Predictive Population Dynamics of Escherichia coli O157:H7 and Salmonella enterica on Plants: a Mechanistic Mathematical Model Based on Weather Parameters and Bacterial State.

Weather affects key aspects of bacterial behavior on plants but has not been extensively investigated as a tool to assess risk of crop contamination with human foodborne pathogens. A novel mechanistic model informed by weather factors and bacterial state was developed to predict population dynamics on leafy vegetables and tested against published data tracking Escherichia coli O157:H7 (EcO157) and Salmonella enterica populations on lettuce and cilantro plants. The model utilizes temperature, radiation, and dew point depression to characterize pathogen growth and decay rates. Additionally, the model incorporates the population level effect of bacterial physiological state dynamics in the phyllosphere in terms of the duration and frequency of specific weather parameters. The model accurately predicted EcO157 and S. enterica population sizes on lettuce and cilantro leaves in the laboratory under various conditions of temperature, relative humidity, light intensity, and cycles of leaf wetness and dryness. Importantly, the model successfully predicted EcO157 population dynamics on 4-week-old romaine lettuce plants under variable weather conditions in nearly all field trials. Prediction of initial EcO157 population decay rates after inoculation of 6-week-old romaine plants in the same field study was better than that of long-term survival. This suggests that future augmentation of the model should consider plant age and species morphology by including additional physical parameters. Our results highlight the potential of a comprehensive weather-based model in predicting contamination risk in the field. Such a modeling approach would additionally be valuable for timing field sampling in quality control to ensure the microbial safety of produce. IMPORTANCE Fruits and vegetables are important sources of foodborne disease. Novel approaches to improve the microbial safety of produce are greatly lacking. Given that bacterial behavior on plant surfaces is highly dependent on weather factors, risk assessment informed by meteorological data may be an effective tool to integrate into strategies to prevent crop contamination. A mathematical model was developed to predict the population trends of pathogenic E. coli and S. enterica, two major causal agents of foodborne disease associated with produce, on leaves. Our model is based on weather parameters and rates of switching between the active (growing) and inactive (nongrowing) bacterial state resulting from prevailing environmental conditions on leaf surfaces. We demonstrate that the model has the ability to accurately predict dynamics of enteric pathogens on leaves and, notably, sizes of populations of pathogenic E. coli over time after inoculation onto the leaves of young lettuce plants in the field.

Weather factors, soil microbiome, and bacteria-fungi interactions as drivers of the epiphytic phyllosphere communities of romaine lettuce.

Lettuce is associated with seasonal outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections. Little is known about how various biotic and abiotic factors affect the lettuce microbiome, which in turn impacts STEC colonization. We characterized the lettuce phyllosphere and surface soil bacterial, fungal, and oomycete communities at harvest in late-spring and -fall in California using metagenomics. Harvest season and field type, but not cultivar, significantly influenced the microbiome composition of leaves and surface soil near plants. Phyllosphere and soil microbiome compositions were correlated with specific weather factors. The relative abundance of Enterobacteriaceae, but not E. coli, was enriched on leaves (5.2%) compared to soil (0.4%) and correlated positively with minimum air temperature and wind speed. Co-occurrence networks revealed seasonal trends in fungi-bacteria interactions on leaves. These associations represented 39%-44% of the correlations between species. All significant E. coli co-occurrences with fungi were positive, while all negative associations were with bacteria. A large proportion of the leaf bacterial species was shared with those in soil, indicating microbiome transmission from the soil surface to the canopy. Our findings provide new insight into factors that shape lettuce microbial communities and the microbial context of foodborne pathogen immigration events in the lettuce phyllosphere.

Weather stressors correlate with Escherichia coli and Salmonella enterica persister formation rates in the phyllosphere: a mathematical modeling study.

Enteric pathogens can enter a persister state in which they survive exposure to antibiotics and physicochemical stresses. Subpopulations of such phenotypic dormant variants have been detected in vivo and in planta in the laboratory, but their formation in the natural environment remains largely unexplored. We applied a mathematical model predicting the switch rate to persister cell in the phyllosphere to identify weather-related stressors associated with E. coli and S. enterica persister formation on plants based on their population dynamics in published field studies from the USA and Spain. Model outputs accurately depicted the bi-phasic decay of bacterial population sizes measured in the lettuce and spinach phyllosphere in these studies. Predicted E. coli persister switch rate on leaves was positively and negatively correlated with solar radiation intensity and wind velocity, respectively. Likewise, predicted S. enterica persister switch rate correlated positively with solar radiation intensity; however, a negative correlation was observed with air temperature, relative humidity, and dew point, factors involved in water deposition onto the phylloplane. These findings suggest that specific environmental factors may enrich for dormant bacterial cells on plants. Our model quantifiably links persister cell subpopulations in the plant habitat with broader physical conditions, spanning processes at different granular scales.

Plant Bioactive Compounds as an Intrinsic and Sustainable Tool to Enhance the Microbial Safety of Crops.

Outbreaks of produce-associated foodborne illness continue to pose a threat to human health worldwide. New approaches are necessary to improve produce safety. Plant innate immunity has potential as a host-based strategy for the deactivation of enteric pathogens. In response to various biotic and abiotic threats, plants mount defense responses that are governed by signaling pathways. Once activated, these result in the release of reactive oxygen and nitrogen species in addition to secondary metabolites that aim at tempering microbial infection and pest attack. These phytochemicals have been investigated as alternatives to chemical sanitization, as many are effective antimicrobial compounds in vitro. Their antagonistic activity toward enteric pathogens may also provide an intrinsic hurdle to their viability and multiplication in planta. Plants can detect and mount basal defenses against enteric pathogens. Evidence supports the role of plant bioactive compounds in the physiology of Salmonella enterica, Escherichia coli, and Listeria monocytogenes as well as their fitness on plants. Here, we review the current state of knowledge of the effect of phytochemicals on enteric pathogens and their colonization of plants. Further understanding of the interplay between foodborne pathogens and the chemical environment on/in host plants may have lasting impacts on crop management for enhanced microbial safety through translational applications in plant breeding, editing technologies, and defense priming.

Seasonality, shelf life and storage atmosphere are main drivers of the microbiome and E. coli O157:H7 colonization of post-harvest lettuce cultivated in a major production area in California.

BACKGROUND: Lettuce is linked to recurrent outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections, the seasonality of which remains unresolved. Infections have occurred largely from processed lettuce, which undergoes substantial physiological changes during storage. We investigated the microbiome and STEC O157:H7 (EcO157) colonization of fresh-cut lettuce of two cultivars with long and short shelf life harvested in the spring and fall in California and stored in modified atmosphere packaging (MAP) at cold and warm temperatures. RESULTS: Inoculated EcO157 declined significantly less on the cold-stored cultivar with short shelf life, while multiplying rapidly at 24 °C independently of cultivar. Metagenomic sequencing of the lettuce microbiome revealed that the pre-storage bacterial community was variable but dominated by species in the Erwiniaceae and Pseudomonadaceae. After cold storage, the microbiome composition differed between cultivars, with a greater relative abundance (RA) of Erwiniaceae and Yersiniaceae on the cultivar with short shelf life. Storage at 24 °C shifted the microbiome to higher RAs of Erwiniaceae and Enterobacteriaceae and lower RA of Pseudomonadaceae compared with 6 °C. Fall harvest followed by lettuce deterioration were identified by recursive partitioning as important factors associated with high EcO157 survival at 6 °C, whereas elevated package CO2 levels correlated with high EcO157 multiplication at 24 °C. EcO157 population change correlated with the lettuce microbiome during 6 °C storage, with fall microbiomes supporting the greatest EcO157 survival on both cultivars. Fall and spring microbiomes differed before and during storage at both temperatures. High representation of Pantoea agglomerans was a predictor of fall microbiomes, lettuce deterioration, and enhanced EcO157 survival at 6 °C. In contrast, higher RAs of Erwinia persicina, Rahnella aquatilis, and Serratia liquefaciens were biomarkers of spring microbiomes and lower EcO157 survival. CONCLUSIONS: The microbiome of processed MAP lettuce evolves extensively during storage. Under temperature abuse, high CO2 promotes a lettuce microbiome enriched in taxa with anaerobic capability and EcO157 multiplication. In cold storage, our results strongly support a role for season and lettuce deterioration in EcO157 survival and microbiome composition, suggesting that the physiology and microbiomes of fall- and spring-harvested lettuce may contribute to the seasonality of STEC outbreaks associated with lettuce grown in coastal California.

Breeding Crops for Enhanced Food Safety.

An increasing global population demands a continuous supply of nutritious and safe food. Edible products can be contaminated with biological (e.g., bacteria, virus, protozoa), chemical (e.g., heavy metals, mycotoxins), and physical hazards during production, storage, transport, processing, and/or meal preparation. The substantial impact of foodborne disease outbreaks on public health and the economy has led to multidisciplinary research aimed to understand the biology underlying the different contamination processes and how to mitigate food hazards. Here we review the knowledge, opportunities, and challenges of plant breeding as a tool to enhance the food safety of plant-based food products. First, we discuss the significant effect of plant genotypic and phenotypic variation in the contamination of plants by heavy metals, mycotoxin-producing fungi, and human pathogenic bacteria. In addition, we discuss the various factors (i.e., temperature, relative humidity, soil, microbiota, cultural practices, and plant developmental stage) that can influence the interaction between plant genetic diversity and contaminant. This exposes the necessity of a multidisciplinary approach to understand plant genotype × environment × microbe × management interactions. Moreover, we show that the numerous possibilities of crop/hazard combinations make the definition and identification of high-risk pairs, such as Salmonella-tomato and Escherichia coli-lettuce, imperative for breeding programs geared toward improving microbial safety of produce. Finally, we discuss research on developing effective assays and approaches for selecting desirable breeding germplasm. Overall, it is recognized that although breeding programs for some human pathogen/toxin systems are ongoing (e.g., Fusarium in wheat), it would be premature to start breeding when targets and testing systems are not well defined. Nevertheless, current research is paving the way toward this goal and this review highlights advances in the field and critical points for the success of this initiative that were discussed during the Breeding Crops for Enhanced Food Safety workshop held 5-6 June 2019 at University of California, Davis.

Formation of Escherichia coli O157:H7 Persister Cells in the Lettuce Phyllosphere and Application of Differential Equation Models To Predict Their Prevalence on Lettuce Plants in the Field.

Escherichia coli O157:H7 (EcO157) infections have been recurrently associated with produce. The physiological state of EcO157 cells surviving the many stresses encountered on plants is poorly understood. EcO157 populations on plants in the field generally follow a biphasic decay in which small subpopulations survive over longer periods of time. We hypothesized that these subpopulations include persister cells, known as cells in a transient dormant state that arise through phenotypic variation in a clonal population. Using three experimental regimes (with growing, stationary at carrying capacity, and decaying populations), we measured the persister cell fractions in culturable EcO157 populations after inoculation onto lettuce plants in the laboratory. The greatest average persister cell fractions on the leaves within each regime were 0.015, 0.095, and 0.221%, respectively. The declining EcO157 populations on plants incubated under dry conditions showed the largest increase in the persister fraction (46.9-fold). Differential equation models were built to describe the average temporal dynamics of EcO157 normal and persister cell populations after inoculation onto plants maintained under low relative humidity, resulting in switch rates from a normal cell to a persister cell of 7.7 × 10-6 to 2.8 × 10-5 h-1 Applying our model equations from the decay regime, we estimated model parameters for four published field trials of EcO157 survival on lettuce and obtained switch rates similar to those obtained in our study. Hence, our model has relevance to the survival of this human pathogen on lettuce plants in the field. Given the low metabolic state of persister cells, which may protect them from sanitization treatments, these cells are important to consider in the microbial decontamination of produce.IMPORTANCE Despite causing outbreaks of foodborne illness linked to lettuce consumption, E. coli O157:H7 (EcO157) declines rapidly when applied onto plants in the field, and few cells survive over prolonged periods of time. We hypothesized that these cells are persisters, which are in a dormant state and which arise naturally in bacterial populations. When lettuce plants were inoculated with EcO157 in the laboratory, the greatest persister fraction in the population was observed during population decline on dry leaf surfaces. Using mathematical modeling, we calculated the switch rate from an EcO157 normal to persister cell on dry lettuce plants based on our laboratory data. The model was applied to published studies in which lettuce was inoculated with EcO157 in the field, and switch rates similar to those obtained in our study were obtained. Our results contribute important new knowledge about the physiology of this virulent pathogen on plants to be considered to enhance produce safety.

Enhanced formation of shiga toxin-producing Escherichia coli persister variants in environments relevant to leafy greens production.

Bacterial persistence is a form of phenotypic heterogeneity in which a subpopulation, persisters, has high tolerance to antibiotics and other stresses. Persisters of enteric pathogens may represent the subpopulations capable of surviving harsh environments and causing human infections. Here we examined the persister populations of several shiga toxin-producing Escherichia coli (STEC) outbreak strains under conditions relevant to leafy greens production. The persister fraction of STEC in exponential-phase of culture varied greatly among the strains examined, ranging from 0.00003% to 0.0002% for O157:H7 strains to 0.06% and 0.08% for STEC O104:H4 strains. A much larger persister fraction (0.1-11.2%) was observed in STEC stationary cells grown in rich medium, which was comparable to the persister fractions in stationary cells grown in spinach lysates (0.6-3.6%). The highest persister fraction was measured in populations of cells incubated in field water (9.9-23.2%), in which no growth was detected for any of the STEC strains examined. Considering the high tolerance of persister cells to antimicrobial treatments and their ability to revert to normal cells, the presence of STEC persister cells in leafy greens production environments may pose a significant challenge in the development of effective control strategies to ensure the microbial safety of fresh vegetables.

Complete Genome Sequences of Three Shiga Toxin-Producing Escherichia coli O111:H8 Strains Exhibiting an Aggregation Phenotype.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are a common source of foodborne illness. STEC O111 is among the most prevalent non-O157 STEC serogroups. Few completed genomes of STEC O111 strains have been reported to date. We report here the complete genomic sequences of three O111:H8 strains that display a distinct aggregation phenotype.

Interactions of Salmonella enterica Serovar Typhimurium and Pectobacterium carotovorum within a Tomato Soft Rot.

Salmonella spp. are remarkably adaptable pathogens, and this adaptability allows these bacteria to thrive in a variety of environments and hosts. The mechanisms with which these pathogens establish within a niche amid the native microbiota remain poorly understood. Here, we aimed to uncover the mechanisms that enable Salmonella enterica serovar Typhimurium strain ATCC 14028 to benefit from the degradation of plant tissue by a soft rot plant pathogen, Pectobacterium carotovorum The hypothesis that in the soft rot, the liberation of starch (not utilized by P. carotovorum) makes this polymer available to Salmonella spp., thus allowing it to colonize soft rots, was tested first and proven null. To identify the functions involved in Salmonella soft rot colonization, we carried out transposon insertion sequencing coupled with the phenotypic characterization of the mutants. The data indicate that Salmonella spp. experience a metabolic shift in response to the changes in the environment brought on by Pectobacterium spp. and likely coordinated by the csrBC small regulatory RNA. While csrBC and flhD appear to be of importance in the soft rot, the global two-component system encoded by barA sirA (which controls csrBC and flhDC under laboratory conditions) does not appear to be necessary for the observed phenotype. Motility and the synthesis of nucleotides and amino acids play critical roles in the growth of Salmonella spp. in the soft rot.IMPORTANCE Outbreaks of produce-associated illness continue to be a food safety concern. Earlier studies demonstrated that the presence of phytopathogens on produce was a significant risk factor associated with increased Salmonella carriage on fruits and vegetables. Here, we genetically characterize some of the requirements for interactions between Salmonella and phytobacteria that allow Salmonella spp. to establish a niche within an alternate host (tomato). Pathways necessary for nucleotide synthesis, amino acid synthesis, and motility are identified as contributors to the persistence of Salmonella spp. in soft rots.

Assessing the Ability of Salmonella enterica to Translocate Type III Effectors Into Plant Cells.

Salmonella enterica serovar Typhimurium, a human enteric pathogen, has the ability to multiply and survive endophytically in plants. Genes encoding the type III secretion system (T3SS) or its effectors (T3Es) may contribute to its colonization. Two reporter plasmids for T3E translocation into plant cells that are based on hypersensitive response domains of avirulence proteins from the Pantoea agglomerans-beet and Xanthomonas euvesicatoria-pepper pathosystems were employed in this study to investigate the role of T3Es in the interaction of Salmonella ser. Typhimurium 14028 with plants. The T3Es of Salmonella ser. Typhimurium, SipB and SifA, which are translocated into animal cells, could not be delivered by Salmonella ser. Typhimurium into cells of beet roots or pepper leaves. In contrast, these effectors were translocated into plant cells by the phytopathogenic bacteria P. agglomerans pv. betae, Erwinia amylovora, and X. euvesicatoria. Similarly, HsvG, a T3E of P. agglomerans pv. gypsophilae, and XopAU of X. euvesicatoria could be translocated into beet roots and pepper leaves, respectively, by the plant pathogens but not by Salmonella ser. Typhimurium. Mutations in Salmonella ser. Typhimurium T3SS genes invA, ssaV, sipB, or sifA, did not affect its endophytic colonization of lettuce leaves, supporting the notion that S. enterica cannot translocate T3Es into plant cells.

Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli.

Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro: it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts.IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the autotransporter protein Cah confers on E. coli DH5α cells a strong autoaggregative phenotype that is inversely correlated with its ability to form biofilms and plays a strain-specific role in plant and animal colonization by STEC. Although cah is widespread in the STEC population, we detected a mutation rate of 31.3% in cah, which is similar to that reported for rpoS and fimH The formation of cell aggregates due to increased bacterium-to-bacterium interactions may be disadvantageous to bacterial populations under conditions that favor a planktonic state in STEC. Therefore, a loss-of-function mutation in cah is likely a selective trait in STEC when autoaggregative properties become detrimental to bacterial cells and may contribute to the adaptability of STEC to fluctuating environments.

Escherichia coli O157:H7 Converts Plant-Derived Choline to Glycine Betaine for Osmoprotection during Pre- and Post-harvest Colonization of Injured Lettuce Leaves.

Plant injury is inherent to the production and processing of fruit and vegetables. The opportunistic colonization of damaged plant tissue by human enteric pathogens may contribute to the occurrence of outbreaks of foodborne illness linked to produce. Escherichia coli O157:H7 (EcO157) responds to physicochemical stresses in cut lettuce and lettuce lysates by upregulation of several stress response pathways. We investigated the tolerance of EcO157 to osmotic stress imposed by the leakage of osmolytes from injured lettuce leaf tissue. LC-MS analysis of bacterial osmoprotectants in lettuce leaf lysates and wound washes indicated an abundant natural pool of choline, but sparse quantities of glycine betaine and proline. Glycine betaine was a more effective osmoprotectant than choline in EcO157 under osmotic stress conditions in vitro. An EcO157 mutant with a deletion of the betTIBA genes, which are required for biosynthesis of glycine betaine from imported choline, achieved population sizes twofold lower than those of the parental strain (P < 0.05) over the first hour of colonization of cut lettuce in modified atmosphere packaging (MAP). The cell concentrations of the betTIBA mutant also were 12-fold lower than those of the parental strain (P < 0.01) when grown in hypertonic lettuce lysate, indicating that lettuce leaf cellular contents provide choline for osmoprotection of EcO157. To demonstrate the utilization of available choline by EcO157 for osmoadaptation in injured leaf tissue, deuterated (D-9) choline was introduced to wound sites in MAP lettuce; LC-MS analysis revealed the conversion of D9-choline to D-9 glycine betaine in the parental strain, but no significant amounts were observed in the betTIBA mutant. The EcO157 ΔbetTIBA-ΔotsBA double mutant, which is additionally deficient in de novo synthesis of the compatible solute trehalose, was significantly less fit than the parental strain after their co-inoculation onto injured lettuce leaves and MAP cut lettuce. However, its competitive fitness followed a different time-dependent trend in MAP lettuce, likely due to differences in O2 content, which modulates betTIBA expression. Our study demonstrates that damaged lettuce leaf tissue does not merely supply EcO157 with substrates for proliferation, but also provides the pathogen with choline for its survival to osmotic stress experienced at the site of injury.

Drying and decontamination of raw pistachios with sequential infrared drying, tempering and hot air drying.

Pistachio nuts have been associated with outbreaks of foodborne disease and the industry has been impacted by numerous product recalls due to contamination with Salmonella enterica. The current hot air drying of pistachios has low energy efficiency and drying rates, and also does not guarantee the microbial safety of products. In the study described herein, dehulled and water-sorted pistachios with a moisture content (MC) of 38.14% (wet basis) were dried in a sequential infrared and hot air (SIRHA) drier to <9% MC. The decontamination efficacy was assessed by inoculating pistachios with Enterococcus faecium, a surrogate of Salmonella enterica used for quality control in the almond industry. Drying with IR alone saved 105min (34.4%) of drying time compared with hot air drying. SIRHA drying of pistachios for 2h with infrared (IR) heat followed by tempering at a product temperature of 70°C for 2h and then by hot air drying shortened the drying time by 40min (9.1%) compared with drying by hot air only. This SIRHA method also reduced the E. faecium cell population by 6.1-logCFU/g kernel and 5.41-logCFU/g shell of pistachios. The free fatty acid contents of SIRHA dried pistachios were on par with that of hot air dried samples. Despite significant differences in peroxide values (PV) of pistachio kernels dried with the SIRHA method compared with hot air drying at 70°C, the PV were within the permissible limit of 5Meq/kg for edible oils. Our findings demonstrate the efficacy of SIRHA drying in achieving simultaneous drying and decontamination of pistachios.

Production of the Plant Hormone Auxin by Salmonella and Its Role in the Interactions with Plants and Animals.

The ability of human enteric pathogens to colonize plants and use them as alternate hosts is now well established. Salmonella, similarly to phytobacteria, appears to be capable of producing the plant hormone auxin via an indole-3-pyruvate decarboxylase (IpdC), a key enzyme of the IPyA pathway. A deletion of the Salmonella ipdC significantly reduced auxin synthesis in laboratory culture. The Salmonella ipdC gene was expressed on root surfaces of Medicago truncatula. M. truncatula auxin-responsive GH3::GUS reporter was activated by the wild type Salmonella, and not but the ipdC mutant, implying that the bacterially produced IAA (Indole Acetic Acid) was detected by the seedlings. Seedling infections with the wild type Salmonella caused an increase in secondary root formation, which was not observed in the ipdC mutant. The wild type Salmonella cells were detected as aggregates at the sites of lateral root emergence, whereas the ipdC mutant cells were evenly distributed in the rhizosphere. However, both strains appeared to colonize seedlings well in growth pouch experiments. The ipdC mutant was also less virulent in a murine model of infection. When mice were infected by oral gavage, the ipdC mutant was as proficient as the wild type strain in colonization of the intestine, but it was defective in the ability to cross the intestinal barrier. Fewer cells of the ipdC mutant, compared with the wild type strain, were detected in Peyer's patches, spleen and in the liver. Orthologs of ipdC are found in all Salmonella genomes and are distributed among many animal pathogens and plant-associated bacteria of the Enterobacteriaceae, suggesting a broad ecological role of the IpdC-catalyzed pathway.

Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation.

Several species of enteric pathogens produce curli fimbriae, which may affect their interaction with surfaces and other microbes in nonhost environments. Here we used two Escherichia coli O157:H7 outbreak strains with distinct genotypes to understand the role of curli in surface attachment and biofilm formation in several systems relevant to fresh produce production and processing. Curli significantly enhanced the initial attachment of E. coli O157:H7 to spinach leaves and stainless steel surfaces by 5-fold. Curli was also required for E. coli O157:H7 biofilm formation on stainless steel and enhanced biofilm production on glass by 19-27 fold in LB no-salt broth. However, this contribution was not observed when cells were grown in sterile spinach lysates. Furthermore, both strains of E. coli O157:H7 produced minimal biofilms on polypropylene in LB no-salt broth but considerable amounts in spinach lysates. Under the latter conditions, curli appeared to slightly increase biofilm production. Importantly, curli played an essential role in the formation of mixed biofilm by E. coli O157:H7 and plant-associated microorganisms in spinach leaf washes, as revealed by confocal microscopy. Little or no E. coli O157:H7 biofilms were detected at 4 °C, supporting the importance of temperature control in postharvest and produce processing environments.

Effect of sulfur dioxide fumigation on survival of foodborne pathogens on table grapes under standard storage temperature.

We examined the fate of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica Thompson inoculated on freshly-harvested table grapes under standard cold storage with initial and weekly sulfur dioxide (SO2) fumigation. L. monocytogenes and S. enterica Thompson were much more sensitive to cold temperature than E. coli O157:H7. Furthermore, L. monocytogenes was highly susceptible to SO2. Initial fumigation with 100 or 200 ppm-hr was sufficient to eliminate this pathogen on grapes with low (10(4) cells/grape) and high (10(6) cells/grape) inocula, respectively. Initial fumigation with 300 ppm-hr reduced S. enterica Thompson population about 300- and 10-fold on grapes with low and high inocula, respectively. Initial fumigation with 300 ppm-hr reduced E. coli O157:H7 population to less than 10-fold, regardless of inoculum density. When grapes were inoculated with the high inoculum and fumigated on days 0 and 7 with 200 or 300 ppm-hr SO2, S. enterica Thompson and E. coli O157:H7 were completely inactivated between days 8 and 14 of cold storage. Standard cold storage combined with SO2 fumigation was effective in reducing and eliminating all three pathogens on table grapes, however, depending on the dose, two or three fumigations were needed for elimination of S. enterica Thompson and E. coli O157:H7.

Downy mildew disease promotes the colonization of romaine lettuce by Escherichia coli O157:H7 and Salmonella enterica.

BACKGROUND: Downy mildew, a plant disease caused by the oomycete Bremia lactucae, is endemic in many lettuce-growing regions of the world. Invasion by plant pathogens may create new portals and opportunities for microbial colonization of plants. The occurrence of outbreaks of Escherichia coli O157:H7 (EcO157) and Salmonella enterica Typhimurium (S. Typhimurium) infections linked to lettuce prompted us to investigate the role of downy mildew in the colonization of romaine lettuce by these human pathogens under controlled laboratory conditions. RESULTS: Whereas both EcO157 and S. Typhimurium population sizes increased 10(2)-fold on healthy leaf tissue under conditions of warm temperature and free water on the leaves, they increased by 10(5)-fold in necrotic lesions caused by B. lactucae. Confocal microscopy of GFP-EcO157 in the necrotic tissue confirmed its massive population density and association with the oomycete hyphae. Multiplication of EcO157 in the diseased tissue was significantly lower in the RH08-0464 lettuce line, which has a high level of resistance to downy mildew than in the more susceptible cultivar Triple Threat. qRT-PCR quantification of expression of the plant basal immunity gene PR-1, revealed that this gene had greater transcriptional activity in line RH08-0464 than in cultivar Triple Threat, indicating that it may be one of the factors involved in the differential growth of the human pathogen in B. lactucae lesions between the two lettuce accessions. Additionally, downy mildew disease had a significant effect on the colonization of EcO157 at high relative humidity (RH 90-100%) and on its persistence at lower RH (65-75%). The latter conditions, which promoted overall dryness of the lettuce leaf surface, allowed for only 0.0011% and 0.0028% EcO157 cell survival in healthy and chlorotic tissue, respectively, whereas 1.58% of the cells survived in necrotic tissue. CONCLUSIONS: Our results indicate that downy mildew significantly alters the behavior of enteric pathogens in the lettuce phyllosphere and that breeding for resistance to B. lactucae may lower the increased risk of microbial contamination caused by this plant pathogen.

Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells.

Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as "super-shedders" (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.