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.

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.

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.

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.

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.

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.

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.

High genotypic and phenotypic similarity among Shiga toxin-producing Escherichia coli O111 environmental and outbreak strains.

Escherichia coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing E. coli (STEC), which are emerging as important foodborne pathogens. We have assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of these strains to gain a better understanding of the epidemiology and biology of this serogroup. Sixty-three percent of the strains (24/38) were of H-type 8, which dominated the environmental- and outbreak-strains group, whereas the sporadic-case strains were more heterogeneous in H-type. All of the environmental and outbreak strains harbored the Shiga toxin 1 gene (stx1), eae, and ehx, and a subset of these also carried the Shiga toxin 2 gene (stx2). Only 9 of 16 sporadic-case strains produced stx1 and/or stx2, and these were mostly of H-type 8 and 10. Pulsed-field gel electrophoresis analysis revealed a cluster of environmental, outbreak, and sporadic illness strains with high phylogenetic similarity. Strains in this pulsogroup were all of the H8 type and STEC pathotype, and carried eae and ehx. Smaller clusters of highly similar STEC O111 strains included outbreak and sporadic illness strains isolated during different time periods or from different geographical locations. A distinct aggregative behavior was observed in the cultures of all environmental and outbreak STEC O111 strains, but not in those of sporadic-case strains. Among environmental and outbreaks strains, aggregation was positively correlated with production of curli fimbriae and RpoS function, and negatively with cellulose synthesis, while the nonaggregative behavior of sporadic-case strains correlated (positively) only with cellulose production. Our results indicate that STEC O111 strains sharing high genotypic similarity and important phenotypic traits with STEC O111 outbreak strains are present in the agricultural environment and may contribute to the burden of foodborne disease.

Effect of the surfactant tween 80 on the detachment and dispersal of Salmonella enterica serovar Thompson single cells and aggregates from cilantro leaves as revealed by image analysis.

Salmonella enterica has the ability to form biofilms and large aggregates on produce surfaces, including on cilantro leaves. Aggregates of S. enterica serovar Thompson that remained attached to cilantro leaves after rigorous washing and that were present free or bound to dislodged leaf tissue in the wash suspension were observed by confocal microscopy. Measurement of S. Thompson population sizes in the leaf washes by plate counts failed to show an effect of 0.05% Tween 80 on the removal of the pathogen from cilantro leaves 2 and 6 days after inoculation. On the contrary, digital image analysis of micrographs of single cells and aggregates of green fluorescent protein (GFP)-S. Thompson present in cilantro leaf washes revealed that single cells represented 13.7% of the cell assemblages in leaf washes containing Tween 80, versus 9.3% in those without the surfactant. Moreover, Tween 80 decreased the percentage of the total S. Thompson cell population located in aggregates equal to or larger than 64 cells from 9.8% to 4.4% (P < 0.05). Regression analysis of the frequency distribution of aggregate size in leaf washes with and without Tween 80 showed that the surfactant promoted the dispersal of cells from large aggregates into smaller ones and into single cells (P < 0.05). Our study underlines the importance of investigating bacterial behavior at the scale of single cells in order to uncover trends undetectable at the population level by bacterial plate counts. Such an approach may provide valuable information to devise strategies aimed at enhancing the efficacy of produce sanitization treatments.

The salmonella transcriptome in lettuce and cilantro soft rot reveals a niche overlap with the animal host intestine.

Fresh vegetables have been recurrently associated with salmonellosis outbreaks, and Salmonella contamination of retail produce has been correlated positively with the presence of soft rot disease. We observed that population sizes of Salmonella enterica serovar Typhimurium SL1344 increased 56-fold when inoculated alone onto cilantro leaves, versus 2,884-fold when coinoculated with Dickeya dadantii, a prevalent pathogen that macerates plant tissue. A similar trend in S. enterica populations was observed for soft-rotted lettuce leaves. Transcriptome analysis of S. enterica cells that colonized D. dadantii-infected lettuce and cilantro leaves revealed a clear shift toward anaerobic metabolism and catabolism of substrates that are available due to the degradation of plant cells by the pectinolytic pathogen. Twenty-nine percent of the genes that were upregulated in cilantro macerates were also previously observed to have increased expression levels in the chicken intestine. Furthermore, multiple genes induced in soft rot lesions are also involved in the colonization of mouse, pig, and bovine models of host infection. Among those genes, the operons for ethanolamine and propanediol utilization as well as for the synthesis of cobalamin, a cofactor in these pathways, were the most highly upregulated genes in lettuce and cilantro lesions. In S. Typhimurium strain LT2, population sizes of mutants deficient in propanediol utilization or cobalamin synthesis were 10- and 3-fold lower, respectively, than those of the wild-type strain in macerated cilantro (P < 0.0002); in strain SL1344, such mutants behaved similarly to the parental strain. Anaerobic conditions and the utilization of nutrients in macerated plant tissue that are also present in the animal intestine indicate a niche overlap that may explain the high level of adaptation of S. enterica to soft rot lesions, a common postharvest plant disease.

Salmonella interactions with plants and their associated microbiota.

The increase in the incidence of gastroenteritis outbreaks linked to the consumption of foods of plant origin has ignited public concern and scientific interest in understanding interactions of human enteric pathogens with plants. Enteric disease caused by nontyphoidal Salmonella is a major public health burden, with the number of cases of illness linked to fresh produce, spices, and nuts surpassing those linked to foods of animal origin. Mounting evidence supports the hypothesis that colonization of plants is an important part of the life cycle of this human pathogen. Although plant responses to human pathogens are distinct from the more specific responses to phytopathogens, plants appear to recognize Salmonella, likely by detecting conserved microbial patterns, which subsequently activates basal defenses. Numerous Salmonella genes have been identified as playing a role in its colonization of plant surfaces and tissues, and in its various interactions with other members of the phyto-microbial community. Importantly, Salmonella utilizes diverse and overlapping strategies to interact with plants and their microflora, and to successfully colonize its vertebrate hosts. This review provides insight into the complex behavior of Salmonella on plants and the apparent remarkable adaptation of this human pathogen to a potentially secondary host.

Distinct transcriptional profiles and phenotypes exhibited by Escherichia coli O157:H7 isolates related to the 2006 spinach-associated outbreak.

In 2006, a large outbreak of Escherichia coli O157:H7 was linked to the consumption of ready-to-eat bagged baby spinach in the United States. The likely sources of preharvest spinach contamination were soil and water that became contaminated via cattle or feral pigs in the proximity of the spinach fields. In this study, we compared the transcriptional profiles of 12 E. coli O157:H7 isolates that possess the same two-enzyme pulsed-field gel electrophoresis (PFGE) profile and are related temporally or geographically to the above outbreak. These E. coli O157:H7 isolates included three clinical isolates, five isolates from separate bags of spinach, and single isolates from pasture soil, river water, cow feces, and a feral pig. The three clinical isolates and two spinach bag isolates grown in cultures to stationary phase showed decreased expression of many σ(S)-regulated genes, including gadA, osmE, osmY, and katE, compared with the soil, water, cow, feral pig, and the other three spinach bag isolates. The decreased expression of these σ(S)-regulated genes was correlated with the decreased resistance of the isolates to acid stress, osmotic stress, and oxidative stress but increases in scavenging ability. We also observed that intraisolate variability was much more pronounced among the clinical and spinach isolates than among the environmental isolates. Together, the transcriptional and phenotypic differences of the spinach outbreak isolates of E. coli O157:H7 support the hypothesis that some variants within the spinach bag retained characteristics of the preharvest isolates, whereas other variants with altered gene expression and phenotypes infected the human host.

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.

Distinct acid resistance and survival fitness displayed by Curli variants of enterohemorrhagic Escherichia coli O157:H7.

Curli are adhesive fimbriae of Enterobacteriaceae and are involved in surface attachment, cell aggregation, and biofilm formation. Here, we report that both inter- and intrastrain variations in curli production are widespread in enterohemorrhagic Escherichia coli O157:H7. The relative proportions of curli-producing variants (C(+)) and curli-deficient variants (C(-)) in an E. coli O157:H7 cell population varied depending on the growth conditions. In variants derived from the 2006 U.S. spinach outbreak strains, the shift between the C(+) and C(-) subpopulations occurred mostly in response to starvation and was unidirectional from C(-) to C(+); in variants derived from the 1993 hamburger outbreak strains, the shift occurred primarily in response to oxygen depletion and was bidirectional. Furthermore, curli variants derived from the same strain displayed marked differences in survival fitness: C(+) variants grew to higher concentrations in nutrient-limited conditions than C(-) variants, whereas C(-) variants were significantly more acid resistant than C(+) variants. This difference in acid resistance does not appear to be linked to the curli fimbriae per se, since a csgA deletion mutant in either a C(+) or a C(-) variant exhibited an acid resistance similar to that of its parental strain. Our data suggest that natural curli variants of E. coli O157:H7 carry several distinct physiological properties that are important for their environmental survival. Maintenance of curli variants in an E. coli O157:H7 population may provide a survival strategy in which C(+) variants are selected in a nutrient-limited environment, whereas C(-) variants are selected in an acidic environment, such as the stomach of an animal host, including that of a human.

Fluorescent viability stains to probe the metabolic status of aflatoxigenic fungus in dual culture of Aspergillus flavus and Pichia anomala.

The metabolic activity of the aflatoxigenic fungus, Aspergillus flavus co-cultured with the biocontrol yeast, Pichia anomala was examined using several viability stains. Both the FUN-1 stain and the combined use of DiBAC(4)(5) with CDFA-AM stains were applied in this study. The results suggest that the ATP-generating system in A. flavus was inactivated as the ratio of yeasts to fungi increased in the dual culture. A decrease in hyphal membrane potential and esterase activity was substantiated by the combined stains of DiBAC(4)(5) and CDFA-AM. Reduced metabolic function in conjunction with cell wall damage of A. flavus hindered the growth and biomass production of this fungus. Viability stains such as FUN-1 and DiBAC(4)(5) with CDFA-AM may assist in elucidating the biocontrol mechanism by allowing for the visualization of the antagonistic effect of yeast species on target fungi in situ, as well as for screening potent biocontrol yeast agents against fungal pathogens.

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.