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Combining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids (eNA). We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ , respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at eNA being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.

Background Shotgun sequencing of microbial communities provides in-depth knowledge of the microbiome by cataloging bacterial, fungal, and viral gene content within a sample, providing an advantage over amplicon sequencing approaches that assess taxonomy but not function and are taxonomically limited. However, mammalian DNA can dominate host-derived samples, obscuring changes in microbial populations because few DNA sequence reads are from the microbial component. We developed and optimized a novel method for enriching microbial DNA from human oral samples and compared its efficiency and potential taxonomic bias with commercially available kits. Results Three commercially available host depletion kits were directly compared with size filtration and a novel method involving osmotic lysis and treatment with propidium monoazide (lyPMA) in human saliva samples. We evaluated the percentage of shotgun metagenomic sequencing reads aligning to the human genome, and taxonomic biases of those not aligning, compared to untreated samples. lyPMA was the most efficient method of removing host-derived sequencing reads compared to untreated sample (8.53 ± 0.10% versus 89.29 ± 0.03%). Furthermore, lyPMA-treated samples exhibit the lowest taxonomic bias compared to untreated samples. Conclusion Osmotic lysis followed by PMA treatment is a cost-effective, rapid, and robust method for enriching microbial sequence data in shotgun metagenomics from fresh and frozen saliva samples and may be extensible to other host-derived sample types.

Background Viability staining with SYTO9 and propidium iodide (PI) is a frequently used tool in microbiological studies. However, data generated by such routinely used method are often not critically evaluated for their accuracy. In this study we aim to investigate the critical aspects of this staining method using Staphylococcus aureus and Pseudomonas aeruginosa as the model microorganisms for high throughput studies in microtiter plates. SYTO9 or PI was added alone or consecutively together to cells and the fluorescence intensities were measured using microplate reader and confocal laser scanning microscope. Results We found that staining of S. aureus cells with SYTO9 alone resulted in equal signal intensity for both live and dead cells, whereas staining of P. aeruginosa cells led to 18-fold stronger signal strength for dead cells than for live ones. After counterstaining with PI, the dead P. aeruginosa cells still exhibited stronger SYTO9 signal than the live cells. We also observed that SYTO9 signal showed strong bleaching effect and decreased dramatically over time. PI intensity of the culture increased linearly with the increase of dead cell numbers, however, the maximum intensities were rather weak compared to SYTO9 and background values. Thus, slight inaccuracy in measurement of PI signal could have significant effect on the outcome. Conclusions When viability staining with SYTO9 and PI is performed, several factors need to be considered such as the bleaching effect of SYTO9, different binding affinity of SYTO9 to live and dead cells and background fluorescence.

A requirement of any foodborne pathogen testing method is that it only detects live bacteria. Ethidium monoazide (EMA) and propidium monoazide (PMA) are dyes that penetrate the membranes of dead cells and form cross-linkages in the DNA, which prevents its amplification in PCR. This study investigated whether treatment with EMA or PMA would inhibit the sequencing of DNA from dead Escherichia coli. Range finding experiments with qPCR were conducted to determine the optimal concentrations of EMA and PMA needed to inhibit the amplification of DNA from dead cells while not influencing live cells. An EMA concentration that differentiated between live and dead cells could not be established. However, a PMA concentration of 25 µM effectively prevented qPCR amplification of DNA from dead E. coli while not impacting the amplification of live E. coli DNA. Sequencing experiments were conducted with PMA-treated live, untreated live, PMA-treated dead, and untreated dead E. coli. There were no significant differences in the detection of virulence genes of interest between the PMA-treated live, untreated live, and untreated dead E. coli. However, no DNA sequencing data were obtained from the PMA-treated dead E. coli. These results suggest that PMA could be incorporated into sample preparation methods prior to sequencing to selectively detect live cells of foodborne pathogens.

Traditional culture-based quantification of Bradyrhizobium diazoefficiens in inoculants presents significant limitations due to its labor-intensive and time-consuming nature. To address this limitation, we aimed to validate a propidium monoazide quantitative PCR (PMA-qPCR) assay as a rapid and reliable alternative for estimating Bradyrhizobium diazoefficiens counts in commercial inoculants. Key experiments optimized PMA concentration (50, 75 and 100 µM) to selectively inhibit DNA amplification from non-viable cells without interfering with viable cell signal. Assay´s efficiency, limit of detection and quantification, intra-assay repeatability and inter-assay reproducibility were determined. The assay demonstrated high efficiency (90–105%), a limit of detection (LOD) of 3.14 log CFU/mL, and a dynamic range from 8.74 to 3.14 log CFU/mL. Robust intra-assay repeatability (SD < 0.3) and inter-assay reproducibility (CV < 10%) were confirmed. The method successfully distinguished quarter-strength and 10-fold serial dilutions of viable bacteria, even in the presence of non-viable cells. Final validation against standard plate counting showed a strong linear correlation with an R² of 0.82. Crucially, this PMA-qPCR assay reduced processing time from 120 hours to just 5 hours, offering a significant improvement in turnaround time while maintaining strong agreement with the reference method. This study marks the first application of PMA-qPCR for Bradyrhizobium diazoefficiens quantification in inoculants, highlighting its potential as a high-throughput tool to enhance efficiency and precision for industrial batch-to-batch quality control.

Mpox, caused by the Monkeypox virus (MPV), is a global public health threat. Virus isolation is the gold standard to confirm MPV infection, but this process can face many challenges. As an alternative, a new method was developed in in vitro settings using 50 µM of propidium monoazide (PMAxx, a DNA-binding agent) coupled with digital droplet PCR (ddPCR). Frozen clinical samples analyzed by PMAxx-ddPCR had a median of 0.8 copies/µL, while untreated samples had a median of 29.8 copies/µL. Since a substantial percentage of reduction was observed in these samples (>80%), it was verified whether this reduction could be due to the freezing process. This hypothesis was confirmed both in vitro and using clinical samples. A gradual increase in the mean percentage of reduction was observed after freezing–thawing cycles of MPV-isolate (59.5−81.4%). Moreover, a different percentage of reduction was observed before (68.2%) and after freezing (97.4%) the specimens, suggesting that the freezing process could reduce the number of complete viral particles. Our study shows strong evidence of the usefulness of PMAxx in clinical settings. PMAxx ensures the detection of intact MPV particles, which improves the accuracy of MPV load measurements. This method not only increases the reliability of MPV diagnosis but also overcomes virus isolation limitations.

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.

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'.

SARS-CoV-2 is a highly infectious virus responsible for the COVID-19 pandemic. Therefore, it is important to assess the risk of SARS-CoV-2 infection, especially in persistently positive patients. Rapid discrimination between infectious and non-infectious viruses aids in determining whether prevention, control, and treatment measures are necessary. For this purpose, a method was developed and utilized involving a pre-treatment with 50 µM of propidium monoazide (PMAxx, a DNA intercalant) combined with a digital droplet PCR (ddPCR). The ddPCR method was performed on 40 nasopharyngeal swabs (NPSs) both before and after treatment with PMAxx, revealing a reduction in the viral load at a mean of 0.9 Log copies/mL (SD ± 0.6 Log copies/mL). Furthermore, six samples were stratified based on the Ct values of SARS-CoV-2 RNA (Ct < 20, 20 < Ct < 30, Ct > 30) and analyzed to compare the results obtained via a ddPCR with viral isolation and a negative-chain PCR. Of the five samples found positive via a ddPCR after the PMAxx treatment, two of the samples showed the highest post-treatment SARS-CoV-2 loads. The virus was isolated in vitro from both samples and the negative strand chains were detected. In three NPS samples, SARS CoV-2 was present post-treatment at a low level; it was not isolated in vitro, and, when detected, the strand was negative. Our results indicate that the established method is useful for determining whether the SARS-CoV-2 within positive NPS samples is intact and capable of causing infection.

Xanthomonas arboricola pv. pruni (Xap) is the causal agent of bacterial spot of stone fruits and almond ( Prunus spp). Detection of Xap is typically carried out using quantitative real-time PCR (qPCR) combined with culture-based isolation. However, qPCR does not differentiate between viable and dead cells, potentially leading to an overestimation of the infective population in a sample. Such overestimation could result in unnecessary phytosanitary measures. The present study aims to develop a specific protocol ideally targeting to detection of only live Xap bacterial cells. To address this challenge, the viable quantitative PCR (v-qPCR) method was evaluated using three nucleic acid-binding dyes: propidium monoazide (PMA), a combination of PMA and ethidium monoazide (EMA), and PMAxx™, an improved version of PMA. PMAxx™ proved to be the most suitable dye for the detection and quantification of living bacterial cells. This methodology was also evaluated in infected plant material over time and can be considered a rapid and reliable alternative to PCR methods for detecting only those putative infective Xap that may pose a risk for Prunus crops. Key points • Protocol to detect biofilm and planktonic viable X. arboricola pv. pruni cells. • Host validated protocol. • Benefits, reduction of chemicals in disease control.