Inhibition of polymerase chain reaction for the detection of Escherichia coli O157:H7 and Salmonella enterica on walnut kernels

The aim of this study was to determine whether Escherichia coli O157:H7 can be reliably detected and isolated from walnut kernels using standard methods of analysis. The limit of detection approached 1 cell per analytical unit (25 g) for E. coli O157:H7 on walnut kernels enriched in modified tryptic soy broth with 20 μg/ml novobiocin and plating onto selective agar media. The presence of PCR inhibitors in walnut kernels was indicated by the failure to detect E. coli O157:H7 from culture positive enrichment broths analysed by PCR, with two separate polymerase and reagent compositions (Dupont BAX E. coli O157:H7 MP system, Promega GoTaq Green for stx) and three methods of template preparation (DuPont BAX, Qiagen DNeasy, Bio-Rad InstaGene). PCR inhibition was overcome by 1:100 dilution in TE buffer of the DNeasy or InstaGene template. PCR inhibition was not relieved by dilution of the BAX template. Similar results were observed for walnut kernels inoculated with Salmonella enterica and analysed for invA, indicating that PCR inhibition is not specific to the organism or primer/template. These results indicate that analysis of walnut kernels for pathogens should be with culture based methods or use protocols for DNA template preparation modified to remove or dilute inhibitors and the need for internal amplification controls in PCR methods.     

Survey of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products by commercial real-time PCR kits

Real-time PCR (RTiPCR) assays including enrichment stage were evaluated for the rapid detection of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products using molecular beacon probes available as commercial kits (WARNEX Genevision, Canada & AES Chemunex detection system, France). The accuracy of the assays was evaluated analyzing 1032 naturally contaminated food samples in combination to the conventional cultural methods. Presence/absence testing of the above pathogens was performed in 25 g samples of each product. In case of L. monocytogenes of 39 positive RTiPCR samples, 37 were confirmed by the cultural method (based on McNemar's test the difference between the two methods is insignificant). The highest incidence of L. monocytogenes in food products was found in desserts and the second highest in frozen pastries. None of the samples were cultural positive but negative in the RTiPCR test. One among the 343 investigated samples was positive for Salmonella spp. by RTiPCR and the cultural method. Out of 333 samples analyzed for E. coli O157:H7 no positive sample was detected. RTiPCR-based methods proved to be powerful tools for fast, sensitive and accurate pathogen detection in raw food ingredients and ready-to-eat products.     

Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on fresh-cut celery

Illnesses from Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella have been associated with the consumption of numerous produce items. Little is known about the effect of consumer handling practices on the fate of these pathogens on celery. The objective of this study was to determine pathogen behavior at different temperatures under different storage conditions. Commercial fresh-cut celery was inoculated at ca. 3 log CFU/g onto either freshly cut or outer uncut surfaces and stored in either sealed polyethylene bags or closed containers. Samples were enumerated following storage for 0, 1, 3, 5, and 7 days when held at 4 °C or 12 °C, and after 0, 8, and 17 h, and 1, and 2 days when held at 22 °C. At 4 °C, all populations declined by 0.5–1.0 log CFU/g over 7 days. At 12 °C, E. coli O157:H7 and Salmonella populations did not change, while L. monocytogenes populations increased by ca. 0.5 log CFU/g over 7 days. At 22 °C, E. coli O157:H7, Salmonella, and L. monocytogenes populations increased by ca. 1, 2, or 0.3 log CFU/g, respectively, with the majority of growth occurring during the first 17 h. On occasion, populations on cut surfaces were significantly higher than those on uncut surfaces. Results indicate that populations are reduced under refrigeration, but survive and may grow at elevated temperatures.     

Diversity of culturable microorganisms and occurrence of Listeria monocytogenes and Salmonella spp. in Tuber aestivum and Tuber melanosporum ascocarps

The aim of this study was to investigate the total mesophilic microorganisms, Pseudomonas genus, Enterobacteriaceae family, mold and yeast counts and the presence of Listeria monocytogenes and Salmonella spp on Tuber aestivum and Tuber melanosporum ascocarps. The results confirmed that the major percentage of the microorganisms, approximately 9.0 log ufc/g, were present in the peridium, the glebas of healthy truffles being practically free of microorganisms. The predominant microbial group was the Pseudomonas averaging 8.3 and 8.4 log cfu/g on T. aestivum and T. melanosporum whole ascocarps, respectively. The Enterobacteriaceae also achieved high populations, especially in T. aestivum truffles, with 6.3 log cfu/g. Molds and yeasts never exceeded 5.0 log cfu/g. The characterization of the isolates revealed that the fluorescens pseudomonads were the most prevalent. Raoultella terrigena and Enterobacter intermedius were the dominant Enterobacteriaceae. The identification of the yeast isolates revealed five species: Debaryomyces hansenii, Issatchenkia scutulata, Rhodotorula aurantiaca, Saccharomyces dairensis and Trichosporon beigelii subspecies A and B. The mold genera detected in both species of truffles were Aspergillus, Cladosporium, Penicillium and Fusarium, Trichoderma being present only in T. aestivum. L. monocytogenes was found in 10% of the samples of T. aestivum analysed but Salmonella spp. was not detected. Knowledge of the microbial population in terms of possible food borne and pathogen microorganisms is very useful for establishing successful disinfection and storage methods to prolong the shelf-life of ascocarps of T. aestivum and T. melanosporum.     

Direct detection of E. Coli O157:H7 in selected food systems by a surface plasmon resonance biosensor

The Spreeta^TM, surface plasmon resonance (SPR)-based biosensor, was used to detect Escherichia coli O157:H7 spiked in milk, apple juice and ground beef extract using specific antibodies. In the Spreeta^TM biosensor light from an LED is reflected off a gold surface, and the angle and intensity corresponding to the SPR minimum is measured and represented as a refractive index (RI) change corresponding to the antigen-antibody coupling at the sensor surface. Milk, apple juice, and ground beef patties spiked with E. coli O157:H7, at varying concentrations, were injected on the sensor surface immobilized with antibodies against the pathogen at a rate of 1 ml/min for a total of 2 min. The change in RI due to the binding of O157:H7 corresponding to each concentration was computed as an average of three replications over a 2 min interaction period. Assays were conducted at near real-time and results obtained after about 30 min of sample injection. Sensitivity of the E. coli O157:H7 assay was 10²-10³ colony forming unit (CFU)/ml. The biosensor assay was also specific to E. coli O157:H7 as other organisms (E. coli K12 and Shigella) did not produce any appreciable change in the sensogram. Further experiments are needed to establish well-defined methods for detecting other food-borne pathogens in more complex and specific food matrices.     

Comparative efficacy of Zataria multiflora Boiss., Origanum compactum and Eugenia caryophyllus essential oils against E. coli O157:H7, feline calicivirus and endogenous microbiota in commercial baby-leaf salads

Ready-to-eat salads using baby-leaf and multi-leaf mixes are one of the most promising developments in the fresh-cut food industry. There is great interest in developing novel decontamination treatments, which are both safe for consumers and more efficient against foodborne pathogens. In this study, emulsions of essential oils (EOs) from Origanum compactum (oregano), Eugenia caryophyllus (clove), and Zataria multiflora Boiss (zataria) were applied by spray (0.8 ml) after the sanitizing washing step. The aim was to investigate their ability to control the growth of potentially cross-contaminating pathogens and endogenous microbiota in commercial baby leaves, processed in a fresh-cut produce company. Zataria EO emulsions of 3%, 5% and 10% reduced Escherichia coli O157:H7 by 1.7, 2.2 and 3.5 log cfu/g in baby-leaf salads after 5 days of storage at 7 °C. By contrast, reductions in E. coli O157:H7 counts remained the same when clove was applied at concentrations of 5% and 10% (2.5 log cfu/g reduction). Oregano (10%) reduced inoculated E. coli O157:H7 counts in baby-leaf salads by a maximum of 0.5 log cfu/g after 5 days of storage. Zataria showed strong antimicrobial efficacy against E. coli O157:H7 and also against the endogenous microbiota of baby-leaf salads stored for 9 days. Feline calicivirus (FCV), a norovirus surrogate, survived on inoculated baby-leaf salads during refrigerated storage (9 days at 7 °C) regardless of treatment. Refrigeration temperatures completely annulled the effectiveness of the EOs against FCV inoculated in baby-leaf salads as occurred in FCV cultures. This study shows that EOs, and zataria in particular, have great potential use as an additional barrier to reduce contamination-related risks in baby-leaf salads. However, further research should be done into foodborne viruses in order to improve food safety.     

The effects of temperature,chlorine and acids on the survival of Listeria and Salmonella strains associated with uncooked shrimp carapace and cooked shrimp flesh

The purpose of this study was to assess the influence of the association of Listeria and Salmonella with shrimp surfaces on the effects of temperature, chlorine and acids on their survival. Planktonic, attached and colonized cells of Listeria monocytogenes Scott A, L. monocytogenes V7, Salmonella Senftenberg 1734b and S. Typhimurium ATCC 14028 were challenged with high (50°, 60° and 70 °C) and low (4 °C) temperature, 100 ppm sodium hypochlorite solution, and acetic, hydrochloric and lactic acids (pH 4.0). Attached and colonized Listeria and Salmonella showed significantly greater (p < 0.05) resistance to heat (∼1.3–2.6 fold increase in D-values), hypochlorite (∼6.6 ≥ 40.0 fold) and acids (∼4.0–9.0 fold) than their planktonic counterparts. There were no significant differences (p > 0.05) in the survival of planktonic, attached or colonized cells of Listeria and Salmonella stored under refrigerated conditions. The association of Listeria and Salmonella with shrimp surfaces enhances their resistance to heat, chlorine and acids. Both attachment to, and subsequent colonization of, shrimp surfaces by pathogens may reduce the efficacy of methods used in their control. Strategies to reduce attachment of these pathogens to shrimp are required to assure safety of this product.     

Control of foodborne pathogens on fresh-cut fruit by a novel strain of Pseudomonas graminis

The consumption of fresh-cut fruit has substantially risen over the last few years, leading to an increase in the number of outbreaks associated with fruit. Moreover, consumers are currently demanding wholesome, fresh-like, safe foods without added chemicals. As a response, the aim of this study was to determine if the naturally occurring microorganisms on fruit are “competitive with” or “antagonistic to” potentially encountered pathogens. Of the 97 and 107 isolates tested by co-inoculation with Escherichia coli O157:H7, Salmonella and Listeria innocua on fresh-cut apple and peach, respectively, and stored at 20 °C, seven showed a strong antagonistic capacity (more than 1-log unit reduction). One of the isolates, CPA-7, achieved the best reduction values (from 2.8 to 5.9-log units) and was the only isolate able to inhibit E. coli O157:H7 at refrigeration temperatures on both fruits. Therefore, CPA-7 was selected for further assays. Dose-response assays showed that CPA-7 should be present in at least the same amount as the pathogen to adequately reduce the numbers of the pathogen. From the results obtained in in vitro assays, competition seemed to be CPA-7's mode of action against E. coli O157:H7. The CPA-7 strain was identified as Pseudomonas graminis. Thus, the results support the potential use of CPA-7 as a bioprotective agent against foodborne pathogens in minimally processed fruit.     

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.     

Prediction of coliforms and Escherichia coli on tomato fruits and lettuce leaves after sanitizing by using Artificial Neural Networks

The objectives of this study were to investigate the efficacy of two sanitizers, i.e. hypochlorous and peracetic acids, in reducing coliforms and Escherichia coli levels on tomato fruits and lettuce leaves, and to mathematically predict the relationship among the initial bacterial load, type of vegetable/fruit, types and concentration of sanitizer and residual microorganism levels after the sanitizing, by applying artificial neural networks (ANNs). The E. coli and coliforms used in this study were isolated from the two food types, and their cultures were activated in Tryptic Soy Broth (ca. 6-7 log₁₀ cfu/ml) before inoculating onto the fruit and vegetable. Both sanitizers reduced the number of the micro-organisms. However, as the hypochlorous acid concentration was increased, the level of viable coliforms and E. coli on the tomato fruits was reduced around 2-3 log₁₀ cfu/g (p ≤ 0.05), compared to only about 1 log₁₀ cfu/g reduction on lettuce leaves (p ≤ 0.05). Conversely, when the peracetic acid concentration was increased, the coliforms and E. coli levels on tomato fruits were reduced by some 3-4 log₁₀ cfu/g (p > 0.05) compared to only about 2 log₁₀ cfu/g on lettuce leaves (p > 0.05).The best sum square error from the neural prediction of residual coliforms and E. coli were 0.50 and 0.84, respectively, and the maximum R² of residual coliforms and E. coli were 0.85 and 0.72, respectively. Only one hidden layer with three hidden neurons for coliforms and five for E. coli, were required to model this data.     

Effects of different sanitizing treatments on biofilms and attachment of Escherichia coli and Listeria monocytogenes on green leaf lettuce

The effects of ozone (2 mg/L), chlorine (100 mg/L) and organic acid (0.25 g/100 g citric acid plus 0.50 g/100 g ascorbic acid) treatments at 10 °C for 2 min on the removal of Escherichia coli and Listeria monocytogenes cells embedded inside biofilms on the surface of lettuce leaves were studied. None of the sanitizing treatments were found effective in removing the bacterial biofilms. Initiation of biofilms was observed after 24 h of incubation. Bacterial cells appeared as individual cells, rather than clusters after 6 h incubation, thus 99.9% reductions in both E. coli and L. monocytogenes counts were achieved with all the three treatments. However, after 48 h incubation, none of the treatments resulted in higher than 90% reduction in microbial counts. Biofilm formation was demonstrated for the 48 h incubated samples with SEM images.     

Lactic acid resistance of Shiga toxin-producing Escherichia coli and multidrug-resistant and susceptible Salmonella Typhimurium and Salmonella Newport in meat homogenate

This study compared lactic acid resistance of individual strains of wild-type and rifampicin-resistant non-O157 Shiga toxin-producing Escherichia coli (STEC) and of susceptible and multidrug-resistant (MDR) and/or MDR with acquired ampC gene (MDR-AmpC) Salmonella against E. coli O157:H7. After inoculation of sterile 10% beef homogenate, lactic acid was added to a target concentration of 5%. Before acid addition (control), after acid addition (within 2 s, i.e. time-0), and 2, 4, 6 and 8 min after addition of acid, aliquots were removed, neutralized, and analyzed for survivors. Of wild-type and of rifampicin-resistant non-O157 STEC strains, irrespective of serogroup, 85.7% (30 out of 35 strains) and 82.9% (29 out of 35 strains), respectively, reached the detection limit within 0–6 min. Of Salmonella strains, 87.9% (29 out of 33 isolates) reached the detection limit within 0–4 min, irrespective of antibiotic resistance phenotype. Analysis of non-log-linear microbial survivor curves indicated that non-O157 STEC serogroups and MDR and susceptible Salmonella strains required less time for 4D-reduction compared to E. coli O157:H7. Overall, for nearly all strains and time intervals, individual strains of wild-type and rifampicin-resistant non-O157 STEC and Salmonella were less (P < 0.05) acid tolerant than E. coli O157:H7.     

Antibacterial activity of oregano and thyme essential oils against Listeria monocytogenes and Escherichia coli O157:H7 in feta cheese packaged under modified atmosphere

The antibacterial activity of the essential oils (EO) of oregano and thyme added at doses of 0.1 or 0.2 and 0.1 ml/100 g, respectively, to feta cheese inoculated with Escherichia coli O157:H7 or Listeria monocytogenes was investigated during cheese storage under modified atmosphere packaging (MAP) of 50% CO₂ and 50% N₂ at 4 °C. Compositional analysis showed that the predominant phenols were carvacrol and thymol for both EO. In control feta inoculated with the pathogens and stored under MAP, results showed that E. coli O157:H7 and L. monocytogenes strains survived up to 32 and 28 days of storage. However, in feta cheese treated with oregano EO at the dose of 0.1 ml/100 g, E. coli O157:H7 or L. monocytogenes survived up to 22 and 18 days, respectively, whereas at the dose of 0.2 ml/100 g up to16 or 14 days, respectively. Feta cheese treated with thyme EO at 0.1 ml/100 g showed populations of E. coli O157:H7 or L. monocytogenes not significantly different (P > 0.05) than those of feta cheese treated with oregano at 0.1 ml/100 g. Although both essential oils exhibited equal antibacterial activity against both pathogens, the populations of L. monocytogenes decreased faster (P < 0.05) than those of E. coli O157:H7 during the refrigerated storage, indicating a stronger antibacterial activity of both essential oils against the former pathogen.     

The effects of X-ray radiation on Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri inoculated on whole Roma tomatoes

In the last two decades several foodborne disease outbreaks associated with produce were reported. Tomatoes, in particular, have been associated with several multi-state Salmonella outbreaks. Inactivation of inoculated Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on whole Roma tomato surfaces by X-ray at 0.1, 0.5, 0.75, 1.0, and 1.5 kGy was studied. The main purpose of this study was to achieve a 5 log reduction in consistent with the recommendations of the National Advisory Committee on Microbiological Criteria for Foods. Moreover, the effect of X-ray on inherent microflora (mesophilic counts, psychrotrophic counts and yeast and mold counts) of untreated and treated Roma tomatoes, during storage at ambient temperature (22 °C) for 20 days was also determined. Mixtures of three or two strains of each tested organism was spot inoculated (100 μl) onto the surface of Roma tomatoes (approximately 7–9 log per tomato), separately, and air-dried, followed by treatment with X-ray doses at 22 °C and 55–60% relative humidity. Surviving bacterial populations on tomato surfaces were evaluated using a nonselective medium (tryptic soy agar) with a selective medium overlay for each bacteria; E. coli O157:H7 (CT-SMAC agar), L. monocytogenes (MOA), and S. enterica and S. flexneri (XLD). Treatment with X-ray significantly reduced the population of the tested pathogens on whole Roma tomato surfaces, compared with the control. Approximately 4.2, 2.3, 3.7 and 3.6 log CFU reduction of E. coli O157:H7, L. monocytogenes, S. enterica and S. flexneri per tomato were achieved by treatment with 0.75 kGy X-ray, respectively. More than a 5 log CFU reduction per tomato was achieved at 1.0 or 1.5 kGy X-ray for all tested pathogens. Furthermore, treatment with X-ray significantly reduced the inherent microflora on Roma tomatoes. Inherent levels were significantly (p < 0.05) lower than the control sample throughout storage for 20 days.     

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.     

Detection of Escherichia coli via VOC profiling using secondary electrospray ionization-mass spectrometry (SESI-MS)

Escherichia coli O157:H7 (EC O157:H7), as well as its recently emerging non-O157 relatives, are a notorious group of pathogenic bacteria associated with foodborne outbreaks. In this study, we demonstrated that secondary electrospray ionization mass spectrometry (SESI-MS) could be a rapid and accurate detection technology for foodborne pathogens. With SESI-MS volatile organic compound (VOC) profiling, we were able to detect and separate a group of eleven E. coli strains from two major foodborne bacteria, Staphylococcus aureus and Salmonella Typhimurium in three food modeling media. In addition, heatmap analysis of relative peak intensity show that there are six core peaks (m/z of 65, 91, 92, 117, 118 and 119) present and at a similar intensity in all eleven E. coli strains at the experimental conditions we tested. These peaks can be considered conserved VOC biomarkers for E. coli species (robustly produced after just 4 h of growth). Bacterial strain-level differentiation was also attempted via VOC profiling, and we found that EC O157:H7 and EC O145 were differentiable from all other EC strains under the conditions investigated.     

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.     

Heat shock effects on the viability of Cronobacter sakazakii during the dehydration,fermentation, and storage of lactic cultured milk products

In the present study, the viability of heat-shocked and non-shocked Cronobacter sakazakii, a foodborne pathogen, after drying and during the fermentation as well as storage of lactic cultured milk was evaluated. It was found that heat shock increased the viability of C. sakazakii. The pure culture of C. sakazakii, regardless of heat shock, grew rapidly in skim milk with a viable population of ca. 8.59–8.70 log cfu/ml after ca. 48 h of cultivation. Thereafter, the viable population of C. sakazakii remained stable. While in the mix culture with Streptococcus thermophilus or Lactobacillus bulgaricus, a marked reduction in the viable population of C. sakazakii was noted after 24 h of cultivation in skim milk. Nevertheless, at the end of fermentation, the heat-shocked C. sakazakii had a viable population of 5.93–6.01 log cfu/ml, which is significantly higher (P < 0.05) than that of non-shocked cells of 4.96–4.99 log cfu/ml. While the presence of C. sakazakii did not affect the growth of lactic acid bacteria in skim milk. Additionally, heat shock was found to enhance the survival of C. sakazakii after freeze-drying or spray-drying and during the storage of the lactic fermented milk products (pH 4.3) at 5 °C for 48 h.     

Population dynamics of Escherichia coli inoculated by irrigation into the phyllosphere of spinach grown under commercial production conditions

Recent outbreaks of food-borne illnesses associated with the consumption of fresh produce have increased attention on irrigation water as a potential source of pathogen contamination. A better understanding of the behaviour of enteric pathogens introduced into agricultural systems during irrigation will aid in risk assessments and support the development of appropriate farm-level water management practices. For this reason, the survival dynamics of two nalidixic acid resistant strains of Escherichia coli after their spray inoculation into the phyllosphere and soil of field spinach were examined over two growing seasons. E. coli strains NAR, an environmental isolate, and DM3n, a non-pathogenic serotype O157:H7, were applied at rates of 10⁴ to 10⁷ cfu/100 ml to the fully developed spinach plants that arose subsequent to the harvesting of their upper leafy portions for commercial purposes (secondary-growth plants). After 72 h, E. coli on spinach were reduced by 3-5 logs. Culturable E. coli were recovered from plants up to 6 days post-inoculation. Survival in soil was greater than in the phyllosphere. Under ambient conditions, the mean 72 h first order decay constant computed by Chick's Law was 0.1 h⁻¹. Although light reduction studies indicated UV irradiation negatively influenced the persistence of E. coli, a simple relationship between UV exposure and phyllosphere E. coli densities could not be established. E. coli introduced to the leafy portions of spinach via spray irrigation displayed rapid declines in their culturability under the open environmental conditions experienced during this study. A 6 day period between the last irrigation and harvest would minimize the risks of E. coli survival in the spinach phyllosphere. E. coli NAR was identified as a possible surrogate for the O157:H7 strain, DM3n.