Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (C_T) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R²) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10⁴) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.     

Specific Detection of Viable Legionella Cells by Combined Use of Photoactivated Ethidium Monoazide and PCR/Real-Time PCR

Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10⁴- to 10⁵-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.     

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms'' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.     

Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by qPCR

A real time quantitative PCR combined with propidium monoazide (PMA) treatment of samples was implemented to quantify live C. jejuni, C. coli and C. lari on broiler chicken carcasses at selected processing steps in the slaughterhouse. The samples were enumerated by culture for comparison. The Campylobacter counts determined with the PMA-qPCR and the culture method were not concordant. We conclude that the qPCR combined with PMA treatment of the samples did not fully reduce the signal from dead cells.     

Rapid Quantification of Viable Campylobacter Bacteria on Chicken Carcasses,Using Real-Time PCR and Propidium Monoazide Treatment,as a Tool for Quantitative Risk Assessment

A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method''s performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (C_T) values (R² = 0.993), with a quantification range of 1 × 10² to 1 × 10⁷ CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R² = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.As Campylobacter remains the leading cause of food-borne bacterial gastrointestinal disease in large parts of the developed world (34), much effort is devoted to improving the detection and elimination of the pathogen, especially in poultry. The ultimate goal is to supply consumers with fresh, Campylobacter-free poultry products, but in order to achieve that goal, it is important to gain more insight into the epidemiology of Campylobacter, to make quantitative risk assessments, and to improve control and intervention strategies.Traditional culture-based detection of Campylobacter bacteria, including enrichment, isolation, and confirmation, is a time-consuming procedure requiring 5 to 6 working days (4, 14). Furthermore, bacterial cells may enter a viable but nonculturable (VBNC) state in which they may have the potential to cause human infection (37) but are not detected by the culture method. The introduction of real-time quantitative PCR (Q-PCR) has enabled faster, more sensitive, and less labor-intensive quantitative detection. Q-PCR methods for food-borne Campylobacter jejuni and C. coli in poultry, which is recognized as an important source of human Campylobacter infections, have been published (11, 12, 15, 38, 46). However, since control strategies mostly focus on reduction of the number of bacterial cells on the chicken carcass, the usefulness of these Q-PCR methods for risk assessment could be limited, since they detect all of the Campylobacter bacteria present in a sample, including the dead cells.The Q-PCR method described in the present study quantifies the three major food-borne Campylobacter species (C. jejuni, C. coli, and C. lari), thereby covering all possible prevalence shifts and coinfections. The PCR assay was previously validated according to the Nordic Organization for Validation of Alternative Microbiological Methods (NordVal) and is certified for detection of Campylobacter bacteria in chickens, cloacal swabs, and boot swabs (7). The present study concerns its suitability for the quantification of Campylobacter bacteria in chicken carcass rinse. Furthermore, a propidium monoazide (PMA) sample treatment step has been incorporated into the method (PMA-PCR), ensuring the quantification of only viable cells with intact membranes. PMA can intercalate into the double-helical DNA available from dead cells with compromised membranes, and upon extensive visible light exposure, cross-linking of the two strands of DNA occurs, leaving it unavailable for PCR amplification (30). PMA is a chemical alteration (additional azide group) of propidium iodide (PI), one of the most frequently applied non-membrane-permeating dyes in flow cytometry, and it can be expected to have the same permeating potential as PI (29). This could be of value from a food safety perspective, since PI penetrates only permeabilized cells and not cells with intact membranes (including the Campylobacter VBNC state), which can still cause infection. Nocker et al. demonstrated that no uptake of PMA occurred in bacterial cells with intact membranes, and PMA was exclusively found in bacteria with compromised membranes (31).PMA sample treatment combined with real-time PCR for detection of viable pathogens has been tested successfully on Listeria monocytogenes and Escherichia coli O157:H7 (31, 36). However, these studies were limited to laboratory-cultured strains and the methods have not been validated on naturally infected samples with the pathogen embedded in a food matrix.This is the first study to establish a correlation between results obtained by PMA-PCR and culture-based enumeration of Campylobacter bacteria for a large number of naturally infected chickens.     

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 method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.     

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

Propidium monoazide (PMA) or ethidium bromide monoazide (EMA) treatment has been used before nucleic acid detection methods, such as PCR, to distinguish between live and dead cells using membrane integrity as viability criterion. The performance of these DNA intercalating dyes was compared in many studies utilizing different microorganisms. These studies demonstrated that EMA and PMA differ in their abilities to identify nonviable cells from mixed cell populations, depending on the microorganism and the nature of the sample. Due to this heterogeneity, both dyes were used in the present study to specifically distinguish dead from live Candida albicans cells using viable quantitative PCR (qPCR). The viable qPCR was optimized, and the best results were obtained when pre-treating the cells for 10 min in the dark with 25 μM EMA followed by continuous photoactivation for 15 min. The suitability of this technique to distinguish clotrimazole- and fluconazole-treated C. albicans cells from untreated cells was then assessed. Furthermore, the antifungal properties of two commercial essential oils (Thymus vulgaris and Matricaria chamomilla) were evaluated. The viable qPCR method was determined to be a feasible technique for assessing the viability of C. albicans after drug treatment and may help to provide a rapid diagnostic and susceptibility testing method for fungal infections, especially for patients treated with antifungal therapies.     

Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into “dead” cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.     

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.     

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

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.     

Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR

A PMA (propidium monoazide) pretreatment protocol, in which PMA is applied directly to membrane filters, was developed for the PCR-based quantification (PMA-qPCR) of viable Legionella pneumophila. Using this method, the amplification of DNA from membrane-damaged L. pneumophila was strongly inhibited for samples containing a small number of dead bacteria.     

Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log₁₀ was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.     

Method to detect only viable cells in microbial ecology

Propidium monoazide can limit the analysis of microbial communities derived from genetic fingerprints to viable cells with intact cell membranes. However, PMA treatment cannot completely suppress polymerase chain reaction (PCR) amplification when the targeted gene is too short. PMA treatment in combination with two-step nested PCR was designed to overcome this problem. Four experiments were performed to determine the limitation of PMA treatment and to evaluate the suitability of the method by applying the following samples: (1) pure cultures of Escherichia coli O157:H7, Enterobacter aerogenes, and Alcaligenes faecalis; (2) pond water samples spiked with heat-killed E. coli O157:H7 and E. aerogenes; (3) anaerobic sludge samples exposed to increasing heat stress; and (4) selected natural samples of estuarine sediment and lake mud. Results from the first two experiments show that PMA treatment cannot efficiently suppress dead cells from PCR amplification when the targeted gene is as short as 190 bp, however, the two-step nested PCR can overcome this problem. The last two experiments indicate the method that PMA treatment in combination with two-step nested PCR is useful for viable cells detection in microbial ecology.     

Detecting the Nonviable and Heat-Tolerant Bacteria in Activated Sludge by Minimizing DNA from Dead Cells

Propidium monoazide (PMA) has been used to determine viable microorganisms for clinical and environmental samples since selected naked DNA which was covalently cross-linked by this dye could not be PCR-amplified. In this study, we applied PMA to the activated sludge samples composed of complex bacterial populations to investigate the viability of human fecal bacteria and to determine the heat-tolerant bacteria by high-throughput sequencing of 16S ribosomal DNA (rDNA) V3 region. The methodological evaluation suggested the validity, and about 2–3 magnitude signals decreasing from the stained DNA were observed. However, the nest PCR, which was previously conducted to further minimize signals from dead cells, seemed not suitable perhaps due to the limitation of the primers. On one hand, for typical human fecal bacteria, less than half of them were viable, and most genera exhibited the similar viable percentages. It was interesting that many “unclassified bacteria” showed low viability, implying their sensitivity to environmental change. On the other hand, after heating at 60 °C for 4 h, the bacteria with high survival rate in activated sludge samples included those reported thermophiles or heat-tolerant lineages, such as Anoxybacillus and diverse species in Actinobacteria, and some novel ones, such as Gp16 subdivision in Acidobacteria. In summary, our results took a glance at the fate of fecal bacteria during sewage treatment and established an example for identifying tolerant species to lethal shocks in a complex community.     

Propidium monoazide–quantitative polymerase chain reaction for viable Escherichia coli and Pseudomonas aeruginosa detection from abundant background microflora

Nucleic acid-based techniques represent a promising alternative to cultivation-based microbial water quality assessment methods. However, their application is hampered by their innate inability to differentiate between living and dead organisms. Propidium monoazide (PMA) treatment was proposed as an efficient approach for alleviating this limitation. In this study, we demonstrate the performance of PMA–quantitative polymerase chain reaction (qPCR) for the detection of indicator organisms (Escherichia coli and Pseudomonas aeruginosa) in a background of a highly abundant and complex microflora. Treatment with 10 μM PMA resulted in the complete or significant reduction of the false positive signal arising from the amplification of DNA from dead cells.     

Discrimination of Viable and Dead Fecal Bacteroidales Bacteria by Quantitative PCR with Propidium Monoazide

Propidium monoazide (PMA) was optimized to discriminate between viable and dead Bacteroides fragilis cells and extracellular DNA at different concentrations of solids using quantitative PCR. Conditions of 100 μM PMA and a 10-min light exposure also excluded DNA from heat-treated cells of nonculturable Bacteroidales in human feces and wastewater influent and effluent.The aim of microbial source tracking (MST) methods is to identify, and in some cases quantify, the dominant sources of fecal contamination in surface waters and groundwater (2, 16). One of the most promising library- and cultivation-independent approaches utilizes fecal Bacteroidales bacteria and quantitative PCR (qPCR) assays to measure gene copies of host-specific genetic markers for 16S rRNA (4, 5, 10, 14). Currently, molecular assays do not directly discriminate between viable and nonviable cells since DNA of both live and dead cells and extracellular DNA can be amplified. Consequently, source tracking data based on detection of genetic markers by PCR cannot distinguish between recent and past contamination events since DNA of selected pathogens can persist after cell death for more than 3 weeks (6). Hence, it would be preferable to detect host-specific markers in viable cells of Bacteroidales bacteria, which are strictly anaerobic microorganisms and unlikely to survive in water.Previous studies have suggested the use of intercalating DNA-binding chemicals combined with PCR to inhibit PCR amplification of DNA derived from dead cells (8, 9, 11, 15). For example, ethidium monoazide (EMA) has been investigated as a means of reducing the PCR signal from DNA originating from dead bacterial cells (7, 15, 19). However, the use of EMA prior to DNA extraction has been found to result in a significant loss of the genomic DNA of viable cells in the case of Escherichia coli 0157:H7, Campylobacter jejuni, and Listeria monocytogenes (3, 7). Recently propidium monoazide (PMA) has been proposed as a more selective agent, penetrating only dead bacterial cells but not viable cells with intact membranes (8). EMA/PMA in combination with PCR or qPCR has been applied to identify viable food-borne pathogens in a simple matrix (3, 7, 8, 11), and possible restrictions in the use of PMA in environmental samples were reported (9, 19). Yet the feasibility of applying PMA in environmental samples or MST studies using fecal Bacteroidales bacteria has not been systematically studied. Any meaningful application of EMA or PMA in stool or natural water samples must consider potential interferences due to particulate matter present in the environmental matrix. Similarly, procedures for the concentration of large volumes of water samples to simultaneously monitor pathogens and MST identifiers can lower the limit of detection (4, 12), but they concentrate solids or other inhibitors of quantitative PCR (qPCR) as well, which might interfere in the covalent binding of PMA to DNA.The objectives of this study were, therefore, the following: (i) to evaluate the applicability of PMA-qPCR methods to detect culturable Bacteroides fragilis, (ii) to determine the feasibility of PMA-qPCR analysis for environmental samples containing different concentrations of solids, and (iii) to validate the utility of the PMA-qPCR method for the detection of fecal Bacteroidales bacteria in defined live and heat-treated mixtures of human feces and in wastewater treatment plant influent and effluent.Pure cultures of Bacteroides fragilis (ATCC 25285) were grown in thioglycolate broth (Anaerobe System, Morgan Hill, CA) under anaerobic conditions in GasPak anaerobic jars (Becton Dickinson Microbiology Systems, Cockeysville, MD). The solids were obtained by hollow-fiber ultrafiltration as described previously (12, 13). Ultrasonification and heat sterilization in an autoclave were used for removing attached bacteria or DNA from solids and inactivating residual DNA. Finally, the solids were resuspended with 1× phosphate-buffered saline (PBS) solution to 100 mg liter⁻¹ or 1,000 mg liter⁻¹ of suspended solids. The concentration of total suspended solids (TSS) was measured using method 2450 C (1). Next, 1 ml of broth medium containing 2 × 10⁹ viable or 2 × 10⁸ heat-treated B. fragilis cells, which had been exposed at 80°C for 20 min, was spiked into 1× PBS buffer solutions containing 0 mg liter⁻¹, 100 mg liter⁻¹, or 1,000 mg liter⁻¹ of TSS. Before the cells were spiked, 1 ml of Bacteroides fragilis cell suspension was enumerated with the Live/Dead BacLight bacterial viability kit (Molecular Probes Inc., Eugene, OR) using a hemacytometer and an Axioskop 2 Plus epifluorescence microscope (Zeiss, Thornwood, NY) equipped with two filter sets (fluorescein isothiocyanate and Texas Red). The inoculated samples were incubated under anaerobic conditions in GasPak anaerobic jars (Becton Dickinson Microbiology Systems, Cockeysville, MD) for 4 h at 20°C to allow sufficient time for the cells to sorb to solids.A fresh human fecal specimen was obtained from a healthy adult. Two grams of feces was suspended in 25 ml 1× PBS. The fecal suspension was diluted 1:10 and 1:100 in a 1× PBS solution, and aliquots were subjected to heat treatment at 80°C for 20 min. The heat-treated fecal portions were mixed with fresh diluted samples (1:10 and 1:100 dilutions) in defined ratios, with fresh feces representing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% of the total, respectively. Effluent and influent water samples were collected in sterile 2-liter bottles from the University of California, Davis, wastewater treatment plant. The effluent samples were concentrated to approximately 200 ml by hollow-fiber ultrafiltration (12).PMA (Biotium Inc., Hayward, CA) was prepared, stored, and used as described in previous studies (8, 9), but PMA concentrations and light exposure time were varied to determine the optimal condition of PMA-qPCR; the PMA concentrations were 2 μM, 6 μM, 20 μM, and 100 μM. Light exposure times were 1 min, 5 min, 10 min, and 20 min. Genomic DNA was extracted using the FastDNA spin kit for soil (Biomedicals, Solon, OH). Cell lysis was achieved by bead beating using a bead mill Minibread beater (Biospec Products Inc., Bartlesville, OK) at 2,400 rpm for 20 s. Otherwise, DNA extraction was performed according to the manufacturer''s instructions. TaqMan probe and primer assays targeting the rRNA genes of all fecal Bacteroidales bacteria (BacUni-UCD) and mixed human-specific Bacteroidales bacteria (BacHum-UCD), developed by Kildare et al. (4), were used to detect and quantify fecal Bacteroidales bacteria present in fecal and (waste)water samples.We explored the ability of PMA-qPCR to discriminate between viable and heat-killed cells at different solids concentrations using Bacteroides fragilis cultures (Fig. ​(Fig.1).1). PMA did not influence the PCR amplification of DNA derived from viable cells when no solids were present (TSS = 0 mg liter⁻¹) (Fig. ​(Fig.1A).1A). The level of PMA concentration slightly affected the mean cycle threshold differences (ΔC_T) of viable cells at higher solids concentrations (TSS = 100 and 1,000 mg liter⁻¹) (Fig. 1C and E). The signal reductions in the amplification of heat-killed cells were a function of both the PMA concentration and exposure time (Fig. 1B, D, and F). Lower solids concentrations did not inhibit the efficacy of discrimination from heat-killed cells. However, solids at 1,000 mg liter⁻¹ affected the amplification of DNA derived from heat-killed cells. Higher solids concentrations affected the suppression of PCR amplification from heat-treated cells by interfering with the cross-linking of PMA. In agreement with previous reports, the number of viable Bacteroides fragilis cells was underestimated in our study when EMA-treated and untreated samples containing only viable cells were compared because mean ΔC_T values were as high as 10 (data not shown). In contrast to EMA, PMA seems to not penetrate live cells, since higher selectivity of PMA is most probably associated with the higher charge of the molecule (8).Open in a separate windowFIG. 1.Effect of PMA on amplification of BacUni-UCD universal marker in viable and dead Bacteroides fragilis cells with different concentrations of solids. The contour lines represented ΔC_T values and were generated by the Origin Pro 8 software program. The mean cycle threshold differences (ΔC_T) were calculated by subtracting C_T values obtained without PMA treatment from C_T values obtained with PMA treatment. (A and B) ΔC_T for viable cells (A) or dead cells (B) in the absence of added solids. (C and D) ΔC_T for viable cells (C) or dead cells (D) at a solids concentration of 100 mg liter⁻¹. (E and F) ΔC_T for viable cells (E) or dead cells (F) at a solids concentration of 1,000 mg liter⁻¹.A factorial three-way analysis of variance including the PMA concentration, exposure time, and TSS concentration was performed to determine the interferences of solids and the optimal PMA-qPCR condition in the differentiation of viable cells from dead cells (Table ​(Table1).1). The mean ΔC_T of viable cells in the PMA experiments was slightly influenced by the PMA concentration (P = 0.05) in the absence of solids (TSS = 0 mg liter⁻¹), but the effect was biologically insignificant (mean ΔC_T = 0.004). The PMA concentration had a significant effect on ΔC_T values for both viable and dead cells in the presence of higher solids concentrations (TSS = 100 and 1,000 mg liter⁻¹), as shown in Table ​Table1.1. However, the effect of exposure time in PMA treatment was insignificant at a TSS concentration of 1,000 mg liter⁻¹ (P > 0.4). The solids concentration caused significantly different ΔC_T values for viable and dead cells in the PMA treatments (P < 0.001) as determined by factorial three-way analysis. The greatest differences in the mean ΔC_T values between viable and dead cells were seen at 100 μM of PMA and with a 10-min exposure time, as determined by Tukey''s comparison test, for TSS concentrations of 100 mg liter⁻¹ and 1,000 mg liter⁻¹. Ideally, shorter light exposure and a lower concentration of dye can minimize the penetration of live cells. However, these conditions were not compatible with sufficient inhibition of amplification of DNA from dead cells for PMA treatment.

TABLE 1.

Statistical analysis for differences (ΔC_T) between nontreatment and PMA treatment for experiments where Bacteroides fragilis was spiked^a

Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide

Aims: The detection of viable Enterobacter sakazakii cells is important due to the association of this pathogen with outbreaks of life-threatening neonatal infections. The aim of this study was to optimize a PCR-based method for selective detection of only viable Ent. sakazakii cells in the presence of dead cells, utilizing propidium monoazide (PMA) or ethidium bromide monoazide (EMA). Methods and Results: PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 μg ml⁻¹) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 μg ml⁻¹, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the amplification from viable cells. PMA was also effective in allowing selective PCR detection of only viable cells in mixtures of varying ratios of viable and dead cells. EMA was equally effective in preventing amplification from dead cells, however, it also inhibited DNA amplification from viable cells. Conclusions: This study demonstrated the efficiency of PMA for viable and dead differentiation of Ent. sakazakii, as well as the lack of selectivity of EMA for this purpose. Significance and Impact of the Study: PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.     

Metabolic Responses of Primary and Transformed Cells to Intracellular Listeria monocytogenes

The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using ¹³C-labelled glucose or glutamine as carbon tracers. The ¹³C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.