Comparative persistence of Salmonella and Escherichia coli O157:H7 in loam or sandy loam soil amended with bovine or swine manure.

The fate of Salmonella and Escherichia coli O157:H7 in swine or dairy manure amended into sandy loam or loam soil under field conditions was studied. Soil was amended with manure inoculated with a Salmonella or E. coli O157:H7 cocktail, then transferred to 0.22 µm pore size membrane walled vials. The vials were then placed on the surface or at 15 cm depth in the test plots. Pathogen numbers, soil moisture, rainfall, and temperature were measured throughout the three trials (20-47 weeks duration) representing spring or fall application. Survival curves were characterized by having an initial rapid decline in pathogen numbers followed by a slower inactivation phase with an occasional increase in culturable cells. The CT99.9 values (time to reach a 3 log CFU reduction) varied from 2 to 120 days, with the most rapid decrease being observed on the surface of sandy loam soil. The persistence of pathogens is primarily governed by variations in moisture and temperature, although season of application along with manure and soil type also contribute. To generate more accurate predictive pathogen models, there is a need for laboratory-based trials to mirror the dynamic variation in temperature and soil moisture encountered within the natural environment.

Inactivation of Listeria monocytogenes on and within Apples Destined for Caramel Apple Production by Using Sequential Forced Air Ozone Gas Followed by a Continuous Advanced Oxidative Process Treatment.

This study evaluated the efficacy of using sequential forced air ozone followed by an advanced oxidative process (AOP) treatment to inactivate Listeria monocytogenes on and within Empire apples. The forced air ozone treatment consisted of a reactor that introduced ozone (6 g/h) into an airstream that flowed through an apple bed (ca. 30 cm in depth). Before treatment, the apples were conditioned at 4°C to ensure that condensate had formed before the apples were transferred to the reactor. The condensate ensured sufficient relative humidity to enhance the antimicrobial action of ozone. Air was passed through the apple bed at 9.3 m/s, and the ozone was introduced after 10 min. The ozone concentration measured after exiting the apple bed reached a steady state of 23 ppm. A 20-min ozone treatment supported a 2.12- to 3.07-log CFU reduction of L. monocytogenes, with no significant effect of apple position within the bed. The AOP-based method was a continuous process whereby hydrogen peroxide was introduced as a vapor into a reactor illuminated by UV-C and ozone-emitting lamps that collectively generated hydroxyl radicals. Operating the AOP reactor with UV-C light (54-mJ cm2 dose), 6% (v/v) hydrogen peroxide, 2 g/h ozone, and a chamber temperature of 48°C resulted in a 3-log CFU reduction of L. monocytogenes on the surface of the apples and internally within the scar tissue. Applying a caramel coating, from a molten solution (at 80°C), resulted in a 0.5-log CFU reduction of L. monocytogenes on the apple surface. In apples treated with the sequential process, L. monocytogenes could only be recovered sporadically by enrichment and did not undergo outgrowth when the caramel apples were stored at 22°C for 19 days. However, growth of L. monocytogenes within the core, but not the surface, was observed from caramel apples prepared from nontreated control fruit.

Dissemination of Clostridium difficile in food and the environment: Significant sources of C. difficile community-acquired infection?

Clostridium difficile is a significant pathogen with over 300 000 cases reported in North America annually. Previously, it was thought that C. difficile was primarily a clinically associated infection. However, through the use of whole genome sequencing it has been revealed that the majority of cases are community acquired. The source of community-acquired C. difficile infections (CDI) is open to debate with foodborne being one route considered. Clostridium difficile fits the criteria of a foodborne pathogen with respect to being commonly encountered in a diverse range of foods that includes meat, seafood and fresh produce. However, no foodborne illness outbreaks have been directly linked to C. difficile there is also no conclusive evidence that its spores can germinate in food matrices. This does not exclude food as a potential vehicle but it is likely that the pathogen is also acquired through zoonosis and the environment. The most significant factor that defines susceptibility to CDI is the host microbiome and functioning immune system. In this respect, effective control can be exercised by reducing the environmental burden of C. difficile along with boosting the host defences against the virulent enteric pathogen.

Persistence of Clostridium difficile in wastewater treatment-derived biosolids during land application or windrow composting.

AIMS: To determine the persistence of Clostridium difficile spores in biosolids during composting or when amended into soil and held under natural environmental climatic conditions. METHODS AND RESULTS: Five log CFU g(-1) Cl. difficile spores (ribotypes 027 or 078) were inoculated into agricultural soils (sandy loam or loam) amended with 10% w/w anaerobically digested biosolids. The inoculated soil : biosolids mixture was then placed into sentinel vials which were introduced at a depth of 15 cm within the field plot consisting of the corresponding soil type. Two trials were performed, the first of which started in late spring (May 2013 through to August 2014) and second from November 2013 through to October 2014 (fall trial). Ribotype 078 endospores in loam or sandy loam soil decreased during the summer but then increased in numbers towards the fall. At the end of the trial, levels of ribotype 078 spores had decreased by 1·5 log CFU g(-1) , with 027 spores decreasing by <1 log CFU g(-1) over the same time period. Windrow composting of biosolids decreased Cl. difficile levels from 3·7 log CFU g(-1) down to 0·3 log CFU g(-1) with the greater reduction occurring during the curing phase. In comparison, Cl. perfringens decreased from 6·3 log CFU g(-1) down to 2·4 log CFU g(-1) but mainly in the thermal phase of the composting process. CONCLUSIONS: Composting of biosolids is a more effective means of inactivating Cl. difficile compared to land application. SIGNIFICANCE AND IMPACT OF THE STUDY: Windrow composting represents an effective method to reduce the environmental burden of Cl. difficile associated with biosolids.

Fate of Clostridium difficile during wastewater treatment and incidence in Southern Ontario watersheds.

AIMS: To investigate the prevalence of Clostridium difficile encountered during sewage treatment and in water sources into which treated effluent was directly or indirectly discharged. METHODS AND RESULTS: Samples from wastewater treatment plants (WWTPs) and rivers were collected and then enriched for Cl. difficile. Each of the isolates was subjected to toxinotyping and DNA typing using ribotyping, in addition to pulse-field gel electrophoresis. Cl. difficile was isolated from 92% (108/117) of the raw sludge and 96% (106/110) of the anaerobic digested sludge samples from two Ontario WWTPs. The pathogen was recovered from 73% (43/59) of dewatered biosolids and effluent discharge, in addition to river sediments 39% (25/64). Ribotype 078 (commonly associated with Community Acquired infections) was recovered from raw sewage (19%; 21/108), digested sludge (8%; 8/106), biosolids (35%; 15/43) and river sediments (60%; 15/25). CONCLUSIONS: Clostridium difficile is commonly encountered in raw sewage and survives the wastewater treatment process. The pathogen can then be disseminated into the wider environment via effluent and land application of biosolids. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has illustrated the wide distribution of toxigenic Cl. difficile in WWTPs and river sediments although the clinical significance still requires to be elucidated.

Concentration and detection of Salmonella in mung bean sprout spent irrigation water by use of tangential flow filtration coupled with an amperometric flowthrough enzyme-linked immunosorbent assay.

The development of a culture-free method for Salmonella screening of spent irrigation water derived from sprouting mung bean beds is described. The system used tangential flow filtration (TFF) to nonspecifically concentrate cells from large (2- to 10-liter) sample volumes. The retentate (100 ml) from the TFF was then flowed over an anti-Salmonella antibody-modified cellulose acetate membrane. The captured Salmonella was detected by reacting with a secondary anti-Salmonella and goat anti-rabbit biotin labeled antibody, followed by avidin-tagged glucose oxidase. The hydrogen peroxide generated from the enzymic oxidation of glucose was amperometrically detected at an underlying platinum electrode. It was found that 10 liters of Salmonella suspensions of 2 log CFU/ml could be concentrated to 4 log CFU/ml with 60% recovery regardless of the flow rate (112 to 511 ml/min) or transmembrane pressure (0 to 20 lb/in2) applied. The solids content of spent irrigation water negatively affected the filtration rate of TFE. This was most evident in spent irrigation water collected in the initial 24 h of the sprouting period, where the solids content was high (4,170 mg/liter) compared with samples collected at 96 h (560 mg/ liter). Trials were performed using mung bean beds inoculated with different Salmonella levels (1.3 to 3.3 log CFU/g). By using the optimized TFF and flowthrough immunoassay it was possible to detect Salmonella in spent irrigation water at levels of 2.43 log CFU/ml within 4 h. The integrated concentration and detection system will provide a useful tool for sprout producers to perform in-house pathogen screening of spent irrigation water.

Microbial population profiles of the microflora associated with pre- and postharvest tomatoes contaminated with Salmonella typhimurium or Salmonella montevideo.

AIMS: To determine the microflora profiles of pre- and postharvest tomatoes contaminated with Salmonella montevideo or S. typhimurium DT104. METHODS AND RESULTS: Salmonella montevideo or S. typhimurium was inoculated onto the flowers of tomato plants with the microflora of the subsequent fruit examined using a combination of Source Carbon Utilization and 16S rDNA-PCR profiling. From 16S rDNA profiles it was evident that tomatoes derived from Salmonella inoculated plants harboured a different microbial population compared to nontreated controls. The same result was observed for tomatoes inoculated at postharvest and subsequently stored for 14 days at 15 degrees C. From sequencing analysis it was found that tomatoes derived from Salmonella inoculated plants but testing negative for the enteric pathogen, frequently harboured Enterobacter and Bacillus spp. In contrast, both bacterial types were not found associated with tomatoes testing positive for Salmonella. CONCLUSIONS: Salmonella introduced onto tomatoes at pre- or postharvest alters the composition of the microbial community. The presence of Enterobacter and Bacillus spp negatively affects the persistence of Salmonella on preharvest tomatoes. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella appears to modify rather than become integrated into the microbial communities associated with tomatoes. Yet, the presence of antagonistic bacteria appears to reduce the persistence of the enteric pathogen.

Assessment of the colorimetric and fluorometric assays for alkaline phosphatase activity in cow's, goat's, and sheep's milk.

Raw milk is a well-established vehicle for the carriage of human pathogens, and many regulatory bodies have consequently mandated compulsory pasteurization as a food safety intervention. The residual activity of alkaline phosphatase (ALP) has historically been used to verify the adequacy of pasteurization of cow's milk. However, there is uncertainty on how the current ALP standards and methods of analysis can be applied to sheep's and goat's milk, which naturally contain different levels of the enzyme than that found in cow's milk. The official ALP methods applied in Canada (colorimetric assay; MFO-3) and in the United States (Fluorophos) were assessed for their ability to detect enzyme activity in raw and pasteurized milk derived from cows, sheep, and goats. The detection limit and the limit of quantitation were 0.8 and 2.02 microg/ml phenol, respectively, for the MFO-3 method and 43 and 85 mU/liter, respectively, for the Fluorophos method. The average ALP levels in raw goat's, cow's, and sheep's milk were 165, 1,562, and 3,512 microg/ml phenol, respectively. Raw milk detection limits, which correspond to raw milk phosphatase levels, were 0.051, 0.485, and 0.023% in cow's, goat's, and sheep's milk, respectively, for the MFO-3 method and 0.007, 0.070, and 0.004%, respectively, for the Fluorophos method. Although both methods can be used for ALP determination in cow's, goat's, and sheep's milk, the Fluorophos assay was superior to the colorimetric MFO-3 method based on sensitivity and time required to complete the analysis.

Inactivation of MS2 F(+) coliphage on lettuce by a combination of UV light and hydrogen peroxide.

The efficacy of a produce decontamination method based on a combination of UV light (254 nm) and hydrogen peroxide (H2O2) to inactivate the MS2 F(+) coliphage inoculated onto iceberg lettuce was evaluated. Lettuce inoculated with 6.57 log PFU of MS2 was reduced by 0.5 to 1.0 log unit when illuminated with UV light alone for 20 to 60 s (12.64 to 18.96 mJ/cm2). In contrast, a 3-log reduction in MS2 was achieved with 2% (vol/vol) H2O2 spray delivered at 50 degrees C. No significant increase in log count reduction (LCR) was observed when H2O2 and UV light were applied simultaneously. However, H2O2 sprayed onto lettuce samples for 10 s, followed by a further 20-s UV illumination, resulted in an LCR of 4.12 that compares with the 1.67 obtained with 200 ppm of calcium hypochlorite wash. No further increase in MS2 inactivation was achieved by the use of either longer H2O2 spray or UV illumination times. The extent of MS2 reduction was significantly (P < 0.05) decreased when the H2O2 spray was delivered at 10 or 25 degrees C compared with 50 degrees C. In the course of aerobic storage at 4 degrees C, lettuce treated with UV light and H2O2 (10 or 25 degrees C) developed discoloration (polyphenol accumulation) within 6 days. In contrast, lettuce treated with UV light and H2O2 at 50 degrees C developed less discoloration within this time period and was comparable to untreated controls. This study demonstrated that the combination of UV light and H2O2 represents an alternative to hypochlorite-based washes to reduce the carriage of viruses on fresh produce.

Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide.

AIMS: To evaluate the efficacy of ultraviolet (UV) light (254 nm) combined with hydrogen peroxide (H(2)O(2)) to inactivate bacteria on and within fresh produce. METHODS AND RESULTS: The produce was steep inoculated in bacterial cell suspension followed by vacuum infiltration. The inoculated samples were sprayed with H(2)O(2) under constant UV illumination. The log count reduction (LCR) of Salmonella on and within lettuce was dependent on the H(2)O(2) concentration, temperature and treatment time with UV intensity being less significant. By using the optimized parameters (1.5% H(2)O(2) at 50 degrees C, UV dose of 37.8 mJ cm(-2)), the surface Salmonella were reduced by 4.12 +/- 0.45 and internal counts by 2.84 +/- 0.34 log CFU, which was significantly higher compared with H(2)O(2) or UV alone. Higher LCR of Escherichia coli O157:H7, Pectobacterium carotovora, Pseudomonas fluorescens and Salmonella were achieved on leafy vegetables compared with produce, such as cauliflower. In all cases, the surface LCR were significantly higher compared with the samples treated with 200 ppm hypochlorite. UV-H(2)O(2)-treated lettuce did not develop brown discolouration during storage but growth of residual survivors occurred with samples held at 25 degrees C. CONCLUSIONS: UV-H(2)O(2) reduce the bacterial populations on and within fresh produce without affecting the shelf-life stability. SIGNIFICANCE OF THE STUDY: UV-H(2)O(2) represent an alternative to hypochlorite washes to decontaminate fresh produce.

Persistence and growth of different Salmonella serovars on pre- and postharvest tomatoes.

The interaction of a range of Salmonella serovars with pre- and postharvest tomatoes was evaluated. Serovars were selected on the basis of previous association in tomato-linked outbreaks of salmonellosis (Salmonella Javiana, Salmonella Montevideo, and Salmonella Newport) or those typically isolated from animal or clinical infections (Salmonella Dublin, Salmonella Enteritidis, Salmonella Hadar, Salmonella Infantis, Salmonella Typhimurium, and Salmonella Senftenberg). Salmonella serovars introduced onto the flowers of growing plants were recovered on and within the developing tomato fruit. Of all the Salmonella serovars tested, Montevideo appeared to be more adapted to survival within tomatoes and was recovered from 90% of the fruit screened. All of the Salmonella serovars could persist and grow when introduced onto unripened (green) tomato fruit. In general, growth (internal and external) was promoted at the high incubation temperature (25 degrees C) and high relative humidity (95%), although this was serovar dependent. The growth and persistence of Salmonella introduced on and into ripened (red) tomatoes was serovar dependent. Salmonella serovars Enteritidis, Typhimurium, and Dublin were less adapted to grow in or on intact red tomatoes than were serovars Hadar, Montevideo, or Newport. The results illustrated that a diverse range of Salmonella serovars can become established within and/or on preharvest tomatoes. The majority of Salmonella can grow and become established both on and within unripened tomatoes, but growth on ripened fruit was serovar dependent. The results provide a possible explanation why only a narrow range of Salmonella serovars are associated with foodborne illness outbreaks linked to tomatoes.

Mode of Salmonella and Escherichia coli O157:H7 inactivation by a stabilized oxychloro-based sanitizer.

AIM: To determine the mechanisms by which a stabilized oxychloro (SOC)-based sanitizer, applied to decontaminate seeds destined for sprout production, inactivates Escherichia coli O157:H7 ph1 and Salmonella serotype Meleagridis. MATERIALS AND RESULTS: The action of SOC on the metabolism, membrane and DNA integrity of Salmonella and E. coli O157:H7 was studied. In both pathogens, there was an oxidative burst and depletion of intracellular glutathione (GSH) upon initial exposure to 200 ppm SOC. Metabolic activity, measured via bioluminescence, decreased over a 4-h period in E. coli O157:H7 ph1 cells exposed to SOC. Membrane integrity, assessed through viability staining, decreased progressively over 23 h when exposed to SOC. The appearance of auxotrophic mutants suggested that DNA damage had also occurred. Enzymes rich in disulfide bonds (alkaline phosphatase and protease) were sensitive to the chlorite-based sanitizer. Through challenging other microbial types, it was found that Gram positive had higher tolerance to SOC than Gram negatives with the exception of Salmonella. MS2 bacteriophage was highly sensitive; however, Bacillus endospores were not inactivated by SOC. CONCLUSIONS: SOC inactivates E. coli O157:H7 and Salmonella through GSH oxidation and disruption of disulfide bonds. Ultimately, membrane damage resulting from prolonged exposure to SOC leads to the loss of cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide a basis for understanding why extended treatment times are required to inactivate bacteria using SOC.

Inactivation of Escherichia coli O157:H7 and Salmonella on artificially or naturally contaminated mung beans (Vigna radiata L) using a stabilized oxychloro-based sanitizer.

AIMS: To evaluate the efficacy of a stabilized oxychloro-based (SOC) sanitizer to decontaminate mung beans artificially or naturally contaminated with Escherichia coli O157:H7 or Salmonella. METHODS AND RESULTS: Naturally contaminated beans were produced by introducing a five-strain cocktail of E. coli O157:H7 or Salmonella onto the flowers of growing mung bean plants. Escherichia coli O157:H7 was only sporadically recovered from sprout lots (three testing positive from 10 tested) derived from harvested beans. In contrast, Salmonella was recovered from 18 of 20 lots screened. Pathogens present on naturally contaminated seed could be successfully inactivated with SOC applied at 200 ppm for 24 h at 28 degrees C. SOC treatment could also decontaminate artificially inoculated mung bean batches containing different levels of contaminated seed. SOC inactivated E. coli O157:H7, but not Salmonella introduced onto damaged (scarified) beans. CONCLUSIONS: SOC sanitizer could inactivate Salmonella or E. coli O157:H7 naturally or artificially introduced onto mung beans. However, the SOC treatment failed to inactivate Salmonella introduced onto damaged mung beans. SIGNIFICANCE AND IMPACT OF THE STUDY: SOC sanitizer represents an effective method for decontaminating undamaged mung beans.

Inactivation of escherichia coli 0157:H7 and Salmonella on mung beans, alfalfa, and other seed types destined for sprout production by using an oxychloro-based sanitizer.

The efficacy of a stabilized oxychloro-based food grade sanitizer to decontaminate seeds destined for sprout production has been evaluated. By using mung bean seeds as a model system, it was demonstrated that the sanitizer could be used to inactivate a five-strain cocktail of Escherichia coli O157:H7 or Salmonella introduced onto beans at 10(3) to 10(4) CFU/g. Salmonella was more tolerant to stabilized oxychloro than was E. coli O157:H7, with sanitizer levels of >150 and >50 ppm, respectively, being required to ensure pathogen-free sprouts. The decontamination efficacy was also found to be dependent on treatment time (>8 h optimal) and the seed-to-sanitizer ratio (>1:4 optimal). Stabilized oxychloro treatment did not exhibit phytotoxic effects, as germination and sprout yields were not significantly (P > 0.05) different as compared with untreated controls. Although human pathogens could be effectively eliminated from mung beans, the aerobic plate count of native microflora on sprouts grown from treated seed was not significantly (P > 0.05) different from the controls. The diversity of microbial populations (determined through 16S rRNA denaturing gradient gel electrophoresis analysis) associated with bean sprouts was not significantly affected by the sanitizer treatment. However, it was noted that Klebsiella and Herbasprillum (both common plant endophytes) were absent in sprouts derived from decontaminated seed but were present in control sprouts. When a further range of seed types was evaluated, it was found that alfalfa, cress, flax, and soybean could be decontaminated with the stabilized oxychloro sanitizer. However, the decontamination efficacy with other seed types was less consistent. It appears that the rate of seed germination and putative activity of sanitizer sequestering system(s), in addition to other factors, may limit the efficacy of the decontamination method.

Application of enterobacterial repetitive intergenic consensus-polymerase chain reaction to trace the fate of generic Escherichia coli within a high capacity pork slaughter line.

The dissemination of enteric contaminants (generic Escherichia coli and Salmonella) associated with pork carcasses and contact surfaces within a high capacity (6,000 carcasses per day) pork slaughter line was evaluated. Sponge samples were taken periodically from the holding area floor and carcasses at different points in the line throughout an 8.75 h production period. E. coli levels within the holding area were high (ca. 6 log cfu 100 cm(-2)) during the initial phase of processing and did not significantly increase throughout the activity period. In the course of dehairing carcasses, the levels of E. coli were significantly (p<0.05) reduced by scalding but increased during the scraping process. A combination of polishing and triple singeing reduced E. coli populations and the bacterium was only recovered sporadically on eviscerated carcasses. The E. coli populations associated with the slaughter line had a low diversity considering the large number of carcasses processed. In Visit I, the 665 E. coli isolates typed using ERIC-PCR could be grouped into 41 genotypes. In Visit II, 141 genotypes were identified among the 855 E. coli isolates tested. This would suggest that contamination on incoming pigs was of only minor significance compared to that present within the slaughterhouse environment. The holding area was shown to act as a reservoir for endemic E. coli genotypes that could be systematically transferred throughout the dressing line on carcasses. Indeed, the majority of genotypes could be re-isolated throughout the 8.75-h processing period. E. coli isolated from carcasses within the evisceration area could be traced to up-stream operations. The holding area and scraper operation were found to be the most important sites of cross-contamination. Fourteen genotypes recovered (primarily within the holding area) on Visit I were re-isolated on Visit II. Despite the presence of endemic E. coli populations, Salmonella was recovered from only two sites (holding area floor and a carcass within the cooler) on a single occasion. The two Salmonella recovered were genetically distinct (similarity index=22%) suggesting that they originated from different sources and were not part of an endemic population. The study has further illustrated the utility of molecular typing of generic E. coli isolates to establish the dynamics of enteric contamination within pork slaughter lines. However, the extent to which the distribution of E. coli can be extrapolated to that of Salmonella remains uncertain.

Spatial distribution of Salmonella, Escherichia coli O157:H7, and other bacterial populations in commercial and laboratory-scale sprouting mung bean beds.

The reliability of testing spent irrigation water to assess the microbiological status of sprouting mung bean beds has been investigated. In commercial trials, the distribution of opportunistic contaminants within 32 bean sprout beds (25 kg of mung beans per bin) was assessed 48 h after germination. The prevalence of generic Escherichia coli, thermotolerant coliforms, and Aeromonas in sprouts (n = 288) was 5, 11, and 39%, respectively, and 57, 70, and 79% in the corresponding spent irrigation water samples (n = 96). Contamination was heterogeneously distributed within the seedbed. In laboratory trials, beans inoculated with a five-strain cocktail of either Salmonella or E. coli O157:H7 (10(3) to 10(4) CFU/g) were introduced (1 g/500 g of noninoculated seeds) at defined locations (top, middle, or base), and the beans were then sprouted for 48 h. When seeds inoculated with pathogens were introduced at the base or top of the seedbed, the pathogens were typically restricted to these sites and resulted in 44% of the spent irrigation water samples returning false-negative results. Introducing inoculated beans into the middle or at the presoak stage enhanced the distribution of both pathogens within the subsequent sprout bed and resulted in comparable levels recovered in spent irrigation water. The study demonstrated that even though screening a single sample of spent irrigation water is more reliable than testing sprouts directly, it does not provide an accurate assessment of the microbiological status of sprouting mung bean beds. Such limitations may be addressed by ensuring that bean batches are mixed prior to use and by taking spent irrigation water samples from multiple sites at the latter stages of the sprouting process.

Internalization of bioluminescent Escherichia coli and Salmonella Montevideo in growing bean sprouts.

AIMS: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts. METHODS AND RESULTS: E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20,000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ beta-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system. CONCLUSIONS: E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts.

Cross-contamination of carcasses and equipment during pork processing.

AIMS: The cross-contamination events within a commercial pork processing line were examined by a combination of ERIC-PCR DNA fingerprinting of Escherichia coli and plate counts. METHODS AND RESULTS: Sponge sampling of environmental surfaces and carcasses was performed over an 8-h processing period. Prior to the start of processing the scraper and dry polisher blades were found to harbour substantial Enterobacteriaceae and Escherichia coli populations. From plate count data the key cross-contamination site for the transfer of bacteria between carcasses occurred during evisceration. However, DNA fingerprints of representative E. coli isolates identified that genotypes initially present on the scraper/dry polisher became distributed on wet polisher blades, band-saw and butcher's hands despite a singeing step being performed post dry polishing. A high proportion of E. coli on post-eviscerated carcasses could be traced to down-stream (pre-singe) environmental contact surfaces. CONCLUSIONS: DNA fingerprinting has demonstrated that E. coli and potential enteric pathogens can be transferred between pork carcasses throughout the processing line. In this respect scalding and singeing cannot be relied upon to control cross-contamination of enteric bacteria between carcasses. SIGNIFICANCE AND IMPACT OF THE STUDY: Sole reliance on indicator organism counts to identify cross-contamination events as currently advocated is limited.

Carton sterilization by u.v.-C excimer laser light: recovery of Bacillus subtilis spores on vegetable extracts and food simulation matrices.

AIMS: To determine the recovery of Bacillus subtilis spores loaded onto preformed cartons and irradiated with u.v.-excimer laser (248 nm) light. METHODS AND RESULTS: Bacillus subtilis spores irradiated with u.v.-excimer laser light retained phase brightness, but were blocked at various stages of germination. In the presence of germinant, the majority of spores began to lose phase brightness but only after an extended lag period (ca 90 min). After 6 h ca 9% of the spores had elongated but failed to form new cells, approx. 12% had undergone partial phase darkening (grey spores), 15% remained phase bright whilst the remainder had turned fully phase dark but failed to elongate. No enhanced recovery of u.v.-treated spores (with intact or permeabilized coats) occurred in media containing hen egg white lysozyme or vegetable extracts (celery, carrot, swede or turnip). However, recovery did occur when irradiated spores were incubated for 26 d, semiaerobically, within cartons containing nutrient broth or milk. CONCLUSIONS: The germination ability of B. subtilis spores is altered following u.v.-excimer laser treatment. Recovery of treated spores was found in liquid systems but not on agar plates supplemented with vegetable extracts or lysozyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential recovery of u.v.-excimer laser-treated spores in a range of carton-packed food systems requires further investigation.

Attachment of bacteria to beef from steam-pasteurized carcasses.

The extent to which a bacterial cocktail containing equal numbers of Pseudomonas fragi NCTC 10689, Listeria monocytogenes BL5/2, Salmonella Typhimurium LT2, and Escherichia coli JM 109 attached to loin surface cuts (7 by 5 cm) derived from steam-pasteurized beef carcasses has been evaluated. The extent of attachment was categorized as loosely attached (removed by rinsing), firmly attached (released by stomaching), and irreversibly bound. No significant difference (P > 0.10) in the attachment of bacteria to steam-pasteurized carcasses was found compared with control loin samples that had received no treatment. No significant difference (P > 0.05) was also found in the attachment strength between the different bacterial species tested. Most bacteria inoculated onto the loin cuts were reversibly bound, since they had been removed by rinsing and stomaching. The irreversible attachment of bacteria to loin cuts was found to vary significantly (P < 0.01) among the different carcass sets used but was independent of whether the carcass had undergone steam pasteurization treatment. Use of a bioluminescent strain of E. coli showed that cells bound preferentially to cut edges and convoluted areas on the loin surface and could not be removed by rinsing. The possible mechanisms of bacterial attachment and the suitability of steam pasteurization to remove contamination incurred during slaughter are discussed.