Propidium Monoazide-quantitative Polymerase Chain Reaction Research Articles

Propidium monoazide (PMA) qPCR assay compared to the plate count method for quantifying the growth of Salmonella enterica serotypes in vacuum-packaged turkey breast combined with a mathematical modeling approach

This study compares the plate count (PC) and the Propidium Monoazide-quantitative Polymerase Chain Reaction (PMA-qPCR) methods to assess the growth of a cocktail of three serotypes of Salmonella enterica (Heidelberg, Typhimurium, and Enteritidis) in cooked, sliced, and vacuum-packaged turkey breast (STB) under isothermal storage temperatures (8 °C–20 °C), using predictive models. Standard curves were developed for PMA-qPCR, demonstrating high efficiency (101%) and sensitivity, with quantification limits ranging from 1 to 2 log10 CFU/g for all temperatures studied. Comparative analysis revealed a significant correlation (R2 = 0.99; 95% CI) between the PC and PMA-qPCR methods; however, the agreement analysis indicated a mean difference (Bias) of −0.11 log10 CFU/g (p < 0.05), suggesting underestimation by the PC method. This indicates the presence of stressed or viable but nonculturable (VBNC) cells, detectable by PMA-qPCR but not by PC. The Baranyi and Roberts model showed a good ability to describe the behavior of S. enterica cocktail in STB for PC and PMA-qPCR data under all isothermal conditions. The exponential secondary model more accurately represented the temperature dependence of the maximum specific growth rate compared to the Ratkowsky square root model, with R2 values ≥ 0.984 and RMSE values ≤ 0.011 for both methods. These results suggest that combining PMA-qPCR with predictive modeling allows for a more accurate prediction of S. enterica growth, compared to PC method. In the event of cold chain disruptions of meat products, the use of PMA-qPCR method allow the quantification of VBNC cells, that can still pose a health risk to consumers, especially in ready-to-eat products.

Preventing proximal enamel caries in neighboring tooth with glass ionomer cement restoration and silver diamine fluoride pretreatment

ObjectiveTo investigate caries preventive effects of 38 % silver diamine fluoride (SDF) pretreatment on neighboring tooth proximal to glass ionomer cement (GIC), including conventional GIC (CGIC) and resin-modified GIC (RMGIC) restorations in an in vitro model. Methods: Human tooth blocks were restored withSDF+CGIC (Group 1), CGIC (Group 2), SDF+RMGIC (Group 3) or RMGIC (Group 4). Enamel specimen simulating proximal surface of neighboring tooth was placed in proximity to the restorations. The specimen underwent cariogenic challenge with cross-kingdom biofilm of Streptococcus mutans, Lacticaseibacillus casei and Candida albicans. After cariogenic challenge, the biofilm's growth kinetics, viability, and morphology were evaluated by propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. The enamel lesion depth, surface morphology and crystal characteristics were determined by micro-computed tomography (micro-CT), SEM and X-ray diffraction (XRD), respectively. ResultsPMA-qPCR demonstrated lower microbial growth in Group 1 and 3 compared with Group 2 and 4 (p < 0.05). CLSM showed the dead−to-live ratio in Groups 1–4 were 1.15±0.12, 0.53±0.13, 1.10±0.24 and 0.63±0.10, respectively (Group 1,3 > 2,4, p < 0.05). SEM revealed Groups 1 and 3 had scattered biofilm whereas Group 2 and 4 had confluent biofilm. Micro-CT showed the enamel lesion depths (µm) were 98±9, 126±7, 103±6 and 128±7 for Group 1 to 4, respectively (Group 1,3 < 2,4, p < 0.05). SEM revealed oriented and ordered enamel prismatic patterns in Group 1 and 3, not in Group 2 and 4. XRD showed the reflections of hydroxyapatite in Groups 1 and 3 were sharper than Groups 2 and 4. ConclusionSDF pretreatment enhances the preventive effect of GIC on proximal enamel surface on neighboring tooth through inhibiting cariogenic biofilm, reducing enamel demineralization and promoting enamel remineralization. Clinical significanceSDF pretreatment of GIC restorations can help prevent caries on neighboring teeth, particular for patients with high caries risk.

Resistance to preservatives and the viable but non-culturable state formation of Asaia lannensis in flavored syrups.

Food security is a crucial issue that has caused extensive concern, and the use of food flavors has become prevalent over time. we used the molecular biological techniques, preservative susceptibility testing, viable but non-culturable (VBNC) state induction testing, and a transcriptome analysis to examine the bacterial contamination of favored syrup and identify the causes and develop effective control measures. The results showed that Asaia lannensis WLS1-1 is a microorganism that can spoil food and is a member of the acetic acid bacteria families. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests showed that WLS1-1 was susceptible to potassium sorbate (PS), sodium benzoate (SB), and sodium sulffte (SS) at pH 4.0. It revealed a progressive increase in resistance to these preservatives at increasing pH values. WLS1-1 was resistant to PS, SB and SS with an MIC of 4.0, 2.0 and 0.5 g/L at pH 5.0, respectively. The MIC values exceed the maximum permissible concentrations that can be added. The induction test of the VBNC state demonstrated that WLS1-1 lost its ability to grow after 321 days of PS induction, 229 days of SB induction and 52 days of SS induction combined with low temperature at 4°C. Additionally, laser confocal microscopy and a propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay showed that WLS1-1 was still alive after VBNC formation. There were 7.192 ± 0.081 (PS), 5.416 ± 0.149 (SB) and 2.837 ± 0.134 (SS) log10(CFU/mL) of viable bacteria. An analysis of the transcriptome data suggests that Asaia lannensis can enter the VBNC state by regulating oxidative stress and decreasing protein synthesis and metabolic activity in response to low temperature and preservatives. The relative resistance of Asaia lannensis to preservatives and the induction of the VBNC state by preservatives are the primary factors that contribute to the contamination of favored syrup by this bacterium. To our knowledge, this study represents the first evidence of the ability of Asaia lannensis to enter the VBNC state and provides a theoretical foundation for the control of organisms with similar types of activity.

Bifidobacterium species viability in dairy-based probiotic foods: challenges and innovative approaches for accurate viability determination and monitoring of probiotic functionality.

Bifidobacterium species are essential members of a healthy human gut microbiota. Their presence in the gut is associated with numerous health outcomes such as protection against gastrointestinal tract infections, inflammation, and metabolic diseases. Regular intake of Bifidobacterium in foods is a sustainable way of maintaining the health benefits associated with its use as a probiotic. Owing to their global acceptance, fermented dairy products (particularly yogurt) are considered the ideal probiotic carrier foods. As envisioned in the definition of probiotics as "live organisms," the therapeutic functionalities of Bifidobacterium spp. depend on maintaining their viability in the foods up to the point of consumption. However, sustaining Bifidobacterium spp. viability during the manufacture and shelf-life of fermented dairy products remains challenging. Hence, this paper discusses the significance of viability as a prerequisite for Bifidobacterium spp. probiotic functionality. The paper focuses on the stress factors that influence Bifidobacterium spp. viability during the manufacture and shelf life of yogurt as an archetypical fermented dairy product that is widely accepted as a delivery vehicle for probiotics. It further expounds the Bifidobacterium spp. physiological and genetic stress response mechanisms as well as the methods for viability retention in yogurt, such as microencapsulation, use of oxygen scavenging lactic acid bacterial strains, and stress-protective agents. The report also explores the topic of viability determination as a critical factor in probiotic quality assurance, wherein, the limitations of culture-based enumeration methods, the challenges of species and strain resolution in the presence of lactic acid bacterial starter and probiotic species are discussed. Finally, new developments and potential applications of next-generation viability determination methods such as flow cytometry, propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), next-generation sequencing, and single-cell Raman spectroscopy (SCRS) methods are examined.

Development of a Novel Peptide with Antimicrobial and Mineralising Properties for Caries Management.

The purpose of the study is to develop a novel peptide for caries management. Gallic-Acid-Polyphemusin-I (GAPI) was synthesised by grafting Polyphemusin I (PI) and gallic acid (GA). Biocompatibility was evaluated using a Cell Counting Kit-8 Assay. Antimicrobial properties were assessed using minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). The bacterial and fungal morphology after GAPI treatment was investigated using transmission electron microscopy (TEM). The architecture of a consortium biofilm consisting of Streptococcus mutans, Lacticaseibacillus casei and Candida albicans was evaluated using scanning electron microscopy (SEM) and confocal laser scanning microscopy. The growth kinetics of the biofilm was examined using a propidium monoazide-quantitative polymerase chain reaction. The surface and calcium-to-phosphorus molar ratio of GAPI-treated enamel after pH cycling were examined with SEM and energy-dispersive X-ray spectroscopy. Enamel crystal characteristics were analysed using X-ray diffraction. Lesion depths representing the enamel's mineral loss were assessed using micro-computed tomography. The MIC of GAPI against S. mutans, L. casei and C. albicans were 40 μM, 40 μM and 20 μM, respectively. GAPI destroyed the biofilm's three-dimensional structure and inhibited the growth of the biofilm. SEM showed that enamel treated with GAPI had a relatively smooth surface compared to that treated with water. The calcium-to-phosphorus molar ratio of enamel treated with GAPI was higher than that of the control. The lesion depths and mineral loss of the GAPI-treated enamel were less than the control. The crystallinity of the GAPI-treated enamel was higher than the control. This study developed a biocompatible, mineralising and antimicrobial peptide GAPI, which may have potential as an anti-caries agent.

The effect of synbiotic-fluoride therapy on multi-species biofilm

ObjectivesThe study objective was to examine the effect of synbiotic-fluoride (SF) therapy within a multi-species cariogenic biofilm model system comprising of S. mutans, S. sanguinis, and S. gordonii. MethodsThe SF therapy was prepared using 2% L-arginine (Arg), 0.2% NaF and probiotic L. rhamnosus GG (LRG). The 8 treatment groups were: Group 1: No treatment, Group 2: 2% Arg, Group 3: 0.2% NaF, Group 4: LRG, Group 5: 2% Arg+0.2% NaF, Group 6: 2% Arg+LRG, Group 7: 0.2% NaF+LRG, and Group 8: SF therapy (2% Arg+0.2% NaF +LRG). Multi-species biofilm model over 96 h comprising Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii was utilized. The biofilms received cariogenic challenge and SF therapy 2 × /day. The extracellular matrix components were analyzed for carbohydrates, proteins, and extra-cellular DNA (eDNA). The live/dead cells were imaged and quantified using confocal microscopy. The viable/dead bacterial concentrations were estimated using propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). The gene expressions for gtfB, sagP, arcA, argG, and argH were measured using real-time reverse transcriptase qPCR. ResultsCarbohydrates and protein content with SF therapy were higher than non-LRG containing groups, while eDNA content was lower than other groups (p<0.05). Live bacterial proportions determined using confocal imaging with SF therapy were the lowest (p<0.05). The 2% Arg+LRG and SF therapy showed higher viable L. rhamnosus GG than 0.2% NaF+LRG (p<0.05). The dead S. mutans with SF therapy were higher than the other groups (p<0.05) with no difference from 2% Arg+0.2% NaF and 2% Arg+LRG (p>0.05). The SF therapy significantly downregulates gtfB and upregulates sagP, arcA, argG, argH gene expression (p<0.05). ConclusionSynbiotic-fluoride therapy effectuates multi-fold changes in the multi-species biofilm matrix and cellular components leading to superior ecological homeostasis than its individual contents, prebiotics (arginine), probiotic (L. rhamnosus GG), and fluorides (NaF). Clinical significanceThe ecological-based synbiotic-fluoride caries-preventive therapy aids in maintaining biofilm homeostasis to preempt/restore dysbiosis thereby sustaining dynamic-diverse health-associated microbial stability significant as a preventive regimen for high caries-risk patients.

Tempeh flour as an excellent source of paraprobiotics

Abstract. Jonesti WP, Prihatna C, Natadiputri GH, Suwanto A, Meryandini A. 2023. Tempeh flour as an excellent source of paraprobiotics. Biodiversitas 24: 1817-1823. Tempeh, a functional fermented food from Indonesia, has many benefits for human health. The presence of oligosaccharides, live and dead microbes in tempeh can function as prebiotics, probiotics, or paraprobiotics. This study aimed to determine the composition of live and dead lactic acid bacteria (LAB) and Enterobacteriaceae in tempeh flour using microbiological and molecular quantifications. Two samples of tempeh flour based on the drying process, i.e., sun-dried and oven-dried, were analyzed. Bacteria were quantified using standard plate count and qPCR, while bacterial cells damaged by the drying process were quantified using Propidium Monoazide-Quantitative Polymerase Chain Reaction (PMA-qPCR). Tempeh flours contained live bacteria as many as 4-5 log CFU/g and intact dead bacterial cells as many as 9 log CFU/g. The number of bacteria only differed slightly between the two drying processes. Based on qPCR analysis, the LAB number was higher than Enterobacteriaceae in tempeh flour. In addition, the bacterial population by qPCR was higher than by cultured analysis. These results indicated that sun-dried or oven-dried tempeh flour could be a functional food because it contained high amounts of live and dead bacteria as candidates for probiotics and paraprobiotics based on PMA-qPCR analysis.

Anti-Porphyromonas gingivalis nanotherapy for maintaining bacterial homeostasis in periodontitis

Periodontitis is caused by oral flora imbalance, which leads to immune imbalance. Porphyromonas gingivalis is a keystone pathogen in periodontitis, causing the blooming of inflammophilic microbes, and becoming dormant to resist antibiotics. Targeted interventions are needed to destroy this pathogen and collapse its inflammophilic flora. Therefore, a targeting nanoagent antibody-conjugated liposomal drug carrier with ginsenoside Rh2 (A-L-R) was developed for pleiotropic benefits. A-L-R showed high quality in high-performance liquid chromatography (HPLC), Fourier transform infrared (FTIR), and transmission electron microscope (TEM) detection. Only P. gingivalis was influenced by A-L-R, as shown by live/dead cell staining and a series of antimicrobial effects assays. With fluorescence in situ hybridization (FISH) staining and in propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), the clearance of P. gingivalis by A-L-R was more than for other groups, and only the proportion of P. gingivalis was reduced by A-L-R in monospecies culture. Moreover, in a periodontitis model, A-L-R targeted P. gingivalis with high efficiency and low toxicity, maintaining homeostasis with a relatively stable oral microflora. This targeting nanomedicine offers new strategies for periodontitis therapy, providing a foundation for the prevention and treatment of periodontitis.

Natural antimicrobial compounds immobilised on silica microparticles as filtering materials: Impact on the metabolic activity and bacterial viability of waterborne microorganisms

The aim of this work was to assess the capability of filtering materials based on silica microparticles functionalised with essential oil components (EOCs) to remove waterborne bacteria from water, and to elucidate the mechanism of action of the inhibitory effect of the filtering materials on the metabolic activity and viability of the studied pathogens. Different silica microparticles (25, 50, 75, 200 or 375 μ m ) were functionalised with carvacrol, eugenol, thymol and vanillin to obtain filtering materials which removal capability was evaluated using distilled water inoculated with Escherichia coli, Helicobacter pylori, Legionella pneumophila or Pseudomonas aeruginosa (10 4 –10 7 cells/mL). Water samples were filtered through different layer thicknesses (0.5, 1 or 1.5 cm) of the filtering materials and the microbial load retained was determined by plate count. In addition, fluorescent viability staining, determination of cellular ATP content, direct viable count–fluorescent in situ hybridisation (DVC–FISH) and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) analyses were performed to prove the materials’ antimicrobial properties. The results exhibited that EOC-functionalised supports were capable of eliminating waterborne microorganisms from water with log reduction values falling within the 3–5 range, whereas the non-functionalised materials did not present relevant inhibitory capacity. The irreversible effect of the EOC-functionalised supports on the viability and metabolic activity of treated bacteria was confirmed by fluorescent staining (absence or red stained cells) and DVC–FISH (no elongated cells). Cellular ATP content was significantly reduced after filtering the inoculated water samples through the EOC-functionalised supports (cATP values below 10 pg/mL). Similarly, the concentration of viable bacteria determined by PMA-qPCR showed the inhibitory effect of the developed materials with negative quantification values for H. pylori and values of 7.98 ⋅ 10 1 –6.07 ⋅ 10 3 GU/mL for L. pneumophila water samples filtered with the EOC-functionalised supports. Thus, the use of the functionalised filtering materials led to loss of bacterial viability of the treated microorganisms with irreversible morphological and metabolic alterations, which confirms their potential use as filtering aids with additional properties for the biological control of water. • Filters based on essential oil components immobilised on silica supports were made. • The developed filters were used to remove and inactivate waterborne bacteria. • As proof of concept study, removal capacity fulfilled water quality requirements. • Viability assays confirmed the antimicrobial properties of filtering materials.

Effects of low pH conditions on decay of methanogenic biomass

Acidic failure is relatively common in anaerobic digesters that receive readily biodegradable food wastes at high loading. Under low pH conditions, the activity of methanogenic biomass decreases resulting in complete failure of the digestion process. In this experimental study, we demonstrated that one of the causes for the digester failure under low pH conditions is due to accelerated decay of methanogenic biomass. When enriched acetate degrading methanogens were exposed to a low pH environment (pH = 5.1 with phosphoric acid) in a batch experiment without external substrate, the specific decay rate was observed to increase as much as 10 times of that at pH 7.0. The specific decay rate for formate degrader was also found to increase under low pH conditions whilst the fermentative microorganisms in the cultures appeared to be tolerant to low pH conditions. A Propidium Mono-Azide-quantitative Polymerase Chain Reaction (PMA-qPCR) analysis revealed that the archaeal biomass dominated by methanogens dropped by 71–79% from the initial concentration after 6 days of the acidic batch experiment whilst the bacterial biomass dominating acidogens decreased by only 25%. The decrease in the number of living cells in the batch experiments at different pH was monitored with time to determine a correlation between decay rate and incubation pH.

Inactivation of antibiotic resistant Escherichia coli and degradation of its resistance genes by glow discharge plasma in an aqueous solution

Emerging contaminants such as antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) are becoming a global environmental problem. In this study, the glow discharge plasma (GDP) was applied for degrading antibiotic resistant Escherichia coli (E. coli) with resistance genes (tetA, tetR, aphA) and transposase gene (tnpA) in 0.9% sterile saline. The results showed that GDP was able to inactivate the antibiotic resistant E. coli and remove the ARGs and reduce the risk of gene transfer. The levels of E. coli determined by 16S rRNA decreased by approximately 4.7 logs with 15 min of discharge treatment. Propidium monoazide - quantitative polymerase chain reaction (PMA-qPCR) tests demonstrated that the cellular structure of 4.8 more logs E. coli was destroyed in 15 min. The reduction of tetA, tetR, aphA, tnpA genes was increased to 5.8, 5.4, 5.3 and 5.5 logs with 30 min discharge treatment, respectively. The removal of ARGs from high salinity wastewater was also investigated. The total abundance of ARGs was reduced by 3.9 logs in 30 min. Scavenging tests indicated that hydroxyl radicals (·OH) was the most probable agents for bacteria inactivation and ARGs degradation. In addition, the active chlorine (Cl· and Cl2) which formed during the discharge may also contribute to the inactivation and degradation.

Viability determination of Ascaris ova in raw wastewater: a comparative evaluation of culture-based, BacLight Live/Dead staining and PMA-qPCR methods.

Accurate evaluation of viable Ascaris ova in wastewater is the key to mitigating Ascaris reinfections in endemic regions. In this study, the viability of Ascaris ova in raw wastewater was determined using three different detection methods: culture-based, BacLight Live/Dead staining and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). Furthermore, comparative assessment of viability utilising the aforementioned detection methods was performed using seeded experiments in wastewater. The percentage of viability was: culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%) respectively. Despite the fact that no statistical difference was shown in the viability determination among the three methods, PMA-qPCR-based viability determination would be preferable over the other two methods for evaluating potential public health risks with A. suum ova due to its accuracy, being least subjective and its rapid reaction time.

Propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay for rapid detection of viable and viable but non-culturable (VBNC) Pseudomonas aeruginosa in swimming pools.

Lack of culturability in the viable but non-culturable (VBNC) bacteria and the ability to regain infectivity in favourable conditions is one of the new challenges of public health providers for Pseudomonas aeruginosa monitoring in environmental samples. Propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) is one of the promising methods for timely detection of VBNC pathogens in environmental samples. We developed and used a method for the first time to detection of VBNC P. aeruginosa in swimming pool water samples using a membrane filter (MF). Moreover, the dominant model of the distribution of colonies on the MF and the effect of the culture medium and MF type on colony recovery by MF were evaluated. Swimming pool samples were subjected to conventional culture-based, qPCR and PMA-qPCR methods and the results were compared for the presence of VBNC P. aeruginosa in the samples. The positivity rate was 21% and 75% for P. aeruginosa in water samples as confirmed by standard culture-based and qPCR methods, respectively. Furthermore, of 24 samples, 9 (37.5%) were positive for VBNC P. aeruginosa. The developed qPCR/PMA-qPCR assay can detect the VBNC bacteria directly from aquatic samples and may result in better monitoring of recreational waters.

New insights into the kinetics of bacterial growth and decay in pig manure-wheat straw aerobic composting based on an optimized PMA-qPCR method.

SummaryAerobic composting is a bacteria‐driven process to degrade and recycle wastes. This study quantified the kinetics of bacterial growth and decay during pig manure–wheat straw composting, which may provide insights into microbial reaction mechanisms and composting operations. First, a propidium monoazide–quantitative polymerase chain reaction (PMA–qPCR) method was developed to quantify the viable bacteria concentration of composting samples. The optimal PMA concentration and light exposure time were 100 μM and 8 min respectively. Subsequently, the concentrations of total and decayed bacteria were quantified. Viable and decayed bacteria coexisted during the entire composting period (experiments A and B), and the proportion of viable bacteria finally fell to only 35.1%. At the beginning, bacteria grew logarithmically and decayed rapidly. Later, the bacterial growth in experiment A remained stable, while that of experiment B was stable at first and then decomposed. The duration of the stable stage was positively related to the soluble sugar content of composting materials. The logarithmic growth and rapid decay of bacteria followed Monod equations with a specific growth (0.0317 ± 0.0033 h−1) and decay rate (0.0019 ± 0.0000 h−1). The findings better identified the bacterial growth stages and might enable better prediction of composting temperatures and the degree of maturation.

Development of a sensitive and false-positive free PMA-qPCR viability assay to quantify VBNC Escherichia coli and evaluate disinfection performance in wastewater effluent

The detection and quantification of viable Escherichia coli cells in wastewater treatment plant effluent is very important as it is the main disinfection efficacy parameter for assessing its public health risk and environmental impact. The aim of this study was to develop a sensitive and false-positive free propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay to quantify the viable but non-culturable (VBNC) E. coli present in secondary wastewater effluent after chlorine disinfection. The qPCR target was the E. coli uidA gene, and native Taq was used to eliminate false positives caused by the presence of contaminant E. coli DNA in recombinant Taq polymerase reagents. Due to issues with qPCR inhibitors in wastewater, this study explored several pre-DNA extraction treatment methods for qPCR inhibitor removal. PMA-qPCR validation was done using salmon testes DNA (Sketa DNA) as an exogenous control added directly to the wastewater samples and amplified using a separate qPCR assay. After disinfection of secondary effluent with 2ppm chlorine at the plant, the mean Log10 CFU reduction in E. coli was 2.85 from a mean CFU of 3.48/10mL compared to 0.21 Log10 CCE mean reduction of the uidA gene from a mean CCE of 3.16/10mL. The VBNC cell concentrations were calculated as 2.32 Log10/10mL by subtracting the colony forming units (CFU) obtained from membrane filtration from the calculated CFU equivalent (CCE) values obtained from PMA-qPCR. These results demonstrate the effective use of a PMA-qPCR method for the quantification of the E. coli uidA gene and indicate there are high numbers (2.01×103CCE/100mL) of VBNC E. coli cells leaving the wastewater treatment plant in the final effluent after chlorine treatment. VBNC bacterial cells are of concern as they have the potential to resuscitate and grow, regain virulence, affect natural microbiome in the discharge sites, and pass on antimicrobial resistant genes to other microorganisms.

Quantitative detection of viable helminth ova from raw wastewater, human feces, and environmental soil samples using novel PMA-qPCR methods.

In this study, we have evaluated the efficacy of propidium monoazidequantitative polymerase chain reaction (PMA-qPCR) to differentiate between viable and non-viable Ancylostoma caninum ova. The newly developed method was validated using raw wastewater seeded with known numbers of A. caninum ova. Results of this study confirmed that PMA-qPCR has resulted in average of 88% reduction (P<0.05) in gene copy numbers for 50% viable +50% non-viable when compared with 100% viable ova. A reduction of 100% in gene copies was observed for 100% non-viable ova when compared with 100% viable ova. Similar reductions (79-80%) in gene copies were observed for A. caninum ova-seeded raw wastewater samples (n=18) collected from wastewater treatment plants (WWTPs) A and B. The newly developed PMA-qPCR method was applied to determine the viable ova of different helminths (A. caninum, A. duodenale, Necator americanus and Ascaris lumbricoides) in raw wastewater, human fecal and soil samples. None of the unseeded wastewater samples were positive for the above-mentioned helminths. N. americanus and A. lumbricoides ova were found in unseeded human fecal and soil samples. For the unseeded human fecal samples (1g), an average gene copy concentration obtained from qPCR and PMA-qPCR was found to be similar (6.8×10(5)±6.4×10(5) and 6.3×10(5)±4.7×10(5)) indicating the presence of viable N. americanus ova. Among the 24 unseeded soil samples tested, only one was positive for A. lumbricoides. The mean gene copy concentration in the positively identified soil sample was 1.0×10(5)±1.5×10(4) (determined by qPCR) compared to 4.9×10(4)±3.7×10(3) (determined by PMA-qPCR). The newly developed PMA-qPCR methods were able to detect viable helminth ova from wastewater and soil samples and could be adapted for health risk assessment.

Microbiomes of the dust particles collected from the International Space Station and Spacecraft Assembly Facilities.

BackgroundThe International Space Station (ISS) is a unique built environment due to the effects of microgravity, space radiation, elevated carbon dioxide levels, and especially continuous human habitation. Understanding the composition of the ISS microbial community will facilitate further development of safety and maintenance practices. The primary goal of this study was to characterize the viable microbiome of the ISS-built environment. A second objective was to determine if the built environments of Earth-based cleanrooms associated with space exploration are an appropriate model of the ISS environment.ResultsSamples collected from the ISS and two cleanrooms at the Jet Propulsion Laboratory (JPL, Pasadena, CA) were analyzed by traditional cultivation, adenosine triphosphate (ATP), and propidium monoazide–quantitative polymerase chain reaction (PMA-qPCR) assays to estimate viable microbial populations. The 16S rRNA gene Illumina iTag sequencing was used to elucidate microbial diversity and explore differences between ISS and cleanroom microbiomes. Statistical analyses showed that members of the phyla Actinobacteria, Firmicutes, and Proteobacteria were dominant in the samples examined but varied in abundance. Actinobacteria were predominant in the ISS samples whereas Proteobacteria, least abundant in the ISS, dominated in the cleanroom samples. The viable bacterial populations seen by PMA treatment were greatly decreased. However, the treatment did not appear to have an effect on the bacterial composition (diversity) associated with each sampling site.ConclusionsThe results of this study provide strong evidence that specific human skin-associated microorganisms make a substantial contribution to the ISS microbiome, which is not the case in Earth-based cleanrooms. For example, Corynebacterium and Propionibacterium (Actinobacteria) but not Staphylococcus (Firmicutes) species are dominant on the ISS in terms of viable and total bacterial community composition. The results obtained will facilitate future studies to determine how stable the ISS environment is over time. The present results also demonstrate the value of measuring viable cell diversity and population size at any sampling site. This information can be used to identify sites that can be targeted for more stringent cleaning. Finally, the results will allow comparisons with other built sites and facilitate future improvements on the ISS that will ensure astronaut health.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0116-3) contains supplementary material, which is available to authorized users.

Effect of Encapsulation on Viability of Pediococcus pentosaceus OZF During Its Passage Through the Gastrointestinal Tract Model.

The main goal of this study was to develop an improved oral delivery system for Pediococcus pentosaceus OZF, a promising probiotic bacterium, and to assess its viability under simulated gastrointestinal (GI) tract model by comparing the efficiency of microbiological and molecular approaches. Encapsulation was carried out using extrusion method and as a result, encapsulation system including 0.75% lactulose, 1.8% sodium alginate, 0.1M CaCl2, and 5min gelling time was shown to have a significantly protective effect against pH 2.0 acid stress over 3h. However, completely loss of viability was exhibited by free OZF cells under similar conditions. To provide an additional barrier for capsules, coating process was investigated using different biopolymers, and the survival rates of free and encapsulated OZF cells upon expose to simulated GI conditions were detected by conventional culture techniques and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) method. No significant differences between the biopolymers were detected, except the chitosan which leads totally 85% protection and extra 25% improvement in the survival of OZF cells compared to uncoated capsules. In conclusion, our findings indicated that chitosan-coated capsules provided an important protective effect on the viability of OZF cells against the GI system conditions encountered during the transit of food. In addition, this study was found successful in monitoring the viable OZF cells in capsules using PMA-qPCR method.

Mixing regime as a key factor to determine DON formation in drinking water biological treatment

Dissolved organic nitrogen (DON) can act as precursor of nitrogenous disinfection by-products formed during chlorination disinfection. The performances of biological fluidized bed (continuous stirred tank reactor, CSTR) and bio-ceramic filters (plug flow reactor, PFR) were compared in this study to investigate the influence of mixing regime on DON formation in drinking water treatment. In the shared influent, DON ranged from 0.71mgL−1 to 1.20mgL−1. The two biological fluidized bed reactors, named BFB1 (mechanical stirring) and BFB2 (air agitation), contained 0.12 and 0.19mgL−1 DON in their effluents, respectively. Meanwhile, the bio-ceramic reactors, labeled as BCF1 (no aeration) and BCF2 (with aeration), had 1.02 and 0.81mgL−1 DON in their effluents, respectively. Comparative results showed that the CSTR mixing regime significantly reduced DON formation. This particular reduction was further investigated in this study. The viable/total microbial biomass was determined with propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) and qPCR, respectively. The results of the investigation demonstrated that the microbes in BFB2 had higher viability than those in BCF2. The viable bacteria decreased more sharply than the total bacteria along the media depth in BCF2, and DON in BCF2 accumulated in the deeper media. These phenomena suggested that mixing regime determined DON formation by influencing the distribution of viable, total biomass, and ratio of viable biomass to total biomass.

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.