PCR quantifies dead cells beside live cells and this makes the judgment of microbial quality of food samples complicated. The objective of this study was comparing the efficacy of ethidium MONOAZIDEqPCR and PROPIDIUM MONOAZIDE-qPCR in quantifying live pathogen cells (E. coli, Staphylococcus aureus, Enterococcus fecalis and Listeria monocyogenes) by real time PCR, in low-fat and high-fat milk, lactic cheese (low-fat) and processed cheese (high-fat). Different proportions of live and heat killed pathogen cells were inoculated into food samples. After separating bacterial pellets, EMA and PMA treatments qPCR was conducted. One-way ANOVA followed by Turkey’ s Multiple Comparison tests were applied to analyze the data. In low-fat milk, EMA treatment resulted in 18, 20, 23 and 30% decrease in cell count of E. coli, S. aureus, E. fecalis and L. monocyogenes, respectively, compared to conventional culturing. Also, following treatment by PMA, 6, 3, 8 and 12% decrease in cell count was obtained for E. coli, S. aureus, E. fecalis and L. monocyogenes, respectively, compared to conventional culturing. In high-fat samples as processed cheese, a reduction of 20, 27, 30 and 25% in EMA treatment and 9, 6, 5 and 10% in PMA treatment was observed in cell count of E. coli, S. aureus, E. fecalis and L. monocyogenes, respectively. The inhibitory potential of EMA and PMA against signal emission was variable in different bacterial species and the fat content of the samples exerted no significant effect (p>0. 05) on PMA and EMA functionality.

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

Background: The aime of this study was to investigate the behavior of Map in Lighvan cheese with special reference to strains of Map, inoculum level, and storage time.Methods: One standard and two native strains of Map were inoculated (2 and 4 log cell/ml) to ewe’s raw milk and were used for cheese making. Behavior of Map throughout the manufacture, ripening and storage of Lighvan cheese was tracked using PROPIDIUM MONOAZIDE (PMA) quantitative realtime PCR (qPCR) and MGIT-MPN assay.Results: PMA-qPCR and culture assay demonstrated comparable outcomes. Based on these results, inoculum level and storage time showed a significant effect on persistency of Map. Furthermore, during the storage period different behavior was observed among the various strains of Map.Conclusion: Map could survive the 6 months of storage period and Lighvan cheese has potential to support the survival of Map.

BACKGROUND: Staphylococcus aureus is one of the most important human pathogens that cause infection and also food intoxication by secreting various enterotoxins. Conventional culturing methods to detect S. aureus have some limitations such as being time-consuming due to bacterial growth and low precision and sensitivity in detecting viable but non-cultivable cells. OBJECTIVES: The objective of this study was to detect and quantify enterotoxigenic (A-E) S. aureus in cream pastry products applying PCR coupling with PROPIDIUM MONOAZIDE (PMA) to distinguish dead and live cells. METHODS: One hundred samples were randomly collected from pastry shops in Amol, in a period of 2 months. After preparing dilutions, bacterial pellets were separated and treated with PMA before DNA extraction. Real time PCR was conducted in order to quantify S. aureus cells and enterotoxigenic strains using specific primers. RESULTS: Results of conventional method were close to PMA-qPCR data (P>0. 05), but data from qPCR that includes live and dead cells shows more bacterial count than two other methods. Sensitivity of the method applied in the present study, detecting low number of S. aureus cells (less than 10/g) seems considerable. CONCLUSIONS: Findings showed that applying PCR coupling with PMA results in more reliable data than conventional culturing method. Regarding this approach being time-effective, considerably sensitive and specific, it is expected that it be used in food quality control labs in monitoring systems in future.

Essential oils (EOs) have long been applied as flavoring agents in foods. Nowadays, due to the antimicrobial properties of EOs, they have been used as natural food preservatives. In this study, initial experiments were performed in order to elucidate the minimum bactericidal concentration of Rosmarinus officinalis L. EO on Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes. Thereafter, PMA-qPCR was applied in order to selectively quantify living cells within a bacterial population treated with rosemary EO. Inactivation was obtained at EO concentrations of 1%, 0.45%, 0.9% for L. monocytogenes, E. coli O157:H7 and S. enterica, respectively. L. monocytogenes were totally killed within 45 min while it took 90 min for E. coli O157:H7 and S. enterica. It was concluded that rosemary EO has the potential to be used as a natural food additive or bio-preservative since it was able to irreversibly inactivate the three tested pathogens at lower concentrations and short exposition times in comparison with the other EOs. In addition, PMA-qPCR approach proved quantitatively precise and specific to selectively detect live pathogenic bacteria in vegetables following inactivation with rosemary EO.

Background and Objectives: Recently, real-time PCR in combination with PMA has successfully been applied to discriminate between live Escherichia coli O157: H7 and dead bacteria killed by cumin, clove, oregano and cinnamon essential oils. In this study, antimicrobial effect of Zataria multiflora Boiss. essential oil on food-borne pathogens in minced beef combining real time-PCR and PROPIDIUM MONOAZIDE has been evaluated.Materials and methods: Essential oil was analyzed by gas chromatography-mass spectrometry equipment. Initial experiments were performed in order to elucidate the minimum bactericidal concentration of Z. multiflora essential oil on E. coli O157: H7, Salmonella enterica and Listeria monocytogenes. Thereafter PMA-qPCR was applied in order to selectively quantify life cells within a bacterial population treated with Z. multiflora essential oil in minced beef.Results: The main component of Z. multiflora Boiss. essential oil was carvacrol (71.12%). Inactivation was obtained at essential oil concentrations of 0.02, 0.035, 0.045 for L. monocytogenes, E. coli O157: H7 and S. enterica, respectively1. L. monocytogenes were totally killed in 30 min while it took 1 h 30 min for the gram negative pathogens. According to the results of PMA-qPCR quantification obtained for the different combinations of live and dead cells in artificially inoculated minced beef, endogenous bacterial counts of the minced beef were 3.27×104 cfu/g. For S. enterica and E. coli O157: H7 PMAqPCR quantification values correlated with plate counts of live cells, but for L. monocytogenes, live cells were overestimated in all cases and in the sample with 100% of dead cells PMA-qPCR still detected a 3.8% of live cells.Conclusion: As a conclusion Z. multiflora essential oil has potential as natural food additive or biopreservative since it was able to irreversibly inactivate the three pathogens tested, at lower concentrations than other essential oils and short exposition times. In addition, the PMA-qPCR approach proved efficient to selectively detect live pathogenic bacteria in raw minced beef following inactivation with Z. multiflora essential oil.

Background and Objectives: Public health protection requires timely evaluation of pathogens in potable water to minimize outbreaks caused by microbial contaminations. The present study was aimed at assessing the microbiological quality of water obtained from Shantinagar (a rural area in the South Goa region of Goa, India) using 5-Bromo-4-Chloro-3-Indoxyl β,-D-glu-curonide-Sorbitol MacConkey agar (BCIG-SMAC) medium and, PROPIDIUM MONOAZIDE-quantitative polymerase chain reac-tion (PMA-qPCR) assay for differential detection and quantification of viable Escherichia coli cells in water samples. Materials and Methods: Membrane filtration method was used for both BCIG-SMAC medium and PMA-qPCR methods. To determine the efficiency of detection of viable cells, we first evaluated the PMA treatment protocol and established the standard calibration curves using previously reported primers. Results: PMA-qPCR detected as low as 7 femtograms of DNA of E. coli per qPCR reaction whereas the limit of detection (LOD) of BCIG-SMAC medium was 1. 8 CFU/100mL. A total of 71 water samples spanning 2017-2018 have been analyzed using BCIG-SMAC medium and PMA-qPCR, of which 95. 77% (68/71) and 7. 04% (5/71) were found to be total E. coli and E. coli O157: H7, respectively. PMA-qPCR study showed the viable counts of total viable E. coli cells ranging from 3 CFU/100mL to 8. 2×102 CFU/100mL. The total E. coli CFU/100mL quantified by PMA-qPCR significantly exceeded (paired t-test,P<0. 05) the number on BCIG-SMAC medium. Conclusion: The present study indicates that the microbiological quality of environmental water samples analyzed do not comply with the regulatory standard. Therefore, special attention is warranted to improve the overall portable quality of water in the perspective of public health.

Background: This novel approach describes a rapid and simple method for identification of necrotic vs. viable cells within a mammalian blastocyst.Materials and Methods: Hatched bovine blastocysts produced in vitro were first incubated for 30 min in pre-equilibrated culture medium containing PROPIDIUM iodide (PI; 300mg/ml) and bisbenzimide (Hoechst: H33342; 5 mg/ml) fluorescent dyes. Embryos were then freed from residual dyes by thoroughly washing in warm phosphate buffer saline free of calcium and magnesium (PBS-), fixed in 2.5% glutharaldehyde and washed again in PBS- Stained embryos afterwards were mounted in a drop of glycerol over a microscopic slide. Prepared samples were examined under an epifluorescent microscope using the same excitation wavelength (330-385nm) and barrier filter (400nm) to distinguish necrosed vs. viable blastomers as being appeared in red and blue, respectively.Results: Obtained results showed that in cells with altered cell membrane such as late apoptotic or necrotic cells, PI and H33342 readily enter through the cytoplasmic barriers and so the chromatin materials are stained by both, but since PI quenches bisbenzimide fluorescence, necrotic blastomeres are seen in red to pinky red, while live cells are seen just as blue.Conclusion: Obtained results clearly indicated that this novel approach can be used as a simple, feasible and precise method for every embryology lab and with all the mammalian blastocysts produced either in vitro or in vivo. The basic assay can be completed in 60 min, and valuable and reliable information can be obtained about the quality of the embryos.

Background and purpose: The aim of this study was to evaluate the in vitro and in vivo anti-Toxoplasma gondii (T. gondii) effect of 5-oxo-hexahydroquinoline compounds. Moreover, molecular docking study of the compounds into the active site of enoyl-acyl carrier protein reductase (ENR) as a necessary enzyme for the vitality of apicoplast was carried out. Experimental approach: A number of 5-oxo-hexahydoquinoline derivatives (Z1-Z4) were synthesized. The T. gondii tachyzoites of RH strain were treated by different concentrations (1-64 μ g/mL) of the compounds. The viability of the encountered parasites with compounds was assessed using flow cytometry and PROPIDIUM iodide (PI) staining. Due to the high mortality effect of Z3 and Z4 in vitro, their chemotherapy effect was assessed by inoculation of tachyzoites to four BALB/c mice groups (n = 5), followed by the gavage of various concentrations of the compounds to the mice. Molecular docking was done to study the binding affinity of the synthesized 5-oxo-hexahydroquinolines into ENR enzyme active site byusing AutoDock Vina® software. Docking was performed by a Lamarckian Genetic Algorithm with 100 runs. Findings / Results: Flow cytometry assay results indicated compounds Z3 and Z4 had relevant mortality effect on parasite tachyzoites. Besides, in vivo experiments were also performed and a partial increase of mice longevity between control and experiment groups was recorded. Molecular docking of Z3 and Z4 in the binding site of ENR enzyme indicated that the compounds were well accommodated within the binding site. Therefore, it could be suggested that these compounds may exert their anti-T. gondii activity through the inhibition of the ENR enzyme. Conclusion and implications: Compounds Z3 and Z4 are good leads in order to develop better anti-T. gondii agents as they demonstrated both in vitro and in vivo inhibitory effects on tachyzoites viability and infection. Further studies on altering the route of administration along with additional pharmacokinetics evaluations are needed to improve the anti-T. gondii impacts of 5-oxo-hexahydroquinoline compounds.

Background and objectives: Plants have been used to treat diseases like cancer for many years and today the trend towards their use is increasing. One of the most effective mechanisms of plants against cancer is inducing apoptosis. Apoptosis is a programmed cell death which acts opposite to cell division. It starts in response to some stimuli. Despite the effectiveness of apoptosis inducing agents, their use has been limited due to side effects and resistance to these treatments; so, applying medicinal herbs due to their lower cost and toxicity has drawn attentions. Recent research at the Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences on two medicinal plants Acanthophyllum bracteatum and A. microcephalum has shown cytotoxic effects of these two species, but the mechanism of their toxicity has remained unknown; thus, the present study was designed to evaluate the apoptotic potential of Acanthophyllum bracteatum and A. microcephalum. Methods: In the present study, the cytotoxic effects of the methanol extract of Acanthophyllum bracteatum and A. microcephalum was evaluated against MCF-7 and MDA-MB-468 cells by MTT assay; furthermore, their apoptosis potential has been evaluated by annexin-V/PROPIDIUM iodide assay and Hoechst 33258 staining in the same cell lines. Results: The methanol extract of A. microcephalum and A. bracteatum showed cytotoxic effects against MCF-7 and MDA-MB-468 cell lines with IC50 values of 64, 159 and 102, 250 μ g/mL, respectively. The results of the apoptosis assays confirmed the potential of the two plants extracts to induce apoptosis in both cell lines while A. microcephalum demonstrated more considerable results. Conclusion: A. microcephalum could be a suitable choice for further breast cancer studies.