Direct extraction of bacterial plasmids from food for polymerase chain reaction amplification.

In this report we describe a simple and rapid technique using DNA affinity columns that permits direct extraction of bacterial plasmids from a variety of foods for polymerase chain reaction amplification. The procedure was used to detect virulent enteroinvasive Escherichia coli in several artificially seeded matrices, including seafoods, greens, dairy products, enrichment media, and water. Polymerase inhibitors present in both foods and enrichment media were removed efficiently.     

Development of a two-step enrichment procedure for recovery of Yersinia enterocolitica from food.

Two new enrichment media were formulated for the recovery of Yersinia enterocolitica from foods: (i) yeast extract-rose bengal broth for preenrichment at 4 or 10 degrees C; and (ii) bile-oxalate-sorbose broth, a selective enrichment incubated at 22 degrees C. Comparison of these media in a two-step enrichment procedure against cold enrichment and modified Rappaport broth showed improved and more rapid recovery of human strains of Y. enterocolitica from inoculated foods. The use of bile-oxalate-sorbose broth as a selective enrichment also improved the performance of cold enrichment with phosphate-buffered saline. Determination of the best enrichment system for recovery of Y. enterocolitica from samples of retail pork and fresh pork tongues depended on whether the criterion was the number of positive samples, the variety of different serotypes recovered, or the ability to recover the important human serotype O:3. A single enrichment system with the widest selectivity would include preenrichment at 4 degrees C with either phosphate-buffered saline for 14 days or yeast extract-rose bengal broth for 9 days followed by selective enrichment with bile-oxalate-sorbose broth at 22 degrees C for 5 days.     

A comparison of immunomagnetic separation plus enrichment with conventional salmonella culture in the examination of raw sausages

Immunomagnetic separation with additional enrichment was used in conjunction with improved selective media to improve the isolation of salmonellae from raw sausages. The isolation rate achieved was almost double that of conventional culture with no increase in processing time. The selective media gave an overall specificity of approximately 74%; all false-positive pick-offs being identified as Citrobacter freundii . It is believed that this method represents a significant advance in the isolation of salmonellae from foods, although the ideal media both for enrichment and selection have yet to be found.     

A System for Detecting Salmonellae in Meat and Meat Products

Leifson's selenite F broth was more selective for salmonellae when incubated at 43° instead of the traditional 37°. Different selective agar media produced different numbers of colonies from similar inocula of salmonella cells, but Difco brilliant green agar consistently gave the highest recoveries when tested in this way. Combined with 43° selenite broth enrichment it provided a useful system for isolating salmonellae from foods. In a short comparative test this system compared favourably with more classical techniques employing enrichment of each sample at 37° in two different enrichment broths, followed by streaking on two selective agars.     

The Examination of Foods for Enterobacteriaceae using a Test of the Type Generally Adopted for the Detection of Salmonellae

S ummary : In countries that have recently increased their bacteriological inspection of foods, many products have shown considerable improvement in microbiological quality; simultaneously, however, discrepancies between salmonella and coli-aerogenes tests, especially on dehydrated and frozen foods, have tended to become more frequent. These discrepancies have been eliminated by applying the following measures: incorporating glucose in culture media so as to reveal all Enterobacteriaceae; placing reliance on growth rather than gas formation so as to avoid missing anaerogenic organisms, and, especially, examining quantities of foods commensurate with those used in salmonella tests.
For this purpose a procedure is recommended in which 10 g of well homogenized food are enriched in 100 ml of buffered brilliant green-bile-glucose broth, with no attention being paid to gas formation; the enrichment cultures are then streaked on to MacConkey's glucose agar. Single colonies so obtained are tested for fermentative attack on glucose and may be further examined for other characteristics. The same enrichment fluid can be used for the so-called 'nonselective pre-enrichment' of samples of food containing salmonellae impaired by periods spent in conditions of low water activity, low pH, etc.     

Listeria monocytogenes: diagnostic problems

The first isolation methods for the detection of Listeria spp. were generally based on the direct culture of samples on simple agar media, but isolation of the pathogenic Listeria monocytogenes was difficult. In time, new techniques were developed, based on a variety of selective and elective agents in isolation and enrichment media, which gained better and quicker results. Current reference methods allow the recovery of L. monocytogenes from a variety of foods with relative ease. However, more comparative studies are needed to select one horizontal method. It is suggested that the procedure of the International Organization for Standardization is a good base for such comparisons.     

The detection of non-O157 E. coli in food by immunomagnetic separation

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.     

The clones of Listeria monocytogenes detected in food depend on the method used

S. LONCAREVIC, W. THAM AND M.-L. DANIELSSON-THAM. 1996. Restriction enzyme analysis (REA) with pulsed-field gel electrophoresis (PFGE) has been used to characterize and compare Listeria monocytogenes strains isolated from foods by two methods, an enrichment procedure and a direct plating procedure. In total 151 isolates from nine foods were investigated. In six of the foods (101 strains investigated) only one clone of L. monocytogenes was found irrespective of the method used. In three foods (50 strains investigated) the direct plating procedure yielded more clones than the enrichment procedure. At the most, five clones were detected in the same food. The results presented here indicate that direct plating from the food reveals more L. monocytogenes clones than revealed by an enrichment procedure.     

Evaluation of new culture media for rapid detection and isolation of salmonellae in foods.

Conventional methods for Salmonella detection in foods can require up to 6 and at least 4 days. We have observed that the total analysis time can be reduced to 48 h by using Salmosyst broth as a liquid medium for both preenrichment and selective enrichment and Rambach agar (RA), a new selective plate medium. In samples of artificially contaminated ground beef Salmonella enteritidis was detected at a concentration of 0.4 CFU/g (10 CFU/25 g) by both a conventional method and the new method. Of 519 samples of foods for sale, 38 were Salmonella positive by both methods while 471 were negative. Nine samples which were negative by the conventional method were positive by the Salmosyst-RA method, while one sample positive by the first method was negative by the last. Therefore, the Salmosyst-RA method showed 97.9% sensitivity compared with the 81.2% sensitivity of the conventional method. The new method was also highly specific (98% specificity) in presumptive identification of Salmonella colonies. Furthermore, a 6-h preenrichment in Salmosyst broth has been proved sufficient for the repair of heat-injured Salmonella cells and for subsequent recovery by selective enrichment. In conclusion, the Salmosyst-RA method shows several advantages over both conventional and rapid noncultural methods: (i) only two media are required instead of the five media for conventional methods; (ii) in real time it is comparable to other rapid noncultural methods, which require 30 to 31 h; (iii) it is highly sensitive and specific; and (iv) it allows the isolation of Salmonella strains which can be characterized by appropriate phenotypic and genotypic typing methods for epidemiological investigations.     

Two modified selenite media for the recovery of Yersinia enterocolitica from meats.

Yersinia enterocolitica is one of the few human pathogens that grows at the proper food refrigeration temperatures of 0 to 5 degrees C. Although the isolation of environmental biotypes of Y. enterocolitica from many types of food and water has been reported in the literature, the recovery of the sensitive strains inoculated into foods has been slow and uncertain. Rapid recovery of several clinical strains inoculated into meats was accomplished by using two modified selenite broths without added nutrients. It was critical to restrict the sample size of the blended meat suspension at the 0.2-g/100 ml level, thereby restricting the growth of the total bacterial population in the selenite enrichment media. Otherwise, the slower growing Y. enterocolitica would be overwhelmed by the faster growing normal bacterial flora from the meat. Both the resistant serotype O:3 and the sensitive O:8 clinical isolates of Y. enterocolitica were recovered from the modified selenite enrichment media after 2 and 3 days of incubation at 22 degrees C.     

Methods for the detection and isolation of Shiga toxin-producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) are an important cause of haemorrhagic colitis and the diarrhoea-associated form of the haemolytic uraemic syndrome. Of the numerous serotypes of E. coli that have been shown to produce Shiga toxin (Stx), E. coli O157:H7 and E. coli O157:NM (non-motile) are most frequently implicated in human disease. Early recognition of STEC infections is critical for effective treatment of patients. Furthermore, rapid microbiological diagnosis of individual patients enables the prompt notification of outbreaks and implementation of control measures to prevent more cases. Most human infections caused by STEC have been acquired by the consumption of contaminated foods, especially those of bovine origin such as undercooked ground beef and unpasteurized cows' milk, and by person-to-person contacts. To identify the reservoirs of STEC and the routes of transmission to man, sensitive methods are needed as these pathogens may only be present in food, environmental and faecal samples in small numbers. In addition, sensitive and rapid detection methods are necessary for the food industry to ensure a safe supply of foods. Sensitive methods are also needed for surveillance programmes in risk assessment studies, and for studies on survival and growth of STEC strains. Cultural methods for the enrichment, isolation and confirmation of O157 STEC are still evolving. Several selective enrichment media have been described, of which modified tryptone soy broth with novobiocin and modified E. coli broth with novobiocin, seem to be the most appropriate. These media are minimally-selective broths that give a somewhat limited differential specificity favouring isolation of O157 STEC, as opposed to other Gram-negative bacteria, in the sample. An incubation temperature of 41-42 degrees C further enhances selectivity. The occurrence of heat-, freeze-, acid- or salt-stressed STEC in foods means that it is important to be able to detect cells that are in a stressed state, as STEC generally have a very low infectious dose, and injured cells mostly retain their pathogenic properties. For the isolation of stressed O157 STEC, pre-enrichment in a non-selective broth is necessary. The most widely used plating medium for the isolation of typical sorbitol-non-fermenting strains of STEC of serogroup O157 is sorbitol MacConkey agar with cefixime and tellurite (CT-SMAC). As some STEC strains are sensitive for tellurite and/or are sorbitol-fermenting, the use of a second isolation medium, such as one of the newer chromogenic media, is recommended. Immunomagnetic separation (IMS) following selective enrichment, and subsequent spread-plating of the concentrated target cells onto CT-SMAC agar, appears to be the most sensitive and cost-effective method for the isolation of E. coli O157 from raw foods. IMS increases sensitivity by concentrating E. coli O157 relative to background microflora, which may overgrow or mimic O157 STEC cells on selective agars. While cultural isolation of O157 STEC from foods and faeces is time-consuming, labour-intensive and hence, costly, rapid immunological detection systems have been developed which significantly reduce the analysis time. These methods include enzyme-linked immunosorbent assays (ELISAs), colony immunoblot assays, direct immunofluorescent filter techniques, and several immunocapture techniques. Both polyclonal and monoclonal antibodies specific for the O and H antigens are used for these methods. Many of these test systems are able to detect less than one O157 STEC cell g(-1) of raw meat after overnight enrichment. Presumptive results are available after just one day, but need to be completed with the isolation of the organisms. The primary use of these procedures is therefore to identify food and faecal samples that possibly contain O157 STEC.     

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.     

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood.

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.     

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 hlyA gene spanning a conserved HindIII 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 10(4) 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.     

Enzyme immunoassay for detection of Salmonellae in foods.

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique.     

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.     

Comparison of Fluorescent-Antibody Methods and Enrichment Serology for the Detection of Salmonella

Four rapid methods for detection of Salmonella, (i) the conventional fluorescent-antibody (FA) technique, (ii) a rapid direct FA technique, (iii) microcolony FA, and (iv) enrichment serology (ES), were compared with conventional cultural procedures. A total of 347 subsamples representing 16 different food prototypes, alleged to be naturally contaminated with Salmonella, were analyzed. From these samples, 52 were found to contain Salmonella by cultural methods. Conventional FA identified all 52 culturally positive samples, ES identified 51, microcolony FA identified 48, and the rapid FA method identified 34. The number of false-positive samples for each procedure was: ES-selenite, 7; tetrathionate, 8; rapid FA, 26; microcolony FA, 33; conventional FA-selenite, 27; tetrathionate, 26. Tetrathionate enrichment was found to be superior to selenite for Salmonella recovery from most foods, but the concurrent use of both media allowed maximum recovery.     

Examination of Prepared Foods in Plastic Packages for Clostridium botulinum

The incidence of Clostridium botulinum organisms was determined in a variety of plastic-packaged "vulnerable" foods (food requiring little or no heating prior to consumption). A total of 113 foods were examined by use of an enrichment recovery procedure followed by toxin testing in animals. Results of the survey indicate that the incidence of C. botulinum organisms in these vulnerable foods is extremely low. The ability of inoculated food products to support growth and toxigenesis of C. botulinum type E was then tested. The 64 packaged foods were inoculated with type E spores and incubated anaerobically at 30 C for 11 days. A slurry of each food was prepared, smears for fluorescent-antibody testing were made, and animal tests were performed for toxin. If the animal tests were negative, enrichment cultures were prepared from the slurry and incubated at 30 C. On direct examination of the slurries for toxin, only samples of turkey roll and soybean cake supported growth and toxigenesis by C. botulinum type E. However, the enrichment culture method was able to induce growth and toxin production in 60 of the remaining 62 samples.     

Timed-Release Capsule Method for the Detection of Salmonellae in Foods and Feeds

A new method was developed for the detection of injured and uninjured salmonellae in foods and feeds. The steps of pre-enrichment in a nonselective broth and selective enrichment in a selective medium were combined into a single procedure. This was achieved by the gradual release of selective agents from wax-coated gelatin capsules added at the time of inoculation of nonselective basal broths. Pre-enrichment in lactose broth was combined with selective enrichment in tetrathionate or selenite-cystine broth by using timed-release capsules containing iodine or selenite. Five different categories of foods and feeds, naturally contaminated with salmonellae, were examined to compare the efficiencies of the capsule methods with conventional procedures. Combination of the separate steps of pre-enrichment and selective enrichment into a single procedure was feasible and resulted in substantial savings of labor and materials.     

Competitive fitness of Listeria monocytogenes serotype 1/2a and 4b strains in mixed cultures with and without food in the U.S. Food and Drug Administration enrichment protocol

Thirteen different serotypes of the food-borne pathogen Listeria monocytogenes have been described. Serotype 4b strains are most often associated with illness, and serotype 1/2a strains are most often isolated from foods and processing plants. Different abilities to respond to stresses have been described for serotype 4b and 1/2a strains. One of the common enrichment protocols used to test foods for the presence L. monocytogenes is described in the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM). We compared three strains of L. monocytogenes serotype 4b and five strains of serotype 1/2a in direct competition with each other in two-strain mixed cultures by using the FDA BAM enrichment protocol, which includes both enrichment broth and selective agar, with and without added food to mimic the conditions that occur during attempts to isolate Listeria species from contaminated foods. Using a colony immunoblot procedure and analyzing over 112,000 colonies, we observed differences in strain fitness, but these differences were not attributable to serotype or genetic lineage.