Detection of Salmonella Enteriditis from Egg Components Using Different Immunomagnetic Beads and Time-resolved Fluorescence

The types of chemical linkage used to bind antibodies to magnetic beads to form immunomagnetic beads (IMB) were compared in the capture and detection of Salmonella Enteriditis from egg white, egg yolk, and whole egg. Egg components were inoculated with outbreak strains of S. Enteriditis. After incubation under different conditions, IMBs derived from linking antibodies to core magnetic beads via biotin–streptavidin interactions, Schiff-base bonds and unspecified proprietary chemistry were used to capture S. Enteriditis. Europium-labeled anti-Salmonella antibodies completed the sandwich, and time-resolved fluorescence served as the means of detection. For the Salmonella isolated in stationary phase and cultured from universal pre-enrichment broth (UPB), the detection signal intensity was affected by the chemistry utilized to link the antibodies to IMB, with results varying among the three test strains. When S. Enteriditis was cultured in egg yolk alone, plating data were similar to those of the growth of S. Enteriditis in UPB. Egg white by itself did not support the growth of S. Enteriditis. The addition of UPB to egg white restored the growth of Salmonella and yielded stronger detection signals than from cultures obtained from UPB with egg yolk. The detection signals obtained from the immunoassay were less intense for cultures grown in egg yolk + UPB than from cultures grown in UPB alone. The lower detection signals elicited by all IMBs suggest the availability of the antigenic groups recognized by the antibodies on IMBs was reduced in the presence of egg yolk.     

Rapid and simultaneous quantification of viable Escherichia coli O157:H7 and Salmonella spp. in milk through multiplex real-time PCR

Escherichia coli O157:H7 and Salmonella spp. in milk are 2 common pathogens that cause foodborne diseases. An accurate, rapid, specific method has been developed for the simultaneous detection of viable E. coli O157:H7 and Salmonella spp. in milk. Two specific genes, namely, fliC from E. coli O157:H7 and invA from Salmonella spp., were selected to design primers and probes. A combined treatment containing sodium deoxycholate (SDO) and propidium monoazide (PMA) was applied to detect viable E. coli O157:H7 and Salmonella spp. only. Traditional culture methods and SDO-PMA-multiplex real-time (mRT) PCR assay were applied to determine the number of viable E. coli O157:H7 and Salmonella spp. in cell suspensions with different proportions of dead cells. These methods revealed consistent findings regarding the detected viable cells. The detection limit of the SDO-PMA-mRT-PCR assay reached 10² cfu/mL for Salmonella spp. and 10² cfu/mL for E. coli O157:H7 in milk. The detection limit of SDO-PMA-mRT-PCR for E. coli O157:H7 and Salmonella spp. in milk was significantly similar even in the presence of 10⁶ cfu/mL of 2 nontarget bacteria. The proposed SDO-PMA-mRT-PCR assay is a potential approach for the accurate and sensitive detection of viable E. coli O157:H7 and Salmonella spp. in milk.     

Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR

The objective of this study was to develop a rapid multiplex PCR (m-PCR) method using pure bacterial cultures and pork that would allow the simultaneous detection of five major foodborne pathogens likely to be found in pork (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella and Yersinia enterocolitica). Five pairs of primers were designed to identify invA gene, hlyA gene, rfbE gene, nuc gene and ail gene for Salmonella, L. monocytogenes, E. coli O157:H7, S. aureus and Y. enterocolitica, respectively. On the basis of determining specificity of the m-PCR, sensitivity was performed with an overnight enrichment step in culture media and pork. The 16S rRNA gene was targeted as an internal control gene of the presence of bacterial DNA. The results suggested that the m-PCR was an effective procedure having high specificity for the simultaneous detection of the five target pathogens. After overnight culture, the detection limit was 10³ cfu/mL for the simultaneous detection of the five target pathogens and less than 10 cfu/mL for detection of a single pathogen. The m-PCR protocol successfully detected all five organisms inoculated overnight together on pork. Salmonella, S. aureus, L. monocytogenes, Y. enterocolitca and E. coli O157:H7 were detected at levels of 142, 51, 9, 33 and 670 cfu/mL, respectively, in the pork. The m-PCR assay developed in this study could provide an effective and informative supplement for routine monitoring for pork safety.     

Microbiological Profile of Goat Meat Inoculated with Lactic Acid Bacteria Cultures and Stored at 30��C for 7?days

The ability of lactic acid bacteria (LAB) cultures to preserve goat meat at 30°C was evaluated in the present study. Strains of Pediococcus pentosaceus GOAT 01 and Lactobacillus plantarum GOAT 012, individually and in combination, were applied as starters on sliced meat samples at 6 log cfu/g and stored for 7 days at 30°C to simulate ambient temperature in Nigeria. They were evaluated for microbiological profile during storage. Reduction in bacterial counts was recorded for enterobacteriaceae, Staphylococcus, yeasts and moulds in starter culture inoculated samples (SCIS), whereas an increase occurred in uninoculated control samples (UCS). In challenge experimental trials, two different sets of meat were inoculated individually with 6 log cfu/g each of pathogenic organisms, Listeria monocytogenes and Salmonella Typhimurium. The inoculated pathogens were monitored during storage to assess the influence of starter cultures on them. Approximately 1 log reduction was recorded in the viable count of L. monocytogenes on day 1, while counts were below detection limit (<2 log) on day 2 in meat samples inoculated with P. pentosaceus alone and in combination with L. plantarum. Counts of Salmonella Typhimurium showed about 2 log reduction in SCIS, inoculated with P. pentosaceus alone and in combination with L. plantarum, on day 2 while an increase by 4 logs was observed in UCS. Our findings suggest that the protective effect of the LAB strains could be exploited in shelf life extension and control of foodborne pathogens in goat meat. If the starter strains could be improved upon, their potential as biopreservatives may be engaged in the preservation of the meat in Nigeria, where storage systems have been very inadequate.     

Poly-l-lysine-functionalized magnetic beads combined with polymerase chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157:H7 in milk

Rapid and credible detection of pathogens is essential to prevent and control outbreaks of foodborne diseases. In this study, a poly-l-lysine-functionalized magnetic beads (PLL-MB) strategy combined with a PCR assay was established to detect Staphylococcus aureus. We also detected Escherichia coli O157:H7 to further verify the strategy for gram-negative bacteria detection. Poly-l-lysine has strong positive charges because of its amino groups, which can conjugate with the carboxyl of carboxyl magnetic beads. Furthermore, it can be used to combine with bacteria through electrostatic adsorption. Under optimum conditions, the developed PLL-MB complexes showed 90% capture efficiency in phosphate-buffered saline and 85% capture efficiency in milk for S. aureus detection. The limit of detection of the PLL-MB-PCR assay was 10² cfu/mL (1.8 × 10² cfu/mL for S. aureus and 7 × 10² cfu/mL for E. coli O157:H7) in phosphate-buffered saline and milk samples. The whole assay can be performed within 4 h. The proposed strategy showed a lower limit of detection when compared with the conventional PCR assay without enrichment. In addition, this method exhibited the advantages of a high-efficient, cost-efficient, and simple operation, indicating its potential applications in foodborne pathogen detection.     

Rapid and miniaturized method for detection of hygiene indicators,Escherichia coli and coliforms,in dairy products

Current investigation was aimed to develop colorimetric tests for rapid detection of Escherichia coli/coliforms. These test (s) for E. coli and coliforms were developed using the modified E. coli selective medium (M-ECSM) and coliform selective medium, respectively. The selective media contain a combination of group specific marker enzymes and selective agents. The marker enzymes were screened using chromogenic substrates wherein β-D-glucuronidase and glutamate decarboxylase were found specific for E. coli while β-D-galactosidase for coliforms. The selectivity of the media was achieved using different concentrations of ampicillin and gentamicin. The optimized test procedures enabled sensitive detection of 0.35 ± 0.10 log cfu/ml of E. coli and 0.57 ± 0.15 log cfu/ml of coliforms at 37°C within 14.30 ± 0.45 and 12.15 ± 0.30 hr, respectively. M-ECSM selectively inhibited major Enterobacteriaceae contaminants (Salmonella, Shigella, and Yersinia) up to 6 log cfu/ml. Moreover, better selectivity of M-ECSM was reported against tested commercial chromogenic media. Field evaluation of developed test (s) reported prevalence of E. coli/coliforms as 57.29/88.54% in 96 raw milk and 16.28/51.16% in 43 pasteurized milk samples. Further, test components were vacuum dried in the form of miniaturized point-of-need test for field application in dairy farms and industries with minimal infrastructural requirements.     

Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples

Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica are the most common foodborne bacterial pathogens and are responsible for many outbreaks. Therefore, multiplex detection of these three using a single assay platform is highly desirable. The objective was to develop and optimize a fiber optic sensor for simultaneous detection of these three from food. The streptavidin coated optical waveguides were immobilized with biotinylated polyclonal antibodies and exposed to the bacterial suspensions or enriched food samples for 2 h. Pathogens were detected after reacting with Alexa-Fluor 647-labeled monoclonal antibodies. Ready-to-eat beef, chicken and turkey meats were inoculated with each pathogen (∼100 cfu/25 g), enriched in SEL (Salmonella, E. coli, Listeria), a multipathogen selective enrichment broth for 18 h and tested with the biosensor. The biosensor was able to detect each pathogen, individually or in a mixture with very little cross-reactivity. The limit of detection for the sensor was ∼10³ cfu/ml for all three pathogens. Furthermore, the biosensor successfully detected each pathogen, grown in a mixture from enriched meat samples under 24 h. The pathogen presence was further verified by PCR and immunofluorescence assay. The multiplex fiber optic sensor shows promise for detection of the three pathogens if present in the same sample eliminating the use of multiple single pathogen detection platforms.     

Changes of microbiological quality in meatballs after heat treatment

The effects of different cooking processes (grilling, oven and microwave cooking) on the microbial flora of the raw meatballs inoculated with E. coli O157:H7 at the level 2×10⁴ cfu/ml were investigated. The meatballs were stored at 4 °C. The flora of the raw meatballs is described in this paper. While Salmonella was found in each sample, none of the samples contained C. perfringens or E. coli O157:H7. The processes of grill cooking or microwave cooking decreased the microbial flora by 2–3 log cycles. E. coli O157:H7 was completely destroyed by all cooking methods. E. coli O157:H7 count of the raw meatball samples increased for 1.5 log cycles at the end of storage compared to beginning.     

Assessment of bacterial biofilm on stainless steel by hyperspectral fluorescence imaging

Hyperspectral fluorescence imaging techniques were investigated for detection of two genera of microbial biofilms on stainless steel material which is commonly used to manufacture food processing equipment. Stainless steel coupons were deposited in nonpathogenic E. coli O157:H7 and Salmonella cultures, prepared using M9 minimal medium with casamino acids (M9C), for 6 days at 37 °C. Hyperspectral fluorescence emission images of the biofilm formations on the stainless coupons were acquired from 416 to 700 nm with the use of ultraviolet-A (320–400 nm) excitation. In general, emission peaks for both bacteria were observed in the blue region at approximately 480 nm and thus provided the highest contrast between the biofilms and background stainless steel coupons. A simple thresholding of the 480 nm image showed significantly larger biofilm regions for E. coli O157:H7 than for Salmonella. Viable cell counts suggested that Salmonella formed significantly higher density biofilm regions than E. coli O157:H7 in M9C medium. On the basis of principal component analysis (PCA) of the hyperspectral fluorescence images, the second principal component image exhibited the most distinguishable morphological differences for the concentrated biofilm formations between E. coli and Salmonella. E. coli formed granular aggregates of biofilms above the medium on stainless steel while Salmonella formed dense biofilm in the medium-air interface region (pellicle). This investigation demonstrated the feasibility of implementing fluorescence imaging techniques to rapidly screen large surface areas of food processing equipment for bacterial contamination. Company and product names are used for clarity and do not imply any endorsement by USDA to the exclusion of other comparable products.     

Application of high hydrostatic pressure to decontaminate green onions from Salmonella and Escherichia coli O157:H7

Consumption of fecally contaminated green onions has been implicated in several major outbreaks of foodborne illness. The objectives of this study were to investigate the survival and growth of Salmonella and Escherichia coli O157:H7 in green onions during storage and to assess the application of high hydrostatic pressure (HHP) to decontaminate green onions from both pathogens. Bacterial strains resistant to nalidixic acid and streptomycin were used to inoculate green onions at low (∼1 log cfu/g) and high (∼2 log cfu/g) inoculum levels which were then kept at 4 or 22 °C for up to 14 days. Both pathogens grew to an average of 5-6 log cfu/g during storage at 22 °C and the bacterial populations were fairly stable during storage at 4 °C. High-pressure processing of inoculated green onions in the un-wetted, wetted (briefly dipped in water) or soaked (immersed in water for 30 min) conditions at 250-500 MPa for 2 min at 20 °C reduced the population of Salmonella and E. coli O157:H7 by 0.6 to >5 log cfu/g, depending on the pressure level and sample wetness state. The extent of pressure inactivation increased in the order of soaked > wetted > un-wetted state. The pressure sensitivity of the pathogens was also higher at elevated treatment temperatures. Overall, after pressure treatment at 400-450 MPa (soaked) or 450-500 MPa (wetted) for a retention time of 2 min at 20-40 °C, wild-type and antibiotic-resistant mutant strains of Salmonella and E. coli O157:H7 inoculated on green onions were undetectable immediately after treatment and throughout the 15-day storage at 4 °C. The pressure treatments also had minimal adverse impact on most sensorial characteristics as well as on the instrumental color of chopped green onions. This study highlights the promising applications of HHP to minimally process green onions in order to alleviate the risks of Salmonella and E. coli O157:H7 infections associated with the consumption of this commodity.     

Inactivation of Escherichia Coli O157:H7 and Salmonella Enterica on Blueberries in Water Using Ultraviolet Light

Ultraviolet light (UV) has antimicrobial effects, but the shadowing effect has limited its application. In this study, a novel setup using UV processing in agitated water was developed to inactivate Escherichia coli O157:H7 and Salmonella on blueberries. Blueberries were dip‐ or spot‐inoculated with E. coli or Salmonella. Blueberries inoculated with E. coli were treated for 2 to 10 min with UV directly (dry UV) or immersed in agitated water during UV treatment (wet UV). E. coli was most easily killed on spot‐inoculated blueberries with a 5.2‐log reduction after 10‐min wet UV treatment. Dip‐inoculated blueberries were the most difficult to be decontaminated with only 1.6‐log reduction after 10‐min wet UV treatment. Wet UV treatment generally showed higher efficacies than dry UV treatment, achieving an average of 1.4 log more reduction for spot‐inoculated blueberries. For dip‐inoculated blueberries, chlorine washing and UV treatments were less effective, achieving <2 log reductions of E. coli. Thus, the efficacy of combinations of wet UV with sodium dodecyl sulfate (SDS), levulinic acid, or chlorine was evaluated. Inoculated blueberries were UV‐treated while being immersed in agitated water containing 100 ppm SDS, 0.5% levulinic acid or 10 ppm chlorine. The 3 chemicals did not significantly enhance the wet UV treatment. Findings of this study suggest that UV treatment could be used as an alternative to chlorine washing for blueberries and potentially for other fresh produce.     

Simultaneous quantitative detection of viable Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. using sodium deoxycholate-propidium monoazide with multiplex real-time PCR

Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. are common food-borne pathogens in milk that may cause serious diseases. In the present study, we established a simple, rapid, and specific method to simultaneously detect viable E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk. Three specific genes, fliC from E. coli O157:H7, cgcA from Cronobacter spp., and invA from Salmonella spp., were selected and used to design primers and probes. False-positive results were eliminated with the use of a combined sodium deoxycholate (SD) and propidium monoazide (PMA) treatment. Using the optimized parameters, this SD-PMA treatment combined with multiplex real-time PCR (SD-PMA-mRT-PCR) detected E. coli O157:H7, Cronobacter spp. and Salmonella spp. respectively, at 10² cfu/mL in pure culture or artificially spiked skim milk samples. A reasonable recovery rate (from 100 to 107%) for detection of viable bacteria using the SD-PMA-mRT-PCR assay was obtained in the presence of dead bacteria at 10⁷ cfu/mL. The SD-PMA-mRT-PCR method developed in this study can accurately detect and monitor combined contamination with E. coli O157:H7, Cronobacter spp., and Salmonella spp. in milk and milk products.     

Evaluation of a Polymerase Chain Reaction–Based System for Detecting Salmonella Species from Pork Carcass Sponge Samples

Pork carcass sponge samples (n = 230) collected from 10 Taiwanese slaughter plants were screened for Salmonella using 2 methods: BAX® for Screening/Salmonella, a polymerase chain reaction‐based detection system and a culture method using SM‐ID agar as the selective plating medium. The BAX method identified 14 samples as positive for Salmonella. The 8 samples identified as Salmonella‐positive by the SM‐ID method were also BAX‐positive. Inoculation studies showed BAX detected Salmonella in samples having initial 1.4 × 10¹ cfu/mL Salmonella inoculum prior to the enrichment process in the presence of 3.0 × 10⁶ cfu/mL of non‐Salmonella florae. BAX provides a rapid screening alternative to the culture method for Salmonella detection.     

Short communication: Detection of stx2 and elt genes in bovine milk by using a multiplex PCR system

The aim of this study was to detect 2 important toxin genes from diarrheagenic Escherichia coli (DEC) in bovine milk using a new multiplex PCR. To standardize the multiplex PCR, the stx2 and elt genes were investigated for the detection of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC), respectively. The DNA template was prepared with a thermal procedure (boiling) and a commercial kit. Samples consisted of UHT and pasteurized milk, both skimmed, and STEC and ETEC were tested in concentrations between 10¹ and 10⁹ cfu/mL. With the thermal procedure, the multiplex PCR system detected both pathotypes of E. coli at 10⁹ cfu/mL in UHT and pasteurized milk. When the commercial kit was used for template preparation, STEC and ETEC could be detected at concentrations as low as 10⁴ cfu/mL in UHT and pasteurized milk. Negative controls (Listeria monocytogenes, Salmonella Typhimurium, Salmonella Enteritidis, and Escherichia coli strain APEC 13) were not amplified with the multiplex PCR. These results indicate that the multiplex PCR was a rapid (less than 6 h) and efficient method to detect STEC and ETEC in milk using different methods for DNA preparation; however, the commercial kit was more sensitive than the thermal procedure.     

Microbiological quality and safety of fresh cultivated and wild mushrooms commercialized in Spain

402 samples of 22 species of cultivated and wild fresh mushrooms sold in retail markets and supermarkets in Zaragoza (Spain) were studied to quantify their microbial load (mesophilic aerobic microorganisms, Pseudomonas genus, Enterobacteriaceae, lactic acid bacteria, total and thermotolerant coliform bacteria, Escherichia coli, yeasts and moulds) and to investigate the presence of E. coli O157:H7, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and Yersinia enterocolitica. The total microbial counts ranged from 4.4 to 9.4 log cfu/g, the genus Pseudomonas being the most prevalent with counts from 3.7 to 9.3 log cfu/g and Auricularia auricula-judae the species with the highest microbial load (9.4 log cfu/g). No significant differences (p > 0.05) were detected between mean counts of wild and cultivated species in all the microbial groups studied. The microbiological safety level of the cultivated mushrooms was excellent since no pathogens were isolated, and the microbial counts of indicator microorganisms were low, being detected in only half of the species. Salmonella spp, E. coli O157:H7 and S. aureus were not isolated from any sample, Y. enterocolitica was detected in only four samples of wild mushrooms whereas twenty-six (6.5%) were positive for L. monocytogenes, their occurrence being relatively high in Calocybe gambosa (40%), Hygrophorus limacinus (40%) and Tuber indicum (100%). These results suggest that a strategy to reduce bacterial populations, and to improve the microbiological safety of some species of fresh mushrooms, should be investigated.     

Microtiter plate-based assay for screening antimicrobial activity of melanoidins against E. coli and S. aureus

A rapid plate reader based method examining the antimicrobial activity of both model and food melanoidins (coffee, beer, sweet wine) is described. Antimicrobial activity against Escherichia coli and Staphylococcus aureus is evaluated as area under the growth curve compared to a control. Method was settled for an aqueous melanoidin concentration of 2 mg/ml inoculated to 10⁶ cfu/ml culture. All tested model and food melanoidins exerted antimicrobial activity in some extent, but inhibition was significantly higher over S. aureus (Gram-positive) than E. coli (Gram-negative). Antimicrobial activity can be further quantified by expressing it as OTEV (oxytetracyclin equivalent value, μg/l) which could serve to compare the results obtained within different laboratories, methodologies and/or compounds. Results indicate that both strains have different sensitivity against the presence of melanoidins and probably different mechanism of inhibition. Procedure can be used for a rapid screening of the potential antimicrobial properties of melanoidins, and subsequently to Maillard reaction products as well, against pathogenic strains in order to isolated substances with biological activity.     

Detection and Isolation of Low Levels of E. coli O157:H7 in Cilantro by Real-Time PCR, Immunomagnetic Separation, and Cultural Methods with and without an Acid Treatment

Abstract: Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real‐time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (～0.02 CFU/g) and slightly higher (～0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow^®agar, R&F^®E. coli O157 Chromogenic medium, TC‐SMAC and CHROMagar? 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms. Practical Application: Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices.     

A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products

A 7-plex PCR assay was developed to achieve an effective detection and identification of serotype Enteritidis of Salmonella spp. and shiga toxin-producing type of Escherichia coli O157 in meat products. Six DNA sequences in the invA and sdfI genes of Salmonella Enteritidis as well as rfbE, eae, stx1, and stx2 genes of E. coli O157:H7 were employed to design primers. The multiplex PCR assay could specifically and sensitively detect and identify target pathogens. Applying the assay to meat samples, the multiplex PCR assay was able to simultaneously detect and identify the two pathogens at a sensitivity of three CFU/10 g raw meats after simple 16 h enrichment in buffered peptone water. Further applying in 21 retail meat samples revealed that two samples were positive for non-shiga toxin producing E. coli O157, one sample was positive for Stx2 producing E. coli O157 and five samples were positive for Salmonella enterica Enteritidis. Taken together, the 7-plex PCR assay is a rapid and reliable method for effectively screening single or multiple pathogens occurrences in various meat products, and could also identify the Salmonella enterica Enteritidis from all Salmonella spp. and shiga toxin producing type from all E. coli strains. Considering as a non expensive screening tool, the multiplex PCR assay has a great potential in complement for food stuff analysis by conventional microbiological tests.     

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.     

Survival ofEscherichia coliO157:H7,Listeria monocytogenesandSalmonella kentuckyin Norwegian fermented,dry sausage

Raw, newly produced sausages containing a mixed starter culture of lactobacilli and micrococci were each inoculated at separate locations (using a syringe) with low (10³–10⁴cfu) and high (10⁵–10⁷cfu) numbers of eitherEscherichia coliO157:H7, Listeria monocytogenes orSalmonella kentucky. Three identically prepared sausages were analysed at each sampling day during fermentation, maturation and storage at 4 and 20°C. In the low-inoculum samples, growth was observed initially (2 days) during fermentation forE. coliO157:H7 (3log₈increase in cfu) andL. monocytogenes(five-fold increase in cfu) but not forS. kentuckywhich decreased below the detection limit (150cfu sample^–1). None of the pathogens was detected after 5.5 months, neither at 4 nor 20°C. In the high-inoculum samples there was a decrease during fermentation and maturation for all the pathogens. After 5.5-months storage at 4°C, there was only about 90% reduction of the original inoculate ofL. monocytogenes, whereasE. coliO157:H7 survived at a low number (500cfu sample^–1) andS. kentuckydisappeared below the detection limit. After 5.5-months storage at 20°C, all the pathogens had disappeared below the detection limit. These results indicate that, from a safety point of view, it may be better to store these kinds of sausages at room temperature than in the cold, provided that the sensory qualities are retained and that similar results are obtained with other food pathogens.