Propidium Monoazide Pretreatment Research Articles

Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. 57 stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) were tested using eqbE SEQ2190 triplex qPCR with (+) and without (-) PMA pre-treatment. Cycle Thresholds (CT) were higher when using PMA indicating a mixture of heat killed (HK) and viable cells. Number of S. equi positive specimens were as follows: 6/57 eqbE +PMA, 13/57 eqbE -PMA (Chi- squared 3.1, p = 0.079); 10/57 SEQ2190 +PMA, 53/57 SEQ2190 -PMA (Chi- squared 65.6, p<0.0001). The mean CT were as follows: 23.88 eqbE -PMA, 29.89 eqbE + PMA (p=0.04); 24.9 SEQ2190 -PMA, 31.9 SEQ2190 +PMA (p<0.0001). PMA qPCR can be used to determine S. equi viability, but testing should be performed on fresh specimens.

Identification of significant live bacterial community shifts in different reclaimed waters during ozone and chlorine disinfection

Ozone and chlorine are the most widely used disinfectants for water and wastewater disinfection. They play important role in microbial inactivation but could also pose a considerable selection effect on the microbial community of reclaimed water. Classical culture-based methods that rely on the assessment of conventional bacterial indicators (e.g., coliform bacteria) could hardly reflect the survival of disinfection residual bacteria (DRB) and hidden microbial risks in disinfected effluents. Hence, this study investigated the shifts of live bacterial community during ozone and chlorine disinfection in three reclaimed waters (i.e., two secondary effluents and one tertiary effluent), adopting Illumina Miseq sequencing technology in combination with a viability assay, propidium monoazide (PMA) pretreatment. Notably, statistical analyses of Wilcoxon rank-sum test confirmed the existance of distinct differences in bacterial community structure between samples with or without PMA pretreatment. On the phylum level, Proteobacteria commonly dominated in three undisinfected reclaimed waters, while ozone and chlorine disinfection posed varied effects on its relative abundance among different influents. On the genus level, ozone and chlorine disinfection significantly changed the bacterial composition and dominant species in reclaimed waters. Specifically, the typical DRB identified in ozone disinfected effluents were Pseudomonas, Nitrospira and Dechloromonas, while for chlorine disinfected effluents, Pseudomonas, Legionella, Clostridium, Mycobacterium and Romboutsia were recognized as typical DRB, which call for much attention. The Alpha and Beta diversity analysis results also suggested that different influent compositions greatly affected the bacterial community structure during disinfection processes. Since the experiments in present study were conducted in a short period and the dataset was relatively limited, prolonged experiment under different operational conditions are needed in future to illustrate the potential long-term effects of disinfection on the microbial community structure. The findings of this study could provide insights into microbial safety concern and control after disinfection for sustainable water reclamation and reuse.

Relic DNA effects on the estimates of bacterial community composition and taxa dynamics in soil

DNA-based analyses have become routine methods in soil microbial research, for their high throughput and resolution in characterizing microbial communities. Yet, concerns arise regarding the interference of relic DNA in estimates of viable bacterial community composition and individual taxa dynamics in soils that recovered from post-gamma irradiation. In this study, different soil samples with varying bacterial diversity but similar soil properties were randomly selected. We split each sample into two parts: one part was treated with propidium monoazide (PMA) before DNA extraction, PMA can bind to relic DNA and inhibit PCR amplification by chemical modification; DNA of the other part was extracted following the same process but without PMA pretreatment. Then, soil bacterial abundance was quantified by quantitative polymerase chain reaction, and bacterial community structure was examined by Illumina metabarcoding sequencing of 16S rRNA gene. The results showed that the higher bacterial richness and evenness were estimated when relic DNA was present. The variation trends of bacterial abundance, alpha diversity, and beta diversity remained the same, as reflected by the significant correlations between PMA-treated and -untreated samples (P < 0.05). Moreover, as the mean abundance increased, the reproducibility of detecting individual taxa dynamics between relic DNA present and absent treatments increased. These findings provide empirical evidence that a more even distribution of species abundance derived from relic DNA would result in the overestimation of richness in the total DNA pools and also have crucial implications for guiding proper application of high-throughput sequencing to estimate bacterial community diversity and taxonomic population dynamic. KEY POINTS: • Relic DNA effects on the bacterial community in sterilized soils were assessed. • More even species abundance distribution in relic DNA overestimates true richness. • The reproducibility of individual taxa dynamics increased with their abundance.

Prospects for the application of infectious virus detection technology based on propidium monoazide in African swine fever management.

African swine fever (ASF) is a hemorrhagic and often fatal disease occurring in domestic pigs and wild boars. ASF can potentially greatly impact the global trade of pigs and pork products and threaten global food security. Outbreaks of ASF must be notified to the World Organization for Animal Health. In this study, we analyzed the feasibility of applying propidium monoazide (PMA) pretreatment-based infectious virus detection technology to ASF prevention and control and investigated the prospects of applying this technology for epidemic monitoring, disinfection effect evaluation, and drug development. PMA as a nucleic acid dye can enter damaged cells and undergo irreversible covalent crosslinking with nucleic acid under halogen light to prevent its amplification. Although this technology has been widely used for the rapid detection of viable bacteria, its application in viruses is rare. Therefore, we analyzed the theoretical feasibility of applying this technology to the African swine fever virus (ASFV) in terms of gene and cell composition. Rapid infectious ASFV detection technology based on PMA pretreatment would greatly enhance all aspects of ASF prevention and control, such as epidemic monitoring, disinfection treatment, and drug development. The introduction of this technology will also greatly improve the ability to prevent and control ASF.

An Improved Method for Quantification of Viable Fusarium Cells in Infected Soil Products by Propidium Monoazide Coupled with Real-Time PCR.

Fusarium is a soil-borne pathogen that causes root rot disease in cucumber. To date, quantitative real-time PCR (qPCR) is a common tool to detect the content of Fusarium in soil. However, qPCR cannot distinguish between viable and nonviable cells. The aim of this study was to develop a detection technique to pretreat tissue fluid with propidium monoazide (PMA) followed by extract DNA, and then to quantify viable Fusarium cells in contaminated soil. In this work, the specific primer pair F8-1/F8-2 was designed based on the translation elongation factor (EF) gene and a PMA-qPCR assay was established to amplify and quantify soils of viable Fusarium cells. The PMA pretreatment test was optimized, which indicated that the optimal PMA concentration and light exposure time were 50 mmol L−1 and 15 min, respectively. The lowest limit of viable cells in suspension detected and soil by PMA-qPCR were 82 spore mL−1 and 91.24 spore g−1, respectively. For naturally contaminated soil, viable Fusarium cells were detected in eight of the 18 samples, and the Fusarium amount ranged from 104 to 106 spore g−1. In conclusion, the PMA-qPCR method has the characteristics of high sensitivity, efficiency, and time saving, which could support nursery plants to avoid Fusarium infection and agro-industry losses.

Virucidal Effects of Dielectric Barrier Discharge Plasma on Human Norovirus Infectivity in Fresh Oysters (Crassostrea gigas).

This study investigates the effects of dielectric barrier discharge (DBD) plasma treatment (1.1 kV, 43 kHz, N2 1.5 L/min, 10~60 min) on human norovirus (HuNoV) GII.4 infectivity in fresh oysters. HuNoV viability in oysters was assessed by using propidium monoazide (PMA) as a nucleic acid intercalating dye before performing a real-time reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Additionally, the impact of the DBD plasma treatment on pH and Hunter colors was assessed. When DBD plasma was treated for 60 min, the HuNoV genomic titer reduction without PMA pretreatment was negligible (<1 log copy number/µL), whereas when PMA treatment was used, HuNoV titer was reduced to >1 log copy number/µL in just 30 min. D1 and D2-value of HuNoV infectivity were calculated as 36.5 and 73.0 min of the DBD plasma treatment, respectively, using the first-order kinetics model (R2 = 0.98). The pH and Hunter colors were not significantly different (p > 0.05) between the untreated and DBD-plasma-treated oysters. The results suggest that PMA/RT-qPCR could help distinguish HuNoV infectivity without negatively affecting oyster quality following >30 min treatment with DBD plasma. Moreover, the inactivation kinetics of nonthermal DBD plasma against HuNoV in fresh oysters might provide basic information for oyster processing and distribution.

Reduction, Prevention, and Control of Salmonella enterica Viable but Non-culturable Cells in Flour Food.

The processing and storage conditions of flour food inevitably pose environmental stress, which promote bacteria to enter a viable but non-culturable (VBNC) state. The existence of VBNC cells causes false-negative detection in traditional culture-based detection methods, resulting in food quality and safety issues. This study aimed at investigating the influence factors including nutrition, acid, salt, and temperature for the entry into a VBNC state of Salmonella enterica and an efficient detection method. During induction with multi-stress conditions, nutrition starvation antagonizes with low-level acidity. Besides, high-level acidity was considered as an inhibitor for VBNC induction. Four inducers including nutrition starvation, salt stress, low-level acidity, and low temperature were concluded for a VBNC state. In addition, the keynote conditions for S. enterica entering a VBNC state included (i) nutrient-rich acidic environment, (ii) oligotrophic low-acidity environment, and (iii) oligotrophic refrigerated environment. Based on the keynote conditions, the environmental conditions of high acidity (1.0% v/v acetate) with low temperature (−20°C) could successfully eliminate the formation of S. enterica VBNC cells in flour food. In addition, combining with propidium monoazide pretreatment, PCR technology was applied to detect S. enterica VBNC cells. The sensitivity of the PMA–PCR technology was 105 CFU/ml in an artificially simulated food system. The results derived from this study might aid in the detection and control of VBNC state S. enterica in flour food products.

How to Count Our Microbes? The Effect of Different Quantitative Microbiome Profiling Approaches.

Next-generation sequencing (NGS) has instigated the research on the role of the microbiome in health and disease. The compositional nature of such microbiome datasets makes it however challenging to identify those microbial taxa that are truly associated with an intervention or health outcome. Quantitative microbiome profiling overcomes the compositional structure of microbiome sequencing data by integrating absolute quantification of microbial abundances into the NGS data. Both cell-based methods (e.g., flow cytometry) and molecular methods (qPCR) have been used to determine the absolute microbial abundances, but to what extent different quantification methods generate similar quantitative microbiome profiles has so far not been explored. Here we compared relative microbiome profiling (without incorporation of microbial quantification) to three variations of quantitative microbiome profiling: (1) microbial cell counting using flow cytometry (QMP), (2) counting of microbial cells using flow cytometry combined with Propidium Monoazide pre-treatment of fecal samples before metagenomics DNA isolation in order to only profile the microbial composition of intact cells (QMP-PMA), and (3) molecular based quantification of the microbial load using qPCR targeting the 16S rRNA gene. Although qPCR and flow cytometry both resulted in accurate and strongly correlated results when quantifying the bacterial abundance of a mock community of bacterial cells, the two methods resulted in highly divergent quantitative microbial profiles when analyzing the microbial composition of fecal samples from 16 healthy volunteers. These differences could not be attributed to the presence of free extracellular prokaryotic DNA in the fecal samples as sample pre-treatment with Propidium Monoazide did not improve the concordance between qPCR-based and flow cytometry-based QMP. Also lack of precision of qPCR was ruled out as a major cause of the disconcordant findings, since quantification of the fecal microbial load by the highly sensitive digital droplet PCR correlated strongly with qPCR. In conclusion, quantitative microbiome profiling is an elegant approach to bypass the compositional nature of microbiome NGS data, however it is important to realize that technical sources of variability may introduce substantial additional bias depending on the quantification method being used.

Formation of viable, but putatively non-culturable (VPNC) cells of beer-spoilage lactobacilli growing in biofilms

Bacteria belonging to the genus Lactobacillus are the most commonly-found beer-spoilage microorganisms in breweries. This work compares the ability of biofilm and planktonic cells of beer-spoilage Lactobacillus sp. to enter a viable, but putatively non-culturable (VPNC) state. Planktonic and biofilm cells of five beer-spoilage species of Lactobacillus were incubated at 26 °C in the lager beer with sub-culturing. Both biofilm and planktonic cells could be induced to enter the VPNC state, however, the state-transition time is variable: it took a minimum of 63 days for the biofilm cells to enter the VPNC state, while the time for planktonic cells was 119–217 days. The presence of VPNC cells in non-culturable biofilms was further confirmed by PCR coupled with propidium monoazide pretreatment that amplified genes 16S rDNA and horA. Taken together, this study provides evidence that Lactobacillus biofilm cells can enter the VPNC state at a rate faster than the planktonic cells under stressful conditions of the beer (low pH, high concentrations of hop bitter compounds and ethanol, and limited carbon/nitrogen sources) in the brewing process.

Interference of Dead Cell DNA on the Analysis of Antibiotic Resistance Genes and Microbial Community in Anaerobic Digestion Sludge

To investigate the potential interference of dead bacteria DNA on the analysis of antibiotic resistance genes (ARGs) and microbial communities in anaerobic digestion sludge, three different types of anaerobic digestion sludge were treated with propidium monoazide (PMA) in this study, and the results of subsequent ARGs and microbial community analysis with the interference of dead cell DNA blocked by PMA was compared to that without PMA treatment. It was found that after PMA treatment, the absolute abundance of the ARGs in the anaerobic digestion sludge from self-anaerobic digestion system of excess sludge and from high solid sludge anaerobic digestion system decreased by 41%-86%, and 74%-98%, respectively. ARGs abundance in the sludge from the anaerobic digestion system with sludge hydrolyzate as a substrate and anaerobic granular sludge as inoculum after 15 days of digestion considerably decreased with PMA treatment. However, its ARGs abundance still declined by up to 34%. PMA treatment influenced the analysis of microbial community of the three types of anaerobic digestion sludge to different degrees, in which the influence was the highest on the community structure analysis of the sludge from high solid anaerobic digestion system. The results of the correlation analysis between the ARGs abundance and the microbial community composition were completely different in the case with PMA treatment and without PMA treatment. This study proved the potential interference of dead cell DNA on the analysis of ARGs and microbial community in anaerobic digestion sludge. PMA pretreatment could achieve a more accurate analysis of the microbial community and ARGs characteristics in anaerobic digestion sludge.

Quantification of Viable Cells of Pseudomonas syringae pv. tomato in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay

Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 μmol liter-1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 102 CFU ml-1 and 11.86 CFU g-1, respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 102 to 104 CFU g-1. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.

Combined propidium monoazide pretreatment with high‐throughput sequencing evaluated the bacterial diversity in chicken skin after thermal treatment

The purpose of this experiment was to study the bacterial diversity and predominance of spoilage bacteria in chicken skin at different thermal treatment temperatures (60, 70, 80, 90, 100, 110, 120°C). Bacteria in chicken skin was collected, then propidium monoazide treatment to remove the DNA of dead cell, total DNA was extracted by Tiandz Bacterial DNA Kit, and investigated by high-throughput sequencing of the v3/v4 regions of the 16S rDNA gene. A total of 796008 high-quality bacterial sequences were obtained for assessing the microbial diversity of chicken skin from seven thermal treatment group and control group. The results showed that the bacterial diversity in chicken skin at 90°C was lowest. And Acinetobacter (25·88%), Clostridium (20·70%), Bacteroides (13·93%) and Myroides (13·13%) were the main flora at 25°C; The Clostridium was dominant genus of the samples heat-treated by 60, 70, 80 and 90°C, the proportion of this genus were up to 64·86, 77·42, 52·22 and 87·30% respectively. The Bacillus was the main flora of the samples heat-treated by 100, 110 and 120°C, and the relative percentages were 39·44, 79·61 and 45·96% respectively. In addition, high-temperature-resistant Serratia was found in chicken skin. The study revealed that the relationship between thermal treatment temperature and bacterial diversity and dominant spoilage bacteria in chicken skin, which had a strong guiding significance for the control and prediction of micro-organisms in foods. The results of this paper could provide a theoretical basis for meat products containing chicken skin, including the safe use of chicken skin, determination of sterilization process parameters and selection of preservatives for compounding, which has strong practicality in China.

Development and characterization of an oral multispecies biofilm implant flow chamber model.

Peri-implant infections are the most common cause of implant failure in modern dental implantology. These are caused by the formation of biofilms on the implant surface and consist of oral commensal and pathogenic bacteria, which harm adjacent soft and hard tissues and may ultimately lead to implant loss. In order to improve the clinical situation, there has to be a better understanding of biofilm formation on abiotic surfaces. Therefore, we successfully developed a system to cultivate an oral multispecies biofilm model in a flow chamber system, optimized for the evaluation of biofilm formation on solid materials by direct microscopic investigation. The model contains four relevant oral bacterial species: Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis in ratios similar to the native situation. The reliability of the developed “Hanoverian Oral Multispecies Biofilm Implant Flow Chamber” (HOBIC) model was verified. Biofilm volume and live/dead distribution within biofilms were determined by fluorescence staining and confocal laser scanning microcopy (CLSM). The individual species distribution was analyzed using quantitative real time PCR with propidium monoazide pretreatment (PMA-qRT-PCR) and by urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH). This in vitro model may be used to analyze biofilm formation on dental implants in more detail and to develop future implant systems with improved material properties.

Viability of Campylobacter spp. in frozen and chilled broiler carcasses according to real-time PCR with propidium monoazide pretreatment

The aim of this study was to evaluate the viability of Campylobacter spp. in frozen and chilled broiler carcasses using real-time PCR with propidium monoazide (PMA) pretreatment. Sixty broiler carcasses were collected: 30 frozen and 30 chilled. Each carcass was submitted to 2 real-time PCR protocols to detect and quantify Campylobacter spp.: one using pretreatment with PMA, which blocks the amplification of DNA from dead bacteria, and the other without PMA. The results showed that PMA-pretreated carcasses, either frozen or chilled, had a lower positivity rate compared to untreated samples (P < 0.001). Regarding storage temperatures, PMA-pretreated frozen carcasses that tested positive were in a lesser number than chilled carcasses (P < 0.05). However, the quantification of total and live bacteria in PMA-pretreated frozen carcasses that tested positive showed no significant difference compared to chilled carcasses. It was concluded that the real-time PCR with PMA pretreatment was a sensitive method for evaluating the viability of Campylobacter spp. in broiler carcasses. Chilled broiler carcasses would represent greater hazard to public health concerning Campylobacter transmission.

Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04 ® via Chip-Based Digital PCR.

The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD) for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours.

Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine

The main disadvantage of DNA-based quantification methods is their inability to distinguish between viable and non-viable cells, as dead cells may also retain a significant amount of DNA. To overcome this limitation, propidium monoazide (PMA) treatment was applied to effectively prevent amplification of DNA from dead cells. The objectives of this study were to develop and evaluate an accurate detection method that combines PMA staining with real-time qPCR to quantify viable dominant microorganisms during the traditional brewing of Hong Qu glutinous rice wine. Firstly, three methodological factors (concentration of PMA, incubation time and photoactivation time) were optimized for representative strains of different microbial species. For the tested microbial strains (Monascus purpureus, Saccharomyces cerevisiae and Lactobacillus plantarum), a treatment of 25 μM PMA for 20 min incubation followed by 10 min photoactivation was considered optimal to achieve a compromise between minimal impact on intact cells and maximal signal reduction for the compromised cells. Compared with qPCR without PMA treatment, results of PMA-qPCR showed that the optimized PMA pretreatment can effectively exclude the dead microbial cells without any negative effects on the intact cells. The concentrations of viable cells calculated by PMA-qPCR were comparable (the same order of magnitude was obtained) with those obtained by culture-based method. Finally, the developed PMA-qPCR methods were successfully applied to monitor the total bacteria, LAB, fungi, yeasts, M. purpureus, S. cerevisiae and L. plantarum during the traditional brewing of Hong Qu glutinous rice wine. Both of the total bacterial and fungal populations increased significantly at the early brewing stage, but they decreased at the later brewing stage. PMA-qPCR results revealed that the predominant microorganisms varied in different brewing phases. L. plantarum and S. cerevisiae grew significantly and became the predominant bacterial and fungal species at the final stage of traditional brewing process, respectively. This study standardized the PMA-qPCR parameters for dominant microorganisms associated with the traditional brewing of Hong Qu glutinous rice wine, which would show great value in scientifically understanding of the brewing mechanism and provide useful information for the selection of beneficial microbial strains to improve wine quality.

Sunlight-Activated Propidium Monoazide Pretreatment for Differentiation of Viable and Dead Bacteria by Quantitative Real-Time Polymerase Chain Reaction

Polymerase chain reaction (PCR)-based methods have been developed and increasingly used for rapid and sensitive detection of pathogens in water samples to better protect public health. A propidium monoazide (PMA) pretreatment can help to differentiate between viable and dead cells, but the photoactivation of PMA normally requires the use of an energy-consuming halogen light, which is not suitable for off-the-grid applications. Herein, we investigate sunlight as an alternative light source. Our results suggest that sunlight can successfully activate PMA, and the sunlight-activated PMA pretreatment can effectively reduce the amplification of DNA derived from dead cells in PCR assays. Potentially, a sunlight-activated PMA pretreatment unit can be integrated into a lab-on-a-chip PCR device for off-the-grid microbial detection and quantification.

Detection of viable enterotoxin-producing Bacillus cereus and analysis of toxigenicity from ready-to-eat foods and infant formula milk powder by multiplex PCR

Bacillus cereus is responsible for several outbreaks of foodborne diseases due to its emetic toxin and enterotoxin. Enterotoxins, cytotoxin K (CytK), nonhemolytic enterotoxin (Nhe), and hemolysin BL (Hbl), have been recorded in several diarrheal cases due to food poisoning from B. cereus. The objective of this study was to develop a rapid and accurate method that combines multiplex PCR with propidium monoazide to selectively detect viable cells of enterotoxin-producing B. cereus in milk powder, noodles, and rice, and investigate the distribution of enterotoxins in 62 strains of B. cereus in Jiangxi province, China. The specificity of primers of 3 enterotoxins (i.e., cytK, nheA, and hblD) of B. cereus was verified by inclusivity and exclusivity tests using single PCR. Upon optimization of multiplex PCR conditions, it was found that the detection limit of viable cells was 102 cfu/mL of B. cereus in pure culture. By enrichment for 3 or 4 h and propidium monoazide pretreatment, a protocol for detection of viable cells as low as 2.2×101 cfu/g in spiked food (e.g., milk powder, noodles, and rice) was established and proved valid even under the interference of non-Bacillus cereus at as high as 105 cfu/g. Moreover, the protocol based on multiplex PCR for detection was applied for the analysis of distribution of toxin gene of B. cereus, and the results showed a regional feature for toxin gene distribution, indicating that potential toxigenicity of B. cereus should be evaluated further.

49 Propidium monoazide (PMA) sample pretreatment impacts the abundance of rare populations in high-throughput sequencing analysis of CF lung mycobiome and bacteriome

Objective Propidium monoazide (PMA) pretreatment has increasingly been used in molecular biology for detecting viable pathogens. Our study aims to determine whether a PMA pretreatment in high-throughput sequencing (HTS) could provide a more realistic picture of lung mycobiome and bacteriome from cystic fibrosis (CF) patients, and consequently reflect more closely the clinical status of patients. Methods We compared HTS data of bacteriome and mycobiome of 15 sputum samples from 5 CF patients that were characterized using the Ion Torrent technique with and without prior PMA treatment of the DNA-extracts. Results PMA pretreatment had no significant effect on the entire and abundant bacterial community (genera expressed as operational taxonomic unit with a relative abundance of ≥1%) but caused a significant difference in the rare biosphere community ( Conclusion To conclude, PMA pretreatment seems to change the relative abundance of bacteria, especially in the rare populations, but not fungi. Given such a cumbersome protocol (PMA pretreatment coupled with HTS), we discuss its potential interest within the follow-up of CF patients. As only few studies suggested the use of this protocol in the characterization of the bacteriome may be clinically relevant, further studies using PMA pretreatment are warranted to improve our omic knowledge.

Detection of viable bacterial pathogens in a drinking water source using propidium monoazide-quantitative PCR

Research Article| September 10 2014 Detection of viable bacterial pathogens in a drinking water source using propidium monoazide-quantitative PCR Avid Banihashemi; Avid Banihashemi 1NSERC Chair in Water Treatment, Department of Civil and Environmental Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1 E-mail: a2baniha@uwaterloo.ca Search for other works by this author on: This Site PubMed Google Scholar Michele I. Van Dyke; Michele I. Van Dyke 1NSERC Chair in Water Treatment, Department of Civil and Environmental Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1 Search for other works by this author on: This Site PubMed Google Scholar Peter M. Huck Peter M. Huck 1NSERC Chair in Water Treatment, Department of Civil and Environmental Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1 Search for other works by this author on: This Site PubMed Google Scholar Journal of Water Supply: Research and Technology-Aqua (2015) 64 (2): 139–148. https://doi.org/10.2166/aqua.2014.063 Article history Received: May 16 2014 Accepted: August 09 2014 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Cite Icon Cite Permissions Search Site Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsThis Journal Search Advanced Search Citation Avid Banihashemi, Michele I. Van Dyke, Peter M. Huck; Detection of viable bacterial pathogens in a drinking water source using propidium monoazide-quantitative PCR. Journal of Water Supply: Research and Technology-Aqua 1 March 2015; 64 (2): 139–148. doi: https://doi.org/10.2166/aqua.2014.063 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex A cell viability assay was applied to measure bacterial enteric pathogens in a river in southern Ontario, Canada that is used as a source of drinking water. Pathogen concentrations were measured using both propidium monoazide (PMA)-quantitative polymerase chain reaction (PCR) and quantitative PCR (qPCR) without PMA pretreatment to compare viable and total (live and dead) cells. The pathogens evaluated were Salmonella enterica, thermophilic Campylobacter, and Escherichia coli O157:H7, and the suspected enteric pathogen Arcobacter butzleri was also investigated. Results showed that for all strains dead cells were detected in few river water samples, and the difference between total and viable cell concentrations for each pathogen group was always less than 0.5 log. A. butzleri was detected at concentrations 2–3 log higher than the other pathogens. S. enterica, Campylobacter, and E. coli O157:H7 were detected at low concentrations at one sample location and at higher concentrations at a second sampling location. Results from this study show qPCR with PMA pretreatment can provide reliable enumeration of viable pathogens in surface water, and that dead cells were rarely present in water used as a source for drinking water treatment. Arcobacter butzleri, PCR, propidium monoazide, river water, viability, waterborne pathogen © IWA Publishing 2015 You do not currently have access to this content.