Effect of the irrigation regime on the susceptibility of pepper and tomato to post-harvest proliferation of Salmonella enterica.

Raw produce is increasingly recognized as a vehicle of human gastroenteritis. Non-typhoidal Salmonella, pathogenic Escherichia coli, and other human pathogens have been isolated from fruits and vegetables in the field and in the marketplace, which led to the hypothesis that these microbes can use plants as alternate hosts. However, environmental and physiological factors that facilitate persistence of these bacteria in the crop production environment and make produce more vulnerable to post-harvest contamination have not been fully delineated. This study tested the effect of irrigation regimes on the susceptibility of peppers and tomatoes to post-harvest proliferation of Salmonella. The experiments were carried out over three experimental seasons in two locations using seven strains of Salmonella. The irrigation regime per se did not affect susceptibility of tomatoes and peppers to post-harvest proliferation of Salmonella; however, in some of the seasons, irrigation regime-dependent differences were observed. Red peppers and tomatoes were more conducive to proliferation of Salmonella than green fruit in all seasons. Inter-seasonal differences were the strongest factors affecting proliferation of Salmonella in peppers.

Ethylene signalling affects susceptibility of tomatoes to Salmonella.

Fresh fruits and vegetables are increasingly recognized as important reservoirs of human pathogens, and therefore, significant attention has been directed recently to understanding mechanisms of the interactions between plants and enterics, like Salmonella. A screen of tomato cultivars for their susceptibility to Salmonella revealed significant differences in the ability of this human pathogen to multiply within fruits; expression of the Salmonella genes (cysB, agfB, fadH) involved in the interactions with tomatoes depended on the tomato genotype and maturity stage. Proliferation of Salmonella was strongly reduced in the tomato mutants with defects in ethylene synthesis, perception and signal transduction. While mutation in the ripening-related ethylene receptor Nr resulted only in a modest reduction in Salmonella numbers within tomatoes, strong inhibition of the Salmonella proliferation was observed in rin and nor tomato mutants. RIN and NOR are regulators of ethylene synthesis and ripening. A commercial tomato variety heterozygous for rin was less susceptible to Salmonella under the greenhouse conditions but not when tested in the field over three production seasons.

Effects of nitrogen and potassium fertilization on the susceptibility of tomatoes to post-harvest proliferation of Salmonella enterica.

Fresh fruits and vegetables are increasingly recognized as vehicles of salmonellosis. Pre- and post-harvest environmental conditions, and physiological, and genetic factors are thought to contribute to the ability of human pathogens to persist in the production environment, attach to, colonize and proliferate in and on raw produce. How field production conditions affect the post-harvest food safety outcomes is not entirely understood. This study tested how varying nitrogen and potassium fertilization levels affected the "susceptibility" of tomatoes to Salmonella infections following the harvest of fruits. Two tomato varieties grown over three seasons under high, medium, and low levels of nitrogen and potassium fertilization in two locations were inoculated with seven strains of Salmonella. Even though the main effects of nitrogen and potassium fertilization on the susceptibility of tomatoes to infections with Salmonella enterica were not statistically significant overall, differences in nitrogen concentrations in plant tissues correlated with the susceptibility of partially ripe tomatoes (cv. Solar Fire) to Salmonella. Tomato maturity and the season in which tomatoes were produced had the strongest effect on the ability of Salmonella to multiply in tomatoes. Tomato phenolics, accumulation of which is known to correlate with rates of the N fertilization, did not inhibit growth of Salmonella in vitro.

Factors that affect proliferation of Salmonella in tomatoes post-harvest: the roles of seasonal effects, irrigation regime, crop and pathogen genotype.

MAIN OBJECTIVES: Fresh fruits and vegetables become increasingly recognized as vehicles of human salmonellosis. Physiological, ecological, and environmental factors are all thought to contribute to the ability of Salmonella to colonize fruits and vegetables pre- and post-harvest. The goal of this study was to test how irrigation levels, fruit water congestion, crop and pathogen genotypes affect the ability of Salmonella to multiply in tomatoes post-harvest. EXPERIMENTAL DESIGN: Fruits from three tomato varieties, grown over three production seasons in two Florida locations, were infected with seven strains of Salmonella and their ability to multiply post-harvest in field-grown tomatoes was tested. The field experiments were set up as a two-factor factorial split plot experiment, with the whole-plot treatments arranged in a randomized complete-block design. The irrigation treatment (at three levels) was the whole-plot factor, and the split-plot factor was tomato variety, with three levels. The significance of the main, two-way, and three-way interaction effects was tested using the (type III) F-tests for fixed effects. Mean separation for each significant fixed effect in the model was performed using Tukey's multiple comparison testing procedure. MOST IMPORTANT DISCOVERIES AND SIGNIFICANCE: The irrigation regime per se did not affect susceptibility of the crop to post-harvest proliferation of Salmonella. However, Salmonella grew significantly better in water-congested tissues of green tomatoes. Tomato maturity and genotype, Salmonella genotype, and inter-seasonal differences were the strongest factors affecting proliferation. Red ripe tomatoes were significantly and consistently more conducive to proliferation of Salmonella. Tomatoes harvested in the driest, sunniest season were the most conducive to post-harvest proliferation of the pathogen. Statistically significant interactions between production conditions affected post-harvest susceptibility of the crop to the pathogen. UV irradiation of tomatoes post-harvest promoted Salmonella growth.

Differential regulation of Salmonella typhimurium genes involved in O-antigen capsule production and their role in persistence within tomato fruit.

Enteric pathogens, including non-typhoidal Salmonella spp. and enterovirulent Escherichia coli, are capable of persisting and multiplying within plants. Yet, little is still known about the mechanisms of these interactions. This study identified the Salmonella yihT gene (involved in synthesis of the O-antigen capsule) as contributing to persistence in immature tomato fruit. Deletion of yihT reduced competitive fitness of S. enterica sv. Typhimurium in green (but not ripe, regardless of color) tomato fruit by approximately 3 logs. The yihT recombinase-based in vivo expression technology (RIVET) reporter was strongly activated in unripe tomato fruit, and fitness of the mutant inversely correlated with the level of the yihT gene expression. Expression of yihT in mature tomato fruit was low, and yihT did not affect competitive fitness within mature fruit. To better understand the molecular basis of the phenotype, behaviors of the yihT RIVET reporter and the yihT mutant were tested in tomato fruit defective in ethylene signaling. These experiments suggest a role for functional ethylene-mediated signaling in the persistence of Salmonella spp. within tomato fruit. Furthermore, jasmonic acid and its precursors strongly reduced expression of yihT.

Spontaneous non-rdar mutations increase fitness of Salmonella in plants.

Proliferation of human enteric pathogens within alternate hosts, like plants, leads to temporal changes in gene expression and also selects for the phenotypic variants of the enterics that are presumed to be more fit within plants. Human enteric pathogens recovered from produce-borne outbreaks exhibit peculiar phenotypes, for example many of them do not display the rdar (red dry and rough) phenotype. The non-rdar phenotype results from mutations in cellulose and/or curli synthesis or regulation. How often these mutants arise, and whether they are more fit within plants is not entirely clear. We addressed this hypothesis by sequentially passaging the type strain of Salmonella enterica sv. Typhimurium ATCC14028 through tomatoes. Two spontaneous mutants defective in their ability to form red dry and rough colonies were further characterized. Even though attachment of the mutants to tomato surfaces was modestly reduced, they were 5- to 50-fold more competitive than the wild-type inside tomato fruits. Because the mutants were outcompeted by the wild-type on common laboratory media, and not in tomatoes, the lack of the rdar phenotype is probably beneficial within tomatoes. Recombinase-based in vivo expression tests indicate that the agfB and yihT genes were regulated differently in the mutants, although the corresponding mutations cannot fully account for the increased competitive fitness of the mutants. One of the variants has a mutated rpoS, which also reduced the expression of a SPI-5 effector encoded by sopB. A survey of the Salmonella strains recovered from produce outbreaks revealed that some were similarly non-rdar, likely containing rpoS mutations. This report indicates that the 'perfect storm' scenario, typically used to model outbreaks of produce-borne gastroenteritis, needs to account for the ability of the pathogen to rapidly evolve and adapt to the crop production environments.

Specific responses of Salmonella enterica to tomato varieties and fruit ripeness identified by in vivo expression technology.

BACKGROUND: Recent outbreaks of vegetable-associated gastroenteritis suggest that enteric pathogens colonize, multiply and persist in plants for extended periods of time, eventually infecting people. Genetic and physiological pathways, by which enterics colonize plants, are still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: To better understand interactions between Salmonella enterica sv. Typhimurium and tomatoes, a gfp-tagged Salmonella promoter library was screened inside red ripe fruits. Fifty-one unique constructs that were potentially differentially regulated in tomato relative to in vitro growth were identified. The expression of a subset of these promoters was tested in planta using recombinase-based in vivo expression technology (RIVET) and fitness of the corresponding mutants was tested. Gene expression in Salmonella was affected by fruit maturity and tomato cultivar. A putative fadH promoter was upregulated most strongly in immature tomatoes. Expression of the fadH construct depended on the presence of linoleic acid, which is consistent with the reduced accumulation of this compound in mature tomato fruits. The cysB construct was activated in the fruit of cv. Hawaii 7997 (resistant to a race of Ralstonia solanacearum) more strongly than in the universally susceptible tomato cv. Bonny Best. Known Salmonella motility and animal virulence genes (hilA, flhDC, fliF and those encoded on the pSLT virulence plasmid) did not contribute significantly to fitness of the bacteria inside tomatoes, even though deletions of sirA and motA modestly increased fitness of Salmonella inside tomatoes. CONCLUSIONS/SIGNIFICANCE: This study reveals the genetic basis of the interactions of Salmonella with plant hosts. Salmonella relies on a distinct set of metabolic and regulatory genes, which are differentially regulated in planta in response to host genotype and fruit maturity. This enteric pathogen colonizes tissues of tomatoes differently than plant pathogens, and relies little on its animal virulence genes for persistence within the fruit.

The diffusive influx and carrier efflux have a strong effect on the bistability of the lac operon in Escherichia coli.

In the presence of gratuitous inducers, the lac operon of Escherichia coli exhibits bistability. Most models in the literature assume that the inducer enters the cell via the carrier (permease), and exits by a diffusion-like process. The diffusive influx and carrier efflux are neglected. However, analysis of the data shows that in non-induced cells, the diffusive influx is comparable to the carrier influx, and in induced cells, the carrier efflux is comparable to the diffusive efflux. Since bistability entails the coexistence of steady states corresponding to both non-induced and induced cells, neither one of these fluxes can be ignored. We present a model accounting for both fluxes, and show that: (1) The thresholds (i.e., the extracellular inducer levels at which transcription turns on or off) are profoundly affected by both fluxes. The diffusive influx reduces the on threshold, and eliminates irreversible bistability, a phenomenon that is inconsistent with data. The carrier efflux increases the off threshold, and abolishes bistability at large permease activities, a conclusion that can be tested experimentally. (2) The thresholds are well approximated by simple analytical expressions obtained by considering two limiting cases (no carrier efflux and no diffusive influx). (3) The simulations are in good agreement with the data for isopropyl thiogalactoside (IPTG), but somewhat discrepant with respect to the data for thiomethyl galactoside (TMG). We discuss the potential sources of the discrepancy.

Gene regulation in continuous cultures: a unified theory for bacteria and yeasts.

During batch growth on mixtures of two growth-limiting substrates, microbes consume the substrates either sequentially (diauxie) or simultaneously. The ubiquity of these growth patterns suggests that they may be driven by a universal mechanism common to all microbial species. Recently, we showed that a minimal model accounting only for enzyme induction and dilution, the two processes that occur in all microbes, explains the phenotypes observed in batch cultures of various wild-type and mutant/recombinant cells (Narang and Pilyugin in J. Theor. Biol. 244:326-348, 2007). Here, we examine the extension of the minimal model to continuous cultures. We show that: (1) Several enzymatic trends, attributed entirely to cross-regulatory mechanisms, such as catabolite repression and inducer exclusion, can be quantitatively explained by enzyme dilution. (2) The bifurcation diagram of the minimal model for continuous cultures, which classifies the substrate consumption pattern at any given dilution rate and feed concentrations, provides a precise explanation for the empirically observed correlations between the growth patterns in batch and continuous cultures. (3) Numerical simulations of the model are in excellent agreement with the data. The model captures the variation of the steady state substrate concentrations, cell densities, and enzyme levels during the single- and mixed-substrate growth of bacteria and yeasts at various dilution rates and feed concentrations. This variation is well approximated by simple analytical expressions that furnish deep physical insights. (4) Since the minimal model describes the behavior of the cells in the absence of cross-regulatory mechanisms, it provides a rigorous framework for quantifying the effect of these mechanisms. We illustrate this by analyzing several data sets from the literature.