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Biofilms are known to play important roles in bacterial survival and persistence in food-processing environments. This study aimed to determine the ability of the top 7 STEC serotypes to form biofilms on polystyrene (POL) and stainless steel (SS) plates and to quantify their survival and transfer from dry-surface biofilms to lettuce pieces. The ability of 14 STEC strains to form biofilms on these two materials at different exposure times and temperatures was assessed using crystal violet, Congo red and SEM. At 10 °C all serotypes were weak biofilm producers on both surfaces. In contrast, serotypes O45-040, O45-445, O103-102, O103-670 and O157-R508 were strong biofilm producers at 25 °C. Strains O103-102, O103-670, O111-CFS, O111–053 and O157:H7-R508 were expressers of curli. Under scanning electron microscopy, strains O103-670, O111-CFS, O157-R508, and O121-083 formed more discernible multilayer, mature biofilms on SS coupons. Regardless of the surface (POL/SS), all STEC strains were able to transfer viable cells onto fresh lettuce within a short contact time (2 min) to varying degrees (up to 6.35 log cfu/g). On POL, viable cell of almost all serotypes exhibited decreased detachment (p = 0.001) over 6 days; while after 30 days on SS, serotypes O45-040, O103-102, O103-670, O111-053, O111-CFS, O121-083, O145-231 O157:H7-R508 and O157:H7-122 were transferred to lettuce. After enrichment, all 14 STEC strains were recovered from dry-surface biofilms on POL and SS plates after 30 days. Results demonstrated that the top 7 STEC remained viable within dry-surface biofilms for at least 30 days, transferring to lettuce within 2 min of exposure and acting as a source of adulteration.

The purpose of this study was to determine the effectiveness of a commercial Salmonella bacteriophage mixture (SalmoFresh™ 6-phage strains) and to compare its effectiveness with a chlorinated water treatment to reduce Salmonella on produce and seeds at different temperatures and storage times. Two sets of experiments were designed to test phage and chlorinated water effectiveness on produce at 2, 10 and 25 °C at different storage times (1, 24, 48 and 72 h). First, SalmoFresh™ was applied to the surface of lettuce, mung bean sprouts and mung bean seeds that were spot-inoculated with a five Salmonella strain mixture (Newport, Braenderup, Typhimurium, Kentucky, and Heidelberg, 105 CFU/mL) by spraying phages onto lettuce (n = 48 pieces, 3×3 cm2 per treatment) and sprouts (n = 48 pieces per treatment). A second set of experiments (scaled-up) consisted in the application of phages by immersion to Salmonella adulterated lettuce (600 g), 300 g sprouts (300 g) or mung bean seeds (30 g) in a phage cocktail (108 PFU/mL) for 15 min (lettuce and sprouts) or 1 h (seeds). Another group of samples was washed with chlorinated water and yet another group was treated with a combination of chlorinated water followed by phage cocktail. Each experiment was repeated three times by quadruplicates. After the treatments for spot-inoculated and scaled-up experiments, lettuce and sprouts were separated into different lots (10 g/lot) and stored at 2, 10 and 25 °C; Salmonella was enumerated after 1, 24, 48 and 72 h. Adulterated phage-treated seeds were packaged and stored dry at 25 °C. Salmonella was enumerated after 72 h of storage. Groups of phage treated mung bean seeds (720 g) were germinated, and the reduction in Salmonella determined. Results of microplate virulence assays indicated that SalmoFresh™ reduced (P = 0.007) Salmonella by an average of 5.34 logs CFU/mL after 5 h at 25 °C. Spraying SalmoFresh™ onto lettuce and sprouts reduced Salmonella by 0.76 and 0.83 log10 CFU/g, respectively (P

Decreasing the health burden caused by foodborne pathogens is challenging and it depends on the identification of the most significant hazards and food sources causing illnesses, so adequate mitigation strategies can be implemented. In this regard, the Canadian Food Inspection Agency (CFIA) has developed the Establishment-based Risk Assessment (ERA) model, so that a more effective and efficient allocation of resources can be assigned to the highest food safety risk areas. To assess risk, the model considers the type of food sub-products being manufactured by establishments and its scope is limited to the 17 most important foodborne pathogens representing the highest level of food safety risk. However, the information on source attribution at the sub-product level based on a structured approach is limited. To overcome this challenge, an expert elicitation was conducted in 2016 to estimate the relative contribution and associated certainty of each sub-product for 31 pathogen-commodity combinations to the total Canadian health burden associated with foodborne illnesses (expressed in DALYs). These DALYs represent 78% of the total Canadian health burden associated with federally-regulated food commodities considered within the model. A total of 49 Canadian experts recruited using a “snow ball” sampling strategy participated in the study by completing an electronic survey. Results of the elicitation displayed variable levels of health burden allocation between the pathogens and the different commodity sub-products. Assessment of the certainty levels showed some combinations being evaluated with more confidence (e.g., Campylobacter and eggs/poultry sub-products) than others, where a bimodal distribution of certainty was observed (e.g., Toxoplasma in pork sub-products). Furthermore, no participant raised concerns on the food classification scheme, suggesting their agreement with the proposed sub-products categorization of the elicitation. Relative contribution estimates will be included in the CFIA ERA model and used to enhance its applicability for risk prioritization and effective resource allocation during food establishment inspections. While substantial uncertainty around the central tendency estimates was found, these estimates provide a good basis for regulatory oversight and public health policy.

Previous studies have reported that the mechanism of bactericidal action of the plant oil aromatics, eugenol, carvacrol and cinnamaldehyde involves inhibition of adenosine triphosphate generation and membrane disruption. In this study the capacity of the aromatics to inhibit the membrane bound ATPase activity of Escherichia coli and Listeria monocytogenes was investigated by experiments on isolated membranes. Inhibition of the ATPase activity of E. coli membranes was observed with 5 mM or 10 mM eugenol or carvacrol. Progressively greater inhibition by cinnamaldehyde was observed as concentration increased from 0.1 to 10 mM. L. monocytogenes ATPase activity was significantly inhibited by eugenol (5 or 10 mM), carvacrol (10 mM) and cinnamaldehyde (10 mM). Lactobacillus sakei is highly resistant to cinnamaldehyde compared to E. coli and L. monocytogenes. To determine whether this resistance was related to the relative hydrophobicity of the cell surface and hence the ability of the cell to take up the aromatics, the percentage of the three organisms partitioning in dodecane was compared. No significant difference was found between the partitioning percentage of L. monocytogenes (17.2%) and L. sakei (13.8%), indicating that surface hydrophobicity does not explain the differing sensitivity to cinnamaldehyde of these two organism. The percent partitioning of E. coli was significantly greater than both other organisms (23.3%) and may explain the greater sensitivity of E. coli to all three aromatics.

The role of membrane disruption in the bactericidal activity of the plant oil aromatic compounds eugenol, carvacrol and cinnamaldehyde was investigated using confocal laser scanning microscopy, changes in ATP levels and cell viability. In 25 mM HEPES buffer pH 7 at 20 degrees C, 10 mM eugenol or carvacrol increased uptake of propidium iodide by Escherichia coli, Listeria monocytogenes and Lactobacillus sakei over a 10-min period. The same treatments resulted in lowered viability, rapid depletion of cellular ATP and release of ATP, with the exception of Lb. sakei treated with carvacrol. Eugenol or carvacrol at 5 mM to 10 mM inhibited E. coli and L. monocytogenes motility. Lb. sakei was resistant to cinnamaldehyde. Thus, its effects were only studied on E. coli and L. monocytogenes. At 10 mM, cinnamaldehyde caused a slight but statistically significant increase in propidium iodide staining of E. coli, but had no effect on L. monocytogenes. Cinnamaldehyde treatment of E. coli at 10 mM and L. monocytogenes at 40 mM resulted in decreased cellular ATP, but there was no concomitant release of ATP. Cinnamaldehyde at 5 and 10 mM inhibited E. coli and L. monocytogenes motility. Results for eugenol and carvacrol are consistent with non-specific permeabilization of the cytoplasmic membrane. Evidence for increased membrane permeability by cinnamaldehyde is less conclusive. The release of ATP from eugenol and carvacrol-treated cells and absence of release from cinnamaldehyde-treated cells could indicate that eugenol and carvacrol possess ATPase inhibiting activity. Secondary effects would also be consistent with membrane disruption.

Commercial allyl isothiocyanate (AIT) was examined for its ability to reduce numbers of Escherichia coli O157:H7 inoculated in fresh ground beef packaged under nitrogen and stored refrigerated or frozen. A five-strain cocktail of E. coli O157:H7 containing 3 or 6 log10 cfu/g was inoculated into 100 g ground beef and formed into 10x1-cm patties. A 10-cm diameter filter paper disk treated with AIT suspended in sterile corn oil was placed on top of a single patty. One patty and paper disk were placed in a bag of Nylon/EVOH/PE with O2 permeability of 2.3 cm3 m(-2) 24 h atm at 23 degrees C. The bags were back-flushed with 100% nitrogen, heat-sealed and stored at 10, 4 and -18 degrees C for 8, 21 or 35 days, respectively. During storage, the AIT levels in the package headspaces were determined by gas liquid chromatography, and mesophilic bacteria and E. coli O157:H7 were counted. The mesophilic aerobic bacteria in ground beef patties were largely unaffected by the addition of AIT. At an initial population of 3 log10 cfu/g, E. coli O157:H7 was reduced by AIT to undetectable levels after 18 days at 4 degrees C or 10 days at -18 degrees C. In samples inoculated with 6 log10 cfu/g, a3 log10 reduction of E. coli O157:H7 was observed after 21 days at 4 degrees C, while a 1 log10 reduction was observed after 8 and 35 days at 10 and -18 degrees C, respectively. The final AIT concentrations in the headspaces after storage at 10, 4, and -18 degrees C were 444, 456, and 112 microg/ml at 8, 21, and 35 days, respectively. Results showed that AIT can substantially reduce numbers of E. coli O157:H7 in fresh ground beef during refrigerated or frozen storage.

This study was undertaken to determine whether the glucosinolates naturally present in non-deheated mustard flour could serve as a source of allyl and other isothiocyanates in sufficient quantity to kill Escherichia coli O157:H7 inoculated in ground beef at three different levels, during refrigerated storage of the meat under nitrogen. Mustard flour was mixed at 5%, 10% or 20% (w/w) with freshly ground beef, then the beef was inoculated with a cocktail of five strains of E. coli O157:H7 at either 3, 6 oror =1.6 log10 cfu/g. The ground beef was formed into 100 g patties and each was placed in a bag of Nylon/EVOH/PE, which was back-flushed with 100% N2, heat-sealed and stored at 4 degrees C foror =21 days. During storage, the allyl isothiocyanate (AIT) levels in package headspaces were determined by gas liquid chromatography. By 21 days, the levels present in treatments were not significantly different. After 21 days storage, there were 0.5, 3 and 5.4 log10 decreases in numbers of E. coli O157:H7 from the initial levels of 6 log10 cfu/g in meat containing 5%, 10% and 20% mustard flour, respectively. When inoculated at 3 log10 cfu/g, E. coli O157:H7 was reduced to undetectable levels after 18, 12 and 3 days with 5%, 10% and 20% mustard flour, respectively. When immunomagnetic separation (IMS) was used for E. coli recovery following its inoculation ator =1.6 log10 cfu/g, 5% mustard did not completely eliminate the pathogen from ground beef stored for 6 days. The natural microflora of the ground beef which developed in vacuum packages was unaffected by the addition of 5% mustard flour but some inhibition was found at higher concentrations. Sensory evaluation of the cooked ground beef showed that there were no significant differences in the acceptability of meat treated with 5 or 10% mustard flour. However, panelists could distinguish untreated controls from mustard treatments, but considered the mustard-treated meat to be acceptable. These results showed that it is possible to use mustard flour at levels of5-10% to eliminate E. coli O157:H7 from fresh ground beef.

Cilantro oil is an essential oil preparation extracted from the plant Coriandrum sativium. A series of experiments were conducted to evaluate the ability of cilantro oil to control the growth of Listeria monocytogenes on vacuum-packed ham. The in vitro minimal inhibitory concentration for five strains of L. monocytogenes was found to vary from 0.074% to 0.018% depending on strain. Cilantro oil treatments were then tested on ham disks inoculated with a cocktail of the five L. monocytogenes strains. The treatments studied were 0.1%, 0.5%, and 6% cilantro oil diluted in sterile canola oil or incorporated into a gelatin gel in which lecithin was used to enhance incorporation of the cilantro oil. Gelatin gel treatments were also conducted with 1.4% monolaurin with or without 6% cilantro oil to determine if an interaction between the antimicrobials could increase inhibition of L. monocytogenes. Treated ham was then vacuum-packed and stored at 10 °C for up to 4 weeks. The only cilantro oil treatment which inhibited growth of L. monocytogenes on the ham samples was 6% cilantro oil gel. Samples receiving this treatment had populations of L. monocytogenes 1.3 log CFU/ml lower than controls at week 1 of storage, though there was no difference between treatments from week 2 onward. It appears that immobilization of the antimicrobial in a gel enhanced the effect of treatments. Cilantro oil does not appear to be a suitable agent for the control of L. monocytogenes on ham. The possible reasons for reduced effectiveness of cilantro oil against L. monocytogenes on ham are discussed.

There is a growing interest in modelling microbial growth as an alternative to time-consuming, traditional, microbiological enumeration techniques. Several statistical models have been reported to describe the growth of different microorganisms, but there are accuracy problems. An alternate technique 'artificial neural networks' (ANN) for modelling microbial growth is explained and evaluated. Published data were used to build separate general regression neural network (GRNN) structures for modelling growth of Aeromonas hydrophila, Shigella flexneri, and Brochothrix thermosphacta. Both GRNN and published statistical model predictions were compared against the experimental data using six statistical indices. For training data sets, the GRNN predictions were far superior than the statistical model predictions, whereas the GRNN predictions were similar or slightly worse than statistical model predictions for test data sets for all the three data sets. GRNN predictions can be considered good, considering its performance for unseen data. Graphical plots, mean relative percentage residual, mean absolute relative residual, and root mean squared residual were identified as suitable indices for comparing competing models. ANN can now become a vehicle whereby predictive microbiology can be applied in food product development and food safety risk assessment.

Carnobacterium spp. are commonly isolated from a variety of foods, especially from meats stored under anaerobic atmospheres at refrigeration temperatures, but the role of these organisms in the spoilage of meat and meat products is yet to be determined. Cresol Red Thallium Acetate Sucrose (CTAS) agar was developed as a selective medium for enumeration of carnobacteria, however problems such as poor recovery of Carnobacterium spp. and interference by other microorganisms have precluded its general use. The aim of this study was to improve CTAS agar by broadening its spectrum of selective recovery for carnobacteria while restricting the ability of interfering species to grow. Ten Carnobacterium spp. (five ATCC cultures and five isolates from fresh pork) and 20 other genera were used in testing the agar. A wider range of Carnobacterium spp. recovery was obtained by modifying concentrations of sucrose, manganese sulphate and thallium acetate. Additions of inulin and thiamine hydrochloride also improved growth response. The additions of vancomycin and Chrisin® (nisin) eliminated interference from other microorganisms. A two-temperature incubation procedure was included to improve the characteristic growth of Carnobacterium spp. on the modified medium, identified as Cresol Red Thallium Sucrose Inulin (CTSI) agar. Lactic acid bacteria and Enterobacteriaceae were unable to grow on CTSI incubated aerobically. Growth of Carnobacterium spp. on CTSI yielded pink colonies, except for Cb. mobile, which formed gray colonies. In some instances, a red precipitate formed in the center of the colony. Yellowing and clearing of the growth medium was also frequently observed. Recovery of carnobacteria using CTSI was identical to that obtained with All Purpose Tween (APT) agar.

The hygienic performances of the processes for the production of cooled carcasses at eight pork packing plants were assessed from small sets of microbiological data. At each plant, a single sample was obtained from a randomly selected site on each of 25 randomly selected carcasses at each of three stages of processing, which were after polishing, after washing at the end of the dressing process, and after cooling. The aerobic bacteria, coliforms and Escherichia coli recovered from each sample were enumerated. When bacteria of one type were recovered fromor = 20 of 25 samples, the log mean number of those bacteria on the population of carcasses undergoing processing was estimated on the assumption that the set of counts was normally distributed. The log of the total number recovered from 25 samples was calculated for each set of counts. The log mean numbers of total aerobic bacteria recovered from the polished carcasses at different plants ranged from about 1.9 to 3.8 log cfu cm(-2). At six of the plants, the log mean numbers of total aerobes on the cooled carcasses did not differ substantially from the log mean numbers on the polished carcasses, but the log mean numbers on the cooled carcasses were substantially higher at one plant and substantially lower at another than on the polished carcasses. Coliforms and E. coli were recovered from too few samples in most sets from cooled carcasses for estimation of their log mean numbers. However, the log total numbers of coliforms and E. coli recovered indicated that substantial numbers of those organisms were added to carcasses during the dressing processes at four of the plants, and that the numbers on the carcasses were substantially reduced by the processes for cooling without spraying at two of the plants. At seven of the plants, the total numbers of coliforms and E. coli recovered from cooled carcasses were3.1 and2.2 log cfu 2500 cm(-2), respectively. The findings indicate that production processes for pig carcasses can be operated to give cooled carcasses with log mean numbers of total aerobes2 cm(-2), and log total numbers of coliforms and E. coli each1 2500 cm(-2).

Unsliced beef pastrami, reformulated ham and bologna held at 6 degrees C were sliced 21, 17, 12 or 7 days before or at the assigned manufacturer's best before date and vacuum packaged. Packages of sliced meats were held at 6 degrees C for another 7, 12, 17 or 21 days, opened and analyses made for total bacteria, lactic acid bacteria, Enterobacteriaceae and Brochothrix thermosphacta. The maximum storage interval was 42 days; half this period unsliced, the remainder as repacked slices. Numbers of bacteria on pastrami were significantly greater than on ham and bologna (pastrami > ham > bologna) with the lactic acid bacteria dominating in all products. As unsliced meats approached their best before date, insignificant increases were generally noted for numbers of lactic bacteria, Enterobacteriaceae and B. thermosphacta. During subsequent storage of slices under vacuum, numbers of total and lactic bacteria increased exponentially at the same rate while B. thermosphacta growth was significantly slower. Numbers of Enterobacteriaceae remained low and were essentially unchanged during sliced meat storage. Within the context of study storage parameters, shelf-life appeared to be determined by length of time after slicing and repackaging rather than by best before date of the unsliced meat. Packages of sliced meat prepared from wholesale unsliced meats with 21 days left until their best before date or from unsliced meats with 12 days left until the best before date showed similar bacterial levels 21 days later. It was probable that the localization of bacterial growth at the meat surface-packaging film interface of the unsliced meats yielded slices with initially lowered bacterial content when repackaged and sampled from the uppermost slice. When Enterobacteriaceae and B. thermosphacta were absent from unsliced meats, extension of sliced meat package shelf-life beyond the best before date of the parent meat was possible. However, these bacterial groups were not always undetected.