Quantitative molecular detection of Salmonella typhimurium in soil and demonstration of persistence of an active but non-culturable population

The effects of three temperatures (5, 15, and 25°C) on the survival of Salmonella enterica serovar Typhimurium in topsoil were investigated in small microcosms by three different techniques: plate counting, invA gene quantification, and invA mRNA quantification. Differences in survival were related to the effect of protozoan predation. Tetracycline-resistant Salmonella serovar Typhimurium was inoculated into soil and manure-amended soil at 1.5 ؋ 10 8 cells g soil ؊1 . Population densities were determined by plate counting and by molecular methods and monitored for 42 days. Simultaneous extraction of RNA and DNA, followed by quantitative PCR, was used to investigate invA gene levels and expression. Analysis by these three techniques showed that Salmonella serovar Typhimurium survived better at 5°C. Comparing DNA and CFU levels, significantly higher values were determined by DNA-based techniques. invA mRNA levels showed a fast decrease in activity, with no detectable mRNA after an incubation period of less than 4 days in any of the soil scenarios. A negative correlation was found between Salmonella serovar Typhimurium CFU levels and protozoan most probable numbers, and we propose the role of the predator-prey interaction as a factor to explain the die-off of the introduced strain by both culture-and DNA quantification-based methods. The results indicate that temperature, manure, and protozoan predation are important factors influencing the survival of Salmonella serovar Typhimurium in soil.

Section: Discussionsupporting

confidence: 84%

“…Numerous methods have been developed for the detection and quantification of Salmonella in different matrices (11,14,35). The introduction of molecular techniques has become an especially important advance in reducing the time required for detection of Salmonella and in detecting active bacteria in environmental samples through their DNA and RNA (10,18,51).…”

mentioning

confidence: 99%

Influence of Temperature and Predation on Survival of Salmonella enterica Serovar Typhimurium and Expression of invA in Soil and Manure-Amended Soil

Bælum

Fredslund

et al. 2010

“…No studies have been done to investigate the impact of environmental conditions on the sensitivity of environmental M. bovis or the vaccine strain Mycobacterium bovis BCG (Pasteur) to these treatments. Methods for the molecular detection of bacterial pathogens in soil have been successfully applied to monitor the fate of salmonellae (10), Escherichia coli O157:H7 (5), and Mycoplasma sp. (9) by either quantitative PCR (Q-PCR) or reverse transcription-PCR (RT-PCR), thus avoiding the problems of selective cultivation.…”

mentioning

confidence: 99%

Molecular Detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in Soil

Young

Gormley

Wellington

2005

123

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 ؋ 10 3 to 3.6 ؋ 10 3 gene copies g of soil ؊1 , depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in 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% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.Environmental and wildlife reservoirs of Mycobacterium bovis are of significance due to the increasing number of bovine tuberculosis breakdowns in cattle herds in both the United Kingdom and Ireland. In Europe, the badger (Meles meles) has been implicated, but it is unclear how the disease is transmitted to cattle from badgers. Few studies have considered dissemination and persistence of environmental M. bovis. Tanner and Michel (12) reported survival of up to 6 weeks for M. bovis cells inoculated into soil and feces, detected by traditional selective cultivation methods. However, cultivation techniques for monitoring M. bovis and other mycobacteria in soil are impeded by the slow growth rates of M. bovis and the need for prolonged incubation of highly selective agars. Pretreatment or decontamination of samples is required involving the addition of 1 to 5% NaOH, often followed by further treatments with H 2

“…Additionally, the ability to disrupt bacterial cells which are tightly adhered to soil particles may also be inefficient, leading to an underestimation of target population size. Molecular techniques, such as quantitative PCR, eliminate some of the biases of culture-based methods for estimation of pathogen abundance in soil, although, in most cases, an enrichment step is still required (22). Despite this, PCR-based approaches rely on direct extraction of nucleic acids or cells (followed by nucleic acid extraction) from soil, which may also be biased depending on the extraction method employed (23).…”

mentioning

confidence: 99%

A Stable Bioluminescent Construct of Escherichia coli O157:H7 for Hazard Assessments of Long-Term Survival in the Environment

Ritchie

Campbell

Shepherd

et al. 2003

A chromosomally lux-marked (Tn5 luxCDABE) strain of nontoxigenic Escherichia coli O157:H7 was constructed by transposon mutagenesis and shown to have retained the O157, H7, and intimin phenotypes. The survival characteristics of this strain in the experiments performed (soil at ؊5, ؊100, and ؊1,500 kPa matric potential and artificial groundwater) were indistinguishable from the wild-type strain. Evaluation of potential luminescence was found to be a rapid, cheap, and quantitative measure of viable E. coli O157:H7 Tn5 luxCDABE populations in environmental samples. In the survival studies, bioluminescence of the starved populations of E. coli O157:H7 Tn5 luxCDABE could be reactivated to the original levels of light emission, suggesting that these populations remain viable and potentially infective to humans. The attributes of the construct offer a cheap and low-risk substitute to the use of verocytotoxin-producing E. coli O157:H7 in long-term survival studies.