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.     

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.     

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.     

Essential Oils Against Pathogen and Spoilage Microorganisms of Fruit Juices: Use of Versatile Antimicrobial Delivery Systems

Essential oils (EO) are increasingly used as natural antimicrobial compounds, however the effect of delivery system to enhance their antimicrobial activity has not been widely studied. Limonene (0 to 10 μL/mL) was added to microbial suspensions (～10⁵ CFU/mL) of selected foodborne pathogens (Listeria monocytogenes Scott A, Salmonella enterica Typhimurium, Escherichia coli and Staphylococcus aureus), and spoilage microorganisms (Lactobacillus plantarum, Saccharomyces cerevisiae, and Candida albicans). S. aureus was found to be the most sensitive foodborne pathogen while Salmonella enterica showed continued growth under all concentrations. Stable nanoemulsions and solid lipid nanoparticles (SLN) (d ～ 170 nm) were prepared using an alkane carrier oil (n‐tetradecane and n‐eicosane, respectively). Interfacial effects and homogenous distribution of limonene in nanoemulsions improved its (8 and 12 μL/mL) antimicrobial effect against S. aureus. Higher aqueous concentrations as a result of expulsion from SLN further enhanced the antimicrobial activity pronounced at higher limonene concentrations. Therefore, our findings confirm that the emulsion‐based delivery systems are able to effectively distribute limonene inside a microbial suspension to improve its antimicrobial activity.     

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.     

Parallel analysis of 7 food-borne pathogens using capillary electrophoresis-based single-strand conformation polymorphism

Capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex polymerase chain reaction (PCR) method was used for the detection of 7 pathogens associated with foodborne illness, including Salmonella enterica, Clostridium perfringens, Bacillus cereus, Listeria monocytogenes, Yersinia enterocolitica, Vibrio parahaemolyticus, and Escherichia coli O157:H7. The method was applied to model food systems, both of culture medium and cooked rice. The detection limit of individual microbes was in the range of 10¹–10³ CFU/g, and that of the mixture of 7 microbes was 10³ CFU/g in the cooked rice sample. This method allowed the detection and identification of all 7 food-borne pathogens within 5 hr without the requirement for enrichment steps.     

Evaluation of sodium dichloroisocyanurate treatment on recovered concentrations of Salmonella enterica,Escherichia coli O157:H7, and Listeria monocytogenes from cattle hide surfaces and culture medium

A major concern of the cattle industry is cross-contamination of meat with pathogens. Cattle are exposed to fecal material, mud, and other contaminants which harbor pathogens that can be shed onto meat and meat processing equipment. Due to increased chances of meat contamination during processing, new antimicrobial formulations for carcass washing before hide removal needs to be identified and tested. Sodium dichloroisocyanurate (SDIC) has biocidal properties and belongs to the N-halamine group of compounds. Disk diffusion assays revealed, 1,000 ppm SDIC effectively reduced pathogen concentrations. SDIC was evaluated for its effects on pathogens in Tryptic Soy Broth and results revealed that 1,000 ppm SDIC had a strong correlation with time and treatment with no bacterial growth in log CFU ml⁻¹ observed at the lowest detection level. Treatment of inoculated hides with 1,000 ppm SDIC for 5 min resulted in reduction of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes at 1.97, 2.02, and 2.84 log CFU cm⁻², respectively.     

Inhibitory effects of UV treatment and a combination of UV and dry heat against pathogens on stainless steel and polypropylene surfaces

Abstract: Pathogens that contaminate the surfaces of food utensils may contribute to the occurrence of foodborne disease outbreaks. We investigated the efficacy of UV treatment combined with dry heat (50 °C) for inhibiting 5 foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Pseudomonas aeruginosa, Listeria monocytogenes, and Staphylococcus aureus) on stainless steel and polypropylene surfaces in this study. We inoculated substrates with each of the 5 foodborne pathogens cultured on agar surface and then UV treatment alone or a combination of both UV and dry heat (50 °C) was applied for 30 min, 1 h, 2 h, and 3 h. The initial populations of the 5 pathogens before treatment were 8.02 to 9.18 and 8.73 to 9.16 log₁₀ CFU/coupon on the surfaces of stainless steel and polypropylene coupons, respectively. UV treatments for 3 h significantly inhibited S. Typhimurium, L. monocytogenes, and S. aureus on the stainless steel by 3.06, 2.18, and 2.70 log₁₀ CFU/coupon, and S. aureus on the polypropylene by 3.11 log₁₀ CFU/coupon, respectively. The inhibitory effects of the combined UV and dry heat treatment (50 °C) increased as treatment time increased, yielding significant reductions in all samples treated for 3 h, with the exception of S. aureus on polypropylene. The reduction level of E. coli O157:H7 treated for 3 h on the surface of stainless steel and polypropylene treated was approximately 6.00 log₁₀ CFU/coupon. These results indicate that combined UV and dry heat (50 °C) treatments may be effective for controlling microbial contamination on utensils and cooking equipment surfaces as well as in other related environments.     

Bactericidal Activities of Health‐Promoting,Food‐Derived Powders Against the Foodborne Pathogens Escherichia coli,Listeria monocytogenes,Salmonella enterica,and Staphylococcus aureus

We evaluated the relative bactericidal activities (BA₅₀) of 10 presumed health‐promoting food‐based powders (nutraceuticals) and, for comparison, selected known components against the following foodborne pathogens: Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus. The relative activities were evaluated using quantitative bactericidal activity [(BA₅₀ value, defined as the percentage of the sample in the assay mixture that resulted in a 50% decrease in colony forming units]. The BA₅₀ values were determined by fitting the data to a sigmoidal curve by regression analysis using concentration–antimicrobial response data. Antimicrobial activity is indicated by a low BA₅₀ value; meaning less material is needed to kill 50% of the bacteria. Olive pomace, olive juice powder, and oregano leaves were active against all 4 pathogens, suggesting that they behave as broad‐spectrum antimicrobials. All powders exhibited strong antimicrobial activity against S. aureus. The following powders showed exceptionally high activity against S. aureus (as indicated by the low BA₅₀ values shown in parentheses): apple skin extract (0.002%); olive pomace (0.008%); and grape seed extract (0.016%). Listeria bacteria were also highly susceptible to apple skin extract (0.007%). The most active substances provide candidates for the evaluation of antimicrobial effectiveness in human food and animal feed. Practical Application : Plant‐derived health‐promoting food supplements, high in bioactive compounds, are candidates for use as antimicrobials in food.     

Effects of X-ray radiation on Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri inoculated on shredded iceberg lettuce

The main goal of this investigation was to study the efficacy of X-ray doses (0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy) on inoculated Escherichia coli O157: H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on shredded iceberg lettuce. The second goal was to study the effect of X-ray on the inherent microflora counts and visual color of shredded iceberg lettuce during storage at 4 °C for 30 days. Treatment with 1.0 kGy X-ray significantly reduced the population of E. coli O157: H7, L. monocytogenes, Salmonella enterica and S. flexneri on shredded iceberg lettuce by 4.4, 4.1, 4.8 and 4.4-log CFU 5 cm⁻², respectively. Furthermore, more than a 5 log CFU reduction of E. coli O157: H7, L. monocytogenes, S. enterica and S. flexneri was achieved with 2.0 kGy X-ray. Treatment with X-ray reduced the initial microflora on iceberg lettuce and kept them significantly (p < 0.05) lower than the control during storage at 4 °C and 90% RH for 30 days. Treatment with X-ray did not significantly (p > 0.05) change the green color of iceberg lettuce leaves. Treatment with X-ray significantly reduced selected pathogens and inherent microorganisms on shredded iceberg lettuce leaves, which could be a good alternative to other technologies for produce (lettuce) industry.     

Variations in Radiation Sensitivity of Foodborne Pathogens Associated with the Suspending Meat

Longissimus dorsi from beef, pork, and lamb and turkey breast and leg meats were inoculated with Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus, and the gamma radiation resistance of the pathogens were determined under identical conditions. At 5°C the respective radiation D-values of E. coli O157:H7 and L. monocytogenes did not vary with the suspending meat. The D-value for a mixture of Salmonella spp. was significantly lower on pork than on beef, lamb, turkey breast, and turkey leg meats. The D-value for S. aureus was significantly lower on lamb and mechanically deboned chicken meat than on the other meats. All values were, nevertheless, within expected ranges.     

Outbreaks and factors influencing microbiological contamination of fresh produce

Fresh fruits and vegetables are nutritionally well‐recognised as healthy components in diets. The microbiological foodborne outbreaks associated with the consumption of fresh produce have been increasing. Salmonella spp., Escherichia coli O157:H7, Staphylococcus aureus, Campylobacter spp. and Listeria monocytogenes are the most common pathogens that contaminate fresh produce. This review discusses recent foodborne outbreaks linked to fresh produce, factors that affect microbiological contamination and measures that could be adopted to reduce the foodborne illnesses. © 2016 Society of Chemical Industry     

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.     

Incorporation of Essential Oils and Nanoparticles in Pullulan Films to Control Foodborne Pathogens on Meat and Poultry Products

The incorporation of essential oils and nanotechnology into edible films has the potential to improve the microbiological safety of foods. The aim of this study was to evaluate the effectiveness of pullulan films containing essential oils and nanoparticles against 4 foodborne pathogens. Initial experiments using plate overlay assays demonstrated that 2% oregano essential oil was active against Staphylococcus aureus and Salmonella Typhimurium, whereas Listeria monocytogenes and Escherichia coli O157:H7 were not inhibited. Two percent rosemary essential oil was active against S. aureus, L. monocytogenes, E. coli O157:H7, and S. Typhimurium, when compared with 1%. Zinc oxide nanoparticles at 110 nm were active against S. aureus, L. monocytogenes, E. coli O157:H7, and S. Typhimurium, when compared with 100 or 130 nm. Conversely, 100 nm silver (Ag) nanoparticles were more active against S. aureus than L. monocytogenes. Using the results from these experiments, the compounds exhibiting the greatest activity were incorporated into pullulan films and found to inhibit all or some of the 4 pathogens in plate overlay assays. In challenge studies, pullulan films containing the compounds effectively inhibited the pathogens associated with vacuum packaged meat and poultry products stored at 4 °C for up to 3 wk, as compared to control films. Additionally, the structure and cross‐section of the films were evaluated using electron microscopy. The results from this study demonstrate that edible films made from pullulan and incorporated with essential oils or nanoparticles may improve the safety of refrigerated, fresh or further processed meat and poultry products.     

Sodium Lactate Affects Pathogens in Cooked Beef

Cooked, quartered beef top rounds containing either 1, 2, 3 or 4% sodium lactate were aseptically sampled and slice sections were inoculated with Listeria monocytogenes (ATCC 43256), Staphylococcus aureus (ATCC 27154), Salmonella typhimurium (ATCC 13311), Clostridium perfringens (ATCC 12924), or Escherichia coli O157:H7 (ATCC 43895). Inoculated slices were stored at 10°C for 0. 7. 14. 21 or 28 days.‘Three and 4% sodium lactate generally displayed lim-ited proliferation of S. typhimurium, L. monocytogenes and E. coli O157:H7 when compared with control roasts (0% sodium lactate) and roasts containing 2% sodium lactate.     

Survival and High‐Hydrostatic Pressure Inactivation of Foodborne Pathogens in Salmorejo,a Traditional Ready‐to‐Eat Food

Salmorejo is a traditional tomato‐based creamy product. Because salmorejo is not heat‐processed, there is a risk of contamination with foodborne pathogens from raw materials. Even though bacterial growth in salmorejo is strongly inhibited because of its acidic pH (close to 3.9), the growth and survival of 3 foodborne pathogens in this food has not been studied before. In this study, 3 cocktails consisting of Escherichia coli O157, Salmonella enterica serovar Enteritidis, and Listeria monocytogenes strains were inoculated in freshly prepared salmorejo. The food was treated by high hydrostatic pressure (HHP) at 400, 500, or 600 MPa for 8 min, or left untreated, and stored at 4 °C for 30 d. Viable cell counts were determined on selective media and also by the triple‐layer agar method in order to detect sublethally injured cells. In control samples, L. monocytogenes viable cells decreased by 2.4 log cycles at day 7 and were undetectable by day 15. S. enterica cells decreased by 0.5 or 2.4 log cycles at days 7 and 15 respectively, but still were detectable at day 30. E. coli O157 cells survived much better in salmorejo, decreasing only by 1.5 log cycles at day 30. Treatments at pressures of 400 MPa or higher reduced viable counts of L. monocytogenes and S. enterica to undetectable levels. HHP treatments significantly (P < 0.05) reduced E. coli counts by approximately 5.2 to 5.4 log cycles, but also yielded surviving cells that apparently were sublethally injured. Only samples treated at 600 MPA for 8 min were devoid of detectable E. coli cells during storage.     

Inhibition of biofilm formation,adhesion and invasion in Caco-2 cells by foodborne pathogens using phyto-mediated synthesised silver nanoparticles from industrial wastes

Certain foodborne diseases are associated with antibiotic resistance, a significant problem throughout the world. Silver nanoparticles (AgNPs) using industrial waste from Eucalyptus camaldulensis and sericin, a protein derived from Bombyx mori, were synthesised by a one-step approach. Spherical-shaped nanoparticles with the average size of 17.19 nm exhibited strong antioxidant activity. The minimum bactericidal concentrations against foodborne pathogens including Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Klebsiella pneumoniae, Salmonella Typhimurium, Shigella sonnei, Vibrio cholerae and Vibrio parahaemolyticus were between 2.96 and 11.83 µg/mL. Killing against L. monocytogenes and E. coli O157:H7 was observed within 4 h. Treatment with AgNPs at 0.25 – 0.5 × MIC significantly reduced biofilm production in all isolates (P < 0.05). AgNPs significantly impeded adhesion to and invasion of human epithelial Caco-2 cells by L. monocytogenes and E. coli O157:H7 (P < 0.05). Biocompatibility assessment of AgNPs with Caco-2 and human red blood cells demonstrated no toxic effects.     

Stability and Antimicrobial Activity of Allyl Isothiocyanate during Long-Term Storage in an Oil-in-Water Emulsion

Abstract: This study investigated the stability and antimicrobial activity of allyl isothiocyanate (AITC) in medium chain triglyceride (MCT) or soybean oil (SBO) dispersed in an oil-in-water (o/w) system during long-term storage. Oil type, content, and oxidative stability affect the stability and antimicrobial activity of AITC during storage. High oil content is favorable for AITC stability in the emulsion. Notably, AITC with MCT is more stable than AITC with SBO with the same oil content. Consequently, AITC with MCT is more effective than AITC with SBO in inhibiting G(−) bacteria (E. coli O157:H7, Salmonella enterica, and Vibrio parahaemolyticus) and G(+) bacteria (Staphylococcus aureus and Listeria monocytogenes).     

Evaluation of Thin Agar Layer Method for Recovery of Heat-Injured Foodborne Pathogens

ABSTRACT The Thin Agar Layer (TAL) method of Kang and Fung 1998 was used to enumerate heat‐injured (55 °C for 10 min) foodborne pathogens. This method involves overlaying 14 mL of nonselective medium (Tryptic Soy Agar, TSA) onto a prepoured, pathogen‐specific, selective medium. The recovery rate with the TAL method was compared with the rate for TSA and pathogen‐specific, selective media. No significant differences occurred between TSA and TAL (P > 0.05) for enumeration of heat‐injured Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Yersinia enterocolitica, and both recovered significantly higher numbers than selective media (P < 0.05). The TAL method is a 1‐step, convenient procedure for recovery of heat‐injured cells.     

A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk

Intoxications and infections caused by food-borne pathogens represent an increasing public health problem, and diagnostic tests in multiplex format are needed for the rapid identification of food contaminations caused by more than one microbial species. We have developed a multiple PCR-based platform for the simultaneous detection of the widespread milk-associated pathogens Salmonella spp., Listeria monocytogenes and Escherichia coli O157. The assay combines an enrichment step in a medium properly formulated for the simultaneous growth of target pathogens, a DNA isolation method, and a multiplex Real-Time PCR detection system based either on dual-labelled probes (mRT-PCR), or on melting curve analysis (mHRM). The second, producing a distinct peak for each amplification product, allows the qualitative assessment of pathogen presence. Moreover, the internal amplification control (IAC) included in the reaction, ensuring the reliability of results, complies with quality management programmes. Inclusivity and exclusivity were 100% each, with a detection limit of 1 CFU for each pathogen in a total of five 25 ml-aliquots of raw milk, and a duration of two working days.The assay represents an alternative approach for the qualitative detection of the cited bacterial species, suitable for a relatively inexpensive screening of several milk samples, reducing the turnaround time and the workload.