Simultaneous Detection Of Total Coliforms Research Articles

Improved detection of Escherichia coli and coliform bacteria by multiplex PCR.

BackgroundThe presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified.ResultsIn this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively.ConclusionsThe assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

To analyse molecular detection of coliforms and shorten the time of PCR. Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN.

Evaluation of a new medium for the enumeration of total coliforms and Escherichia coli in Japanese surface waters

A new medium, EC-Blue-10, containing chromogenic and fluorogenic substrates, KNO(3) and sodium pyruvate has been developed for the rapid simultaneous detection and enumeration of total coliforms and Escherichia coli in water. Two evaluations of EC-Blue-10 were carried out. Firstly, EC-Blue-10 was compared with Colilert-MPN for 96 water samples using MPN for total coliforms and E. coli. Secondly, the detection of coliforms and E. coli were compared using 2400 tubes of EC-Blue-10 and Colilert-MPN. The regression coefficients between EC-Blue-10 and Colilert-MPN for total coliforms and E. coli were 0.91 and 0.89, respectively. For the detection results, the Cohen's kappa values between the two media were 0.79 for coliforms and 0.72 for E. coli. EC-Blue-10 is almost same as Colilert-MPN for the detection of coliforms and E. coli in surface waters. Further evaluation for EC-Blue-10 is needed to verify in different geographical areas. EC-Blue-10 is useful method for the rapid and simultaneous detection of total coliforms and E. coli in water sample.

Detection of Escherichia coli in the sewage influent by fluorescent labeled T4 phage

For a precise estimation of sanitary condition, Escherichia coli detection system using E. coli-specific bacteriophage T4 was constructed. To facilitate E. coli detection, T4e − phage which did not produce the lysozyme was constructed and green fluorescent protein (GFP) was displayed on T4e − small outer capside (SOC) protein. This T4e −/GFP can detect E. coli K12 without cell lysis. In this study, we applied T4e −/GFP to the detection of E. coli in the sewage influent. Chromocult ® coliform (CC) agar plates are generally used for simultaneous detection of total coliforms and E. coli. We investigated the number of coliforms and E. coli in the municipal sewage influent by using CC agar plates for 1 year. There were 10 5–10 6 CFU/ml of total coliforms and 10 4–10 5 CFU/ml of E. coli throughout the year. More than 20 strains of E. coli selected from CC agar were infected by T4e −/GFP. The ratio of clear plaque forming E. coli was different every month and very low (annual average 8%). Most of the E. coli showed turbid plaque or no plaque. E. coli formed the turbid plaque showed no-fluorescence, fluorescence only on cell surface due to phage adsorption, or heterogeneous fluorescence among the cells, while E. coli formed clear plaque could be detected because of T4e −/GFP amplification in the cell.

Analysis of Bottled Water for Escherichia coli and Total Coliforms

U.S. Food and Drug Administration regulations governing bottled water include microbiological quality guidelines based on coliform counts. Recently, a new MF medium for simultaneous detection of total coliforms and Escherichia coli was developed. This medium, m-ColiBlue24 (m-CB) was compared to m-Endo medium and an International Organization for Standardization standard coliform medium, lactose agar with Tergitol 7. Coliform analysis was conducted on 104 brands of bottled water from 10 countries. Some samples were additionally analyzed for heterotrophic plate count and Pseudomonas sp. populations, including P. aeruginosa. Presumptive coliform colonies were found in 5.8% of the samples with m-CB, 1.9% with m-Endo and 11.5% with lactose agar with Tergitol 7. None of the presumptive coliforms from any of the three media were verified as true coliforms in subsequent analysis. Consequently, the presumptive recovery rates actually represented false-positive error (FPE) rates. The FPE for m-CB and m-Endo were not statistically different (P < 0.05) but the FPE for lactose agar with Tergitol 7 was significantly larger.

Detection and occurrence of waterborne bacterial and viral pathogens

Detection and occurrence of waterborne bacterial and viral pathogens

New Medium for the Simultaneous Detection of Total Coliforms and Escherichia coli in Water

New Medium for the Simultaneous Detection of Total Coliforms and <i>Escherichia coli</i> in Water

New medium for the simultaneous detection of total coliforms and Escherichia coli in water

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.

Rapid, Specific Autoanalytical Method for the Simultaneous Detection of Total Coliforms and E. Coli from Drinking Water

The Autoanalysis Colilert (AC) test is a modification of the defined substrate technology designed to enumerate specific target microbe(s) from a mixture of bacteria. The AC test simultaneously enumerates total coliforme and Escherichia coli directly from a water sample. A national United States evaluation of the method, covering a wide range of surface and subsurface water sources and water processing modalities, was conducted. The AC test was compared with Multiple Tube Fermentation (MTF) (quantitative) and presence-absence (P-A) (qualitative) Standard Methods procedures. The quantitative comparison showed that the AC test was slightly more sensitive than MTF. For two of the five sites it was more precise; for the other three it was equal. For the qualitative P-A the AC and Standard Methods analyses agreed 94%. Specificity of the AC method was established by subculturing a species of total coliform or E. coli from positives. The Autoanalysis Colilert method showed equivalent sensitivity and specificity to currently available standard methods.

Rapid, specific, defined substrate technology for the simultaneous detection of total coliforms and Escherichia coli

AbstractThe Autoanalysis Colilert (AC) test is a modification of the defined substrate technology designed to enumerate specific target microbe(s) from a mixture. All ingredients are present in the tube or vessel in powdered form. The AC test is simple to perform—a measured amount of water is added to test tubes (for the most probable number test) or vessel [Presence/Absence (P/A) test]. After incubation, the development of a yellow color is specific for total coliforms. Fluorescence in the same tube(s) is specific for Escherichia coli. No confirmatory or completed tests need be performed. A national United States evaluation covering all sources and processing of water was conducted. The AC test was compared to multiple tube fermentation (quantitative) and P/A (qualitative) Standard Methods procedures. Testing protocol was that of the U.S. Environmental Protection Agency's Environmental Monitoring and Support Laboratory for establishing equivalency between methods. The quantitative comparison showed that the AC test was slightly more sensitive than MTF. For two of five sites it was more precise; for the other three it was equal. For the qualitative P/A the AC and Standard Methods analysis agreed 94%. Specificity of the AC method was established by subculturing a species of total coliform or E. coli from positives. The AC test was easy to use, can be performed by large and small utilities, and is less expensive than current methods. It can simultaneously, specifically enumerate total coliforms and E. coli without the need for confirmatory tests.