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Through recent advances in our understanding of microbial:plant interactions it is becoming apparent that human pathogens, principally, Escherichia coli O157:H7 and Salmonella are adapted to survive in the plant environment. The aforementioned pathogens have surface epitopes that can bind to plant structures such as stomata to aid attachment. In addition, Salmonella is attracted and able to metabolize nutrients contained within the apoplastic fluid of plants. The question of internalization into the inner tissue of plants remains inconclusive largely because of the problems encountered in detecting low pathogen levels. Nevertheless, once internalized human pathogens can trigger and potentially evade plant defenses that are typically induced by phytopathogens. Although more research in this area is required, the hypothesis that human pathogens have adapted to the plant environment as part of their natural lifecycle appears to be supported., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The following reports on the application of a combination of antagonistic bacteria and lytic bacteriophages to control the growth of Salmonella on sprouting mung beans and alfalfa seeds. Antagonistic bacteria were isolated from mung bean sprouts and tomatoes by using the deferred plate assay to assess anti-Salmonella activity. From the isolates screened, an Enterobacter asburiae strain (labeled "JX1") exhibited stable antagonistic activity against a broad range of Salmonella serovars (Agona, Berta, Enteritidis, Hadar, Heidelberg, Javiana, Montevideo, Muenchen, Newport, Saint Paul, and Typhimurium). Lytic bacteriophages against Salmonella were isolated from pig or cattle manure effluent. A bacteriophage cocktail prepared from six isolates was coinoculated with E. asburiae JX1 along with Salmonella in broth culture. The combination of E. asburiae JX1 and bacteriophage cocktail reduced the levels of Salmonella by 5.7 to 6.4 log CFU/ml. Mung beans inoculated with Salmonella and sprouted over a 4-day period attained levels of 6.72 + or - 0.78 log CFU/g. In contrast, levels of Salmonella were reduced to 3.31 + or - 2.48 or 1.16 + or - 2.14 log CFU/g when the pathogen was coinoculated with bacteriophages or E. asburiae JX1, respectively. However, by using a combination of E. asburiae JX1 and bacteriophages, the levels of Salmonella associated with mung bean sprouts were only detected by enrichment. The biocontrol preparation was effective at controlling the growth of Salmonella under a range of sprouting temperatures (20 to 30 degrees Celsius) and was equally effective at suppressing the growth of Salmonella on sprouting alfalfa seeds. The combination of E. asburiae JX1 and bacteriophages represents a promising, chemical-free approach for controlling the growth of Salmonella on sprouting seeds.

A biocontrol preparation based on a combination of Enterobacter asburiae JX1 and a cocktail of five lytic bacteriophages was evaluated for control of Salmonella Javiana within the rhizosphere of plants and in pre- and postharvest tomatoes. The biocontrol preparation introduced into the rhizosphere of growing tomato plants reduced the persistence of Salmonella, although no synergistic action was observed between E. asburiae JX1 or the bacteriophage cocktail when used in combination. When the biocontrol preparation was coinoculated with Salmonella onto the blossom of tomato plants, the prevalence of the enteric pathogen both on the surface and in internal tissues of the subsequent tomatoes was significantly reduced compared with controls. Tomatoes derived from plants inoculated with Salmonella alone had a prevalence of 92% surface contamination (22 of 24 tomato batches were positive for Salmonella) and 43% internal contamination (31 of 72 batches positive). This Salmonella prevalence was reduced to 0% (0 of 38 positive) and 2% (1 of 57 positive), respectively, when the biocontrol preparation was applied. Although bacteriophages reduced the prevalence of internalized Salmonella, the main growth suppressing effect was via the antagonistic activity of E. asburiae JX1. No bacteriophages were recovered from tomatoes despite being introduced at 6 log PFU onto the blossom of plants. The biocontrol preparation was not effective for controlling the growth of Salmonella introduced onto postharvest tomatoes that were stored for 7 days at 15 degrees C. The application of E. asburiae JX1 is a promising approach for controlling Salmonella encountered in tomato production, and there was no evidence to suggest that the antagonistic activity could be enhanced by the coinoculation of bacteriophages.

Foodborne illness outbreaks linked to fresh produce are becoming more frequent and widespread. High impact outbreaks, such as that associated with spinach contaminated with Escherichia coli O157:H7, resulted in almost 200 cases of foodborne illness across North America and >$300 m market losses. Over the last decade there has been intensive research into gaining an understanding on the interactions of human pathogens with plants and how microbiological safety of fresh produce can be improved. The following review will provide an update on the food safety issues linked to fresh produce. An overview of recent foodborne illness outbreaks linked to fresh produce. The types of human pathogens encountered will be described and how they can be transferred from their normal animal or human host to fresh produce. The interaction of human pathogens with growing plants will be discussed, in addition to novel intervention methods to enhance the microbiological safety of fresh produce.

The fabrication of Bacillus subtilis endospore imprinted conducting polymer films and subsequent electrochemical detection of bound spores is reported. Imprinted films were prepared by absorbing spores on the surface of glassy carbon electrodes upon which a polypyrrole, followed by a poly(3-methylthiophene), layer were electrochemically deposited. Spore template release was achieved through soaking the modified electrode in DMSO. Binding of endospores to imprinted films could be detected via impedance spectroscopy by monitoring changes in Y'' (susceptance) using Mn(II)Cl2 (0.5M pH 3) as the supporting electrolyte. Here, the change in Y'' could be correlated to spore densities between 10(4) and 10(7)cfu/ml. More sensitive detection of absorbed spores was achieved by following endospore germination via changes in film charge as measured using cyclic voltammetry. Here, imprinted films were submerged in spore suspensions to permit absorption, heat activated at 70 degrees C for 10 min prior to transferring to an electrochemical cell containing germination activators. By using the assay format it was possible to detect 10(2)cfu/ml. The observed changes in film charge could be attributed to the interaction of the supporting conducting polymer with dipicolinic acid (DPA) and other constituents released from the core in the course of germination. In all cases, it was not possible to regenerate the imprinted films without losing electrode response. In summary, the study has provided proof-of-concept for fabricating microbial imprinted films using conducting polymers.

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.

The objective of this study was to determine whether the shedding and antibody titre to Salmonella was lower for pig herds provided liquid-feed compared to those on traditional dry rations. Twenty liquid-feeding farms and 61 dry-feeding farms were selected. The amount of antibodies to Salmonella in sera from 15 finisher pigs on each of 80 Ontario swine farms was analyzed by means of enzyme-linked immunosorbent assay (ELISA). In addition, the presence of Salmonella on the 20 liquid-feeding farms and 21 of the dry-feeding farms was assessed by culture of 15 fecal samples taken directly from finisher pigs and five pooled pen-fecal samples at each farm. A cut-off of OD% 10 was used. The Salmonella sero-prevalence differed between the two groups of farms. At least one pig tested sero-positive on 98% of the dry-feeding farms and 84% of the liquid-feeding farms (P<0.05). A multi-variable mixed linear regression model with the farm as a random variable and farm factors as the fixed effects was fitted. Crude optical density (OD) of the individual pig was considered as the continuous dependent variable. Dry-feeding and antimicrobial daily usage was associated with crude OD (P<0.05). In addition, crude OD increased with increasing herd size (P<0.05). Salmonella was isolated from 25 out of 420 fecal samples (6%) from dry-feeding farms compared to three out of 400 samples (0.8%) from liquid-feeding farms. Eight of the dry-feeding farms (38%) tested positive compared to only three of the liquid-feeding farms (15%). Salmonella was also recovered from the pen environment on five dry-feeding farms but were not isolated from the facilities using liquid-feeding. Salmonella Typhimurium was isolated from four farms in the dry-feed group and on one farm with liquid-feeding. The one S. Typhimurium isolate from the liquid-feeding farm exhibited no antimicrobial resistance, but those from dry-feeding farms were resistant to four or more antimicrobial agents. The results of the logistic regression, with farm as a random effect showed that dry-feeding [OR=2.7 (1.1-15.1)] and continuous flow system [OR=2.3 (1.2-12.7)] increased risk of finding Salmonella in the individual pig. These findings indicate that liquid-feeding and all-in all-out management of the grower-finisher barns can reduce the Salmonella prevalence.

The internalization and persistence of a bioluminescent Escherichia coli O157:H7 Ph1 was investigated in growing spinach plants that had been either biologically or mechanically damaged. In control (undamaged) plants cultivated in soil microcosms inoculated with E. coli O157:H7 Phl, the bacterium was recovered from surface-sterilized root tissue but not from leaves. Mechanical disruption of the seminal root and root hairs of the plants did not result in the internalization of the pathogen into the aerial leaf tissue. When imprints of the root tissue were made on plates of tryptic soy agar plus ampicillin, no colonies of E. coli O157:H7 were observed around damaged tissue. The roots of growing plants were exposed to the northern root-knot nematode, Meloidogyne hapla, in the presence of E. coli O157:H7. Although this treatment caused knot formation on the roots, it did not enhance the internalization of the bacterium into the plant vascular system. Coinoculation of intact leaves with E. coli O157:H7 and the phytopathogen Pseudomonas syringae DC3000 resulted in localized necrosis, but the persistence of the human pathogen was not affected. The mechanical disruption of roots does not result in the internalization of E. coli O157:H7 into the aerial tissue of spinach, and there does not appear to be any effect of P. syringae in terms of enhancing the persistence of E. coli O157:H7 in spinach leaves.

The aim of this study was to determine whether the attachment strength to pork skin, quaternary ammonium salt resistance, and thermal inactivation kinetics (at 65 degrees C) of a range of Escherichia coli isolates could be correlated with their temporal stability (persistence) within a pork slaughter line. The genetic lineage of the E. coli isolates was determined using enterobacterial repetitive intergenic consensus-PCR. The genotypes were divided into transient and endemic populations based on the number of times they were recovered within and across sampling visits made to a pork slaughterhouse. No significant variation in the D-value at 65 degrees C (0.27 to 0.51 min) was observed among the genotypes tested. However, differences in D-values were found for 100 ppm quaternary ammonium salt (3.0 to 6.0 min). All of the E. coli genotypes attached strongly to pork skin, and a high proportion of cells were irreversibly bound (39 to 42% of the initial inoculum). However, variation among genotypes was found with respect to loose attachment (21 to 33% of inoculated cells). No correlation between persistence of E. coli genotypes within the slaughter line and attachment strength or quaternary ammonium salt resistance was found. Variation in either physiological attribute could not be predicted based on genetic lineage. Additional or alternative factors may contribute to the ability of E. coli populations to become endemic within pork processing facilities. More studies should be conducted to elucidate the underlying factors that promote the formation of endemic populations of E. coli and other enteric bacteria (e.g., Salmonella) within slaughter lines.

The growth and persistence of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes on a diverse range of plant types over extended cultivation periods was studied. When introduced on the seed of carrot, cress, lettuce, radish, spinach and tomato all the pathogens became rapidly established shortly after germination, attaining cell densities of the order of 5.5-6.5 log cfu/g. In general, Es. coli O157:H7 and L. monocytogenes became established and persisted at significantly higher levels on seedlings (9 days post-germination) than Salmonella. Es. coli O157:H7 became internalized in cress, lettuce, radish and spinach seedlings but was not recovered within the tissues of mature plants. Internalization of Salmonella was also observed in lettuce and radish but not cress or spinach seedlings. In contrast, L. monocytogenes did not internalize within seedlings but did persist on the surface of plants throughout the cultivation period. Co-inoculation of isolates recovered from the rhizosphere of plants did not significantly affect the numbers or persistence of human pathogens. The only exception was with Enterobacter cloacae, which reduced Es. coli O157:H7 Ph1 and L. monocytogenes levels by ca. 1 log cfu/g on lettuce. With the bioluminescent phenotype of Es. coli O157:H7 Ph1, it was demonstrated that the human pathogen became established on the roots of growing plants. Scanning electron micrographs of root seedlings suggested that Es. coli O157:H7 Ph1 preferentially colonized the root junctions of seedlings. It is proposed that such colonization sites enhanced the persistence of Es. coli O157:H7 on plants and facilitated internalization within developing seedlings. The results suggest that the risk associated with internalized human pathogens in salad vegetables at harvest is low. Nevertheless, the introduction of human pathogens at an early stage of plant development could enhance their persistence in the rhizosphere. The implications of the study with regards to on-farm food safety initiatives are discussed.

In this study, the interaction of a bioluminescence-labeled Escherichia coli strain with growing spinach plants was assessed. Through bioluminescence profiles, the direct visualization of E. coli growing around the roots of developing seedlings was accomplished. Subsequent in situ glucuronidase (GUS) staining of seedlings confirmed that E. coli had become internalized within root tissue and, to a limited extent, within hypocotyls. When inoculated seeds were sown in soil microcosms and cultivated for 42 days, E. coli was recovered from the external surfaces of spinach roots and leaves as well as from surface-sterilized roots. When 20-day-old spinach seedlings (from uninoculated seeds) were transferred to soil inoculated with E. coli, the bacterium became established on the plant surface, but internalization into the inner root tissue was restricted. However, for seedlings transferred to a hydroponic system containing 10(2) or 10(3) CFU of E. coli per ml of the circulating nutrient solution, the bacterium was recovered from surface-sterilized roots, indicating that it had been internalized. Differences between E. coli interactions in the soil and those in the hydroponic system may be attributed to greater accessibility of the roots in the latter model. Alternatively, the presence of a competitive microflora in soil may have restricted root colonization by E. coli. The implications of this study's findings with regard to the microbiological safety of minimally processed vegetables are discussed.

Ultraviolet (u.v.) laser irradiation has been used to inactivate Bacillus subtilis spores deposited on to planar aluminium- and polyethylene-coated packaging surfaces. Kill kinetics were found to be diphasic, with an initial rapid inactivation phase followed by tailing. Although no definitive evidence was obtained, it is thought that spores located within packaging crevices/pores were primarily responsible for the observed tailing. Surviving spores were also found on the unexposed underside of cards and, to a lesser extent, within clumps. The log count reduction in B. subtilis was dependent on spore loading and total u.v. dose. In comparison, packaging surface composition, fluence (2-18 Jm-2) and frequency (40-150 Hz) had only a negligible effect. By irradiating boards carrying 106 spores, with a dose of 11.5 J cm-2, a log count reduction >5 was obtained. The mode of spore inactivation was primarily through DNA disruption. This was confirmed by the high sensitivity of spores lacking protective, small, acid-soluble proteins, in addition to the high frequency of auxotrophic and asporogenous mutations found amongst survivors.