Use of real-time PCR with propidium monoazide for enumeration of viable Escherichia coli in anaerobic digestion

2016 ◽  
Vol 74 (5) ◽  
pp. 1243-1254 ◽  
Author(s):  
Wataru Ruike ◽  
Atsushi Higashimori ◽  
Junichi Yaguchi ◽  
Yu-you Li
Keyword(s):  
Escherichia Coli ◽  
Anaerobic Digestion ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Fecal Coliforms ◽  
Most Probable Number ◽  
Propidium Monoazide ◽  
Probable Number ◽  
E Coli

A combination of propidium monoazide (PMA) with real-time quantitative polymerase chain reaction (PMA-qPCR) was optimized to enumerate only viable Escherichia coli in anaerobic digestion processes. Repeating the PMA treatment twice and a final concentration of 100 μM resulted in an effective exclusion of DNA from heat-treated E. coli cells. In three anaerobic digestion processes, real-time PCR, PMA-qPCR, and the most probable number method (MPN) were used to estimate the numbers of total, viable, and culturable E. coli cells, respectively. Culturable concentrations of fecal coliforms were also measured by the membrane filter method. For thermophilic digestion, the reductions in total and viable E. coli cells from the digester influent to the effluent were significantly lower than those in culturable cells and fecal coliforms by two to four orders of magnitude. For mesophilic digestion, the differences in the reductions in E. coli and fecal coliforms counts were less than two orders of magnitude. Based on the measurements of viable E. coli determined by the PMA-qPCR method, the microbial quality of digester effluents was discussed for agricultural application, and pasteurization after anaerobic digestion was suggested for the destruction of viable pathogens.

Enumeration of viable Escherichia coli by real-time PCR with propidium monoazide

2012 ◽  
Vol 66 (10) ◽  
pp. 2065-2073 ◽  
Author(s):  
N. Yokomachi ◽  
J. Yaguchi
Keyword(s):  
Escherichia Coli ◽  
Wastewater Treatment ◽  
Real Time ◽  
Real Time Pcr ◽  
Wastewater Treatment Plants ◽  
Propidium Monoazide ◽  
E Coli ◽  
Cell Counts ◽  
Viable Cells ◽  
Heat Treated

A photo-inducible DNA-binding dye, propidium monoazide (PMA), was used to distinguish viable and dead Escherichia coli cells. Microscopic observations using a combination of the dyes 4′,6-diamidino-2-phenylindole and PMA indicated that PMA stained only dead cells, with membrane damage, red. Mixtures of viable and heat-treated E. coli cells were subjected to real-time polymerase chain reaction (PCR) with PMA treatment. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells in the mixtures of viable and heat-treated cells, as long as the ratio of dead cells to viable cells was no greater than 10. In the wastewater treatment plants, total, viable and culturable E. coli were enumerated by real-time PCR, real-time PCR coupled with PMA treatment and the most probable number method using EC-MUG medium, respectively. The concentrations of viable E. coli in the wastewater treatment plants were much higher than those of culturable cells. In addition, viable cells were even more chlorine resistant than culturable ones.

Comparison of culturable fecal coliforms and Escherichia coli enumeration in freshwaters

2007 ◽  
Vol 53 (6) ◽  
pp. 798-801 ◽  
Author(s):  
Tamara Garcia-Armisen ◽  
Josué Prats ◽  
Pierre Servais
Keyword(s):  
Escherichia Coli ◽  
Microbiological Quality ◽  
Fecal Coliforms ◽  
Quality Data ◽  
Plate Count ◽  
Most Probable Number ◽  
Tetrazolium Chloride ◽  
Probable Number ◽  
E Coli ◽  
Glucuronidase Activity

Fecal coliforms (FC) counts were compared with Escherichia coli counts in differently contaminated freshwater samples (n = 166). FC were enumerated by plate count on triphenyl 2,3,5-tetrazolium chloride Tergitol medium. Escherichia coli were enumerated by the most probable number microplate method based on the detection of glucuronidase activity. FC and E. coli counts were highly correlated; an average E. coli/FC ratio equal to 0.77 was found, meaning that on average, 77% of FC were E. coli. Knowing the E. coli/FC ratio allows us to convert the historical microbiological quality data expressed in FC counts into E. coli abundance and thus to compare with present and future monitoring data that are (or will be) based on E. coli enumeration.

Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

2007 ◽  
Vol 70 (12) ◽  
pp. 2774-2781 ◽  
Author(s):  
I-CHEN YANG ◽  
DANIEL YANG-CHIH SHIH ◽  
JAN-YI WANG ◽  
TZU-MING PAN
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Probable Number ◽  
Cooked Rice ◽  
Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

scholarly journals Lessons from a large outbreak of Escherichia coli O157[ratio ]H7 infections: insights into the infectious dose and method of widespread contamination of hamburger patties

1999 ◽  
Vol 122 (2) ◽  
pp. 185-192 ◽  
Author(s):  
J. TUTTLE ◽  
T. GOMEZ ◽  
M. P. DOYLE ◽  
J. G. WELLS ◽  
T. ZHAO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Most Probable Number ◽  
Strong Argument ◽  
Probable Number ◽  
Infectious Dose ◽  
E Coli ◽  
Beef Patties ◽  
Large Outbreak ◽  
Microbiological Testing

Between November 1992 and February 1993, a large outbreak of Escherichia coli O157[ratio ]H7 infections occurred in the western USA and was associated with eating ground beef patties at restaurants of one fast-food chain. Restaurants that were epidemiologically linked with cases served patties produced on two consecutive dates; cultures of recalled ground beef patties produced on those dates yielded E. coli O157[ratio ]H7 strains indistinguishable from those isolated from patients, confirming the vehicle of illness. Seventy-six ground beef patty samples were cultured quantitatively for E. coli O157[ratio ]H7. The median most probable number of organisms was 1·5 per gram (range, <0·3–15) or 67·5 organisms per patty (range, <13·5–675). Correlation of the presence of E. coli O157[ratio ]H7 with other bacterial indicators yielded a significant association between coliform count and the presence of E. coli O157[ratio ]H7 (P=0·04). A meat traceback to investigate possible sources of contamination revealed cattle were probably initially colonized with E. coli O157[ratio ]H7, and that their slaughter caused surface contamination of meat, which once combined with meat from other sources, resulted in a large number of contaminated ground beef patties. Microbiological testing of meat from lots consumed by persons who became ill was suggestive of an infectious dose for E. coli O157[ratio ]H7 of fewer than 700 organisms. These findings present a strong argument for enforcing zero tolerance for this organism in processed food and for markedly decreasing contamination of raw ground beef. Process controls that incorporate microbiological testing of meat may assist these efforts.

scholarly journals Mechanically Ventilated Broiler Sheds: a Possible Source of Aerosolized Salmonella, Campylobacter, and Escherichia coli

2009 ◽  
Vol 75 (23) ◽  
pp. 7417-7425 ◽  
Author(s):  
H. N. Chinivasagam ◽  
T. Tran ◽  
L. Maddock ◽  
A. Gale ◽  
P. J. Blackall
Keyword(s):  
Escherichia Coli ◽  
Most Probable Number ◽  
Production Cycle ◽  
Airborne Bacteria ◽  
Probable Number ◽  
Mechanically Ventilated ◽  
The Public ◽  
E Coli ◽  
Poultry Farms ◽  
Low Levels

ABSTRACT This study assessed the levels of two key pathogens, Salmonella and Campylobacter, along with the indicator organism Escherichia coli in aerosols within and outside poultry sheds. The study ranged over a 3-year period on four poultry farms and consisted of six trials across the boiler production cycle of around 55 days. Weekly testing of litter and aerosols was carried out through the cycle. A key point that emerged is that the levels of airborne bacteria are linked to the levels of these bacteria in litter. This hypothesis was demonstrated by E. coli. The typical levels of E. coli in litter were ∼108 CFU g−1 and, as a consequence, were in the range of 102 to 104 CFU m−3 in aerosols, both inside and outside the shed. The external levels were always lower than the internal levels. Salmonella was only present intermittently in litter and at lower levels (103 to 105 most probable number [MPN] g−1) and consequently present only intermittently and at low levels in air inside (range of 0.65 to 4.4 MPN m−3) and once outside (2.3 MPN m−3). The Salmonella serovars isolated in litter were generally also isolated from aerosols and dust, with the Salmonella serovars Chester and Sofia being the dominant serovars across these interfaces. Campylobacter was detected late in the production cycle, in litter at levels of around 107 MPN g−1. Campylobacter was detected only once inside the shed and then at low levels of 2.2 MPN m−3. Thus, the public health risk from these organisms in poultry environments via the aerosol pathway is minimal.

scholarly journals GREEN SYNTHESIS OF MAGNETIC IRON NANOPARTICLES USING MEDICINAL PLANT TRIDAX PROCUMBENS LEAF EXTRACTS AND ITS APPLICATION AS AN ANTIMICROBIAL AGENT AGAINST E. COLI

2020 ◽  
pp. 34-39
Author(s):  
YOJANA Y. PATIL ◽  
VAISHNVI B. SUTAR ◽  
ARPITA P. TIWARI
Keyword(s):  
Escherichia Coli ◽  
Magnetic Nanoparticles ◽  
Iron Nanoparticles ◽  
Most Probable Number ◽  
Leaf Extracts ◽  
Probable Number ◽  
Gram Negative ◽  
E Coli ◽  
Tridax Procumbens ◽  
Escherichia Coli Bacteria

Objective: The present study was aimed at the biological synthesis of magnetic iron nanoparticles by using the plant extract of Tridax procumbens and also to study their antimicrobial property against gram-negative bacteria (Escherichia coli). Methods: The synthesis of magnetic iron nanoparticles was carried out by the co-precipitation method using biological methods like plant extract as reducing agent and capping agents are biocompatible and non-hazardous. These nanoparticles were characterized by UV-Visible spectroscopy, XRD (X-Ray Diffraction), and SEM (Scanning Electron Microscope). As well as antibacterial activity of the nanoparticles was carried out by agar well diffusion method and Most Probable Number (MPN) method against gram-negative E. coli (Escherichia coli) bacteria. Results: The average crystallite size of Magnetic Nanoparticles (MNPs) was found to be 72 nm by X-ray diffraction. The optical absorption band at wavelengths of 240 nm and 402 nm was obtained from the UV Visible spectrum. Spherical shape morphology was observed in SEM studies. The antibacterial assay clearly expressed that E. coli showed a maximum zone of inhibition (15±0.15 mm) at 2 mg/ml and 1 mg/ml concentration was found for Magnetic Nanoparticles. In the Most Probable Number (MPN) test it is seen that the bacterial count is reduced after adding synthesized NPs into the water sample. Conclusion: The results of the present study conclude that the Magnetic Nanoparticles synthesized using Tridax procumbens leaf extracts is found to be stable and show good antibacterial activity against gram-negative (Escherichia coli) bacteria.

scholarly journals Escherichia coli O157:H7 in Drin Prevalence of Escherichia coli O157:H7 in Drink from Different Food Premises in Kota Samarahan Sarawak

2019 ◽  
Vol 2 (2) ◽  
pp. a13-19
Author(s):  
ELEXSON NILLIAN ◽  
AMIZA NUR ◽  
DIYANA NUR ◽  
AMIRAH ZAKIRAH ◽  
GRACE BEBEY
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Coliform Bacteria ◽  
Most Probable Number ◽  
Plain Water ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
E Coli ◽  
Preventive Actions ◽  
Stx1 Gene

Contamination of drinks with E. coli O157:H7 served in food premises such as restaurants can cause haemorrhagic colitis and haemolytic uremic syndrome to humans. The presence or absence of faecal pathogen was demonstrated using coliform group as indicator microorganisms. Therefore, this study was conducted to detect the presence of E. coli O157:H7 in drinking water from food restaurant premise in Kota Samarahan and Kuching to ensure safe and potable drinking water is served to the consumer. A total of thirty (n=30) drink samples including six types of each of the samples are cold plain water, iced tea, iced milo, syrup and iced milk tea. Most Probable Number (MPN) procedure was used in this study to enumerate the MPN values of coliform bacteria in each drink collected. A total of 53.33% (16/30) of the drink samples showed positive E. coli detection. Then, the PCR assay showed 6.25% (one out of 16 isolates) samples were positive and carried stx1 gene produced by E. coli O157:H7 in iced milo sample types. This study showed the drinks collected from food premises was contaminated with faecal contamination, which was not safe to drink by the consumer. Therefore, preventive actions should be taken to prevent foodborne illness outbreak in future

Comparison of a New Enrichment Procedure for Shiga Toxin–Producing Escherichia coli with Five Standard Methods

2005 ◽  
Vol 68 (8) ◽  
pp. 1593-1599 ◽  
Author(s):  
MICHAEL A. GRANT
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Target Cells ◽  
Macconkey Agar ◽  
Standard Methods ◽  
E Coli ◽  
Canadian Health ◽  
Health Products

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin–producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g−1. Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 × 108 CFU ml−1 and 1.80 × 106 CFU ml−1 after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.

Potential pitfalls in the quantitative molecular detection ofEscherichia coliO157:H7 in environmental matrices

2006 ◽  
Vol 52 (5) ◽  
pp. 482-488 ◽  
Author(s):  
Rebekka R.E Artz ◽  
Lisa M Avery ◽  
Davey L Jones ◽  
Ken Killham
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Soil System ◽  
E Coli ◽  
Animal Wastes ◽  
Pcr Assays

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene–based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.Key words: Escherichia coli O157:H7, real-time PCR, animal waste, soil, VBNC.

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