequenced. The amino acid sequence of the RecA protein is highly homologous to other RecA proteins. Three other potential open reading frames were identified. One of these showed extensive homology to a protein, HypB, involved in the incorporation of nickel into hydrogenases. Another, found downstream of and overlapping recA, was similar to a gene, recX, which has been proposed to play a regulatory role related to recA function. The homology between the M. smegmatis sequence and that of Mycobacterium tuberculosis extended upstream of the recA coding region for 140 bp including a motif identical to the Cheo-box consensus sequence which has been shown to bind LexA. In addition, the transcriptional start sites were found to be identical to those identified previously for M. tuberculosis. Transcriptional fusions to the reporter gene chloramphenicol acetyltransferase (CAT) revealed that recA was DNA-damage inducible and that expression required sequences at some distance from the mapped transcriptional start sites. Although a motif with only one mismatch to the Cheo box was found in the intergenic region between orf1 and orf2 these open reading frames were not DNA-damage inducible, nor was this motif required for regulation of recA expression. Gel retardation assays revealed that the reason for this was that LexA did not bind to this sequence containing a mismatch. Reverse transcription/polymerase chain reaction analysis of M. smegmatis RNA demonstrated that recA and orf3 (recX) are within the same transcriptional unit.

Use of polymerase chain reaction to assess efficacy of leprosy chemotherapy.

The assessment of chemotherapy efficacy in leprosy is difficult, since the only reliable method for determining whether the causative organism, Mycobacterium leprae, is viable depends on its growth in mouse foot pads. In an attempt to replace this expensive, time-consuming test, methods based on the polymerase chain reaction (PCR) have been developed. These methods depend on detection of DNA, which is more susceptible to degradation on cell death than are other cell components, so should be a more accurate indicator of viability. We have used a specific PCR assay to detect M leprae DNA in skin biopsy samples from leprosy patients. By use of limiting dilution PCR (LD-PCR), the concentration of M leprae DNA in the original sample could be measured. The DNA concentration was more closely correlated with the morphological index (derived from a staining technique that distinguishes morphologically intact and damaged bacteria) than with the number of bacteria visible (bacterial index, BI, which counts both alive and dead bacteria). In a longitudinal study of multibacillary patients on multi-drug therapy, skin biopsy samples were collected before treatment and 3, 6, 12, and 24 months after the start of therapy. While the BI showed little or no change during treatment, the number of genomes detected by PCR fell sharply, in parallel with the MI. We propose that PCR can be used as a rapid measure of M leprae viability and that this approach can be used for monitoring individual leprosy patients and for assessment of existing and new regimens. The method may be applicable to other infectious diseases in which culture of the causative organism is slow or impossible.

Physical mapping of 2000 kb of the mouse X chromosome in the vicinity of the Xist locus.

A physical map encompassing approximately 2.0 megabases (Mb) in the region of the mouse X-inactivation center has been constructed. The map extends from the Gjb-1 locus to the Xist locus and demonstrates the order of probes inseparable by genetic analysis. The deduced locus order is as follows: Gjb-1, Ccg-1, DXCrc171, Rps4, Phka, DXCrc177, DXCrc318, Xist. Detailed physical mapping in the region between the Phka and Xist loci indicates the position of CpG-rich islands associated with the 5' end of genes. The DXCrc177 and DXCrc318 loci, both defined by probes derived from linking clones, are associated with CpG-rich islands. The map provides a framework for the isolation of underlying sequences in the mouse X-inactivation center region.

Construction of a mouse yeast artificial chromosome library in a recombination-deficient strain of yeast.

We have constructed a new generation yeast artificial chromosome (YAC) library from female C57BL/10 mice in a recombination-deficient strain of Saccharomyces cerevisiae carrying a mutation in the RAD52 gene. The YAC library contains 41,568 clones with an average insert size of 240 kilobases, representing a greater than threefold coverage of the mouse genome. Currently, the library can be screened by polymerase chain reaction and we have isolated positive clones at a number of loci in the mouse genome. This rad52 library should enable a long-term assessment of the effect of one of the yeast recombination pathway genes on both, genome-wide YAC clone stability and the frequency of chimaeric clones.

Rps4 maps near the inactivation center on the mouse X chromosome.

RPS4Y, a Y-linked gene in humans, appears to encode an isoform of ribosomal protein S4. A homologous locus on the human X chromosome, RPS4X, lies close to the X-inactivation center but fails to undergo X-inactivation. We have isolated a genomic clone from the mouse Rps4 locus, the homolog of human RPS4X. We derived an intron probe that hybridizes to the functional Rps4 locus but does not cross-hybridize to related sequences elsewhere in the mouse genome. Genetic mapping utilizing interspecific mouse backcrosses and the intron-specific probe demonstrates that Rps4 maps close to the Phka locus on the mouse X chromosome and in the vicinity of the X-inactivation center. The gene order Ccg-1-Rps4/Phka-Xist-Pgk-1 is conserved between mouse and human.

High-density molecular map of the central span of the mouse X chromosome.

A total of 17 linking clones previously sublocalized to the central span of the mouse X chromosome have been ordered by detailed analysis through interspecific Mus spretus/Mus musculus domesticus backcross progeny. These probes have been positioned with respect to existing DNA markers utilizing a new interspecific backcross segregating for the Tabby (Ta) locus. The density of clones within this 11.5-cM interval is now, on average, one clone every 1000 kb. This high-density map provides probes in the vicinity of a number of important genetic loci in this region which include the X-inactivation center, the Ta locus, and the mottled (Mo) locus, and therefore provides a molecular framework for identification of the genes encoded at these loci.

Genetic mapping in the region of the mouse X-inactivation center.

The mouse X-inactivation center lies just distal to the T16H breakpoint. Utilizing pedigree analysis of backcross progeny from a Mus domesticus/Mus spretus interspecific cross, we have mapped a number of genetic loci, gene probes, microclones, and EagI linking clones distal to the T16H breakpoint. The genetic analysis provides a detailed genetic map in the vicinity of the mouse X-inactivation center. Comparative mapping data from the human X chromosome indicate that the most probable location of the mouse X-inactivation center is distal to Ccg-1 and in the region of the Pgk-1 locus. We report the assignment of two new loci, EM13 and DXSmh44, to the Ccg-1/Pgk-1 interval.