Prevalence,serotype diversity,and antimicrobial resistance of Salmonella in imported shipments of spice offered for entry to the United States,FY2007–FY2009

In response to increased concerns about spice safety, the U.S. FDA initiated research to characterize the prevalence of Salmonella in imported spices. Shipments of imported spices offered for entry to the United Sates were sampled during the fiscal years 2007–2009. The mean shipment prevalence for Salmonella was 0.066 (95% CI 0.057–0.076). A wide diversity of Salmonella serotypes was isolated from spices; no single serotype constituted more than 7% of the isolates. A small percentage of spice shipments were contaminated with antimicrobial-resistant Salmonella strains (8.3%). Trends in shipment prevalence for Salmonella associated with spice properties, extent of processing, and export country, were examined. A larger proportion of shipments of spices derived from fruit/seeds or leaves of plants were contaminated than those derived from the bark/flower of spice plants. Salmonella prevalence was larger for shipments of ground/cracked capsicum and coriander than for shipments of their whole spice counterparts. No difference in prevalence was observed between shipments of spice blends and non-blended spices. Some shipments reported to have been subjected to a pathogen reduction treatment prior to being offered for U.S. entry were found contaminated. Statistical differences in Salmonella shipment prevalence were also identified on the basis of export country.     

Assessment of the microbiological safety of edible dried seeds from retail premises in the United Kingdom with a focus on Salmonella spp.

Sesame seed products have recently been associated with a number of Salmonella outbreaks in the UK and elsewhere. Aside from sesame seeds, there is little published information on the prevalence of Salmonella spp. in edible seeds. A study of 3735 samples of retail edible dried seeds in the UK was therefore carried out between October 2007 and March 2008 to assess their microbiological safety in relation to Salmonella contamination and levels of Escherichia coli, an indicator of faecal contamination. Overall, Salmonella was detected in 23 samples (0.6%), of which over half (57%) were sesame seeds. Other seeds contaminated with Salmonella were linseed (1 sample), sunflower (1 sample), alfalfa (1 sample), melon (4 samples) and mixed seeds (3 samples). E. coli was detected in 9% of samples, with 1.5% containing unsatisfactory levels (≥10²/g). These included melon, pumpkin, sesame, hemp, poppy, linseed, sunflower and mixed seeds. The UK retailers affected by the detection of Salmonella in their products recalled the contaminated batches, and Food Standards Agency food alerts were issued to advise against the consumption of affected seed products. This study highlights the importance of good hygiene practices and effective decontamination procedures during the production of these products.     

Slow-release chlorine dioxide gas treatment as a means to reduce Salmonella contamination on spices

Salmonella enterica is a major food safety issue for the spice industry. Slow-releasing chlorine dioxide (ClO₂) gas from self-contained sachets, which can be employed in a small-scale operation, was evaluated for its effectiveness in reducing Salmonella contamination on black pepper, cumin, and sesame seed. Three levels of chlorine dioxide gas (100, 200, or 500 mg ClO₂/kg spice) were applied to Salmonella-inoculated spices. Spices were sampled immediately after treatment, and at 1, 10, and 30-days post-treatment. The combined effect of ClO₂ gas treatment and storage time on Salmonella numbers on spices was evaluated. Salmonella reductions on black pepper, cumin, and sesame seed samples ranged from 0.81 to 2.74 log CFU/g after ClO₂ treatment. Storage time also had a significant impact on Salmonella numbers, as numbers decreased by up to 2.50 log CFU/g over 30 days of storage. Self-contained ClO₂ releasing sachets could provide small food processors a post-lethality treatment alternative for spices.     

Inactivation of Salmonella and Escherichia coli O157:H7 on artificially contaminated alfalfa seeds using high hydrostatic pressure

Alfalfa sprouts contaminated with Salmonella and Escherichia coli O157:H7 have been implicated in several outbreaks of foodborne illnesses in recent years. The seed used for sprouting appears to be the primary source of pathogens. Seed decontamination prior to sprouting presents a unique challenge for the sprouting industry since cells of the pathogenic survivors although undetectable after sanitizing treatments, can potentially multiply back to hazardous levels. The focus of this study was to therefore test the efficacy of high hydrostatic pressure to eliminate a ∼5 log CFU/g load of Salmonella and E. coli O157:H7 on alfalfa seeds. Pressure treatment of 600 MPa for up to 25 min at 20 °C could not result in complete inactivation of Salmonella. High-pressure treatment was then carried out either at sub-ambient (4 °C) or elevated (40, 45 and 50 °C) temperatures to test the ability of high pressure to eliminate Salmonella. Pressure treatment at 4 and 20 °C did not deliver any satisfactory inactivation of Salmonella while high pressure at elevated temperatures achieved complete kill. Pre-soaking seeds prior to high-pressure treatment also enhanced pressure inactivation of Salmonella but at the expense of seed viability. High-pressure treatment of 500 MPa for 2 min at 45 °C was able to eliminate wild-type Salmonella and E. coli O157:H7 strains without bringing about any appreciable decrease in the seed viability.     

Differences in biofilm formation of produce and poultry Salmonella enterica isolates and their persistence on spinach plants

Spinach plants were irrigated biweekly with water containing 2.1 log CFU Salmonella/100 ml water (the maximum Escherichia coli MPN recommended by the Leafy Greens Marketing Agreement; LGMA), or 4.1 CFU Salmonella/100 ml water to determine Salmonella persistence on spinach leaves. Green Fluorescent protein expressing Salmonella were undetectable by most-probable number (MPN) at 24 h and 7 days following each irrigation event. This study indicates that Salmonella are unlikely to persist on spinach leaves when irrigation water is contaminated at a level below the LGMA standards. In a parallel study, persistence of Salmonella isolated from poultry or produce was compared following biweekly irrigation of spinach plants with water containing 6 log CFU Salmonella/100 ml. Produce Salmonella isolates formed greater biofilms on polystyrene, polycarbonate and stainless steel surfaces and persisted at significantly higher numbers on spinach leaves than those Salmonella from poultry origin during 35 days study. Poultry Salmonella isolates were undetectable (<1 log CFU/g) on spinach plants 7 days following each irrigation event when assayed by direct plating. This study indicates that Salmonella persistence on spinach leaves is affected by the source of contamination and the biofilm forming ability of the strain.     

Salmonella, Escherichia coli O157:H7 and E. coli biotype 1 in a pilot survey of imported and New Zealand pig meats

A pilot survey for the pathogens Salmonella and Escherichia coli O157:H7, and E. coli biotype 1 was conducted on 100 New Zealand-produced (domestic) pig carcasses and 110 imported pig meat samples over an 8-month period to assess the likelihood of introduction of novel pathogen strains into New Zealand (NZ), and as a guide for development of a domestic pork National Microbiological Database programme. Salmonella was not isolated from domestic pig carcasses or from pig meat imported from Canada and the USA. The prevalence of Salmonella in imported pig meat was 3.6% (95% CI 1.0–9.0) with positive samples detected from Australian pig meat. The prevalence of E. coli O157:H7 on domestic pig carcasses was 1% (95% CI 0.03–5.4) while the overall prevalence of E. coli O157:H7 in imported pig meat was 1.8% (95% CI 0.2–6.4), detected mainly from Australian but not from Canadian or US pork. All except three samples have an E. coli biotype 1 count of <100 CFU cm⁻² or g⁻¹, indicating good hygiene quality of domestic and imported pig meat. The results demonstrated that importation of uncooked pig meat is a potential route for the introduction of new clones of Salmonella and E. coli O157:H7 into New Zealand.     

Prevalence and counts of Salmonella spp. in minimally processed vegetables in São Paulo, Brazil

Minimally processed vegetables (MPV) may be important vehicles of Salmonella spp. and cause disease. This study aimed at detecting and enumerating Salmonella spp. in MPV marketed in the city of São Paulo, Brazil. A total of 512 samples of MPV packages collected in retail stores were tested for Salmonella spp. and total coliforms and Escherichia coli as indication of the hygienic status. Salmonella spp. was detected in four samples, two using the detection method and two using the counting method, where the results were 8.8 × 10² CFU/g and 2.4 × 10² CFU/g. The serovars were Salmonella Typhimurium (three samples) and Salmonella enterica subsp. enterica O:47:z4,z23:- (one sample). Fourteen samples (2.7%) presented counts of E. coli above the maximum limit established by the Brazilian regulation for MPV (10² CFU/g). Therefore, tightened surveillance and effective intervention strategies are necessary in order to address consumers and governments concerns on safety of MPV.     

Examination of the internalization of Salmonella serovar Typhimurium in peanut,Arachis hypogaea,using immunocytochemical techniques

A variety of products of plant origin, such as tomatoes, melons, peppers, and peanuts, have been implicated in Salmonella spp. associated outbreaks in recent years. Although these bacteria have been found to internalize within some plants associated with foodborne-related outbreaks, the internalization in peanut plants has not been examined to date. To investigate internalization and where the bacteria localize within the plant, intact peanut seeds were contaminated with Salmonella serovar Typhimurium expressing green fluorescent protein (GFP) for 30 min and immunocytochemical techniques were used to localize the bacterium within the stem tissue of 16 day-old peanut plants. An average of 13.6 bacteria/mm³ were localized within the sampled tissue. The bacteria were found to be associated with every major tissue (cortical, vascular, epidermal, and pith) and corresponding cell type. The cortical cells located to the outside of the vascular bundles contained the majority of the Salmonella cells (72.4%). Additional growth experiments demonstrated peanut seedlings could support the reproduction of Salmonella to high levels (10⁹ CFU/plant) after 2 days following seed contamination. Together these results show that Salmonella Typhimurium can internalize within many different plant tissue types after a brief seed contamination event and that the bacteria are able to grow and persist within the plant.     

The microbiological safety of ready-to-eat specialty meats from markets and specialty food shops: A UK wide study with a focus on Salmonella and Listeria monocytogenes

From 2359 specialty meats (continental sausages, cured/fermented, dried meats) sampled from markets and specialty food shops, 98.9% of samples were of satisfactory or acceptable microbiological quality. However, 16 (0.7%) were unsatisfactory as a result of Escherichia coli, Staphylococcus aureus or Listeria spp. contamination (≥10² CFU/g), and nine (0.4%) were unacceptable due to presence of Salmonella spp. or Listeria monocytogenes (>10² CFU/g). Meats with unacceptable levels of L. monocytogenes were within shelf life (range: 8–143 days remaining). Nine different subtypes of L. monocytogenes were detected with sero/AFLP type 1/2c VII predominating (37%), although this subtype was not overrepresented in any particular meat type (P > 0.05). Ninety-six percent of continental sausages and cured/fermented products were stored at <8 °C at premises, including seven of the nine unacceptable samples. These nine meats were all pre-packed prior to supply to retail premises (OR = 0.1 P = 0.003) indicating that contamination with bacterial pathogens occurred earlier in the production chain. Most samples (72.7%, 8/11) with unsatisfactory levels of E. coli were sliced on request, suggesting cross-contamination at point of sale. This study highlights the importance of ensuring that products do not become contaminated before final packaging, that storage conditions are controlled, and that durability dates are an accurate indication of the shelf life of the product so as to minimise the potential for L. monocytogenes to be present at levels hazardous to health at the point of sale.     

Distribution of Salmonella typhimurium in romaine lettuce leaves

Leafy greens are occasionally involved in outbreaks of enteric pathogens. In order to control the plant contamination it is necessary to understand the factors that influence enteric pathogen-plant interactions. Attachment of Salmonella enterica serovar typhimurium to lettuce leaves has been demonstrated before; however, only limited information is available regarding the localization and distribution of immigrant Salmonella on the leaf surface. To extend our knowledge regarding initial pathogen-leaf interactions, the distribution of green-fluorescent protein-labeled Salmonella typhimurium on artificially contaminated romaine lettuce leaves was analyzed. We demonstrate that attachment of Salmonella to different leaf regions is highly variable; yet a higher attachment level was observed on leaf regions localized close to the petiole (7.7 log CFU g⁻¹) compared to surfaces at the far-end region of the leaf blade (6.2 log CFU g⁻¹). Attachment to surfaces located at a central leaf region demonstrated intermediate attachment level (7.0 log CFU g⁻¹). Salmonella displayed higher affinity toward the abaxial side compared to the adaxial side of the same leaf region. Rarely, Salmonella cells were also visualized underneath stomata within the parenchymal tissue, supporting the notion that this pathogen can also internalize romaine lettuce leaves. Comparison of attachment to leaves of different ages showed that Salmonella displayed higher affinity to older compared to younger leaves (1.5 log). Scanning electron microscopy revealed a more complex topography on the surface of older leaves, as well as on the abaxial side of the examined leaf tissue supporting the notion that a higher attachment level might be correlated with a more composite leaf landscape. Our findings indicate that initial attachment of Salmonella to romaine lettuce leaf depends on multiple plant factors pertaining to the specific localization on the leaf tissue and to the developmental stage of the leaf.     

A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood

Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1–10³ cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1 cfu/g, in enriched samples, and higher sensitivity (10²–10³ cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.     

Assessment of Microbiological and Bio-chemical Quality of Fish in a Supply Chain in Negombo,Sri Lanka

This study aimed to investigate quality of fish landed in Negombo area and distributed in suburban areas in Western province of Sri Lanka. Hundred samples of large fish (Katsuwonus pelamis and Euthynnus affinis) and 60 samples of small fish (Amblygaster sirm, Pterocaesio chrysozona, Stolephorus commersoni, and Sardinella albella) were sampled from different stages of a supply chain at five and six sampling visits, respectively. All fish samples (N=160) were analysed for aerobic plate counts (APC) at 37^oC, Coliforms, feacal coliforms, Escherichia coli, Salmonella spp., Listeria monocytogenes, total volatile base nitrogen (TVB-N) while 130 were analysed for histamine. Water from fishery harbor basin, fishery harbour, ice manufcaturing plants and ice used in multiday boats were also analysed for microbiological parameters. Large and small fish contained APC in the range of 2.0 x10² - 2.0 x 10⁶ and 8.0×10³ - 2.0×10⁸ cfu/g, respectively. Faecal coliform counts ranged between not detected (ND) and 90 MPN/g in large fish and between ND and >1100 MPN/g in small fish. 5% of large fish were contaminated with E.coli and ranged from ND to 15 MPN/g. E.coli was present in 70% of small fish samples and ranged from ND to >1100 MPN/g. Of the 160 fish samples, tested Salmonella spp were detected in nine occassions. Of the 160 fish samples, L. monocytogenes was found in eight Katsuwonus pelamis and one Sardinella albella fish. TVB-N of large fish were found at range of 1-67 mgN/100 g and 79% samples contained unaccptable levels. Small fish contained about 25.10-104.30 mgN/100 g while 78% samples exceeded acceptable levels. Histamine level of large and small fish, 26% and 83% of samples exceeded the maximum acceptable levels, respectively. Harbour basin water was heavily contaminated with Salmonella spp. (50%), Faecal streptococci (100%), Faecal coliforms and E.coli (100%). Ice samples (20%) from one ice plant were found contaminated with Salmonella spp.     

Individual and combined application of dry heat with high hydrostatic pressure to inactivate Salmonella and Escherichia coli O157:H7 on alfalfa seeds

Alfalfa sprouts are recurrently implicated in outbreaks of food-borne illnesses as a result of contamination with Salmonella or Escherichia coli O157:H7. In the majority of these outbreaks, the seeds themselves have been shown to be the most likely source of contamination. The aims of this study were to comparatively assess the efficacy of dry heat treatments alone or in conjunction with high hydrostatic pressure (HHP) to eliminate a ∼5 log CFU/g load of Salmonella and E. coli O157:H7 on alfalfa seeds. Dry heat treatments at mild temperatures of 55 and 60 °C achieved ≤1.6 and 2.2 log CFU/g reduction in the population of Salmonella spp. after a 10-d treatment, respectively. However, subjecting alfalfa seeds to more aggressive temperatures of 65 °C for 10 days or 70 °C for 24 h eliminated a ∼5 log population of Salmonella and E. coli O157:H7. We subsequently showed that the sequential application of dry heating followed by HHP could substantially reduce the dry heating exposure time while achieving equivalent decontamination results. Dry heating at 55, 60, 65 and 70 °C for 96, 24, 12 and 6 h, respectively followed by a pressure treatment of 600 MPa for 2 min at 35 °C were able to eliminate a ∼5 log CFU/g initial population of both pathogens. Finally, we evaluated the impact of selected treatments on the seed germination percentages and yield ratios and showed that dry heating at 65 °C for 10 days did not bring about any considerable decrease in the germination percentage. However, the sprout yield of treated alfalfa seeds was reduced by 21%. Dry heating at 60 and 65 °C for 24 and 12 h respectively followed by the pressure treatment of 600 MPa for 2 min at 35 °C did not significantly (P > 0.05) affect the germination percentage of alfalfa seeds although a reduction in the sprouting yield was observed.     

Antagonistic effect of Pseudomonas graminis CPA-7 against foodborne pathogens in fresh-cut apples under simulated commercial conditions

Recently, we reported that the application of the strain CPA-7 of Pseudomonas graminis, previously isolated from apple, could reduce the population of foodborne pathogens on minimally processed (MP) apples and peaches under laboratory conditions. Therefore, the objective of the present work was to find an antioxidant treatment and a packaging atmosphere condition to improve CPA-7 efficacy in reducing a cocktail of four Salmonella and five Listeria monocytogenes strains on MP apples under simulated commercial processing. The effect of CPA-7 application on apple quality and its survival to simulated gastric stress were also evaluated. Ascorbic acid (2%, w/v) and N-acetyl-l-cysteine (1%, w/v) as antioxidant treatments reduced Salmonella, L. monocytogenes and CPA-7 recovery, meanwhile no reduction was observed with NatureSeal^® AS1 (NS, 6%, w/v). The antagonistic strain was effective on NS-treated apple wedges stored at 10 °C with or without modified atmosphere packaging (MAP). Then, in a semi-commercial assay, efficacy of CPA-7 inoculated at 10⁵ and 10⁷ cfu mL⁻¹ against Salmonella and L. monocytogenes strains on MP apples with NS and MAP and stored at 5 and 10 °C was evaluated. Although high CPA-7 concentrations/populations avoided Salmonella growth at 10 °C and lowered L. monocytogenes population increases were observed at both temperatures, the effect was not instantaneous. No effect on apple quality was detected and CPA-7 did not survived to simulated gastric stress throughout storage. Therefore, CPA-7 could avoid pathogens growth on MP apples during storage when use as part of a hurdle technology in combination with disinfection techniques, low storage temperature and MAP.     

Transfer of Listeria innocua from contaminated compost and irrigation water to lettuce leaves

Many foodborne outbreaks of some pathogens such as Escherichia coli O157:H7, Salmonella or Listeria have been associated with the consumption of contaminated vegetables. Contaminated manure and polluted irrigation water are probable vehicles for the pathogens. The aim of this study was to determine the potential transfer of Listeria innocua from soil fertilized with contaminated compost or irrigated with contaminated water to the edible parts of lettuce grown on these soils together with its survival in lettuce and in soil under field conditions during two different seasons. Moreover, its survival on lettuce sprinkled with contaminated irrigation water was evaluated. L. innocua survived in soil samples for 9 weeks at high concentrations, 10⁵ cfu gdw⁻¹ in fall and 10³ cfu gdw⁻¹ in spring. Pathogen survived better in fall, indicating an important influence of temperature and humidity. L. innocua population in lettuce leaves was very high on lettuce leaves after sprinkling, but decreased to undetectable levels at field conditions. There was also transfer of L. innocua from soil contaminated with compost or irrigated with contaminated water to lettuce leaves, mainly to the outer ones. Survival profiles of L. innocua on lettuce and soil samples contaminated either by application of contaminated compost or surface irrigation water was similar. Our results indicated that contaminated compost and contaminated irrigation water can play an important role in the presence of foodborne pathogens on vegetables.     

Development of multiplex real-time PCR with Internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter spp. and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of Cronobacter and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 10³ to 10⁸ CFU/ml for both Salmonella and Cronobacter. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 10³ CFU/ml for Salmonella and Cronobacter without enrichment. The commercial PIF was then inoculated with Salmonella and Cronobacter at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. At 12 h post-enrichment, we could detect Salmonella and Cronobacter at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of Cronobacter and Salmonella in PIF.     

Application of bacteriophages during depuration reduces the load of Salmonella Typhimurium in cockles

As bivalve molluscs are filter feeder, often consumed raw or lightly cooked and are frequently cultivated in contaminated waters, they are implicated in food-borne disease transmission to human. The present study investigated the potential application of bacteriophage (or phage) phSE-2, phage phSE-5 and phage cocktail phSE-2/phSE-5 to decrease the concentration of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) during the depuration of natural and artificially contaminated cockles (Cerastoderma edule). Cockles were artificially infected with 10⁵ and 10⁶ colony-forming units (CFU)/mL of S. Typhimurium in static seawater and infected group were treated with phages at four different MOI values: 0.1, 1, 10 and 100. Depuration in static seawater at multiplicity of infection (MOI) of 0.1 with single phage suspensions of phSE-2 and phSE-5 provided the best results, as it decreased by ~ 1.3 and 1.7 log CFU/g, respectively, the concentration of Salmonella spp. after a 4 h treatment. At a MOI of 0.1, the rate of inactivation with single phage suspensions was higher when compared with the results obtained using the phage cocktail. However, in naturally contaminated cockles treated in static seawater with single phage suspensions and phage cocktail phSE-2/phSE-5, similar decreases in cultivable bacteria concentration (~ 0.7–0.9 log CFU/g) were achieved after 6 h of treatment. When artificially contaminated cockles were depurated with phage phSE-5 in a recirculated seawater system (mimicking industrial depuration conditions), a 0.9 and 2.0 log CFU/g reduction of Salmonella spp. was reached after 4 and 6 h treatment. Once the depuration process was performed without phage, a 6 h treatment was needed to obtain a 1.1 log CFU/g reduction of Salmonella spp. Results indicated that combining phage biocontrol with depuration procedures enhance bivalve microbial safety for human consumption by improving decontamination efficiency, proving that this technology can be transposed to the bivalves industry. Moreover, this approach also displays the advantage of reducing the time required for depuration and consequently its associated costs.     

Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella

Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5 ng–50 fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 10⁴ CFU/g, this was improved to 10 CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food.     

Survival of Salmonella Newport in oysters

Salmonella enterica is the leading cause of laboratory-confirmed foodborne illness in the United States and raw shellfish consumption is a commonly implicated source of gastrointestinal pathogens. A 2005 epidemiological study done in our laboratory by Brands et al., showed that oysters in the United States are contaminated with Salmonella, and in particular, a specific strain of the Newport serovar. This work sought to further investigate the host-microbe interactions between Salmonella Newport and oysters. A procedure was developed to reliably and repeatedly expose oysters to enteric bacteria and quantify the subsequent levels of bacterial survival. The results show that 10 days after an exposure to Salmonella Newport, an average concentration of 3.7 × 10³ CFU/g remains within the oyster meat, and even after 60 days there still can be more than 10² CFU/g remaining. However, the strain of Newport that predominated in the market survey done by Brands et al. does not survive within oysters or the estuarine environment better than any other strains of Salmonella we tested. Using this same methodology, we compared Salmonella Newport's ability to survive within oysters to a non-pathogenic strain of E. coli and found that after 10 days the concentration of Salmonella was 200-times greater than that of E. coli. We also compared those same strains of Salmonella and E. coli in a depuration process to determine if a constant 120 L/h flux of clean seawater could significantly reduce the concentration of bacteria within oysters and found that after 3 days the oysters retained over 10⁴ CFU/g of Salmonella while the oysters exposed to the non-pathogenic strain of E. coli contained 100-times less bacteria. Overall, the results of this study demonstrate that any of the clinically relevant serovars of Salmonella can survive within oysters for significant periods of time after just one exposure event. Based on the drastic differences in survivability between Salmonella and a non-pathogenic relative, the results of this study also suggest that unidentified virulence factors may play a role in Salmonella's interactions with oysters.