Andreas Nocker

Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology

ABSTRACT One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.

Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification

The ideal scenario in most applications of microbial diagnostics is that only viable cells are detected. Bacteria were traditionally considered viable when they could be cultured, whereas todays viability concept tends to be alternatively based on the presence of some form of metabolic activity, a positive energy status, responsiveness, detection of RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion, although conservative, was the focus of one of the most successful recent approaches to detect viable cells in combination with DNA amplification techniques. The technology is based on sample treatment with the photoactivatable, and cell membrane impermeant, nucleic acid intercalating dyes ethidium monoazide (EMA) or propidium monoazide (PMA) followed by light exposure prior to extraction of DNA and amplification. Light activation of DNA-bound dye molecules results in irreversible DNA modification and subsequent inhibition of its amplification. Sample pretreatment with viability dyes has so far been mainly used in combination with PCR (leading to the term viability PCR, v-PCR), and increasingly with isothermal amplification method. The principle is not limited to bacteria, but has also successfully been applied to fungi, protozoa and viruses. Despite the success of the method, some practical limitations have been identified, especially when applied to environmental samples. In part they can be minimized by choice of experimental parameters and conditions adequate for a particular sample. This review summarizes current knowledge and presents aspects which are important when designing experiments employing viability dyes.

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the methods capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.

Effect of PCR amplicon length on suppressing signals from membrane-compromised cells by propidium monoazide treatment

Treatment of microbiological samples with viability dyes prior to extraction of DNA and PCR amplification for downstream analysis has evolved into a commonly applied method. The addition of this easy-to-perform step to the sample analysis procedure inhibits the amplification of DNA from dead cells with compromised cell membranes. The method is currently used both in combination with quantitative PCR (qPCR), end-point PCR, and isothermal amplification. We present here a detailed study of the effect of amplicon size on amplification signals from unstressed and heat-exposed cells after treatment with propidium monoazide (PMA). PMA treatment was shown to be more efficient in excluding dead cells from the analysis both in combination with qPCR (PMA-qPCR) and denaturing gradient gel electrophoresis (PMA-DGGE), when longer amplicons were used. When applied to pure cultures of the fish pathogens Vibrio anguillarum and Flavobacterium psychrophilum exposed to a heat gradient ranging from mild to lethal, qPCR product lengths did not influence PMA-qPCR results at low temperatures, whereas an increasingly strong impact was seen at higher temperatures. Membrane permeability as a result of heat exposure might however have to be considered a conservative parameter for cell death for these pathogens as culturability and redox activity were lost at lower stress intensities than membrane integrity. When applying PMA-DGGE to an environmental water sample which was either left untreated or was exposed to heat, differences to non-PMA treated samples tended to slightly increase when amplified fragments in the first round of the nested PCR were longer, whereas the impact of 1st-round cycle numbers remains unclear.

Improving efficiency of viability-PCR for selective detection of live cells.

Viability PCR (v-PCR) as a method to selectively detect intact live cells has gained considerable interest over the last years with an increasing number of applications. The principle is based on treatment of microbiological samples with a viability dye prior to extraction of genomic DNA and its amplification. The dye is selectively taken up by membrane-compromised dead cells resulting in the degradation of their DNA upon light exposure and therefore inhibition of amplification. Although the treatment greatly helps to generate more meaningful data, one of the main drawbacks of the technique is currently that the exclusion of dead cell signals can be incomplete leading to false-positive signals. The resulting overestimation of live cell population is especially problematic for the detection of pathogens. We assessed in this study different conditions to increase the penetration of propidium monoazide (PMA) into dead cells of Salmonella Typhimurium and Listeria monocytogenes as representatives of gram-negative and gram-positive bacteria. When working with a low dye concentration of 10μM, a strong relationship of PMA treatment efficiency with temperature and incubation time was observed. Exposing cells to PMA at a temperature exceeding the growth temperature by 10°C for 30min proved greatly beneficial. Co-incubation of cells with PMA and deoxycholate on the other hand was only beneficial for Salmonella, but resulted in a strong undesired uptake of PMA by live Listeria cells. This difference is in agreement with the gram-specific effect of the bile salt during growth.

Evaluation of engineered nanoparticle toxic effect on wastewater microorganisms: current status and challenges.

The use of engineered nanoparticles (ENPs) in a wide range of products is associated with an increased concern for environmental safety due to their potential toxicological and adverse effects. ENPs exert antimicrobial properties through different mechanisms such as the formation of reactive oxygen species, disruption of physiological and metabolic processes. Although there are little empirical evidences on environmental fate and transport of ENPs, biosolids in wastewater most likely would be a sink for ENPs. However, there are still many uncertainties in relation to ENPs impact on the biological processes during wastewater treatment. This review provides an overview of the available data on the plausible effects of ENPs on AS and AD processes, two key biologically relevant environments for understanding ENPs-microbial interactions. It indicates that the impact of ENPs is not fully understood and few evidences suggest that ENPs could augment microbial-mediated processes such as AS and AD. Further to this, wastewater components can enhance or attenuate ENPs effects. Meanwhile it is still difficult to determine effective doses and establish toxicological guidelines, which is in part due to variable wastewater composition and inadequacy of current analytical procedures. Challenges associated with toxicity evaluation and data interpretation highlight areas in need for further research studies.

Microbiological tap water profile of a medium-sized building and effect of water stagnation

Whereas microbiological quality of drinking water in water distribution systems is routinely monitored for reasons of legal compliance, microbial numbers in tap water are grossly understudied. Motivated by gross differences in water from private households, we applied in this study flow cytometry as a rapid analytical method to quantify microbial concentrations in water sampled at diverse taps in a medium size research building receiving chlorinated water. Taps differed considerably in frequency of usage and were located in laboratories, bathrooms, and a coffee kitchen. Substantial differences were observed between taps with concentrations (per mL) in the range from 6.29×103 to 7.74×105 for total cells and from 1.66×103 to 4.31×105 for intact cells. The percentage of intact cells varied between 7% and 96%. Water from taps with very infrequent use showed the highest bacterial numbers and the highest proportions of intact cells. Stagnation tended to increase microbial numbers in water from those taps which were otherwise frequently used. Microbial numbers in other taps that were rarely opened were not affected by stagnation as their water is probably mostly stagnant. For cold water taps, microbial numbers and the percentage of intact cells tended to decline with flushing with the greatest decline for taps used least frequently whereas microbial concentrations in water from hot water taps tended to be somewhat more stable. We conclude that microbiological water quality is mainly determined by building-specific parameters. Tap water profiling can provide valuable insight into plumbing system hygiene and maintenance.

Assessing microbiological water quality in drinking water distribution systems with disinfectant residual using flow cytometry.

Flow cytometry (FCM) as a diagnostic tool for enumeration and characterization of microorganisms is rapidly gaining popularity and is increasingly applied in the water industry. In this study we applied the method to obtain a better understanding of total and intact cell concentrations in three different drinking water distribution systems (one using chlorine and two using chloramines as secondary disinfectants). Chloramine tended to result in lower proportions of intact cells than chlorine over a wider residual range, in agreement with existing knowledge that chloramine suppresses regrowth more efficiently. For chlorinated systems, free chlorine concentrations above 0.5 mg L(-1) were found to be associated with relatively low proportions of intact cells, whereas lower disinfectant levels could result in substantially higher percentages of intact cells. The threshold for chlorinated systems is in good agreement with guidelines from the World Health Organization. The fact that the vast majority of samples failing the regulatory coliform standard also showed elevated proportions of intact cells suggests that this parameter might be useful for evaluating risk of failure. Another interesting parameter for judging the microbiological status of water, the biological regrowth potential, greatly varied among different finished waters providing potential help for investment decisions. For its measurement, a simple method was introduced that can easily be performed by water utilities with FCM capability.

Effect of air drying on bacterial viability: A multiparameter viability assessment.

The effect of desiccation on the viability of microorganisms is a question of great interest for a variety of public health questions and industrial applications. Although viability is traditionally assessed by plate counts, cultivation-independent methods are increasingly applied with the aim to gain more insight into why cells might not form colonies and to optimize production processes. To evaluate their usefulness, we applied in this study a multiparameter viability assay to selected bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterococcus hirae, and Staphylococcus aureus) subjected to air-drying in the absence or presence of supplements. Tests included growth on solid culture medium and the measurement of membrane integrity, membrane potential, esterase and respiratory activities using fluorescent dyes. All measured parameters were responsive to desiccation stress. Results suggested that extending plate count analysis with cultivation-independent methods can greatly enhance resolution especially for moderate stress conditions, which do not get reflected in plate counts due to cellular recovery. Whereas plate counts reflect the final effect on viability, immediate measurement of cellular functions provides a snapshot picture of the fitness status at a specific point in time. Special emphasis was given to MgCl(2) which in concentrations≥50mM dramatically increased the bacterial susceptibility to desiccation in the case of the gram-negative bacteria and to a lesser extent also for the gram-positive bacteria. The study in addition confirmed a good agreement of results obtained with the recently developed real-time viability (RTV) assay and the BacLight LIVE/DEAD method in combination with a fluorescence plate reader.

Multiparameter Viability Assay for Stress Profiling Applied to the Food Pathogen Listeria monocytogenes F2365

ABSTRACT A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This food-borne pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters.