Salmonella Testing of Pooled Pre-Enrichment Broth Cultures for Screening Multiple Food Samples

A method has been described for testing multiple food samples for Salmonella without loss in sensitivity. The method pools multiple pre-enrichment broth cultures into single enrichment broths. The subsequent stages of the Salmonella analysis are not altered. The method was found applicable to several dry food materials including nonfat dry milk, dried egg albumin, cocoa, cottonseed flour, wheat flour, and shredded coconut. As many as 25 pre-enrichment broth cultures were pooled without apparent loss in the sensitivity of Salmonella detection as compared to individual sample analysis. The procedure offers a simple, yet effective, way to increase sample capacity in the Salmonella testing of foods, particularly where a large proportion of samples ordinarily is negative. It also permits small portions of pre-enrichment broth cultures to be retained for subsequent individual analysis if positive tests are found. Salmonella testing of pooled pre-enrichment broths provides increased consumer protection for a given amount of analytical effort as compared to individual sample analysis.     

Application of an automated immunomagnetic separation-enzyme immunoassay for the detection of Salmonella spp during an outbreak associated with a retail premises

AIMS: The application of an automated immunomagnetic separation-enzyme immunoassay (AIMS-EIA) during the investigation of a suspected outbreak of Salmonella food poisoning at a retail premises. METHODS AND RESULTS: Six food samples and 24 environmental swabs were taken from the retail premises and six food handlers' submitted faecal samples during the investigation of the outbreak. Isolation and identification of Salmonella from these samples was performed according to established standard operating procedures and by AIMS-EIA. Twelve of the 18 (67%) Salmonella culture positive samples were AIMS-EIA positive on testing pre-enrichment samples after 24 h, whilst 17 (94%) samples were AIMS-EIA positive following selective enrichment for a further 48 h. One food handler was found to be positive for Salmonella by both culture and AIMS-EIA. All Salmonella isolates were confirmed as Salmonella Enteritidis phagetype 21c. CONCLUSIONS: The AIMS-EIA protocol compliments the conventional culture approach to produce more timely results for the management of the risk to public health without significantly increasing the workload of the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: The food production premise investigated in this study was heavily contaminated with Salmonella Enteritidis. Application of the AIMS-EIA was significant in the effective intervention of control measures for the protection of public health.     

Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction

Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.     

Application of BAX system, Tecra Unique Salmonella test, and a conventional culture method for the detection of Salmonella in ready-to-eat and raw foods

AIMS: To compare the BAX system, the Tecra Unique Salmonella test, and a conventional culture method for the detection of Salmonella in various foods. METHODS AND RESULTS: Ready-to-eat and raw foods were inoculated with Salmonella serotype Typhimurium, Salmonella serotype Enteritidis, Salmonella serotype Typhi, or Salmonella serotype Derby. Incubated pre-enrichment cultures were examined using the BAX system, the Tecra Unique Salmonella test, and a conventional culture method. Salmonella could be detected in all ready-to-eat food samples inoculated with S. Typhimurium, S. Enteritidis, or S. Derby, with any of the three test methods. However, false negatives were obtained with the Tecra test and the culture method when samples with higher background flora were inoculated with S. Typhi. Sensitivity test results suggested the two rapid tests performed as well as the culture method in the detection of 10(1) CFU of S. Typhimurium in 25-g cooked or raw food. CONCLUSIONS: The BAX system and the Tecra Unique Salmonella test demonstrated results comparable with those of the culture method in the detection of Salmonella serotypes used except S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first evaluation of the BAX system, the Tecra Unique Salmonella test, and a culture method in the detection of Salmonella in a variety of western and oriental foods.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum. The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

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 degrees 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 H2S indicator, and incubated at 42 degrees 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.     

A Rapid, Presumptive Procedure for the Detection of Salmonella in Foods and Food Ingredients

A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory.     

Improved bioluminescent enzyme immunoassay for the rapid detection of Salmonella in chicken meat samples

AIMS: To evaluate an improved bioluminescent enzyme immunoassay (BEIA) using biotinylated firefly luciferase for the rapid detection of Salmonella in naturally contaminated chicken meat samples. METHODS AND RESULTS: Capture agents and lipopolysaccharide (LPS) extraction reagents for Salmonella were investigated to improve the sensitivity of the BEIA. Also, the use of Oxoid SPRINT (Simple Pre-enrichment and Rapid Isolation New Technology) as a pre-enrichment and selective medium for 26-h BEIA detection of Salmonella in chicken meat samples was examined. The use of polymyxin B as a capture agent on solid support and 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS) for extraction of the LPS facilitated sensitive detection of Salmonella. Of 120 chicken meat samples, 25 samples were positive using the improved BEIA with the SPRINT and 25 samples were positive using the SPRINT followed by the standard isolation methods. CONCLUSIONS: The improved BEIA, in which polymxin B was used as a capture agent and CHAPS was used for extraction of the antigen, had a sensitivity of 96.0% and a specificity of 98.9% for the detection of Salmonella in chicken meat. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved BEIA combined with the SPRINT medium for the detection of Salmonella in chicken meat samples produced comparable results to the culture methods in 26 h.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

APPLICATION OF POLYMERASE CHAIN REACTION-OLIGONUCLEOTIDE LIGATION ASSAY FOR THE DETECTION OF SALMONELLAE IN PROCESSED MEAT, POULTRY, FISH, AND PET FOODS

We have tested a rapid and sensitive DNA-based assay for the detection of Salmonella serovars in a number of different processed meat, fish, poultry, and pet food samples. This technique uses an enrichment broth cultivation followed by a Salmonella-specific polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) to specifically detect amplified PCR products in an ELISA-based microtiter plate format. The combined cultivation and PCR-OLA techniques were compared with a conventional culture method and with DNA hybridizations of PCR products for the detection of Salmonella bacteria. Eighty-one different processed meat, poultry, and pet food samples were screened for the presence of Salmonella serovars after 24 h and 48 h of enrichment broth cultivation. After 24 h of incubation, one ground turkey sample was positive by both culture and PCR-OLA (100% sensitivity and 100% specificity). After 48 h of incubation, two additional samples (ground beef and a dog food sample) were positive by both culture and PCR-OLA (100% sensitivity and 100% specificity), and three other samples (two ground beef samples and one ground turkey) were positive only by PCR-OLA (96.1% specificity). All positive PCR-OLA results were confirmed in DNA hybridizations with an oligonucleotide specific for the amplified PCR product. When compared to conventional culture, the combined 48 h enrichment and PCR-OLA had a positive predictive value of 50% and a negative predictive value of 100%. We concluded that a combined cultivation and PCR-OLA could be used as a sensitive and specific presumptive screening method for detecting Salmonella serovars in processed meat, fish, poultry, and pet foods.     

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.     

Improved immunological membrane filter method for detection of food-borne Salmonella strains.

An improved membrane filter method that involves the use of an enzyme-labeled antibody stain has been developed for the rapid detection of Salmonella species in foods. The procedure is carried out directly on a hydrophobic grid-membrane filter without requiring transfer by blotting to nitrocellulose. Pure cultures of 54 Salmonella species and 10 foods artificially contaminated with Salmonella colindale gave a positive reaction in which Salmonella colonies were visible as purple dots. Of 11 nonsalmonella organisms, only Citrobacter freundii reacted with Spicer-Edwards antiserum. Of 22 naturally contaminated food samples, 10 were positive for both the hydrophobic grid-membrane filter procedure of the Association of Official Analytical Chemists and the improved enzyme-labeled antibody stain method, and there was perfect agreement between the methods. Of these 10 positive samples, one was negative by the Health Protection Branch method; of the negative samples, two were positive by this latter method. The improved enzyme-labeled antibody stain method allows detection of Salmonella spp. in foods within 48 h, requires little equipment, and is inexpensive, easy to perform, and suitable for automated detection.     

Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project

A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).     

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity

AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.     

Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods

AIMS: The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods. METHODS AND RESULTS: A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens(R) Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 101 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification. CONCLUSIONS: For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 102 and 101 CFU per 25 g food sample. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.     

Accelerated Immunofluorescence Procedure for the Detection of Salmonella in Foods and Animal By-Products

An accelerated, direct immunofluorescent-antibody procedure was developed for the detection of Salmonella in food products. This method includes pre-enrichment and selective enrichment but eliminates many of the washing and smear treatments present in existing methods. Commercially available fluorescein-conjugated somatic antiserum was used in comparing this method with conventional culture, biochemical, and serological procedures. The 894 samples tested represented 39 different products. The fluorescent-antibody procedure detected Salmonella in 216 test samples as compared to 205 positives recovered by using the standard culture procedures. In no instance did the fluorescent-antibody procedure fail to detect a Salmonella positive which had been detected by the standard procedure. With a three-tube, most-probable-number procedure, the fluorescent-antibody method was able to detect Salmonella at a level of 0.036 organism per g. In addition to being a more rapid method for the detection of Salmonella, it has proven to be comparable to conventional culture procedures.     

Improved immunological membrane filter method for detection of food-borne Salmonella strains

An improved membrane filter method that involves the use of an enzyme-labeled antibody stain has been developed for the rapid detection of Salmonella species in foods. The procedure is carried out directly on a hydrophobic grid-membrane filter without requiring transfer by blotting to nitrocellulose. Pure cultures of 54 Salmonella species and 10 foods artificially contaminated with Salmonella colindale gave a positive reaction in which Salmonella colonies were visible as purple dots. Of 11 nonsalmonella organisms, only Citrobacter freundii reacted with Spicer-Edwards antiserum. Of 22 naturally contaminated food samples, 10 were positive for both the hydrophobic grid-membrane filter procedure of the Association of Official Analytical Chemists and the improved enzyme-labeled antibody stain method, and there was perfect agreement between the methods. Of these 10 positive samples, one was negative by the Health Protection Branch method; of the negative samples, two were positive by this latter method. The improved enzyme-labeled antibody stain method allows detection of Salmonella spp. in foods within 48 h, requires little equipment, and is inexpensive, easy to perform, and suitable for automated detection.     

Rapid radiometric method for detection of Salmonella in foods.

A radiometric method for the detection of Salmonella in foods has been developed which is based on Salmonella poly H agglutinating serum preventing Salmonella from producing 14CO2 from [14C]dulcitol. The method will detect the presence or absence of Salmonella in a product within 30 h compared to 4 to 5 days by routine culture methods. The method has been evaluated against a routine culture method using 58 samples of food. The overall agreement was 91%. Five samples negative for Salmonella by the routine method were positive by the radiometric method. These may have been false positives. However, the routine method may have failed to detect Salmonella due to the presence of large numbers of lactose-fermenting bacteria which hindered isolation of Salmonella colonies on the selective agar plates.     

A rapid, sensitive and automated method for detection of Salmonella species in foods using AG-9600 AmpliSensor Analyzer

The AG-9600 AmpliSensor Analyzer is an automated fluorescence-based system for detection of polymerase chain reaction (PCR) products. The principle of the AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to a target sequence within a 284-bp amplified fragment of the Salmonella invA gene. Since the assay is homogenous, the data can be obtained by direct measurement of fluorescence of the amplification mixture. The accumulation of the amplified product, reflected by the fluorescence index, is monitored cycle by cycle by the AG-9600 Analyzer. The detection limit of the assay was less than 2 colony-forming units (cfu) per PCR reaction using a pure culture of Salmonella typhimurium. In post-spiking experiments in which Salmonella was added to the overnight pre-enriched samples (chicken carcass rinses, ground beef, ground pork and raw milk), the detection limit of the assay was 2–6 cfu per PCR reaction. In pre-spiking experiments in which Salmonella was added to the samples prior to overnight pre-enrichment, the detection limit was less than 3 cfu per 25 g or 25 ml of food. The assay was up to 2 orders of magnitude more sensitive than detection by ethidium bromide-stained agarose gel electrophoresis. To further evaluate assay performance, 54 naturally contaminated chicken carcass rinses, 65 raw milk and six ground pork samples were tested in the study. Thirty-eight Salmonella- positive samples confirmed by the Modified Semi-solid Rappaport-Vassiliadis (MSRV) culture assay were found positive using the AmpliSensor assay. Two chicken carcass rinses found positive using the assay were MSRV-negative. In addition, relative quantification using the AmpliSensor assay was linear up to 3 logs of initial target concentration in artificially contaminated food samples.     

Addition of Novobiocin in pre-enrichment step can improve Salmonella culture protocol of modified semisolid Rappaport-Vassiliadis

The aim was to investigate the effect of addition of Novobiocin to the non-selective buffered peptone water (BPW) for pre-enrichment of Salmonella in connection with plating on modified semisolid Rappaport-Vassiliadis (MSRV). In a semi-quantitative study, the level of Salmonella following pre-enrichment of 32 presumably naturally contaminated swine fecal samples were assessed for BPW with and without addition of Novobiocin (22 microg/ml). In another experiment, a total of 400 swine fecal samples were screened for the presence of Salmonella spp., in order to compare the performance of the non-selective pre-enrichment broth with BPW made semi-selective by addition of Novobiocin. The semi-quantitative assessment of the Salmonella level showed that addition of Novobiocin in the pre-enrichment step on average increased the level of Salmonella 1.2 log dilution steps. When growth was scored at five levels, 90 samples opposed to 50 yielded a strong positive reading (+++) when Novobiocin was applied. Growth was on average 0.3 scores higher when pre-enriched with Novobiocin. The difference in growth score medians of the two methods was highly significant (Sign test; p<0.001). Despite the increased sensitivity, 13 culture-positive samples were missed when using the Novobiocin-containing BPW. In conclusion, a simple addition of Novobiocin in the BPW pre-enrichment step of fecal samples may facilitate reading and thereby detection of Salmonella on MSRV. The increase of Salmonella in the semi-quantitative study may be caused by a reduction in the number of competitive microorganisms.