Molecular Detection of Functional Gene Signatures for Detecting Pathogens in Soil

Detection of pathogens and assessment of their activity in the environment is important for ecological studies as well as for public health. A variety of microbial pathogens that infect humans and animals are known to survive in soil, e.g. enteric pathogens such as Salmonella sp., Vibrio cholerae, Shigella sp., Campylobacter jejuni, Yersinia sp., Escherichia coli O157:h7 (Santamaria and Toranzos 2003) and other pathogens such as Mycobacterium bovis (Young et al. 2005). Pathogens enter the soil via excreta of infected wild and domesticated animals, and animal and human waste applied to agricultural land as fertiliser. Landfill leachates present an additional route for human and animal pathogens to enter the soil; in 1993, the US generated 156 million tons of waste, which was sequestered in landfills. Household waste can contain pathogens present in human and animal faeces, food waste and sewage sludge (Santamaria and Toranzos 2003).

Gene Detection

A number of studies have used PCR primers designed to amplify functional genes specific to Salmonella spp. Ziemer and Steadham (2003) assessed nine sets of PCR primers targeting Salmonella for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. Functional gene targets of PCR primers included a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, /ur-regulated gene, histi-dine transport operon, junction between sipB and sipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Way et al. (1993) used three sets of oligonucleotide primers to detect Salmonella spp. including from inoculated soil samples. PhoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter spp. served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella spp. and are not present in Shigella, E. coli, or Citrobacter spp. Further studies on molecular detection of S. typhimurium in soil (Marsh et al.1998) using Way's primers illustrated H-Li PCR products at >103 cells g-1. It was also demonstrated using QPCR that S. typhimurium numbers decreased over time in non-sterile microcosms, from 108 copies of the H-Li gene g-1 soil at day 0 to 105 copies g-1 soil at day 60.

Faecal coliforms were detected in inoculated soil samples by amplification of a 179-bp fragment of lamb, a gene that encodes an outer membrane protein present specifically in faecal coliforms. Following cell extraction with CaCl2 and purification using sucrose density gradient centrifugation, 1cfu of E. coli was detectable in 1 g of soil (Josephson et al. 1991).

Different soils require different methods to efficiently extract DNA. Poussier et al. (2003) applied a wide range of published methods, modifications and novel methods in order to detect Ralstonia solanacearum, the causative agent of bacterial wilt in solanaceous plants, from four different soil types. Protocols using the QIAamp kit (Qiagen, Courtaboeuf, France) were found to be most reliable.

E. coli O157:H7 is an important human pathogen found in soil, and the possibility of prolonged survival has led to the development of novel methods used to detect virulence genes which differentiate it from non-enterohaemorrhagic strains. E. coli O157:H7 has been detected in inoculated soil and wetland samples using real-time PCR (Ibekwe et al. 2002). Shiga-like toxin genes stx1, stx2 and the intimin gene eae were detected in inoculated soils. DNA was extracted using an UltraClean soil DNA kit (MO BIO). Sensitivity of the multiplex PCR was established as 7.9x 10-5 pg of starting DNA ml-1 equating to 6.4x103 cfu ml-1 in genomic DNA; in DNA extracted from seeded soil samples, the detection limit ranged from 3.5x103 cfu ml-1 for the stx1 gene and 3.5x104 cfu ml-1 for the stx2 and eae genes.

PCR primers specific for the Mycobacterium tuberculosis complex have been used to detect the presence and survival of M. bovis BCG (Pasteur) in soil microcosms and subsequently Mycobacterium bovis in the environment (Young et al. 2005). PCR detection of the M. tuberculosis complex in clinical specimens has been achieved using antigen genes, such as mpb70 (Gormley et al. 1999) and also the insertion sequence IS6110 (Broccolo et al. 2003). The former provides a highly specific and quantifiable target for molecular detection, as it is a single copy gene found only in members of the tuberculosis complex. Environmental samples were collected from a farm in Ireland with a history of bovine tuberculosis and M. bovis was detected in soil up to 21 months after initial contamination. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in the Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 °C with moist soil (-20 kPa, 30% v/w).

The use of microarrays for the detection of pathogens has become more common in recent years, although few have applied the technology to detection in complex environments such as soil. Chizhikov et al. (2001) used microarray analysis to discriminate strains of E. coli and other pathogenic enteric bacteria harbouring various virulence factors. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors was monitored by multiplex PCR followed by hybridisation of the denatured PCR product to the gene-specific oligonucleotides on a microchip. The array was able to detect virulence factors in 15 Salmonella, Shigella, and E. coli strains. The advantage of this technique over conventional visualisation using gel electrophoresis was that the latter technique produced unexpected and uncharacterised PCR products giving ambiguous results. Lievens et al. (2003) used a DNA array for the rapid detection of multiple tomato wilt pathogens using internal transcribed spacer (ITS) sequences from rDNA. Seeded potting compost wasinoculatedwithfungalculturesandthen tomato seedlingswereplanted and grown for 7 and 10 weeks. The pathogens were subsequently detected in compost using the DNA array.

Metabolic Activity and Infectivity of Bacterial Pathogens in Soil

The physiological state of pathogens in soil is largely unknown. There is evidence that organisms such as salmonellae become "viable but non-culturable" (VBNC) in soil (Turpin et al. 1993) with a reduction in cell size. However, laboratory microcosm studies have shown that different serovars of Salmonella and E. coli are able to grow in composts at 37 °C. Agricultural soils are highly heterogeneous, with areas of variable organic matter (e.g. faeces, decaying plant material) so the possibility remains that an active subpopulation of pathogenic bacteria may exist in the soil environment. Intracellular growth within soil protozoan vacuoles has been demonstrated for some bacterial pathogens, from where bacterial cells can be released and provide an active subpopulation of cells in soil (Barker et al. 1999; Gaze et al. 2003).

The detection of genes from pathogenic organisms in soil gives limited information regarding the viability, metabolic activity or infectivity of the organisms. DNA remains amplifiable by PCR for some time after cell death. Romanowski et al. (1993) reported that, after 60 days, 0.2, 0.05, and 0.01% of added genes on plasmids were detectable by PCR in a loamy sand soil, a clay soil, and a silty clay soil, respectively. However, in seeding experiments with M. bovis DNA, lysed cells and dead cells, no DNA could be detected after 10 days indicating that detection of DNA was a useful indicator for the presence of live cells in soil from fields known to have been grazed by infected cattle (Young et al. 2005). The half-life of mRNA molecules is considerably shorter than that of DNA, and many studies have concentrated on detection of these molecules as evidence of pathogen activity in soil. Laboratory studies have shown that mRNA in E. coli becomes undetectable after 2-16 h after being heat-killed (Sheridan et al. 1998); mRNA has been extracted directly from soil and has proved useful in investigating microbial activity (Sessitsch et al. 2002; Burgmann et al. 2003; Anukool et al. 2004). However, very little work has been undertaken applying this technique to pathogens in soil.

In situ RT-PCR has allowed the physiological state of individual S. ty-phimurium cells to be monitored. Holmstrom et al. (1999) used groEL mRNA (which is induced by heat shock) and tsf mRNA (which is expressed as a function of growth rate) to monitor different physiological functions. Intracellular mRNA was amplified using biotin-labelled primers in fixed cells and PCR products were detected using a streptavidin-horseradish peroxidase conjugate; this type of approach has considerable potential for elucidation of pathogen activity in soils. Nucleic acid based fluorescent probes can also be used to monitor bacterial cells extracted from soil in conjunction with flow cytometry; several reviews are available on the subject (Davey and Kell 1996; Amann and Kuhl 1998; Porter and Pickup 2000; Shapiro 2000; Steen 2000).

The expression of virulence genes may not occur when a pathogen is outside its host, and the retention of virulence can only be confirmed by use of infectivity assays using animal models. The virulence of cells extracted from soil and identified using oligonucleotide probes and FACS could theoretically be tested. Some research to date suggests that pathogens such as Vibrio spp. which appear to be in a VBNC form are capable of resuscitation and retain their infectivity (Baffone et al. 2003).

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