Salmonellae In Foods Research Articles

Automated simultaneous detection of low levels of listeriae and salmonellae in foods

Salmonella spp. and Listeria monocytogenes continue to be major pathogens of concern to food processors. However, routine screening of food samples to detect these pathogens is generally labor intensive and costly. Automated optical procedures for the detection of salmonellae and listeriae in foods were developed in our laboratory. In the present study we report their adaptation to a simultaneous recovery and detection procedure. Milk, shell eggs, fresh and ready-to-eat (RTE) meats or raw chicken contaminated with a combination of sub-lethally injured salmonellae and listeriae (10–50 cells each) were incubated for 6 h at 35°C in modified universal pre-enrichment broth (MUPB). Volumes (4 ml) were then transferred to vials containing selective liquid media for these pathogens (4 ml), and incubated overnight at 35°C in a BioSys instrument. The presence of the pathogens was identified by a black coloration of the media and a sharp drop in light transmittance caused by hydrogen sulfide production ( Salmonella organisms), or esculin hydrolysis ( Listeria organisms). There was no difference in the detection time of salmonellae when incubated alone or with listeriae, but listeriae grew at a slower rate in the presence of salmonellae, resulting in a delay of ≤1 h in their detection. Overall, the detection of 10–50 salmonellae and 10–50 listeriae in 25 g of the tested foods required a total of 24 h. Confirmation of the pathogens by PCR-based assay (6 h) was completed the following day directly from positive vials, requiring a total of ≤30 h for detection and confirmation. Negative samples required no confirmation. The testing system was confirmed in 70 naturally contaminated foods.

Automated Rapid Screening of Foods for the Presence of Salmonellae

We previously reported an accelerated method to detect salmonellae in foods. It consisted of a preenrichment step (6 h) followed by immunomagnetic separation (IMS) and automated detection, based on positive biochemical characteristics, during overnight incubation. We describe here a simplified procedure with a detection limit of 2 CFU/25 g of food, a procedure that does not require the IMS step. Liquid eggs, milk, ice cream, or poultry were inoculated with Salmonella or non-Salmonella Enterobacteriaceae organisms (2 to 100 CFU/25 g). Following preenrichment for 6 h at 35°C, samples (3 ml) were added to selective liquid media (6 ml). Automated light transmittance was recorded during incubation at 42°C in the BioSys instrument. Salmonella-positive samples were identified by a sharp drop in transmittance (caused by black discoloration of the media) within 18 h of incubation following the formation of hydrogen sulfide and its reaction with iron ions. Detection of 2 to 10 CFU/25 g of the tested foods was completed in 24 h. A total of 24 Salmonella spp., including heat-injured cells, and 37 non-Salmonella Enterobacteriaceae were tested using the procedure.

Automated detection of Salmonella spp. in foods

An automated method to detect salmonellae in foods was developed and tested in food samples intentionally contaminated with the test organisms. Liquid eggs, shell eggs, dry eggs, skim milk and chicken were spiked with Salmonella enteritidis, S. typhimurium or S. newport to yield 2 to 25 CFU per 25 g or ml of sample. Following pre-enrichment in universal pre-enrichment broth at 42°C for 6 h (eggs and milk) or 16 h (chicken), Salmonella cells were captured by immunomagnetic beads coated with Salmonella antibody (Vicam, Watertown, MA). The beads were transferred to selective liquid media containing carbohydrate (dulcitol or xylose), amino acid (lysine or ornithine), and H 2S indicator, and incubated at 42°C in the BioSys instrument (MicroSys, Ann Arbor, MI). Salmonella positive samples were identified by black discoloration of the media during incubation, while negative samples remained colorless. These color changes were recorded by the instrument. All the artificially contaminated samples tested positive within 15–18 h, while control samples remained negative during 24 h incubation. The results agreed with standard identification procedures. A total of 24 h was required to detect 2 to 25 CFU of the pathogen in 25 g or ml of eggs and milk, and up to 36 h in chicken, compared to 72 h in the standard methods.

The use of a Rapid Salmonella Latex Serogrouping Test (Spectate) to assist in the confirmation of ELISA-based Rapid Salmonella Screening tests

Spectate, a 10 min, simple, latex-based agglutination test for serogrouping of salmonellae, has been investigated as a tool to assist in the confirmation of ELISA presumptive positive broth samples when screening for salmonellae in foods. When obtaining a combined ELISA and Spectate positive result, there was a 90% (27/30) confidence limit of a genuine positive result, some one or two working days quicker than the traditional methods. Of the 10% (3/30) that were not confirmed as salmonellas, two gave a Spectate result which is advised as being a possible Citrobacter spp. and one sample gave a positive confirmation by Spectate only, which suggested a failure of the traditional confirmation process, a finding confirmed at other sites. Extensive studies performed at several food company microbiology laboratories showed Spectate to be a useful additional tool in the confirmation process of ELISA screening techniques for salmonellae in food. Additionally the concept of a "false positive' may need to be refined to that of a "not culturally' positive, given the apparent possible failure of the traditional confirmation methods.

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.

Rapid Detection of Salmonellae by Means of a New Impedance-Splitting Method

An Impedance-Splitting method is proposed for the rapid detection of salmonellae in foods. The measuring System, BacTrac™ 4100, permits the registration of changes, caused by bacterial metabolism, not only of the impedance of the culture medium but also of changes in the ionic layers at the measuring electrodes, which has advantages in case of high salt concentrations. These changes are expressed as percentage decreases of the initial values, M-value and E-value, respectively. Food samples were pre-enriched 14 to 16 h at 37°C in peptone water by addition of mannitol, which facilitated the detection of salmonellae on selective culture media. Following this, 0.1 mi of the preenrichment culture was transferred to 9.9 ml of Impedance-Splitting Salmonellae (ISS) medium which consisted of magnesium chloride (hydrated), malachite green oxalate, novobiocin, phosphate buffer, mannitol, peptone and yeast extract. Despite the high magnesium chloride concentration in this medium, salmonellae produced changes of the E-value up to 100%, while the changes in M-values were limited to a few percent. The impedance changes were automatically recorded during incubation in the measuring system for up to 22 h at 40°C, and the time required to exceed a threshold value of 15% (E reaction time) was evaluated. Comparative testing of the ISS method with standard cultural analysis of 250 unknown food samples showed high sensitivity and selectivity in detecting salmonellae. From all of the 122 Salmonella-positive samples, the largest number (119) was obtained by the ISS method, as compared to that obtained by conventional testing with the selenite-cystine (106), Rappaport Vassiliadis soya (95), Rappaport Vassiliadis (92) and tetrathionate brilliant green medium (64). Six samples were false positive by Enterobacter cloaceae. One strain each of Salmonella enteritidis PT8 and Salmonella panama were not recorded. The ISS method is very suitable as a screening test, all the more since a negative investigation result will be obtained within 38 h. In view of the practicability, this method is superior to the enzyme-immunological and molecular-biological procedures.

Fluorescent detection‐polymerase chain reaction (FD‐PCR) assay on microwell plates as a screening test for salmonellas in foods

This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100-extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS200. The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55 degrees C, the microwells were washed and an alkaline phosphatase-labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 microliters of 1 mmol 1(-1) AttoPhos (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1-10 cfu. A total of 172 food samples were tested, both by culture and FD-PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD-PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD-PCR assay described here can be useful to screen a large number of food samples for contamination by salmonellas.

Rapid and alternative methods for the detection of salmonellas in foods.

Rapid and alternative methods for the detection of salmonellas in foods.

Experimental enzyme-linked amperometric immunosensors for the detection of salmonellas in foods.

Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10(4) cfu/ml (phenyl phosphate system) or 10(5) cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1-5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).

Salmonella Detection in Meat and Fish by Membrane Hybridization with Chromogenic/Phosphatase/Biotin DNA Probe

ABSTRACTThe hybridization specificity of a biotin labeled 1.8 kb HindIII DNA fragment was confirmed by colony hybridization with Salmonella and non‐Salmonella isolates. Culture conditions were then tested for the enrichment of salmonellae in foods with large populations of competitive flora. Different conditions of preenrichment and selective enrichment could be used for detecting low populations of salmonellae in foods. Enrichment using lactose combined tetrathionate (CTET) broth and Salmonella‐Shigella (SS) agar followed by spotting the suspected isolates on a nitrocellulose membrane [CETE → SS (S)] was better. As low as 1.6 × 100 salmonellae/g food could be detected.

Comparative Study of Colorimetric DNA Hybridization Method and Conventional Culture Procedure for Detection of Salmonella in Foods

A second generation nucleic acid hybridization assay has been developed and evaluated against the conventional culture method for detection of salmonellae in foods. The assay involves a liquid hybridization with Salmonella-specific oligonucleotide probes, capture of probe:target hybrids onto a solid support (plastic dipstick), and a colorimetric end point detection. The assay can be completed in 2.5 h, following approximately 44 h of culture enrichment. One thousand samples representing 20 food types were analyzed in parallel by both methods. Samples included uninoculated test product, and product inoculated with Salmonella at 2 levels. Eighteen Salmonella serotypes were used as inocula. The data demonstrate that the colorimetric hybridization method and the conventional culture method are equivalent in their ability to detect Salmonella contamination of foods.

Rapid detection of salmonella in foods—a convenient two-day procedure

A new method for detecting salmonellas in foods within 42 h is described. This highly specific and sensitive selective motility procedure gave an efficiency of 96.8% when tested against traditional methods using more than 800 food samples. It is stable at ambient temperatures and can be prepared for use in 5 min.

Immunodiffusion Screening Method for Detection of Motile Salmonella in Foods: Collaborative Study

A collaborative study was performed to validate the performance of the 1-2 TEST for detection of motile salmonellae in foods. Detection is based on observation of an immobilized band of cells. Twenty-three laboratories participated in the study. The 1-2 TEST (immunodiffusion test) was compared with the standard culture procedure (BAM/AOAC; FDA Bacteriological Analytical Manual) for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole egg, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. After the tests on the 6 foods were completed, analysis of the data for turkey and soy flour showed that certain collaborators obtained data inconsistent with the data from the majority of collaborators. No specific method deviations to account for the inconsistencies were reported by those collaborators, so the collaborative testing of these 2 foods was repeated. Analysis of data for pepper, chocolate, nonfat dry milk, dried whole egg, and the second set of soy flour and turkey indicated 96.1% agreement between the BAM/AOAC and immunodiffusion test methods. The false negative rates for the immunodiffusion test and BAM/AOAC methods were 3.6 and 1.7%, respectively. There was no significant difference in the productivity of the immunodiffusion test and BAM/AOAC method at the 5% level for any of the 6 foods. The immunodiffusion screening method has been approved official first action for detection of motile Salmonella in foods.

Assessment of an enzyme immunoassay for the detection of salmonellas in foods and animal feeding stuffs.

The Salmonella Bio-EnzaBead Screening Kit, in its modified form with both the MOPC 467 and the 6H4 antibodies, was used for the detection of salmonellas in naturally contaminated foods and animal feeding stuffs in parallel with a traditional cultural procedure. Initial results showed an 82% agreement between the enzyme immunoassay (EIA) and cultural methods when using the criterion recommended by the manufacturer as a cut-off for all types of foods. By adjusting the cut-off for each type of food, the number of EIA positive, culture negative samples was reduced although the number of EIA negative, culture positive samples increased. The EIA may be more sensitive than the cultural methods as in many cases the EIA positive, culture negative results could be real positives which were not detected by the cultural methods. The screening kit provides a simple and convenient method for the detection of salmonella in foods and feeds and a presumptive positive result can be reported within 48 h. The advantages and disadvantages of the method are discussed.

Direct immunoassay for detection of salmonellae in foods and feeds.

A direct enzyme immunoassay (EIA) with polyclonal antibodies was developed for detecting salmonellae in foods and feeds. Salmonella cells were attached firmly to the wells of polystyrene microtitration plates with a capture-antibody technique. Spicer-Edwards anti-H immunoglobulin G was bound to protein A-beta-D-galactosidase to serve as the signal; 4-methylumbelliferyl-beta-D-galactoside was used as the substrate. The sensitivity threshold was 10(7) cells per ml. Direct EIA, indirect EIA, and pure-culture techniques were compared by using 48 samples of naturally contaminated foods and feeds. The direct EIA was more sensitive than the indirect EIA or pure-culture technique. Food samples were analyzed within 3 working days, and 32 samples were tested simultaneously in a single 96-well microtitration plate. False-positive or false-negative results did not pose a problem. This direct EIA is sensitive, rapid, and amenable to automation.

An enzyme immunoassay for the detection of all Salmonella using a combination of a myeloma protein and a hybridoma antibody

An enzyme immunoassay was developed for the detection of salmonellae in foods and stool samples. The test is sensitive and specific, showing virtually no cross-reactivity to other enteric organisms. The sensitivity is such that <10 6 Salmonella organisms can be detected, and this is not influenced even by a 3 log 10 overgrowth of other bacteria. This assay can be performed routinely in any laboratory, and can be automated as much as desired. It can be performed from growth in liquid or on solid media. A myeloma protein (M467) and a hybridoma antibody (6H4) were attached to a polycarbonate coated metal bead. The beads were then incubated in a heat extract of the organisms for 20 min. After thorough washing by a magnetic transfer device, the beads were then incubated in a mixture of the 2 antibodies which were labeled with horseradish peroxidase. After another thorough washing and incubation in substrate, the reactions were either read by the naked eye or by spectrophotometry. The entire process following the culture takes less than 2 h.

Salmonella detection in foods and feeds in 27 hours by an enzyme immunoassay

A heterogeneous enzyme immunoassay (EIA), which could be completed within 27 h, was developed for the detection of salmonellae in foods. Samples were subjected to the usual non-selective enrichment for 16–18 at 35°C and to a short (6 h) post-enrichment in a moderately selective broth. The EIA was carried out on polystyrene microtitration plates. A pooled polyvalent Salmonella flagellar antiserum and a protein A-alkaline phosphatase conjugate were used. The sensitivity of the enzyme immunoassay compared favorably with that of the conventional cultural technique for detection of Salmonella in 40 naturally contaminated food and feed samples. No sample was positive only by the cultural technique; samples positive only by the enzyme immunoassay were observed for feeds. Some specimens yielded high background values indicating possible interference from food proteins.

An Improved Enzyme Immunoassay for the Detection of Salmonella

ABSTRACTAn enzyme immunoassay (EIA) was developed for the detection of salmonellae in foods or other samples. This assay detected Salmonella contamination within two working days after initiation, and the EIA required &lt;2 hr to perform. The test was sensitive and specific, showing virtually no cross reactivity to other organisms. No selective enrichment was found to be necessary, as the presence of other organisms, even in much greater numbers, had no effect on the reaction. The sensitivity is such that &lt;106Salmonella/mL were detected, and the food samples studied showed no effect on the background values and did not cause false readings.

Recovery of Salmonellae from Foods using a Combined Enrichment Technique

ABSTRACTThe separate steps of preenrichment in lactose broth and selective enrichment in tetrathionate (TET) or selenite‐cystine (SC) broths were combined into single procedures for the detection of both injured and uninjured salmonellae in foods. Following 4 hr of incubation, iodine or selenite were added to combined lactose‐tetrathionate (CTET) or selenite basal broths, forming selective environments. CTET broth was as efficient as conventional TET and SC broths but the combined selenite media were not. Salmonellae were detected in foods in 30 hr using direct fluorescent antibody and combined enrichment techniques. The combined procedure resulted in savings of assay time, labor and materials.

A Semi‐Automatic Method for the Detection of Salmonellas in Food Products

A semi‐automatic method for the detection of salmonellas in food products is described. This method involves measurement of the fluorescence intensity of preparations stained with fluorescent antibody. An overall agreement of 89·6% was obtained when the method was compared with a standard cultural technique for the detection of salmonellas in foods, feeds and environmental samples, although false negatives were a problem with raw meat products. A saving in time of at least 18 h was achieved.