scholarly journals Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

2011 ◽  
Vol 77 (13) ◽  
pp. 4329-4335 ◽  
Author(s):  
Bilgin Taskin ◽  
Ayse Gul Gozen ◽  
Metin Duran
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Membrane Integrity ◽  
Dna Amplification ◽  
Live Cells ◽  
Dead Cell ◽  
Target Sequence ◽  
Propidium Monoazide ◽  
Indicator Organisms ◽  
Content Type

ABSTRACTQuantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed withEscherichia coliATCC 25922 as the model organism and theuidAgene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter−1.

scholarly journals Effect of Long-Term Starvation on the Survival, Recovery, and Carbon Utilization Profiles of a Bovine Escherichia coli O157:H7 Isolate from New Zealand

2014 ◽  
Vol 80 (14) ◽  
pp. 4383-4390 ◽  
Author(s):  
Ron N. Xavier ◽  
Hugh W. Morgan ◽  
Ian R. McDonald ◽  
Helen Withers
Keyword(s):  
Escherichia Coli ◽  
New Zealand ◽  
Membrane Integrity ◽  
Nutrient Level ◽  
Carbon Utilization ◽  
Content Type ◽  
E Coli ◽  
Significant Difference ◽  
Carbon Utilization Profiles ◽  
Phosphate Buffered Saline

ABSTRACTThe ability to maintain a dual lifestyle of colonizing the ruminant gut and surviving in nonhost environments once shed is key to the success ofEscherichia coliO157:H7 as a zoonotic pathogen. Both physical and biological conditions encountered by the bacteria are likely to change during the transition between host and nonhost environments. In this study, carbon starvation at suboptimal temperatures in nonhost environments was simulated by starving a New Zealand bovineE. coliO157:H7 isolate in phosphate-buffered saline at 4 and 15°C for 84 days. Recovery of starved cells on media with different nutrient availabilities was monitored under aerobic and anaerobic conditions. We found that the New Zealand bovineE. coliO157:H7 isolate was able to maintain membrane integrity and viability over 84 days and that the level of recovery depended on the nutrient level of the recovery medium as well as the starvation temperature. In addition, a significant difference in carbon utilization was observed between starved and nonstarved cells.

scholarly journals Appropriate Regulation of the σE-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Hervé Nicoloff ◽  
Saumya Gopalkrishnan ◽  
Sarah E. Ades
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Stress Responses ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Point Mutations ◽  
Content Type ◽  
Envelope Stress ◽  
Outer Membrane Porins

ABSTRACT The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σE-directed cell lysis and the role of σE in stationary phase, we investigated the effects of elevated σE activity in cultures grown for 10 days. We demonstrate that high σE activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σE activity, due primarily to point mutations in the region of σE that binds the −35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σE activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the σE-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σE activity. Our results demonstrate that appropriate regulation of σE activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability. IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σE response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σE is tightly regulated to match the production of σE regulon members to the needs of the cell. In E. coli, loss of σE results in lethality. Here we demonstrate that excessive σE activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

scholarly journals Determination of viability in foodborne bacteria with inter-calating dyes: ethidium monoazide (EMA) and propidium monoazide (PMA)

2017 ◽  
Vol 5 (2) ◽  
pp. 59 ◽  
Author(s):  
Arife Ezgi Telli ◽  
Yusuf Doğruer
Keyword(s):  
Covalent Binding ◽  
Membrane Integrity ◽  
Bright Light ◽  
Covalent Bond ◽  
Dna Amplification ◽  
Molecular Techniques ◽  
Dead Cell ◽  
Mixed Cell ◽  
Intercalating Dye

The ability to distinguish between living and dead cells is considered to be very important for biological researches. It is an important problem that the technology used up to day does not allow the quantitative differentiation of specific cells in a mixed cell community. Determination of whether the microorganisms present in the foods are in a viable form is an important phenomenon in determining the disease-forming potential.It is a fact that DNA, which is found in cells that lose their viability, can maintain its activity for a long time. Discrimination of live-dead cell occurs when the intercalating dye is covalently bound to DNA that is cleaved in the dead cell where membrane integrity is impaired. The formation of the covalent bond is activated by photoactivation. Inter-collating dyes only affect dead cells that are damaged by cell wall or membrane integrity. Due to the covalent binding of the inter-collating dye, DNA amplification cannot occur in PCR and other molecular techniques based on PCR.Among the non-permeable stains, it is accepted that PI is the most commonly used. PMA is identical to PI and additionally contains azide groups. Azide groups allow PMA to cross-covalently bond with DNA in bright light. Another inter-collating dyes with an azide group is ethidium mono azide (EMA).The The PMA molecule provides a higher selectivity on discrimination of live-dead cells by virtue of its’ higher charge when compared to EMA. Many researchers have combined EMA and PMA with PCR, Real-time PCR and LAMP in order to differentiate the live population of bacterial, viral, fungal and parasitic food-borne pathogens because they are claimed to be more successful in complex samples than in fluorescence based techniques.

scholarly journals Survival of Host-Associated Bacteroidales Cells and Their Relationship with Enterococcus spp., Campylobacter jejuni, Salmonella enterica Serovar Typhimurium, and Adenovirus in Freshwater Microcosms as Measured by Propidium Monoazide-Quantitative PCR

2011 ◽  
Vol 78 (4) ◽  
pp. 922-932 ◽  
Author(s):  
Sungwoo Bae ◽  
Stefan Wuertz
Keyword(s):  
Quantitative Pcr ◽  
Campylobacter Jejuni ◽  
Salmonella Enterica ◽  
Sunlight Exposure ◽  
Propidium Monoazide ◽  
Content Type ◽  
Significant Difference ◽  
Serovar Typhimurium ◽  
Dark Conditions ◽  
Realistic Information

ABSTRACTThe ideal host-associated genetic fecal marker would be capable of predicting the presence of specific pathogens of concern. Flowthrough freshwater microcosms containing mixed feces and inocula of the pathogensCampylobacter jejuni,Salmonella entericaserovar Typhimurium, and adenovirus were placed at ambient temperature in the presence and absence of diurnal sunlight. The totalEnterococcusDNA increased during the early periods (23 h) under sunlight exposure, even though cultivableEnterococcusand DNA in intact cells, as measured by propidium monoazide (PMA), decreased with first-order kinetics during the entire period. We found a significant difference in the decay of host-associatedBacteroidalescells between sunlight exposure and dark conditions (Pvalue < 0.05), whereas the persistence of host-associatedBacteroidalesDNA was comparable. The 2-log reduction times of adenovirus were 72 h for sunlight exposure and 99 h for dark conditions with similar decay rate constants (Pvalue = 0.13). The persistences of fecalBacteroidalescells andCampylobactercells exposed to sunlight were similar, and host-associatedBacteroidalesDNA and waterborne pathogen DNA were degraded at comparable rates (Pvalues > 0.05). Overall, the ratio of quantitative PCR (qPCR) cycle threshold (CT) values with and without PMA treatment was indicative of the time elapsed since inoculation of the microcosm with (i) fecal material from different animal sources based on host-associatedBacteroidalesand (ii) pure cultures of bacterial pathogens. The use of both PMA-qPCR and qPCR may yield more realistic information about recent sources of fecal contamination and result in improved prediction of waterborne pathogens and assessment of health risk.

scholarly journals Thermal and Nonthermal Effects of Discontinuous Microwave Exposure (2.45 Gigahertz) on the Cell Membrane of Escherichia coli

2014 ◽  
Vol 80 (16) ◽  
pp. 4832-4841 ◽  
Author(s):  
Carole Rougier ◽  
Audrey Prorot ◽  
Philippe Chazal ◽  
Philippe Leveque ◽  
Patrick Leprat
Keyword(s):  
Escherichia Coli ◽  
Cell Membrane ◽  
Cell Membranes ◽  
Membrane Integrity ◽  
Threshold Effect ◽  
Cell Suspensions ◽  
Conventional Heating ◽  
Content Type ◽  
Cell Membrane Integrity ◽  
Average Temperature

ABSTRACTThe aim of this study was to investigate the effects on the cell membranes ofEscherichia coliof 2.45-GHz microwave (MW) treatment under various conditions with an average temperature of the cell suspension maintained at 37°C in order to examine the possible thermal versus nonthermal effects of short-duration MW exposure. To this purpose, microwave irradiation of bacteria was performed under carefully defined and controlled parameters, resulting in a discontinuous MW exposure in order to maintain the average temperature of the bacterial cell suspensions at 37°C.Escherichia colicells were exposed to 200- to 2,000-W discontinuous microwave (DW) treatments for different periods of time. For each experiment, conventional heating (CH) in a water bath at 37°C was performed as a control. The effects of DW exposure on cell membranes was investigated using flow cytometry (FCM), after propidium iodide (PI) staining of cells, in addition to the assessment of intracellular protein release in bacterial suspensions. No effect was detected when bacteria were exposed to conventional heating or 200 W, whereas cell membrane integrity was slightly altered when cell suspensions were subjected to powers ranging from 400 to 2,000 W. Thermal characterization suggested that the temperature reached by the microwave-exposed samples for the contact time studied was not high enough to explain the measured modifications of cell membrane integrity. Because the results indicated that the cell response is power dependent, the hypothesis of a specific electromagnetic threshold effect, probably related to the temperature increase, can be advanced.

scholarly journals Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model

2019 ◽  
Vol 85 (20) ◽  
Author(s):  
Thomai P. Lazou ◽  
Eleni G. Iossifidou ◽  
Athanasios I. Gelasakis ◽  
Serafeim C. Chaintoutis ◽  
Chrysostomos I. Dovas
Keyword(s):  
Quantitative Pcr ◽  
Osmotic Shock ◽  
Campylobacter Coli ◽  
Propidium Monoazide ◽  
Content Type ◽  
Viable Cells ◽  
Signal Suppression ◽  
Nonculturable State ◽  
Bacterial Model ◽  
Qpcr Quantification

ABSTRACT A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml−1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria. IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.

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