Propidium Monoazide Treatment Research Articles

Improved Canker Processing and Viability Droplet Digital PCR Allow Detection of Erwinia amylovora Viable Nonculturable Cells in Apple Bark.

The bacterium Erwinia amylovora causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen cells in fire blight cankers. Several factors may prevent E. amylovora from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 102 to 7.6 × 107 cells mL-1, as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of E. amylovora viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the E. amylovora life cycle, and elucidate E. amylovora physiology, epidemiology, and new options for canker management.

Real-time and visual detection of viable Salmonella in milk from remote pasture via IMS-LAMP-NALFS

Salmonella is a prevalent foodborne pathogen commonly found in dairy products. Controlling the presence of Salmonella during dairy product production is of utmost concern due to the potential for contamination at any stage of the production chain, particularly at the source. Moreover, the unique location of pastures and limited testing resources further highlight the necessity of establishing a rapid and precise detection method to ensure milk quality control and enhance overall farm safety. To address these issues, in this study, we constructed a method as well as a portable toolbox for rapid on-site detection of Salmonella. The method involves immunomagnetic separation (IMS) using streptavidin-coupled magnetic nanoparticles and biotinylated antibodies. The reaction conditions for magnetic capture and enrichment of Salmonella were optimized to efficiently remove matrix interference from milk and enhance detection sensitivity. Additionally, an improved propidium monoazide (PMAxx) treatment was employed to eliminate false positive results caused by dead bacteria. This treatment was combined with loop-mediated isothermal amplification (LAMP) and nucleic acid lateral flow strip (NALFS) (LAMP-NALFS) for on-site detection of viable bacteria in milk. The resulting IMS-LAMP-NALFA assay achieved rapid and sensitive detection of viable Salmonella, with a detection sensitivity of 1 × 102 CFU/mL in milk samples, completed within 90 min under optimal conditions. Based on this achievement, a portable toolbox was designed for easy transportation and on-site testing in pastures. It is equally applicable for rapid detection of viable Salmonella contamination in food products in resource-limited areas such as pastures, where the observed detection rate of Salmonella in unpasteurized raw milk was 7.93 % (28 out of 353 samples tested). Consequently, the IMS-LAMP-NALFA method and the associated portable toolbox represent highly effective means of enhancing the detection of viable Salmonella in dairy products.

Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method.

The identification and quantification of viable bacteria at the species/strain level in compound probiotic products is challenging now. Molecular biology methods, e.g., propidium monoazide (PMA) combination with qPCR, have gained prominence for targeted viable cell counts. This study endeavors to establish a robust PMA-qPCR method for viable Lacticaseibacillus rhamnosus detection and systematically validated key metrics encompassing relative trueness, accuracy, limit of quantification, linear, and range. The inclusivity and exclusivity notably underscored high specificity of the primers for L. rhamnosus, which allowed accurate identification of the target bacteria. Furthermore, the conditions employed for PMA treatment were fully verified by 24 different L. rhamnosus including type strain, commercial strains, etc., confirming its effective discrimination between live and dead bacteria. A standard curve constructed by type strain could apply to commercial strains to convert qPCR Cq values to viable cell numbers. The established PMA-qPCR method was applied to 46 samples including pure cultures, probiotics as food ingredients, and compound probiotic products. Noteworthy is the congruity observed between measured and theoretical values within a 95% confidence interval of the upper and lower limits of agreement, demonstrating the relative trueness of this method. Moreover, accurate results were obtained when viable L. rhamnosus ranging from 103 to 108 CFU/mL. The comprehensive appraisal of PMA-qPCR performances provides potential industrial applications of this new technology in quality control and supervision of probiotic products.

Establishment and application of a multiplex real-time PCR assay coupled with propidium monoazide for the simultaneous detection of viable Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus

A propidium monoazide (PMA) - multiplex real-time PCR (mRT-PCR) assay was established for the simultaneous detection of viable Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus. According to the unique sequences of the target genes identified by our laboratory for the above pathogenic bacteria, an efficient and specific mRT-PCR method based on TaqMan probes was developed, and then PMA was introduced into the mRT-PCR system. The optimization of the PMA treatment conditions successfully achieves the purpose of distinguishing viable and dead bacteria in the cell suspension, and effectively eliminates false positive results caused by dead cells. The method's specificity was verified by 54 strains, exhibiting 100% specificity without cross-reactivity with other pathogens. Furthermore, the developed method could accurately detect the target bacteria in the presence of non-target bacteria at a concentration range of 100-107 CFU/mL, demonstrating excellent anti-interference ability. The detection limits of V. parahaemolyticus, V. vulnificus, and V. alginolyticus were 2.0 × 101 CFU/mL, 3.2 × 101 CFU/mL, and 3.75 × 101 CFU/mL, respectively. To reflect the practicability of the method, the established method was developed as a detection kit, and the evaluation results showed that the kit has good stability and good reproducibility. The applicability of this kit was satisfying after evaluating in artificially spiked aquatic products (shrimp, yellow croakers and oysters) with recoveries ranging from 90.22% to 109.35%. The PMA-mRT-PCR detection assay is a feasible method for the rapid, sensitive, and quantitative detection of bacterial pathogens. And the novel kit developed based on this method has significant application value for the large-scale screening and identification of harmful Vibrios in food matrices.

DNA from non-viable bacteria biases diversity estimates in the corals Acropora loripes and Pocillopora acuta

BackgroundNucleic acid-based analytical methods have greatly expanded our understanding of global prokaryotic diversity, yet standard metabarcoding methods provide no information on the most fundamental physiological state of bacteria, viability. Scleractinian corals harbour a complex microbiome in which bacterial symbionts play critical roles in maintaining health and functioning of the holobiont. However, the coral holobiont contains both dead and living bacteria. The former can be the result of corals feeding on bacteria, rapid swings from hyper- to hypoxic conditions in the coral tissue, the presence of antimicrobial compounds in coral mucus, and an abundance of lytic bacteriophages.ResultsBy combining propidium monoazide (PMA) treatment with high-throughput sequencing on six coral species (Acropora loripes, A. millepora, A. kenti, Platygyra daedalea, Pocillopora acuta, and Porites lutea) we were able to obtain information on bacterial communities with little noise from non-viable microbial DNA. Metabarcoding of the 16S rRNA gene showed significantly higher community evenness (85%) and species diversity (31%) in untreated compared with PMA-treated tissue for A. loripes only. While PMA-treated coral did not differ significantly from untreated samples in terms of observed number of ASVs, > 30% of ASVs were identified in untreated samples only, suggesting that they originated from cell-free/non-viable DNA. Further, the bacterial community structure was significantly different between PMA-treated and untreated samples for A. loripes and P. acuta indicating that DNA from non-viable microbes can bias community composition data in coral species with low bacterial diversity.ConclusionsOur study is highly relevant to microbiome studies on coral and other host organisms as it delivers a solution to excluding non-viable DNA in a complex community. These results provide novel insights into the dynamic nature of host-associated microbiomes and underline the importance of applying versatile tools in the analysis of metabarcoding or next-generation sequencing data sets.

Response of viable bacteria to antibiotics in aerobic granular sludge: Resistance mechanisms and behaviors, bacterial communities, and driving factors

The assessment of antimicrobial resistance (AMR) risk by DNA-based techniques mainly relies on total bacterial DNA. In this case, AMR risk recognition is restricted to the genotype level, lacking crucial phenotypic information, such as the distribution of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in dead and viable bacteria. This limitation hinders the recognition of AMR behavior. Herein, based on propidium monoazide (PMA) shielding method, this work firstly quantified the intracellular ARGs/MGEs in viable and dead bacteria, and the impact of viable bacteria composition on the formation of intracellular/extracellular polymeric substance-related /cell-free ARGs (i/e/cARGs) and MGEs (i/e/cMGEs) in aerobic granular sludge (AGS). The shielding efficiency of PMA against dead bacteria was optimized to be as high as 97.5% when the MLSS of AGS was 2.0 g/L. Under antibiotic stimulation, 29.0% ∼ 49.0% of iARGs/iMGEs were carried by viable bacteria, and the remaining proportion were carried by dead bacteria. 18 out of the top 20 dominant genera showed a change in abundance by more than 1% after PMA treatment. 29 viable hosts were identified to associate with 52 iARGs, of which 28 and 15 hosts were also linked to 40 eARGs and 26 cARGs. Also, partial least-squares path model and variance partitioning analysis disclosed that viable bacteria and i/e/cMGEs had a positive effect on i/e/cARGs, with both contributing as much as 64.5% to the total ARGs enrichment. These results better visualized the AMR risk carried by viable bacteria and the categories of viable hosts. This work provides a novel insight into analyzing the actual AMR risk and viable hosts, helping to the reduction and control of AMR in wastewater treatment plants.

A rapid and reliable method for the determination of Lactiplantibacillus plantarum during wine fermentation based on PMA-CELL-qPCR.

Real-time monitoring of microbial dynamics during fermentation is essential for wine quality control. This study developed a method that combines the fluorescent dye propidium monoazide (PMA) with CELL-qPCR, which can distinguish between dead and live microbes for Lactiplantibacillus plantarum. This method could detect the quantity of microbes efficiently and rapidly without DNA extraction during wine fermentation. The results showed that (1) the PMA-CELL-qPCR enumeration method developed for L. plantarum was optimized for PMA treatment concentration, PMA detection sensitivity and multiple conditions of sample pretreatment in wine environment, and the optimized method can accurately quantify 104-108 CFU/mL of the target strain (L. plantarum) in multiple matrices; (2) when the concentration of dead bacteria in the system is 104 times higher than the concentration of live bacteria, there is an error of 0.5-1 lg CFU/mL in the detection results. The optimized sample pretreatment method in wine can effectively reduce the inhibitory components in the qPCR reaction system; (3) the optimized PMA-CELL-qPCR method was used to monitor the dynamic changes of L. plantarum during the fermentation of Cabernet Sauvignon wine, and the results were consistent with the plate counting method. In conclusion, the live bacteria quantification method developed in this study for PMA-CELL-qPCR in L. plantarum wines is accurate in quantification and simple in operation, and can be used as a means to accurately monitor microbial dynamics in wine and other fruit wines.

Relic DNA obscures DNA-based profiling of multiple microbial taxonomic groups in a river-reservoir ecosystem.

Numerous studies have investigated the spatiotemporal variability in water microbial communities, yet the effects of relic DNA on microbial community profiles, especially microeukaryotes, remain far from fully understood. Here, total and active bacterial and microeukaryotic community compositions were characterized using propidium monoazide (PMA) treatment coupled with high-throughput sequencing in a river-reservoir ecosystem. Beta diversity analysis showed a significant difference in community composition between both the PMA untreated and treated bacteria and microeukaryotes; however, the differentiating effect was much stronger for microeukaryotes. Relic DNA only resulted in underestimation of the relative abundances of Bacteroidota and Nitrospirota, while other bacterial taxa exhibited no significant changes. As for microeukaryotes, the relative abundances of some phytoplankton (e.g. Chlorophyta, Dinoflagellata and Ochrophyta) and fungi were greater after relic DNA removal, whereas Cercozoa and Ciliophora showed the opposite trend. Moreover, relic DNA removal weakened the size and complexity of cross-trophic microbial networks and significantly changed the relationships between environmental factors and microeukaryotic community composition. However, there was no significant difference in the rates of temporal community turnover between the PMA untreated and treated samples for either bacteria or microeukaryotes. Overall, our results imply that the presence of relic DNA in waters can give misleading information of the active microbial community composition, co-occurrence networks and their relationships with environmental conditions. More studies of the abundance, decay rate and functioning of nonviable DNA in freshwater ecosystems are highly recommended in the future.

Absolute quantification of viable Vibrio cholerae in seawater samples using multiplex droplet digital PCR combined with propidium monoazide.

Toxigenic Vibrio cholerae serogroup O1 and O139 are the pathogens responsible for the global cholera epidemic. V. cholerae can settle in the water and spread via the fecal-oral route. Rapid and accurate monitoring of live V. cholerae in environmental water has become an important strategy to prevent and control cholera transmission. Conventional plate counting is widely used to detect viable bacteria but requires time and effort. This study aims to develop a new assay that combines triplex droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment for quantitatively detecting live V. cholerae O1/O139 and cholera enterotoxin. Specific primers and probes were designed according to the conserved regions of gene rfb O1, rfb O139, and ctxA. The amplification procedures and PMA treatment conditions were optimized. The specificity, sensitivity, and ability of PMA-ddPCR to detect viable bacteria-derived DNA were evaluated in simulated seawater samples. The results revealed that the optimal primer concentrations of rfb O1, rfb O139, and ctxA were 1 μM, while the concentrations of the three probes were 0.25, 0.25, and 0.4 μM, respectively. The best annealing temperature was 58°C to obtain the most accurate results. The optimal strategy for distinguishing dead and live bacteria from PMA treatment was incubation at the concentration of 20 μM for 15 min, followed by exposure to a 650-W halogen lamp for 20 min. In pure culture solutions, the limit of detection (LODs) of V. cholerae O1 and O139, and ctxA were 127.91, 120.23 CFU/mL, and 1.5 copies/reaction in PMA-triplex ddPCR, respectively, while the LODs of the three targets were 150.66, 147.57 CFU/mL, and 2 copies/reaction in seawater samples. The PMA-ddPCR sensitivity was about 10 times higher than that of PMA-qPCR. When detecting spiked seawater samples with live bacterial concentrations of 1.53 × 102 and 1.53 × 105 CFU/mL, the assay presented a higher sensitivity (100%, 16/16) than qPCR (50.00%, 8/16) and a perfect specificity (100%, 9/9). These results indicate that the developed PMA-triplex ddPCR is superior to the qPCR regarding sensitivity and specificity and can be used to rapidly detect viable toxigenic V. cholerae O1 and O139 in suspicious seawater samples.

Correlation between Bacterial Cell Density and Abundance of Antibiotic Resistance on Milking Machine Surfaces Assessed by Cultivation and Direct qPCR Methods

The relative abundance of antibiotic-resistant bacteria and antibiotic-resistance genes was surveyed for different parts of a milking machine. A cultivation approach based on swab samples showed a highly diverse microbiota, harboring resistances against cloxacillin, ampicillin, penicillin, and tetracycline. This approach demonstrated a substantial cloxacillin resistance of numerous taxa within milking machine microbiota coming along with regular use of cloxacillin for dry-off therapy of dairy cows. For the less abundant tetracycline-resistant bacteria we found a positive correlation between microbial cell density and relative abundance of tetracycline-resistant microorganisms (R2 = 0.73). This indicated an accelerated dispersion of resistant cells for sampling locations with high cell density. However, the direct quantification of the tetM gene from the swap samples by qPCR showed the reverse relation to bacterial density if normalized against the abundance of 16S rRNA genes (R2 = 0.88). The abundance of 16S rRNA genes was analyzed by qPCR combined with a propidium monoazide treatment, which eliminates 16S rRNA gene signals in negative controls.

Rapid Monitoring of Viable Genetically Modified Escherichia coli Using a Cell-Direct Quantitative PCR Method Combined with Propidium Monoazide Treatment.

The commercialization of industrial genetically modified microorganisms (GMMs) has highlighted their impact on public health and the environment. Rapid and effective monitoring methods detecting live GMMs are essential to enhance current safety management protocols. This study aims to develop a novel cell-direct quantitative polymerase chain reaction (qPCR) method targeting two antibiotic-resistant genes, KmR and nptII, conferring resistance against kanamycin and neomycin, along with propidium monoazide, to precisely detect viable Escherichia coli. The E. coli single-copy taxon-specific gene of D-1-deoxyxylulose 5-phosphate synthase (dxs) was used as the internal control. The qPCR assays demonstrated good performance, with dual-plex primer/probe combinations exhibiting specificity, absence of matrix effects, linear dynamic ranges with acceptable amplification efficiencies, and repeatability for DNA, cells, and PMA-treated cells targeting KmR/dxs and nptII/dxs. Following the PMA-qPCR assays, the viable cell counts for KmR-resistant and nptII-resistant E. coli strains exhibited a bias% of 24.09% and 0.49%, respectively, which were within the acceptable limit of ±25%, as specified by the European Network of GMO Laboratories. This method successfully established detection limits of 69 and 67 viable genetically modified E. coli cells targeting KmR and nptII, respectively. This provides a feasible monitoring approach as an alternative to DNA processing techniques to detect viable GMMs.

Effect of Propidium Monoazide in the Detection of Enterotoxigenic Escherichia coli in the Pediatric

Objectives: The purpose of this study is to determine the viability of enterotoxigenic Escherichia coli (ETEC) in a sample of diarrhea. The investigation focuses specifically on the lt gene and utilizes propidium monoazide (PMA) and quantitative real-time polymerase chain reaction (qPCR) to differentiate between live and dead bacteria. Methods: Propidium monoazide is a chemical that can bind to and inhibit the amplification of free DNA during qPCR analysis. In this study, in addition to analyzing diarrhea samples, artificially spiked samples were used to assess the sensitivity and accuracy of the PMA treatment. The qPCR results were compared to the gold standard of culture-based methods both with and without PMA treatment. Results: The method’s limit of detection was 8 CFU/mL, and it exhibited linearity from a 10-1 to a 10-9 dilution. The qPCR approach revealed a higher bacterial count than the culture method due to the detection of DNA released from dead bacteria. However, when PMA was employed, the bacterial count was similar to that obtained using colony count agar, which is attributed to the elimination of free DNA during investigation. Conclusions: The present study developed a PMA-based qPCR approach that enables the detection of live bacterial DNA. This method involves PMA and real-time PCR and offers several advantages, including faster detection times (a few hours vs. several days with the traditional culture method) and the ability to exclusively detect live bacteria without interference from free DNA released by dead bacteria. Additionally, the use of real-time PCR enables precise quantification of the live bacterial load. Overall, this approach is cost-effective, rapid, highly sensitive, and specific, making it a valuable tool for various applications.

QMRA of beach water by Nanopore sequencing-based viability-metagenomics absolute quantification

The majority of the current regulatory practices for routine monitoring of beach water quality rely on the culture-based enumeration of faecal indicator bacteria (FIB) to develop criteria for promoting the general public's health. To address the limitations of culture methods and the arguable reliability of FIB in indicating health risks, we developed a Nanopore metagenomic sequencing-based viable cell absolute quantification workflow to rapidly and accurately estimate a broad range of microbes in beach waters by a combination of propidium monoazide (PMA) and cellular spike-ins. Using the simple synthetic bacterial communities mixed with viable and heat-killed cells, we observed near-complete relic DNA removal by PMA with minimal disturbance to the composition of viable cells, demonstrating the feasibility of PMA treatment in profiling viable cells by Nanopore sequencing. On a simple mock community comprised of 15 prokaryotic species, our results showed high accordance between the expected and estimated concentrations, suggesting the accuracy of our method in absolute quantification. We then further assessed the accuracy of our method for counting viable Escherichia coli and Vibrio spp. in beach waters by comparing to culture-based method, which were also in high agreement. Furthermore, we demonstrated that 1 Gb sequences obtained within 2 h would be sufficient to quantify a species having a concentration of ≥ 10 cells/mL in beach waters. Using our viability-resolved quantification workflow to assess the microbial risk of the beach water, we conducted (1) screening-level quantitative microbial risk assessment (QMRA) to investigate human illness risk and site-specific risk patterns that might guide risk management efforts and (2) metagenomics-based resistome risk assessment to evaluate another layer of risk caused by difficult illness treatment due to antimicrobial resistance (AMR). In summary, our metagenomic workflow for the rapid absolute quantification of viable bacteria demonstrated its great potential in paving new avenues toward holistic microbial risk assessment.

Inactivation of Human Norovirus GII.4 and Vibrio parahaemolyticus in the Sea Squirt (Halocynthia roretzi) by Floating Electrode-Dielectric Barrier Discharge Plasma.

Human norovirus (HNoV) GII.4 and Vibrio parahaemolyticus may be found in sea squirts. Antimicrobial effects of floating electrode-dielectric barrier discharge (FE-DBD) plasma (5-75 min, N2 1.5 m/s, 1.1 kV, 43 kHz) treatment were examined. HNoV GII.4 decreased by 0.11-1.29 log copy/μL with increasing duration of treatment time, and further by 0.34 log copy/μL when propidium monoazide (PMA) treatment was added to distinguish infectious viruses. The decimal reduction time (D1) of non-PMA and PMA-treated HNoV GII.4 by first-order kinetics were 61.7 (R2 = 0.97) and 58.8 (R2 = 0.92) min, respectively. V. parahaemolyticus decreased by 0.16-1.5 log CFU/g as treatment duration increased. The D1 for V. parahaemolyticus by first-order kinetics was 65.36 (R2 = 0.90) min. Volatile basic nitrogen showed no significant difference from the control until 15 min of FE-DBD plasma treatment, increasing after 30 min. The pH did not differ significantly from the control by 45-60 min, and Hunter color in "L" (lightness), "a" (redness), and "b" (yellowness) values reduced significantly as treatment duration increased. Textures appeared to be individual differences but were not changed by treatment. Therefore, this study suggests that FE-DBD plasma has the potential to serve as a new antimicrobial to foster safer consumption of raw sea squirts.

Shifts and importance of viable bacteria in treatment of DSS-induced ulcerative colitis mice with FMT.

Ulcerative colitis (UC) has become a global public health concern, and is in urgent need of novel therapies. Fecal microbiota transplantation (FMT) targeting gut microbiota has recently been applied to the treatment of UC. Despite its recent successes, it is still largely unknown how FMT functionally modulates the gut microbiota and improves the disease. We prospectively collected fecal samples from the 40 mice (30 mice for dextran sulfate sodium (DSS)-induced, 10 for controls), followed by Propidium monoazide treatment for 16S rRNA gene sequencing. These 30 mice were divided equally into 3 groups, which were transplanted with original donor microbiota (DO), inactivated donor microbiota (DI) and saline, respectively. Subsequently, we used 16S rRNA gene sequencing to analyze the viable gut bacteria of ulcerative colitis (UC) mice and histological analysis to evaluate the effects of fecal microbiota transplantation (FMT) with viable microbiota. We demonstrated that the community structure of viable bacteria was significantly different from fecal bacteria based on total DNA. Furthermore, the intestinal viable microbiota and colonic mucosal structure of mice were significantly changed by DSS induction. The histological analysis showed that only the mice treated with original donor microbiota group (HF) achieved a significant improvement. Compared with inactivated donor microbiota group (IF) and saline (NF), Lactobacillus and Halomonas were significantly enriched in the HF group. We inferred that only live bacteria from human donor reversed the histopathology and symptoms of UC in mice and altered the gut microbiota. The activity of gut microbiota in donor samples should be considered in FMT and that detailed analysis of viable microbiota is essential to understand the mechanisms by which FMT produces therapeutic effects in the future.

Evaluation of Bacterial Composition and Viability of Equine Feces after Processing for Transplantation

Fecal microbiota transplantation (FMT) has been used empirically for decades in equine medicine to treat intestinal dysbiosis but evidence-based information is scarce. This in vitro study aimed at assessing the effect of a commonly used pre-FMT processing method on the bacterial composition and viability of the fecal filtrate. Three samples of fresh equine manure (T0) were processed identically: the initial manure was mixed with 1 L of lukewarm water and chopped using an immersion blender to obtain a mixture (T1), which was left uncovered during 30 min (T2) and percolated through a sieve to obtain a fecal filtrate (T3). Samples were taken throughout the procedure (Tn) and immediately stored at 4 °C until processing. The 16S rDNA amplicon profiling associated with propidium monoazide treatment was performed on each sample to select live bacteria. Analyses of α and β diversity and main bacterial populations and quantitative (qPCR) analysis were performed and statistically compared (significance p < 0.05) between time points (T0-T3). No significant differences in ecological indices or mean estimated total living bacteria were found in the final fecal filtrate (T3) in regard to the original manure (T0); however, relative abundances of some minor genera (Fibrobacter, WCHB1-41_ge and Akkermansia) were significantly different in the final filtrate. In conclusion, the results support the viability of the major bacterial populations in equine feces when using the described pre-FMT protocol.

An improved workflow for accurate and robust healthcare environmental surveillance using metagenomics

BackgroundEffective surveillance of microbial communities in the healthcare environment is increasingly important in infection prevention. Metagenomics-based techniques are promising due to their untargeted nature but are currently challenged by several limitations: (1) they are not powerful enough to extract valid signals out of the background noise for low-biomass samples, (2) they do not distinguish between viable and nonviable organisms, and (3) they do not reveal the microbial load quantitatively. An additional practical challenge towards a robust pipeline is the inability to efficiently allocate sequencing resources a priori. Assessment of sequencing depth is generally practiced post hoc, if at all, for most microbiome studies, regardless of the sample type. This practice is inefficient at best, and at worst, poor sequencing depth jeopardizes the interpretation of study results. To address these challenges, we present a workflow for metagenomics-based environmental surveillance that is appropriate for low-biomass samples, distinguishes viability, is quantitative, and estimates sequencing resources.ResultsThe workflow was developed using a representative microbiome sample, which was created by aggregating 120 surface swabs collected from a medical intensive care unit. Upon evaluating and optimizing techniques as well as developing new modules, we recommend best practices and introduce a well-structured workflow. We recommend adopting liquid-liquid extraction to improve DNA yield and only incorporating whole-cell filtration when the nonbacterial proportion is large. We suggest including propidium monoazide treatment coupled with internal standards and absolute abundance profiling for viability assessment and involving cultivation when demanding comprehensive profiling. We further recommend integrating internal standards for quantification and additionally qPCR when we expect poor taxonomic classification. We also introduce a machine learning-based model to predict required sequencing effort from accessible sample features. The model helps make full use of sequencing resources and achieve desired outcomes.DVg4DtPBM9AbUXUoj_2n2mVideo ConclusionsThis workflow will contribute to more accurate and robust environmental surveillance and infection prevention. Lessons gained from this study will also benefit the continuing development of methods in relevant fields.

Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study.

Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared different sequencing-based methods to assess their relative accuracy with respect to distinguishing between viable and non-viable cells, using a live and heat-inactivated model community spiked into bovine milk. The methods used were shotgun metagenomics with and without propidium monoazide (PMA) treatment, RNA-based 16S rRNA sequencing and metatranscriptomics. The results showed that methods were generally accurate, though significant differences were found depending on the library types and sequencing technologies. Different molecular targets were the basis for variations in the results generated using different library types, while differences in the derived composition data from Oxford Nanopore Technologies-and Illumina-based sequencing likely reflect a combination of different sequencing depths, error rates and bioinformatics pipelines. Although PMA was successfully applied in this study, further optimisation is required before it can be applied in a more universal context for complex microbiomes. Overall, these methods show promise and represent another important step towards the ultimate establishment of approaches that can be applied to accurately identify live microorganisms in milk and other food niches.

Quantitative detection of Vibrio parahaemolyticus in aquatic products by duplex droplet digital PCR combined with propidium monoazide

Based on the combination of droplet digital PCR (ddPCR) and propidium monoazide (PMA), a new method called PMA-duplex ddPCR was developed for the quantitative determination of viable Vibrio parahaemolyticus in aquatic products. Specific primers and probes targeting a new gene, vp0488, coding for putative DNA polymerase III beta chain, which was self-screened by this laboratory, and a traditional target gene, thermolabile hemolysin (tlh), were designed and verified. The duplex ddPCR system and conditions and PMA treatment conditions were optimized. The limit of detection (LOD) for PMA-duplex ddPCR was evaluated and applied to detect V. parahaemolyticus in artificially contaminated samples and actual samples. The results indicate that the optimal duplex ddPCR annealing temperature was 60 °C, the optimal number of amplification cycles was 40, and the optimal ratio of primer to probe was 1:0.5. The optimal PMA treatment conditions were as follows: incubation time 5 min, concentration 30 μmol/L, and exposure time 15 min. The LOD of PMA-duplex ddPCR (7.32 × 101 CFU/mL) was lower than that of duplex quantitative PCR (qPCR) (7.32 × 102 CFU/mL). The lowest detectable V. parahaemolyticus in oysters was 8.15 × 101 CFU/g without any pre-enrichment. The method established in this study has the advantages of good specificity, high sensitivity and accuracy, which provides an effective method for the detection of viable Vibrio parahaemolyticus in aquatic foods.

Dynamics of Starter and Non-Starter Lactic Acid Bacteria Populations in Long-Ripened Cheddar Cheese Using Propidium Monoazide (PMA) Treatment.

The microbial community of industrially produced Canadian Cheddar cheese was examined from curd to ripened cheese at 30–32 months using a combination of viable plate counts of SLAB (GM17) and NSLAB (MRSv), qPCR and 16S rRNA gene amplicon sequencing. Cell treatment with propidium monoazide excluded DNA of permeable cells from amplification. The proportion of permeable cells of both Lactococcus spp. and Lacticaseibacillus spp. was highest at 3–6 months. While most remaining Lacticaseibacillus spp. cells were intact during later ripening stages, a consistent population of permeable Lactococcus spp. cells was maintained over the 32-month period. While Lactococcus sequence variants were significant biomarkers for viable cheese curd communities at 0–1 m, Lacticaseibacillus was identified as a distinctive biomarker for cheeses from 7 to 20 months. From 24 to 32 months, Lacticaseibacillus was replaced in significance by four genera (Pediococcus and Latilactobacillus at 24 m and at 30–32 m, Secundilactobacillus and Paucilactobacillus). These results underscore the importance of monitoring potential defects in cheeses aged over 24 months, which could be diagnosed early through microbial DNA profiling to minimize potential waste of product. Future perspectives include correlating volatile flavor compounds with microbial community composition as well as the investigation of intra-species diversity.