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PubMed1993

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.

Antibodies, Monoclonal↗

PubMed2005

Simultaneous detection of listeria monocytogenes in chicken meat enrichments by PCR and reverse-transcription PCR without DNA/RNA isolation.

Environmental and food samples can be analyzed using PCR and reverse transcription (RT)-PCR techniques to discriminate between viable and nonviable cells of bacterial pathogens. Here, we describe the use of a commercial lysis buffer, initially designed for mammalian cells, that permits the rapid extraction of bacterial DNA and RNA. The buffer is an RT-PCR-compatible lysis solution in which RNA is stable and can be frozen for later use. RT-PCR is carried out directly after DNase I treatment of crude bacterial lysates using rTth polymerase for RT-PCR in a single tube. Untreated lysate is used for standard PCR. The procedure permits the amplification of either mRNA or DNA of Listeria monocytogenes at a level similar to that obtained with purified nucleic acids. Using lysates obtained with this buffer, nested PCR and RT-PCR assays detected low numbers of L. monocytogenes cells from artificially contaminated chicken meat samples. The simplicity of this system may foster the development of similar buffers specifically designed for bacteria to improve RNA detection methods that can be performed in parallel with DNA analysis. The use of a single buffer decreases the time needed for analysis, is amenable to automation and real-time assays, and might be adaptable to all bacteria and amplification methods.

Animals↗

PubMed1982

Antibiotic resistance and R plasmids in coliforms isolated from some Malaysian cooked foods.

Forty samples of Malaysian cooked foods were examined for the presence of antibiotic-resistant coliforms and R plasmids. Twenty seven (68%) of the foods had antibiotic-resistant coliforms and 5 (13%) had R plasmids. Nineteen samples (48%) had total bacterial counts over 10(6) per gm and in 5 samples, no coliforms were detected. Our findings show that cooked food may be one possible way by which R plasmids are spread. The control of the spread of R plasmids is discussed.

Anti-Bacterial Agents↗

PubMed2005

Analysis of PCR-based methods for characterization of Listeria monocytogenes strains isolated from different sources.

Listeria monocytogenes strains, isolated from various sources (food, environment, and animals), were used to test different PCR-based methods to investigate their capability to define the strain origin. RAPD-PCR with three primers and the SAU-PCR method, in which the DNA was first digested with the Sau3A restriction endonuclease and then amplified with a primer designed on the restriction site, were carried out, and the profiles obtained were used to perform cluster analysis. Based on the cluster analysis of Listeria spp. strains, obtained from international collections, the coefficient of similarity was selected. The results obtained showed that the methods tested in the study gave different levels of differentiation between the strains tested. The RAPD protocol using the P1254 primer and the SAU-PCR gave appreciable results only for strains isolated from animals and from a food processing plant in two different periods of the year 2003. Better differentiation was observed using the RAPD-PCR with primer D8635. As a matter of fact, it was able to distinguish L. monocytogenes obtained from different species of animals, different food samples and strains from the same production plant isolated in different periods of the year. Also primer M13 gave positive results, but the coefficient of similarity to use had to be increased to 80%. On the basis of the results observed, RAPD-PCR with primers D8635 and M13 should be considered reliable tools for epidemiological investigations focusing on L. monocytogenes.

Cluster Analysis↗

PubMed2006

Comparison of two chromogenic media for the detection of Listeria monocytogenes with the plating media recommended by EN/DIN 11290-1.

The efficacy of two selective chromogenic culture media, Agar Listeria Ottaviani and Agosti (ALOA) and RAPID' L. mono for the detection of Listeria monocytogenes in food, was compared with that of an official culture method according to the EN/DIN 11290-01 and -02 protocols [corresponding to the section 35 LMBG (German Food Act) method]. A total of 310 pre-packed ready-to-eat food samples (100 of graved and cold smoked salmon, 130 of different raw and cooked sausages and 80 of delicatessen and mixed salads) were examined. L. monocytogenes was identified in 52 investigated salmon samples. Using two chromogenic media, 50 samples were found positive for L. monocytogenes. Compared to the reference method there were no false-positive results. By the EN/DIN 11290-01 culture procedure after the selective enrichment in Fraser broth 12 out of 130 samples of sausages were positive for L. monocytogenes. These 12 samples were also positive for L. monocytogenes with the chromogenic medium RAPID' L. mono. One sample was false negative with ALOA. Three additional samples were found positive with ALOA and four with RAPID' L. mono. The standard method was inadequate to confirm these samples as positive. Listeria spp. were isolated from 7 samples of mixed salads with both methods. One, 3 and 3 samples were found to contain L. monocytogenes, L. innocua and L. seeligeri, respectively. Both chromogenic media enabled a rapid and specific detection of L. monocytogenes within 24h after enrichment. Visual detection of pathogenic L. monocytogenes and other Listeria spp. was easier on chromogenic media.

Agar↗

PubMed1990

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods.

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.

Enzyme-Linked Immunosorbent Assay↗

PubMed1995

Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods.

DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non-Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non-Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37 degrees C and/or 43 degrees C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella.

Base Sequence↗

PubMed2002

Practical lessons from the management of an outbreak of small round structured virus (Norwalk-like virus) gastroenteritis.

An outbreak of viral gastroenteritis caused illness in 92 people during October 2000. All the cases had an association with one hotel and 80% of cases identified had attended one of two buffet meals on 18th October 2000. Cohort analysis did not implicate any particular foodstuff in this outbreak. All bacteriological tests on food samples were negative. Small round structured virus (SRSV)/Norwalk-like virus (NLV) particles were seen by electron microscopy in 6 out of 27 stool samples submitted for analysis. This paper describes the outbreak and in particular the lessons learned from the management and control of it. These include practical points with regard to the outbreak control team, liaison with the microbiology laboratory, handling data, agreeing a case definition, and occupational health issues. All can be applied elsewhere by those responsible for managing outbreaks at a local level.

Caliciviridae Infections↗

PubMed2003

Immediate-type hypersensitivity reaction to ingestion of mycoprotein (Quorn) in a patient allergic to molds caused by acidic ribosomal protein P2.

BACKGROUND: Quorn is the brand name for a line of foods made with so-called "mycoprotein," which springs from the mold Fusarium venenatum. Since the introduction on the food market, there have been complaints from consumers reporting adverse gastrointestinal reactions after ingestion of mycoprotein. To date, it is not clear whether the reported symptoms are IgE-mediated. OBJECTIVE: The aim of the study was to describe for the first time a case history of an asthmatic patient with severe hypersensitivity reactions to ingested mycoprotein and to identify and characterize the potential allergen that might be responsible for this. METHODS: The sensitization pattern of the asthmatic subject was characterized, and food allergy to mycoprotein was assessed by double-blinded placebo-controlled food challenge. Afterward, specific IgE antibodies of the serum of this patient were used to screen a Fusarium culmorum cDNA expression library. The coding sequence of one enriched cDNA-clone was expressed in Escherichia coli to produce a recombinant protein that was further purified and immunologically characterized. RESULTS: The patient showed high sensitization to many known aeroallergens but apart from Quorn not to any other tested food samples. The deduced amino acid sequence of the enriched cDNA-clone (Fus c 1) showed large identity to the 60S acidic ribosomal protein P2 which is highly conserved among several species and also described as minor allergen in other mold species. The frequency of IgE reactivity of sera from F culmorum -sensitized subjects to rFus c 1 was approximately 35%. By enzyme allergosorbent test inhibition, we found 65% inhibition of mycoprotein IgE reactivity by rFus c 1. On the opposite we found reduced IgE reactivity of rFus c 1 of 68% by using mycoprotein as inhibitor. CONCLUSIONS: Sensitization to mold allergens by the respiratory tract and subsequent oral ingestion of cross-reactive proteins may lead to severe food-allergic reactions. Thus, the 60S acidic ribosomal protein P2 of F venenatum probably is the reason for the described severe hypersensitivity reactions of the patient to Quorn-mycoprotein because of its potential cross-reactivity to the F culmorum allergen Fus c 1.

Adult↗

PubMed1993

Dose assessment for recent inhabitants living adjacent to zones heavily contaminated from the Chernobyl fallout.

Within the framework of the "International Chernobyl Project," selected areas in the Republics of Ukraine, Belarus, and Russia of the former USSR, contaminated by radioactive fallout from the Chernobyl accident in 1986, were investigated by international teams. In addition, environmental studies were carried out in areas officially declared as "uncontaminated regions" in order to corroborate this classification and to provide reference baseline data for the simultaneously performed medical investigations on health effects in the contaminated areas. Altogether, 141 measurements of the gamma dose rate, both outdoors and indoors, were carried out. Also, the radionuclide concentration in 58 soil and food sample was determined. In addition, results from 1,620 individual film dosimeter readings were analyzed. The results show that, in areas adjacent to those officially designated as contaminated regions (137Cs surface ground contamination > or = 37 kBq m-2), levels of environmental fallout contamination are insignificant. The additional resulting committed effective dose over the next 70 years due to the fallout is only a fraction of the corresponding value from the natural radiation environment.

Accidents↗

PubMed2006

The alphaPROX assay: fluorescence screening of the inhibitory effects of hydrophilic antioxidants on protein oxidation.

We report on a new and convenient high-throughput fluorescence technique for determining antioxidant capacities of hydrophilic food samples. The new method is called alphaPROX (anti protein oxidation) and is based on an equimolar complex of diphenylhexatriene propionic acid (DPHPA) and bovine serum albumin (BSA) in aqueous buffer at pH 7.4. DPHPA is a reporter fluorophore that becomes nonfluorescent upon free radical-induced oxidation. In a typical assay, the DPHPA/BSA complex is challenged with peroxyl radicals and shows almost the same susceptibility to oxidation as unlabeled BSA. The progress of protein oxidation and its inhibition by antioxidants at physiological pH is determined from the time-dependent decrease in DPHPA fluorescence intensity. The alphaPROX method was compared to other techniques frequently used to measure antioxidant capacities. In this article, representative results are provided for the inhibitory effects of pure food components, fruit juices, wines, and various polar plant extracts on protein oxidation.

Antioxidants↗

PubMed2005

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques.

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.

Bacterial Proteins↗

PubMed2006

Indirect measurement of the lag time distribution of single cells of Listeria innocua in food.

The distribution of log counts at a given time during the exponential growth phase of Listeria innocua measured in food samples inoculated with one cell each was applied to estimate the distribution of the single-cell lag times. Three replicate experiments in broth showed that the distribution of the log counts is a linear mapping of the distribution of the detection times measured by optical density. The detection time distribution reflects the lag time distribution but is shifted in time. The log count distribution was applied to estimate the distributions of the lag times in a liquid dairy product and in liver paté after different heat treatments. Two batches of ca. 100 samples of the dairy product were inoculated and heated at 55 degrees C for 45 min or at 62 degrees C for 2 min, and an unheated batch was incubated at 4 degrees C. The final concentration of surviving bacteria was ca. 1 cell per sample. The unheated cells showed the shortest lag times with the smallest variance. The mean and the variance of the lag times of the surviving cells at 62 degrees C were greater than those of the cells treated at 55 degrees C. Three batches of paté samples were heated at 55 degrees C for 25 min, 62 degrees C for 81 s, or 65 degrees C for 20 s. A control batch was inoculated but not heated. All paté samples were incubated at 15 degrees C. The distribution of the lag times of the cells heated at 55 degrees C was not significantly different from that of the unheated cells. However, at the higher temperatures, 62 degrees C and 65 degrees C, the lag duration was longer and its variance greater.

Animals↗

PubMed1986

Conversion of pyridoxine into 6-hydroxypyridoxine by food components, especially ascorbic acid.

In experiments designed to examine interactions between pyridoxine (PN) and food components, PN was found to be converted into an unidentified compound in the presence of the homogenates of various plant foods under mild conditions. The formation of the compound tended to be higher when food samples had a higher ascorbic acid (AsA) content. The reaction was neither thermal decomposition nor photodecomposition. This compound was also formed by incubating PN with AsA in the dark. Conversion of PN into the compound proceeded with oxidation of AsA, and was negligible under anaerobic conditions. The pH optimum for the reaction was between 4 and 7, and the temperature optimum was between 30 and 50 degrees C. The compound was purified by ion-exchange chromatography, isolated as colorless needles, and identified as 6-hydroxypyridoxine from UV, PMR, IR and MS spectral data. 6-Hydroxypyridoxine had neither vitamin B6 nor antivitamin B6 activity for Saccharomyces carlsbergensis 4228 (ATCC 9080). From these results, we inferred that hydroxylation of PN in the presence of food components, especially AsA, caused loss of vitamin B6 in plant foods during food processing, storage and cooking.

Ascorbic Acid↗

PubMed1993

Comparison and genomic sizing of Escherichia coli O157:H7 isolates by pulsed-field gel electrophoresis.

Genomic DNAs of Escherichia coli O157:H7 strains isolated from patients and food samples were analyzed by pulsed-field gel electrophoresis. The rare-cutting endonucleases SfiI and XbaI generated 6 and 10 distinct genomic profiles, respectively, for the 22 strains analyzed, indicating that this technique may find application for epidemiologic studies. Summation of XbaI fragments from five E. coli O157:H7 strains estimated the genomic length at ca. 4.7 Mb.

DNA, Bacterial↗

PubMedDate not supplied

Efficiency of two commercial ELISA kits compared with the BAM culture method for detecting Listeria in naturally contaminated foods.

The efficiency of 2 commercial enzyme-linked immunosorbent assay (ELISA) kits (Listeria-Tek and Tecra) for detecting Listeria in naturally contaminated foods was evaluated and compared with that of the culture method described in the Bacteriological Analytical Manual (BAM). Both ELISAs use modified University of Vermont (UVM-1) medium as a primary enrichment; the BAM method uses Listeria enrichment broth. Secondary enrichments for Listeria-Tek and Tecra, respectively, were Fraser broth and UVM-2, which contains additional acriflavin-HCl. When ELISA test results differed, secondary enrichments were tested against the other ELISA; Fraser broth was used to determine recovery rates because of its superiority over UVM-2. Of the 178 food samples examined, the presence of Listeria was detected and culturally confirmed in 38, 37, and 40 samples by the BAM, Listeria-Tek, and Tecra methods, respectively. Differences in results of the ELISAs compared with those of the BAM method were not statistically significant; however, differences between results of the 2 ELISA methods were significant. It was concluded that as rapid screening methods, the Listeria-Tek and the Tecra kits qualify as alternative methods to the BAM cultural method.

Bacteriological Techniques↗

PubMed1988

Rapid hydrophobic grid membrane filter-enzyme-labeled antibody procedure for identification and enumeration of Escherichia coli O157 in foods.

An O-antigen-specific monoclonal antibody, labeled by horseradish peroxidase-protein A, was used in a hydrophobic grid membrane filter-enzyme-labeled antibody method for rapid detection of Escherichia coli O157 in foods. The method yielded presumptive identification within 24 h and recovered, on average, 95% of E. coli O157:H7 artificially inoculated into comminuted beef, veal, pork, chicken giblets, and chicken carcass washings. In food samples from two outbreaks involving E. coli O157:H7, the organism was isolated at levels of up to 10(3)/g. The lower limit of sensitivity was 10 E. coli O157 per g of meat. Specific typing for E. coli O157:H7 can be achieved through staining with labeled H7 antiserum or tube agglutination.

Animals↗

PubMed1991

Mean daily intake of volatile N-nitrosamines from foods and beverages in West Germany in 1989-1990.

The levels of volatile N-nitrosamines were determined in 38 alcoholic drinks and 215 food samples prepared for human consumption using standard culinary practices. The analyses used gas chromatography-thermal energy analysis. Detectable levels were found in 80 (31.5%) of the individual samples. The average daily intake of volatile N-nitrosamines in West Germany in 1989-1990 was found to be: 0.28 microgram N-nitrosodimethylamine (NDMA)/day, 0.011 microgram N-nitrosopyrrolidine (NPYR)/day and 0.015 microgram N-nitrosopiperidine (NPIP)/day for men, and 0.17 microgram NDMA/day, 0.011 microgram NPYR/day and 0.015 microgram/NPIP/day for women. For men, 31% of the daily NDMA exposure results from the consumption of beer.

Adult↗