ABSTRACT
Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR) has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique's inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA) has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells) were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that using PMA is not an appropriate method for discriminating between live and dead cells to enumerate Legionella for regulatory purposes.
ABSTRACT
Rhizoremediation involves the breakdown of contaminants in soil resulting from microbial activity that is enhanced in the plant root zone. The objective of this study was to assess Australian native grasses for their ability to stimulate removal of aliphatic hydrocarbons from a mine site soil. Time-course pot experiments were conducted in a greenhouse with three grass species (Cymbopogon ambiguus, Brachiaria decumbens, and Microlaena stipoides) in a mine site soil experimentally contaminated with a 60:40 diesel:oil mixture at 1% (w/w) concentration. Plants were cultivated for 100days with periodic evaluation of changes in soil total petroleum hydrocarbon (TPH) concentration, soil lipase activity, and abundance of hydrocarbon-degrading microorganisms. Results were compared to unplanted control treatments. Significantly lower endpoint TPH concentrations were recorded in planted soil compared to unplanted soil (p=0.01). Final TPH concentrations and rates of TPH removal varied between grass species, with total TPH removal of between 50% and 88% achieved in planted treatments. The presence of grasses significantly increased the abundance of hydrocarbon-degrading microorganisms and soil lipase activity relative to unplanted soil (p<0.05). Residual TPH concentration was found to be closely (negatively) correlated with abundance of hydrocarbon-degrading microorganisms and to a lesser extent with soil lipase activity. Australian native grass species were identified that effectively enhance the remediation of diesel/oil contaminated soil, without any requirement for nutrient supplementation. Results may have extensive application to the nationwide problems associated with hydrocarbon contaminated sites.
