propidium-monoazide and 8-azidoethidium

As a self-protection mechanism, the viable but non-culturable (VBNC) state provides the ability against conventional detection methods among various foodborne pathogens. The ability of forming colonies is lost while metabolism is still maintaining in VBNC state cells. Recently, ethidium monoazide (EMA) and propidium monoazide (PMA) have been widely applied on the detection of foodborne pathogens in VBNC state. Combined with loop-mediated isothermal amplification (LAMP), the PMA/EMA-LAMP showed a significant priority in high sensitivity, specificity and rapidity over conventional PCR based assays. Particularly, PMA/EMA-LAMP has been proved as an effective method in the detection of Escherichia coli, Vibrio parahaemolyticus and Staphylococcus in VBNC state. Based on the current investigations, the VBNC mechanism and current detection method for VBNC-state foodborne pathogens were introduced and discussed in this review.

Topics: Azides; Bacteria; Bacteriological Techniques; DNA, Bacterial; Escherichia coli; Food Microbiology; Microbial Viability; Nucleic Acid Amplification Techniques; Propidium; Sensitivity and Specificity; Staphylococcus; Vibrio parahaemolyticus

This article elaborates on possible future directions for microbial viability assessment using nucleic acid-modifying compounds in combination with DNA- (and potentially RNA-) amplification technologies. Bacteria were traditionally considered viable when they could be cultured, whereas today's viability concept is based on the presence of some form of metabolic activity, responsiveness, RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion was the focus of recent approaches to limit detection to intact cells using ethidium monoazide or propidium monoazide. Membrane integrity must, however, be considered as a very conservative criterion for microbial viability. The new concept presented here aims at limiting nucleic acid-based detection to cells with an active metabolism, which might be a more appropriate viability criterion. To selectively detect only cells with metabolic and respiratory activity (while excluding inactive dead cells from detection), we suggest the use of 'activity-labile compounds'. In addition to their potential usefulness for viability assessment, these new compounds could also be beneficial for selectively amplifying nucleic acids of cells that have metabolic activities of interest. This preferential detection of microorganisms with certain metabolic capabilities is referred to as 'molecular enrichment' in distinction to 'growth enrichment'.

Topics: Azides; Microbial Viability; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Propidium

Vibrio sp., ubiquitous in the aquatic ecosystem, are bacteria of interest because of their involvement in human health, causing gastroenteritis after ingestion of seafood, as well as their role in vibriosis leading to severe losses in aquaculture production. Their ability to enter a viable but non-culturable (VBNC) state under stressful environmental conditions may lead to underestimation of the Vibrio population by traditional microbiological enumeration methods. As a result, using molecular methods in combination with EMA or PMA allows the detection of viable (VBNC and culturable viable) cells. In this study, the impact of the EMA and PMA was tested at different concentrations on the viability of several Vibrio species. We compared the toxicity of these two DNA-binding dyes to determine the best pretreatment to use with qPCR to discriminate between viable and dead Vibrio cells. Our results showed that EMA displayed lethal effects for each strain of V. cholerae and V. vulnificus tested. In contrast, the concentrations of PMA tested had no toxic effect on the viability of Vibrio cells studied. These results may help to achieve optimal PMA-qPCR methods to detect viable Vibrio sp. cells in food and environmental samples.

Topics: Anti-Bacterial Agents; Azides; Ecosystem; Gastroenteritis; Humans; Microbial Sensitivity Tests; Microbial Viability; Propidium; Real-Time Polymerase Chain Reaction; Vibrio cholerae; Vibrio vulnificus; Water Microbiology

Ethidium monoazide (EMA) quantitative polymerase chain reaction (qPCR), propidium monoazide (PMA)-qPCR and DNase treatment in combination with qPCR were compared for the determination of microbial cell viability. Additionally, varying EMA and PMA concentrations were analysed to determine which dye and concentration allowed for the optimal identification of viable cells. Viable, heat treated (70 °C for 15 min) and autoclaved cultures of Legionella pneumophila, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus and Enterococcus faecalis were utilised in the respective viability assays. Analysis of the viable and heat-treated samples indicated that variable log reductions were recorded for both EMA [log reductions ranging from 0.01 to 2.71 (viable) and 0.27 to 2.85 (heat treated)], PMA [log reductions ranging from 0.06 to 1.02 (viable) and 0.62 to 2.46 (heat treated)] and DNase treatment [log reductions ranging from 0.06 to 0.82 (viable) and 0.70 to 2.91 (heat treated)], in comparison to the no viability treatment controls. Based on the results obtained, 6 μM EMA and 50 μM PMA were identified as the optimal dye concentrations as low log reductions were recorded (viable and heat-treated samples) in comparison to the no viability treatment control. In addition, the results recorded for the 6 μM EMA concentration were comparable to the results obtained for both the 50 μM PMA and the DNase treatment. The use of EMA-qPCR (6 μM) may therefore allow for the rapid identification and quantification of multiple intact opportunistic pathogens in water sources, which would benefit routine water quality monitoring following disinfection treatment.

Topics: Azides; Deoxyribonucleases; DNA, Bacterial; Enterococcus faecalis; Legionella pneumophila; Microbial Viability; Polymerase Chain Reaction; Propidium; Pseudomonas aeruginosa; Salmonella typhimurium; Staphylococcus aureus; Water Microbiology

Quantitative PCR methods are commonly used to monitor enteric viruses in the aquatic environment because of their high sensitivity, short reaction times and relatively low operational cost. However, conclusions for public health drawn from results of such molecular techniques are limited due to their inability to determine viral infectivity. Ethidium monoazide (EMA) and propidium monoazide (PMA) are capable to penetrate the damaged or compromised capsid of the inactivated viruses and bind to the viral nucleic acids. We assessed whether dye treatment is a suitable approach to improve the ability of qPCR to distinguish between infectious and non-infectious human adenovirus, enterovirus and rotavirus A in surface water of an urban river and sewage before and after UV disinfection. Like the gold standard of cell culture assays, pretreatment EMA-/PMA-qPCR succeeded in removing false positive results which would lead to an overestimation of the viral load if only qPCR of the environmental samples was considered. A dye pretreatment could therefore provide a rapid and relatively inexpensive tool to improve the efficacy of molecular quantification methods in regards to viral infectivity.

Topics: Azides; Enterovirus; Humans; Lakes; Propidium; Real-Time Polymerase Chain Reaction; Rivers; Sewage; Urban Renewal; Water Microbiology

Despite the great sensitivity of PCR in monitoring enteric viruses in an aquatic environment, PCR detects viral nucleic acids of both infectious and noninfectious viruses, limiting the conclusions regarding significance for public health. Ethidium monoazide (EMA) and propidium monoazide (PMA) are closely related membrane impermeant dyes that selectively penetrate cells with compromised membranes. Inside the cells, the dye can intercalate into nucleic acids and inhibit PCR amplification. To assess whether EMA and PMA pretreatment is a suitable approach to inhibit DNA amplification from noninfectious viruses upon heat treatment, UV exposure or chlorine treatment, viruses were measured by qPCR, EMA-qPCR, PMA-qPCR and cell culture titration. EMA/PMA-qPCR of UV- and heat-treated viruses did not correlate with the results of the cell culture assay. However, the data from EMA/PMA-qPCR of chlorine-inactivated viruses was consistent with the cell culture infectivity assay. Therefore, a dye treatment approach could be a rapid and inexpensive tool to screen the efficacy of chlorine disinfection, but it is not able to distinguish between infectious and noninfectious viruses inactivated via heat treatment or UV irradiation. Indeed, different viruses may have different trends and mechanisms of inactivation; thus, the assay must be evaluated for each virus separately.

Topics: Adenoviruses, Human; Azides; Biological Assay; Cell Culture Techniques; Chlorine; Disinfection; DNA, Viral; Ethidium; Hot Temperature; Humans; Polymerase Chain Reaction; Propidium; Public Health; Ultraviolet Rays; Virus Inactivation; Water Microbiology

Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. In vitro growth of the bacterium is usually limited to viable eukaryotic host cells imposing experimental constraints for molecular studies, such as the identification and characterisation of major virulence factors. Studies of pathogenicity may benefit from the recent development of an extracellular growth medium for C. burnetii. However, it is crucial to investigate the consistency of the virulence phenotype of strains propagated by the two fundamentally different culturing systems. In the present study, we assessed the viability of C. burnetii and the lipopolysaccaride (LPS) encoding region of the bacteria in both culture systems as indirect but key parameters to the infection potential of C. burnetii. Propidium monoazide (PMA) treatment-based real-time PCR was used for enumeration of viable C. burnetii which were validated by fluorescent infectious focus forming unit counting assays. Furthermore, RNA isolated from C. burnetiipropagated in both the culture systems was examined for LPS-related gene expression. All thus far known LPS-related genes were found to be expressed in early passages in both culturing systems indicating the presence of predominantly the phase I form of C. burnetii. Finally, we used immune-competent mice to provide direct evidence, that the relative virulence of different C. burnetii strains is essentially the same for both axenic and cell-based methods of propagation.

Topics: Animals; Azides; Bacteriological Techniques; Biological Assay; Coxiella burnetii; Electrophoresis, Polyacrylamide Gel; Female; Gene Deletion; Gene Dosage; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Lipopolysaccharides; Mice; Microbial Viability; Propidium; Q Fever; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Virulence

Viability PCR uses cell membrane integrity to differentiate live cells from dead. Our new approach improves viability PCR by enabling it to also discriminate between cells with an intact cell membrane and the ability to actively maintain bacterial homeostasis and cells that have an intact membrane but are metabolically inactive.

Topics: Azides; Bacteria; Cell Membrane; Microbial Viability; Polymerase Chain Reaction; Propidium

Human norovirus (HuNoV) is the most common cause of gastroenteritis worldwide. The lack of a virus culture system makes it difficult to determine the viability of norovirus by only reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR). The aim of this study was to investigate the detection of viable murine norovirus (MNV) by combining propidium monoazide (PMA) or ethidium monoazide (EMA) with qRT-PCR. MNV (5.21log10PFU/mL) was subjected to heat treatment at room temperature, 65, 70, 75, 80, 85, or 90°C in a water bath for 1min. The plaque assay, qRT-PCR, PMA-combined qRT-PCR, and EMA-combined qRT-PCR were then performed with heat exposed MNV samples. The MNV titer was reduced by 0.38, 1.34, and 3.71log10PFU/mL at temperatures of 65, 70, and 75°C, respectively. MNV was reduced >4.21log10PFU/mL at 80, 85, and 90°C heat inactivation. PMA (EMA) value equation for the interpretation of the viability of MNV was derived as follows: PMA (EMA) value=-logRN-logRP (RN: the relative quantity value of the not-treated sample, and RP: the relative quantity value of the PMA- or EMA-treated sample as determined by qRT-PCR). By PMA-combined qRT-PCR, the viable PMA value was 0.32, 0.83, and 2.62 for the 65, 70, and 75°C preheated MNVs, respectively. The viable PMA values for the viruses heated at 80, 85, and 90°C were all greater than 3.0, which was the cutoff value for discriminating between live and dead MNVs. The results of EMA-combined qRT-PCR were similar to those of qRT-PCR. Thus, PMA-combined qRT-PCR correlated well with the plaque assay in detecting viable MNVs.

Topics: Animals; Azides; Cell Line; Macrophages; Mice; Microbial Viability; Norovirus; Propidium; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Temperature; Viral Plaque Assay; Virology

Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses.

Topics: Animals; Azides; Ethidium; High-Throughput Screening Assays; Lung; Molecular Diagnostic Techniques; Oligonucleotide Array Sequence Analysis; Porcine Reproductive and Respiratory Syndrome; Porcine respiratory and reproductive syndrome virus; Propidium; Sensitivity and Specificity; Serum; Swine

More than two decades after its discovery, contaminant microbial DNA in PCR reagents continues to impact the sensitivity and integrity of broad-range PCR diagnostic techniques. This is particularly relevant to their use in the setting of human sepsis, where a successful diagnostic on blood samples needs to combine universal bacterial detection with sensitivity to 1-2 genome copies, because low levels of a broad range of bacteria are implicated.. We investigated the efficacy of ethidium monoazide (EMA) and propidium monoazide (PMA) treatment as emerging methods for the decontamination of PCR reagents. Both treatments were able to inactivate contaminating microbial DNA but only at concentrations that considerably affected assay sensitivity. Increasing amplicon length improved EMA/PMA decontamination efficiency but at the cost of assay sensitivity. The same was true for UV exposure as an alternative decontamination strategy, likely due to damage sustained by oligonucleotide primers which were a significant source of contamination. However, a simple combination strategy with UV-treated PCR reagents paired with EMA-treated primers produced an assay capable of two genome copy detection and a <5% contamination rate. This decontamination strategy could have important utility in developing improved pan-bacterial assays for rapid diagnosis of low pathogen burden conditions such as in the blood of patients with suspected blood stream infection.

Topics: Azides; Decontamination; DNA Contamination; DNA Primers; DNA, Bacterial; Gene Dosage; Humans; Indicators and Reagents; Molecular Sequence Data; Propidium; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Ultraviolet Rays

A rapid and efficient method for quantification and discrimination of Salmonella enterica ser. Enteritidis between viable and dead cells killed by heat was developed using ethidium bromide monoazide (EMA) in combination with a real-time loop amplified (Rti-LAMP) DNA assay. The use of 8.0 μg/ml or less of EMA did not inhibit DNA amplification in Rti-LAMP assays derived from viable cells. However, 8.0 μg/ml of EMA notably inhibited DNA amplification and significantly increased the Tp values with dead cells. When the DNA from 2000 viable CFU was subjected to EMA-Rti-LAMP the resulting Tp value was 13 min. In contrast, the DNA from 2000 CFU completely heat destroyed CFU still yielded a Tp value, which was greatly increased to 33.1 min. When the DNA from viable plus heat killed CFU at a ratio of 7:1993 was subjected to EMA-Rti-LAMP, the resulting Tp value was 19.3 min, which was statistically identical (P<0.05) to the Tp value of 19.9 min. obtained with the DNA from only 7 viable CFU. These results indicate that even though 2000 dead cells yielded a Tp value of 33.1 min., low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tp values. In addition, propidium monoazide (PMA) was found to be ineffective in distinguishing between low numbers of viable and heat killed cells of S. enterica.

Topics: Azides; Colony Count, Microbial; Microbial Viability; Nucleic Acid Amplification Techniques; Propidium; Salmonella enterica

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 (CT) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R(2)) 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 (>10(4)) 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.

Topics: Animals; Azides; Bacterial Load; Campylobacter coli; Campylobacter jejuni; Cell Survival; Chickens; Enzyme Inhibitors; Propidium; Real-Time Polymerase Chain Reaction; Staining and Labeling

The unsuitability of the "CFU" parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.

Topics: Animals; Azides; Campylobacter; Chickens; Colony Count, Microbial; DNA, Bacterial; Microbial Viability; Poultry; Propidium; Proton-Motive Force; Real-Time Polymerase Chain Reaction

Diagnostic methods of TB, nowadays, are prone to delay in diagnosis, increased false negative results and are not sensitive to many forms of paucibacillary disease. The aims of this study were to implement a quantitative nucleic acid-based diagnostic test for paucibacillary tuberculosis, enabling the identification and quantification of viable Mycobacterium tuberculosis bacilli by quantitative Real-Time PCR (qRT-PCR). The intergenic region of the single-copy inhA-mabA gene was chosen as the target region for design of primers and probes conjugated with fluorophores. The construction of synthetic DNA flanking the target region served as standards for absolute quantification of nucleic acids. Using the intercaling dye, propidium monoazide, we were able to discriminate between viable and dead cells of M. tuberculosis. The diagnosis method showed a broad sensitivity (96.1%) when only compared to samples of smear-positive sputum and ROC analyses shows that our approach performed well and yielded a specificity of 84.6% and a sensitivity of 84.6% when compared to M. tuberculosis colony-forming units counting.

Topics: Affinity Labels; Azides; Colony Count, Microbial; Coloring Agents; DNA, Bacterial; DNA, Intergenic; Dose-Response Relationship, Drug; Humans; Microbial Viability; Mycobacterium tuberculosis; Propidium; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Sputum; Tuberculosis, Pulmonary

This paper concerns the formation of biofilm in bacteria of the genus Arcobacter. A multiplex polymerase chain reaction (PCR) method was introduced and optimized for detecting biofilm while using the intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA), first for analysis of strains of the genus Arcobacter from a collection, and then applied to samples of prepared biofilms. The results of the study indicate considerable variability among species of bacteria within the genus Arcobacter. The EMA-PMA PCR method can distinguish viable cells from dead cells and is therefore suitable for determining the viability of cells.

Topics: Azides; Biofilms; Campylobacter; Intercalating Agents; Microbial Viability; Multiplex Polymerase Chain Reaction; Propidium

DNA-based methodology for the identification and detection of specific bacteria in dental plaque offers advantages over culturing techniques. One drawback of current molecular techniques like real-time quantitative polymerase chain reaction (RT-QPCR) is that they are not able to distinguish between live or dead bacteria. To overcome this problem an assay was assessed to discriminate between viable or dead bacteria using DNA intercalating substances, propidium monoazide (PMA) and ethidium monoazide (EMA) in combination with RT-QPCR. The assay was tested on oral pathogens: Streptococcus mutans, Prevotella intermedia and Aggregatibacter actinomycetemcomitans. To determine the effectiveness of EMA and PMA, different concentrations (from 5 to 100 μg ml(-1)) of the substances were added to viable or heat-killed suspensions of both organisms (ranging from 10(8) to 10(4) colony-forming units ml(-1)). Afterwards, PMA was tested on mixtures of varying ratios of viable and dead cells. After DNA extraction, RT-QPCR was performed using species-specific primers. Both compounds inhibited PCR amplification from dead cells. The EMA treatment resulted in the largest signal decrease but EMA also inhibited DNA amplification from viable cells. For this reason, PMA was selected for use in further experiments. It was shown to be efficient in allowing selective PCR detection of only viable cells in mixtures containing both viable and dead cells. The amount of amplified DNA corresponded to the percentage of viable cells in the sample. The developed assay will potentially be useful for assessing bacterial loads remaining after disinfection protocols without interference by non-viable bacteria.

Topics: Aggregatibacter actinomycetemcomitans; Azides; Bacteriological Techniques; Dental Plaque; DNA, Bacterial; Intercalating Agents; Microbial Viability; Polymerase Chain Reaction; Prevotella intermedia; Propidium; Streptococcus mutans

Because Helicobacter pylori has a role in the pathogenesis of gastric cancer, chronic gastritis and peptic ulcer disease, detection of its viable form is very important. The objective of this study was to optimize a PCR method using ethidium monoazide (EMA) or propidium monoazide (PMA) for selective detection of viable H. pylori cells in mixed samples of viable and dead bacteria. Before conducting the real-time PCR using SodB primers of H. pylori, EMA or PMA was added to suspensions of viable and/or dead H. pylori cells at concentrations between 1 and 100 μM. PMA at a concentration of 50 μM induced the highest DNA loss in dead cells with little loss of genomic DNA in viable cells. In addition, selective detection of viable cells in the mixtures of viable and dead cells at various ratios was possible with the combined use of PMA and real-time PCR. In contrast, EMA penetrated the membranes of both viable and dead cells and induced degradation of their genomic DNA. The findings of this study suggest that PMA, but not EMA, can be used effectively to differentiate viable H. pylori from its dead form.

Topics: Affinity Labels; Azides; Cell Membrane; Colony Count, Microbial; DNA, Bacterial; Helicobacter pylori; Microbial Viability; Permeability; Propidium; Real-Time Polymerase Chain Reaction

To optimize ethidium monoazide (EMA) coupled with real-time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems.. EMA (0.9-45.5 microg ml(-1)) and propidium monoazide (PMA, 0.9 and 2.3 microg ml(-1)) combined with qPCR (i.e. EMA-qPCR and PMA-qPCR, respectively) were applied to unheated and heated (70 degrees C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight-EM). The effects of nontarget microflora and sample matrix on the performance of EMA-qPCR were also evaluated. In comparison with BacLight-EM results, qPCR with EMA at 2.3 microg ml(-1) was determined as the optimal EMA-qPCR assay, which performed equally well as PMA-qPCR for unheated Leg. pneumophila but better than PMA-qPCR for heated Leg. pneumophila (P < 0.05). Moreover, qPCR with EMA at 2.3 microg ml(-1) accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella-like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0.05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR.. The qPCR with EMA at 2.3 microg ml(-1) may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems.. The EMA-qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.

Topics: Azides; Biofilms; Legionella; Legionella pneumophila; Microbial Viability; Microscopy, Fluorescence; Polymerase Chain Reaction; Propidium; Water Microbiology

Ethidium monoazide (EMA) and propidium monoazide (PMA) have been utilized for selective PCR amplification of DNA from viable bacterial cells. In this study, we compared the abilities of EMA and PMA, together with real-time PCR, to specifically distinguish dead Legionella cells from viable cells. Several experiments showed that PMA or EMA treatment could specifically prevent the PCR amplification of DNA from dead Legionella cells in water samples. However, a 4-fold higher concentration of PMA than EMA was required to achieve this effect. EMA may therefore be more useful for practical environmental investigations of Legionella.

Topics: Animals; Azides; Bacteriological Techniques; Enzyme Inhibitors; Humans; Legionella; Microbial Viability; Polymerase Chain Reaction; Propidium; Sensitivity and Specificity

The detection and quantification of wine yeast can be misleading due to under or overestimation of these microorganisms. Underestimation may be caused by variable growing rates of different microorganisms in culture media or the presence of viable but non-cultivable microorganisms. Overestimation may be caused by the lack of discrimination between live and dead microorganisms if quantitative PCR is used to quantify with DNA as the template. However, culture-independent methods that use dyes have been described to remove the DNA from dead cells and then quantify the live microorganisms. Two dyes have been studied in this paper: ethidium monoazide bromide (EMA) and propidium monoazide bromide (PMA). The technique was applied to grape must fermentation and ageing wines. Both dyes presented similar results on yeast monitoring. Membrane cell recovery was necessary when yeasts were originated from ethanol-containing media. When applied to grape must fermentation, differences of up to 1 log unit were seen between the QPCR estimation with or without the dye during the stationary phase. In ageing wines, good agreement was found between plating techniques and QPCR. Most of the viable cells were also culturable and no differences were observed with the methods, except for Zygosaccharomyces bailii and Dekkera bruxellensis where much higher counts were occasionally detected by QPCR. The presence of excess dead cells did not interfere with the quantification of live cells with either of the dyes.

Topics: Azides; Coloring Agents; DNA, Fungal; Ethanol; Fermentation; Microbial Viability; Polymerase Chain Reaction; Propidium; Saccharomyces cerevisiae; Wine; Yeasts; Zygosaccharomyces

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).. PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 microg ml(-1)) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 microg 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.. 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.. PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.

Topics: Affinity Labels; Azides; Bacteriological Techniques; Cronobacter sakazakii; DNA Primers; Food Microbiology; Humans; Infant; Infant Food; Infant, Newborn; Microbial Viability; Polymerase Chain Reaction; Propidium

The differentiation between live and dead bacterial cells presents an important challenge in many microbiological applications. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based detection methods cannot differentiate whether positive signals originate from live or dead bacterial targets. We present here a novel chemical, propidium monoazide (PMA), that (like propidium iodide) is highly selective in penetrating only into 'dead' bacterial cells with compromised membrane integrity but not into live cells with intact cell membranes/cell walls. Upon intercalation in the DNA of dead cells, the photo-inducible azide group allows PMA to be covalently cross-linked by exposure to bright light. This process renders the DNA insoluble and results in its loss during subsequent genomic DNA extraction. Subjecting a bacterial population comprised of both live and dead cells to PMA treatment thus results in selective removal of DNA from dead cells. We provide evidence that this chemical can be applied to a wide range of species across the bacterial kingdom presenting a major advantage over ethidium monoazide (EMA). The general application of EMA is hampered by the fact that the chemical can also penetrate live cells of some bacterial species. Transport pumps actively export EMA out of metabolically active cells, but the remaining EMA level can lead to substantial loss of DNA. The higher charge of PMA might be the reason for the higher impermeability through intact cell membranes, thus avoiding DNA loss.

Topics: Azides; Cell Survival; DNA, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Intercalating Agents; Light; Microscopy, Fluorescence; Polymerase Chain Reaction; Propidium