scholarly journals PCR detection of Vibrio cholerae, Escherichia coli, and Salmonella sp. from bottled drinking water in Iran

2018 ◽  
Vol 12 (09) ◽  
pp. 700-705
Author(s):  
Mojtaba Bonyadian ◽  
Hamdallah Moshtaghi ◽  
Hanie Nadi
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Vibrio Cholerae ◽  
Screening Method ◽  
Chain Reaction ◽  
Culture Methods ◽  
E Coli ◽  
Drinking Waters ◽  
Polymerase Chain

Introduction: The quality of drinking water has an important role in human health. This study was aimed to detect Escherichia. coli, Salmonella sp. and Vibrio cholerae from bottled drinking waters produced in Iran. Methodology: A total of 240 samples of bottled water of different brands were collected for testing between March 2015 to December 2015 in Shahrekord-Iran. Samples were examined by polymerase chain reaction (PCR) combined with culture methods for the detection of E. coli, Salmonella sp., and V. cholerae. Results: The results of PCR revealed that the uidA gene from E. coli, IpaB gene from Salmonella sp, and epsM gene from V. cholerae were detected in 6 (2.5%), 1 (0.4 %), 0 (0%) of the samples, respectively. But in culture methods, only E. coli 5 (2.1%) were isolated from the samples. The contamination with E. coli was significantly higher (P < 0.05) in water produced during the hot seasons than the cold seasons. Conclusions: This study confirmed the presence of Escherichia coli as the main microorganism in bottle drinking water in Iran. Also, our study showed that PCR can be used as a screening method for monitoring the enteric pathogens in drinking water.

scholarly journals Detection of virulence genes of diarrheagenic Escherichia coli strains from drinking water in Khartoum State

2020 ◽  
Author(s):  
Ehssan H. Moglad ◽  
Omima Abdl El Jalil Adam ◽  
Maram M. Alnosh ◽  
Hisham N. Altayb
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Genomic Dna ◽  
Multiplex Polymerase Chain Reaction ◽  
Virulence Genes ◽  
Chain Reaction ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Polymerase Chain ◽  
Drinking Water Samples

Abstract This study aimed to determine the prevalence of virulence genes in all the diarrheagenic Escherichia coli DEC strains (EAEC, EHEC, EIEC, EPEC, and ETEC) isolated from drinking water from Khartoum State, Sudan. A total of 46 drinking water samples obtained from different water sources were analyzed for the presence of E. coli as fecal contamination indicator and the antimicrobial-resistant pattern of isolated E. coli DEC strain was investigated. The bacterial genomic DNA was used as a template for multiplex polymerase chain reaction (MPCR) for the detection of the EHEC (stx gene), EIEC (ipaH gene), EPEC (eae gene), and EAEC (aggR gene) as virulence and biomarker genes. Our results showed that ipaH gene was found in 41.3% (19/46) of isolates, and aggR gene detected in 30.4% (14/46) of isolates. Both aggR and ipaH were found positive in 9 (19.5%) isolates and as well the combination of aggR and stx genes were detected in 2 (4.3%) isolates. In conclusion, this report confirmed the presence of DEC strains in drinking water from different resources and locations. Such findings require separate future clinical research studies to examine waterborne pathogens that exist in this state's water and find a management solution to stop or avoid potential outbreaks.

Evaluation of a Polymerase Chain Reaction–Based System for Detection of Salmonella Enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on Fresh Fruits and Vegetables†

2001 ◽  
Vol 64 (6) ◽  
pp. 788-795 ◽  
Author(s):  
ADRIENNE E. H. SHEARER ◽  
CHRISTINE M. STRAPP ◽  
ROLF D. JOERGER
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Salmonella Enteritidis ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Culture Methods ◽  
E Coli ◽  
Polymerase Chain ◽  
Alfalfa Sprouts

A polymerase chain reaction (PCR)-based detection system, BAX, was evaluated for its sensitivity in detecting Salmonella Enteritidis, Escherichia coli O157:H7, Listeria sp., and Listeria monocytogenes on fresh produce. Fifteen different types of produce (alfalfa sprouts, green peppers, parsley, white cabbage, radishes, onions, carrots, mushrooms, leaf lettuce, tomatoes, strawberries, cantaloupe, mango, apples, and oranges) were inoculated, in separate studies, with Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes down to the predicted level of 1 CFU per 25-g sample. Detection by BAX was compared to recovery of the inoculated bacteria by culture methods according to the Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). BAX was essentially as sensitive as the culture-based method in detecting Salmonella Enteritidis and L. monocytogenes and more sensitive than the culture-based method for the detection of E. coli O157:H7 on green pepper, carrot, radish, and sprout samples. Detection of the pathogenic bacteria in samples spiked with a predicted number of less than 10 CFU was possible for most produce samples, but both methods failed to detect L. monocytogenes on carrot samples and one of two mushroom and onion samples spiked with less than 100 CFU. Both BAX and the culture method were also unable to consistently recover low numbers of E. coli O157:H7 from alfalfa sprouts. The PCR method allowed detection of Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes at least 2 days earlier than the conventional culture methods.

scholarly journals Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products

2014 ◽  
Vol 12 (4) ◽  
pp. 763-771 ◽  
Author(s):  
Jingrang Lu ◽  
Tammie L. Gerke ◽  
Helen Y. Buse ◽  
Nicholas J. Ashbolt
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Drinking Water ◽  
Polymerase Chain Reaction Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
The Novel ◽  
Water Pipe ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABITM internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 103 colony-forming units (CFU) E. coli K12 mL−1, but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms.

scholarly journals On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

2005 ◽  
Vol 37 (4) ◽  
pp. 265-269 ◽  
Author(s):  
Xi-Qiang Zhu ◽  
Su-Xia Li ◽  
Hua-Jun He ◽  
Qin-Sheng Yuan
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Mass Ratio ◽  
Chain Reaction ◽  
Expression Plasmid ◽  
Protein Subunits ◽  
E Coli ◽  
Metal Affinity ◽  
Polymerase Chain ◽  
Escherichia Coli Expression

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

scholarly journals Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil

2007 ◽  
Vol 59 (2) ◽  
pp. 508-512 ◽  
Author(s):  
B.R. Paneto ◽  
R.P. Schocken-Iturrino ◽  
C. Macedo ◽  
E. Santo ◽  
J.M. Marin
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Antimicrobial Agents ◽  
Raw Milk ◽  
Diffusion Method ◽  
Chain Reaction ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Polymerase Chain ◽  
Raw Milk Cheese

The occurrence of toxigenic Escherichia coli in raw milk cheese was surveyed in Middle Western Brazil. Fifty samples of cheese from different supermarkets were analyzed for E.coli. The isolates were serotyped and screened for the presence of verotoxigenic E. coli (VTEC) and enterotoxigenic E. coli (ETEC) by Polymerase Chain Reaction (PCR). The susceptibility to thirteen antimicrobial agents was evaluated by the disk diffusion method. E.coli were recovered from 48 (96.0%) of the samples. The serogroups identified were O125 (6.0%), O111 (4.0%), O55 (2.0%) and O119 (2.0%). Three (6.0%) and 1(2.0%) of the E.coli isolates were VTEC and ETEC, respectively. Most frequent resistance was observed to the following antimicrobials: cephalothin (60.0%), nalidixic acid (40.0%), doxycyclin (33.0%), tetracycline (31.0%) and ampicillin (29.0%).

scholarly journals Multiplex Polymerase Chain Reaction Assay for Identification of Enterotoxigenic Escherichia Coli Strains

2001 ◽  
Vol 13 (4) ◽  
pp. 308-311 ◽  
Author(s):  
Jacek Osek
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Direct Determination ◽  
Enteric Bacteria ◽  
Enterotoxigenic Escherichia Coli ◽  
Chain Reaction ◽  
Gram Negative ◽  
E Coli ◽  
Polymerase Chain

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein ( uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non- E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.

scholarly journals Eliminating false positives in a qPCR assay for the detection of the uidA gene in Escherichia coli

2013 ◽  
Vol 11 (3) ◽  
pp. 382-386 ◽  
Author(s):  
Richard Kibbee ◽  
Natalie Linklater ◽  
Banu Örmeci
Keyword(s):  
Escherichia Coli ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Uida Gene ◽  
Chain Reaction ◽  
Taq Polymerase ◽  
E Coli ◽  
Gene Copies ◽  
Polymerase Chain ◽  
Low Levels

Due to contaminant Escherichia coli DNA present in recombinant Taq polymerase reagents, it is not possible to reliably detect low levels of E. coli in samples using the quantitative polymerase chain reaction (qPCR) assay. Native Taq polymerase was successfully used in this study to detect five uidA gene copies (5 fg of genomic DNA) of the uidA gene.

scholarly journals IDENTIFICATION OF AMPC Β-LACTAMASE-PRODUCING CLINICAL ISOLATES OF ESCHERICHIA COLI

2017 ◽  
Vol 10 (12) ◽  
pp. 357
Author(s):  
Tanushree Barua Gupta ◽  
Malini Shariff ◽  
Thukral Ss ◽  
S.s Thukral
Keyword(s):  
Escherichia Coli ◽  
Detection Method ◽  
Clinical Isolates ◽  
Confirmatory Test ◽  
Chain Reaction ◽  
E Coli ◽  
Resistance To Penicillin ◽  
Polymerase Chain ◽  
Third Generation Cephalosporins

  Objective: Indiscriminate use of β-lactam antibiotics has resulted in the emergence of β-lactamase enzymes. AmpC β-lactamases, in particular, confer resistance to penicillin, first-, second-, and third-generation cephalosporins as well as monobactams and are responsible for antibiotic resistance in nosocomial pathogens. Therefore, this study was undertaken to screen nosocomial Escherichia coli isolates for the presence and characterization of AmpC β-lactamases. The study also envisaged on the detection of inducible AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) in AmpC β-lactamase-producing E. coli.Methods: A total of 102 clinical isolates of E. coli, were subjected to cefoxitin screening, and screen-positive isolates were further subjected to inhibitor-based detection method, phenotypic confirmatory test, disc antagonism test, polymerase chain reaction (PCR), and isoelectric focusing (IEF).Results: In this study, 33% of E. coli were resistant to cefoxitin, of which 35% were found to be positive for AmpC β-lactamase by inhibitor-based phenotypic test. Of the AmpC-positive isolates, 83% were positive for ESBLs, whereas 25% were producing inducible AmpC β-lactamases. PCR and IEF showed CIT and EBC types of AmpC β-lactamases present in the tested isolates.Conclusion: Our study showed the presence of inducible AmpC enzymes and ESBLs in E. coli isolates and PCR identified more isolates to be AmpC producers.

scholarly journals Prevalence and molecular detection of shiga toxin producing Escherichia coli from diarrheic cattle

2016 ◽  
Vol 14 (1) ◽  
pp. 63-68 ◽  
Author(s):  
MM Akter ◽  
S Majumder ◽  
KH MNH Nazir ◽  
M Rahman
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Molecular Detection ◽  
Chain Reaction ◽  
Specific Primers ◽  
Fecal Samples ◽  
E Coli ◽  
Uremic Syndrome ◽  
Polymerase Chain ◽  
Positive Isolate

Shiga toxin-producing Escherichia coli (STEC) are zoonotically important pathogen which causes hemorrhagic colitis, diarrhea, and hemolytic uremic syndrome in animals and humans. The present study was designed to isolate and identify the STEC from fecal samples of diarrheic cattle. A total of 35 diarrheic fecal samples were collected from Bangladesh Agricultural University (BAU) Veterinary Teaching Hospital. The samples were primarily examined for the detection of E. coli by cultural, morphological and biochemical characteristics, followed by confirmation of the isolates by Polymerase Chain Reaction (PCR) using gene specific primers. Later, the STEC were identified among the isolated E. coli through detection of Stx-1 and Stx-2 genes using duplex PCR. Out of 35 samples, 25 (71.43%) isolates were confirmed to be associated with E. coli, of which only 7 (28%) isolates were shiga toxin producers, and all of them were positive for Stx-1. However, no Stx-2 positive isolate could be detected. From this study, it may be concluded that cattle can act as a reservoir of STEC which may transmit to human or other animals.J. Bangladesh Agril. Univ. 14(1): 63-68, June 2016

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