DIRECT DETECTION OF ESCHERICHIA COLI 0157:H7 IN UNPASTEURIZED APPLE JUICE WITH AN EVANESCENT WAVE BIOSENSOR

An evanescent wave biosensor was used to detect Escherichia coli O157:H7 in unpasteurized apple juice. Light is launched from a 635 nm laser diode into silica or polystyrene optical waveguides, generating an evanescent field which extends from the waveguide surface. Fluorescent molecules within the evanescent field are excited resulting in an emission signal that the biosensor then detects and quantifies. A sandwich immunoassay was performed on the waveguides using cyanine 5 dye-labeled anti-E. coli O157:H7 antibodies for generation of the specific fluorescent signal. The lower limit of detection was between 6.0 × 10² and 6.0 × 10⁴ CFU/mL with silica waveguides and between 3.2 × 10⁴ and 3.2 × 10⁴ CFU/mL using polystyrene waveguides. One-hundred percent correct identification of true positive samples occurred at 6.0 × 10⁴ and 3.2 × 10⁴ CFU/mL for silica and polystyrene waveguides, respectively. Signals from a variety of non-E. coli O157 bacteria, including closely related enterotoxigenic strains of E. coli at concentrations of ˜ 10⁶ CFU/mL, were below the limits of detection. Assays were conducted in near real-time with results obtained within 15 min of sample processing.     

Bacteriological studies on Iraqi milk products

F. ABBAR AND H. KH. KADDAR. 1991. Samples from different types of domestic milk products including cheese, kishfa and gaymer were assessed for bacteriological quality over a 4-month period. A total of 400 samples were randomly selected across Mosul city and tested for faecal coliform counts, enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E. coli (ETEC). Faecal coliforms were present at levels greater than 10² cfu/g in 72.5% and less than 10² cfu/g in 27.5% of samples. Of the 430 E. coli strains isolated from the 400 samples of milk products, 138 were serotypes of EPEC. These were found in 81 (40.5%) samples of cheese, 35 (23.8%) of kishfa and 22 (29.7%) of gaymer. During this period, 26 strains of ETEC were also isolated, all of which demonstrated heat-labile or heat-stable toxins. The high proportion of strains of three groups of E. coli showing resistance to antibiotics is discussed in relation to widespread use of antibiotics and the possible public health implication.     

Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples

Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 10², by PCR amplification; 10³, by whole cell luminescence; and 10³−10⁴, by measurement of luminescence in cell extracts.     

COMPARISON OF THREE POLYMERASE CHAIN REACTION-BASED METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD

Three PCR-based methods for the detection of Listeria monocytogenes in food (BAX for Screening, Probelia and a method according to Kaclíková et al. (2003) were compared on the basis of the determination of detection limits for 15 artificially contaminated food products. Detection limits of all methods for all samples were 10⁰ cfu per 10 g, with the exception of three cheese samples which did not produce valid results because of the inhibition of Probelia PCR. Detection limits for nonviable L. monocytogenes cells were sufficiently high ( 10⁹ cfu per 10 g) for BAX and the method according to Kaclíková et al. (2003), but considerably low ( 10⁶ cfu per 10 g) for Probelia. The results demonstrate that BAX for Screening as well as the Kaclíková et al. (2001) method fulfill the sensitivity requirements for a rapid alternative method for the detection of L. monocytogenes in food, which would be equivalent to the standard method EN ISO 11290–1.     

Aflatoxin content and number of fungi in poultry feedstuffs from Indonesia

The content of aflatoxin and associated fungi was determined in 56 samples, including 34 of corn, 10 of soybean meal, nine of rice bran and three of broken rice, collected from different poultry farms and poultry feedmills situated around Jakarta-Bogor, Indonesia.
Ninety-one per cent of the corn samples contained aflatoxins and the total concentration ranged from 22 to 6171 μ g/kg. With rice bran, 100% of the samples were positive for aflatoxin B₁, ranging from 36 to 71 μ g/kg. No aflatoxin was detected in samples of soybean meal or broken rice. All the samples were contaminated by several fungi (8 times 10³–5 times 10⁶cfu/g) and further identification was limited to Aspergillus flavus and A. parasiticus. The dominant species was A. flavus (2 times 10³–4 times 10⁶cfu/g in corn samples, 1·0 times 10³–1·0 times 10⁵cfu/g in soybean meal, 2 times 10⁴–4·4 times 10⁵cfu/g in rice bran and 2 times 10⁴–6 times 10⁴cfu/g in broken rice). Some of the corn samples also contained A. parasiticus (2 times 10³–9·5 times 10⁴cfu/g).     

Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium

A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 10⁵ when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro-colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100-fold, or 10-fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 10¹–10² cells ml⁻¹ and with the latter ca 10¹ cells ml⁻¹, when different numbers of streptomycin-resistant Eca strain were added to peel extract from three Eca-free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers.     

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non- E. coli bacteria. Its detection limit was 10²–10³ cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10⁰ cells 100 ml⁻¹ of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples

A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism. A two primer set and an oligonucleotide detection probe were used to specifically detect Cl. botulinum type A neurotoxin gene (BoNT/A). After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization. This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction. The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10³ bacteria per g according to the type of food samples. No cross-reactions were observed with the other Cl. botulinum toxinotypes and other bacteria found routinely in food. This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl. botulinum type A in food samples.     

A note on the use of microwaves in an improved non-radioactive DNA hybridization procedure for the detection of bacteria in foodstuffs

Foodstuffs were artificially contaminated with Escherichia coli carrying plasmid pBR322, dot blotted onto nylon membranes and briefly subjected to microwaves in the presence of 1·5 mol/l NaC1/0·5 mol/l NaOH. Subsequent hybridization with a biotin-labelled probe specific for pBR322 enabled the detection of cell concentrations > 10⁴ cells/dot blot, equivalent to 2 × 10⁷ cells/g food tested. This shortened and simplified method was effective for all ten foods tested, generated low background levels and should be applicable to a wide range of bacteria.     

THE USE OF STREPTAVIDIN COATED MAGNETIC BEADS FOR DETECTING PATHOGENIC BACTERIA BY LIGHT ADDRESSABLE POTENTIOMETRIC SENSOR (LAPS)

A modified procedure for magnetic capture of antibody-conjugated bacteria for light addressable potentiometric sensor (LAPS) detection using the Threshold System was developed. Streptavidin coated magnetic beads, partially labeled with biotinylated anti Escherichia coli O157 antibodies, were used to capture Escherichia coli O157:H7. Captured bacteria were further labeled with fluorescein-conjugated anti -E. coli O157:H7 antibodies and urease-labeled. anti-fluorescein antibody. Magnetically concentrated bacteria-containing complexes were then immobilized through streptavidin-biotin interactions on 0.45 μ biotinylated nitro-cellulose membranes assembled as sample sticks for the Threshold instrument. The rate of pH change associated with the production of NH₃ by the urease in urea-containing solution was measured by a LAPS incorporated in the Threshold instrument. This approach allowed us to detect 10³ to 10⁴ CPU of cultured E. coli O157:H7 in PBS solutions. Furthermore, detectable LAPS signals of the sample sticks remained relatively constant for at least 24 h at 4C. The developed approach was applied to detect the E. coli in beef hamburger spiked with the bacteria. After a 5 to 6-h enrichment at 37C, as low as 1 CFU/g of E. coli O157:H7 in beef hamburger could be detected.     

Persistence of Escherichia coli O157:H7 on the rhizosphere and phyllosphere of lettuce

Aims:  The major objective of this study was to determine the effects of low levels of Escherichia coli O157:H7 contamination on plant by monitoring the survival of the pathogen on the rhizosphere and leaf surfaces of lettuce during the growth process.
Methods and Results:  Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in the rhizosphere and leaf surfaces after planting. Real-time PCR assays were designed to amplify the stx 1, stx 2 and the eae genes of E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real-time PCR was 2·4 × 10³ CFU g⁻¹ of starting DNA in rhizosphere and phyllosphere samples and about 10² CFU g⁻¹ by plate count. The time for pathogens to reach detection limits on the leaf surface by plate counts was 7 days after planting in comparison with 21 days in the rhizosphere. However, real-time PCR continued to detect stx 1, stx 2 and the eae genes throughout the experimental period.
Conclusion:  Escherichia coli O157:H7 survived throughout the growth period as was determined by real-time PCR and by subsequent enrichment and immunomagnetic separation of edible part of plants.
Significance and impact of the Study:  The potential presence of human pathogens in vegetables grown in soils contaminated with E. coli O157:H7 is a serious problem to our national food supply as the pathogen may survive on the leaf surface as they come in contact with contaminated soil during germination.     

A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes

Development of a routine detection assay for Listeria monocytogenes in foods that uses the polymerase chain reaction (PCR) and enrichment cultures was investigated. Oligonucleotide primers were chosen to amplify a 3'region of L. monocytogenes hly A gene spanning a conserved Hin dIII site. PCR detection sensitivity for L. monocytogenes in dilutions of pure enrichment cultures was between 50 and 500 colony forming units. A short enrichment period before PCR amplification allowed detection of the organisms in a range of complex foods contaminated with 10⁴ cfu/g. Detection sensitivity for the assay in the presence of chicken skin and soft cheese was determined at 10–100 cfu/g. Utilization of enrichment cultures and PCR allowed identification of the organism within 24 h or 2 days.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

F eresu , S. & N yati , H. 1990. Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products. Journal of Applied Bacteriology 69 , 814–821.
The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20C for 24 h and then stored at either 20C or 5C for 96 h.
Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20C and 5C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10⁷-10⁹/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10³-10⁶ and 10²-10⁵ during storage of the fermented product at 20C and 5C respectively. Higher maximum numbers, 10⁹-10¹⁰, of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10⁵-10⁸ and 10⁴-10⁷ during storage of the fermented product at 20C and 5C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.     

Random mutagenesis of Legionella pneumophila with mini-Tn10

Abstract The degradation of sheets of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (BIOPOL^®) by aerobic sewage sludge was analyzed. Degradation of the polymer was highly dependent on the pH of the culture medium and was maximal between pH 7 and pH 8.5. Below pH 6 and above pH 9 the degradation rate was very low. Agitation of the culture fluid had relatively little influence on the rates of degradation. 1.2×10⁵ aerobic polymer-degrading bacteria per ml sewage sludge were identified by halo formation on solid poly(3-hydroxybutyrate) (PHB)-containing media. The number of PHB-degrading bacteria in other ecosystems amounted to 3.8×10³ per ml sludge of a fresh-water lake, 9.2×10⁵ per g garden-soil, 1.3×10⁶ per g field-soil and 4.3×10⁶ per g compost.     

A demonstration of bacterial conjugation within the alimentary canal of Rhabditis nematodes

Abstract: Rhabditis nematodes fed a diet of Escherichia coli defecate viable undigested bacteria. These bacteria retain phenotypic characteristics, including those encoded on plasmids. Nematodes can survive a 2-min surface sterilization with 2% chlorine bleach; internalized bacteria also survive this treatment and are released in the nematode wastes. Bacteria alone or on the surface of dead nematodes are unable to survive incubation with this solution. There were 3.2 × 10⁵ viable bacteria per nematode, indicating that sufficient bacteria were present for gene transfer. Transconjugants ( lac ⁻ nal ^R str ^R cm ^R) were recovered in the nematode fecal material following a protocol where nematodes were initially fed a plasmidless lac ⁻ nal ^R str ^S cm ^S E. coli and then, after surface sterilization, a lac ⁺ nal ^S E. coli plasmid donor containing the conjugative R100JA ( str ^R cm ^R) plasmid. The presence of plasmids in the transconjugants was confirmed by gel electrophoresis. The occurrence of conjugation in the gut was confirmed by dissection of individual surface-sterilized nematodes and isolation of transconjugants.     

Characterization of glycerol kinase and NAD-independent glycerol-3-phosphate dehydrogenase from Pediococcus pentosaceus N5p

The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157 : H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean^® purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g⁻¹ faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157 : H7 in less than 8 h, but with a sensitivity of approximately 10³ cfu g⁻¹ faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers.     

ASSESSMENT OF IMPEDANCE MICROBIOLOGICAL METHOD FOR THE DETECTION OF ESCHERICHIA COLI IN FOODS

This study was conducted to determine the sensitivity and specificity of the impedance-based microbiological method for the detection of Escherichia coli in foods within 24 h of testing. A Malthus Microbiological Analyzer system (Malthus System V, Malthus Instruments Ltd., Bury, United Kingdom), and a modified Malthus Coliform Broth Medium (MCBM), and an incubation temperature of 44C were used. The sensitivity of the impedance method was determined by testing E. coli-negative food samples spiked with different concentrations of E. coli. The specificity of the method was determined by testing E. coli -negative food samples spiked with Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The test results were compared with those obtained by the Most Probable Number (MPN) method. Milk, milk products, raw and ready-to-eat meats, and vegetables were tested for the presence of E. coli by both methods. The sensitivity of the impedance method and the MPN method for the detection of foods containing 10¹ CFU/g was 100% and 84.4%, respectively. Both methods had a specificity of 100% for food samples spiked with 10¹ CFU/g E. coli. The specificity of the impedance and the MPN methods for the detection of E. coli in naturally contaminated milk and meat samples was 100% and 95.7% respectively. E. coli was detected in foods by the impedance method within 4–24 h of testing at a detection limit of 1 CFU/mL. These results demonstrate that the impedance method can be used as a rapid and sensitive method for the detection of E. coli in foods.     

A Simple and Fast Extraction Procedure to Obtain Amplifyable DNA from Ralstonia (Pseudomonas) Solanacearum and Clavibacter michiganensis ssp. sepedonicus Inoculated Potato Tuber Extracts and Naturally Infected Tubers to Conduct a Polymerase Chain Reaction (PCR)

A modified NaOH alkaline boiling procedure using a mixture of lysozyme and proteases combined with minimized TRIS/HCl/BSA buffer volume was applied to extract amplifyable DNA from the two quarantine bacteria Ralstonia (Pseudomonas) solanacearum and Clavibacter michiganensis ssp. sepedonicus artificially added to potato tuber extracts of a low and a high starch potato variety. A PCR detection threshold of 10⁴−10⁵ colony forming units per ml extract of each quarantine bacterium was obtained by using the two potato varieties, the high starch potato variety resulting in a lower detection threshold.
Similar sensitivities could be obtained from potato tubers naturally infected with both quarantine bacteria. When comparing a published DNA extraction procedure suitable for Clavibacter michiganensis ssp. sepedonicus with the alkaline extraction the latter is much faster and simpler with similar detection thresholds and representing an inexpensive method to obtain suitable template DNA for routine PCR tests.     

The effect of cryptolepine on the morphology and survival of Escherichia coli, Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of the indoloquinoline alkaloid, cryptolepine, isolated from Cryptolepis sanguinolenta (Fam. Periplocaceae) was determined against selected micro-organisms. The minimum inhibitory concentration (MIC) ranges obtained, expressed as μg ml⁻¹, were: 5–10 for Saccharomyces cerevisiae NCPF 3139; 10–20 for S. cerevisiae NCPF 3178; 20–40 for Escherichia coli NCTC 10418; 40–80 for E. coli NCTC 11560, Candida albicans ATCC 10231 and C. tropicalis NCPF; and 80–160 for C. albicans NCPF 3242 and NCPF 3262.
Biocidal effects were noted at concentrations 2–4 times those of the MIC of the alkaloid following challenge with 10⁶ cfu ml⁻¹ of micro-organisms. Time-kill studies showed a reduction in viable count from 10⁶ to < 10 cfu ml⁻¹ in 4 h in C. albicans ATCC 10231 exposed to 320 μg ml⁻¹ of the agent; 3 log cycle reductions were recorded for the 6 h counts of E. coli NCTC 10418 and S. cerevisiae NCPF 3139 exposed to 40μg ml⁻¹ and 160 μg ml⁻¹ of the alkaloid respectively.
These results were consistent with findings using scanning electron microscopy. Exposure of cells to biocidal concentrations of cryptolepine produced filamentation prior to lysis in E. coli NCTC 10418 and extreme disturbance of surface structure, including partial and total collapse, followed by lysis in C. albicans ATCC 10231 and S. cerevisiae NCPF 3139.     

Mobilization of CS fimbriae-associated plasmids of enterotoxigenic Escherichia coli of serotype O6: K15: H16 or H- into various wild-type hosts

Abstract CS fimbriae-associated plasmids of two enterotoxigenic Escherichia coli strains of serotype O6: K15: H16 or H- (biotypes A and F) with M _r values of 51 × 10⁶ and 72 × 10⁶, respectively, were mobilized into various alternative host bacteria. Expression of CS1 or CS2 fimbriae was obtained when either of the CS fimbriae-associated plasmids was introduced into CS Fim⁻, O6: K15: H16 or H- recipients with rhamnose-negative and rhamnose-positive fermentation phenotypes, respectively, whereas CS3 fimbriae were expressed irrespective of the biotype of the recipient. On transfer into a CS Fim⁻ variant of an enterotoxigenic O8: H9 strain and into two K-12 strains, a CS3-fimbriae-only phenotype was conferred by the presence of either of the plasmids. When a CS Fim⁻ variant of a Rha⁺ CS2-fimbriae-only strain of serotype O6: K15: H16 harboured either of the plasmids, both CS2 and CS3 fimbriae were expressed, indicating that the rare CS2-fimbriae-only wild-type phenotype is probably due to the presence of a defective plasmid in such strains. Mobilization of the 51 MDa CS fimbriae-associated plasmid into five non-enterotoxigenic Rha⁺ porcine isolates of E. coli with O6 serotypes other than O6: K15: H16 or H- yielded CS3-fimbriae-only transconjugants. Thus the correlation between a Rha⁺ fermentation phenotype and expression of CS2 fimbriae does not hold in general for O-group 6 strains.