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      Factors That Affect Proliferation of Salmonella in Tomatoes Post-Harvest: The Roles of Seasonal Effects, Irrigation Regime, Crop and Pathogen Genotype

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          Abstract

          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.

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          Most cited references39

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          Regulation of enteric endophytic bacterial colonization by plant defenses.

          Bacterial endophytes reside within the interior of plants without causing disease or forming symbiotic structures. Some endophytes, such as Klebsiella pneumoniae 342 (Kp342), enhance plant growth and nutrition. Others, such as Salmonella enterica serovar Typhimurium (S. typhimurium), are human pathogens that contaminate raw produce. Several lines of evidence are presented here to support the hypothesis that plant defense response pathways regulate colonization by endophytic bacteria. An ethylene-insensitive mutant of Medicago truncatula is hypercolonized by Kp342 compared to the parent genotype. Addition of ethylene, a signal molecule for induced systemic resistance in plants, decreased endophytic colonization in Medicago spp. This ethylene-mediated inhibition of endophytic colonization was reversed by addition of the ethylene action inhibitor, 1-methylcyclopropene. Colonization of Medicago spp. by S. typhimurium also was affected by exogenous ethylene. Mutants lacking flagella or a component of the type III secretion system of Salmonella pathogenicity island 1 (TTSS-SPI1) colonize the interior of Medicago spp. in higher numbers than the wild type. Arabidopsis defense response-related genotypes indicated that only salicylic acid (SA)-independent defense responses contribute to restricting colonization by Kp342. In contrast, colonization by S. typhimurium is affected by both SA-dependent and -independent responses. S. typhimurium mutants further delineated these responses, suggesting that both flagella and TTSS-SPI1 effectors can be recognized. Flagella act primarily through SA-independent responses (compromising SA accumulation still affected colonization in the absence of flagella). Removal of a TTSS-SPI1 effector resulted in hypercolonization regardless of whether the genotype was affected in either SA-dependent or SA-independent responses. Consistent with these results, S. typhimurium activates the promoter of PR1, a SA-dependent pathogenesis-related gene, while S. typhimurium mutants lacking the TTSS-SPI1 failed to activate this promoter. These observations suggest approaches to reduce contamination of raw produce by human enteric pathogens and to increase the number of growth-promoting bacteria in plants.
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            Recurrent multistate outbreak of Salmonella Newport associated with tomatoes from contaminated fields, 2005.

            Salmonella Newport causes more than an estimated 100,000 infections annually in the United States. In 2002, tomatoes grown and packed on the eastern shore of Virginia contaminated with a pan-susceptible S. Newport strain caused illness in 510 patients in 26 states. In July-November 2005, the same strain caused illness in at least 72 patients in 16 states. We conducted a case-control study during the 2005 outbreak, enrolling 29 cases and 140 matched neighbourhood controls. Infection was associated with eating tomatoes (matched odds ratio 9.7, 95% confidence interval 3.3-34.9). Tomatoes were traced back to the eastern shore of Virginia, where the outbreak strain was isolated from pond water used to irrigate tomato fields. Two multistate outbreaks caused by one rare strain, and identification of that strain in irrigation ponds 2 years apart, suggest persistent contamination of tomato fields. Further efforts are needed to prevent produce contamination on farms and throughout the food supply chain.
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              Prevalence, distribution, and diversity of Salmonella enterica in a major produce region of California.

              A survey was initiated to determine the prevalence of Salmonella enterica in the environment in and around Monterey County, CA, a major agriculture region of the United States. Trypticase soy broth enrichment cultures of samples of soil/sediment (n = 617), water (n = 252), wildlife (n = 476), cattle feces (n = 795), and preharvest lettuce and spinach (n = 261) tested originally for the presence of pathogenic Escherichia coli were kept in frozen storage and later used to test for the presence of S. enterica. A multipathogen oligonucleotide microarray was employed to identify a subset of samples that might contain Salmonella in order to test various culture methods to survey a larger number of samples. Fifty-five of 2,401 (2.3%) samples yielded Salmonella, representing samples obtained from 20 different locations in Monterey and San Benito Counties. Water had the highest percentage of positives (7.1%) among sample types. Wildlife yielded 20 positive samples, the highest number among sample types, with positive samples from birds (n = 105), coyotes (n = 40), deer (n = 104), elk (n = 39), wild pig (n = 41), and skunk (n = 13). Only 16 (2.6%) of the soil/sediment samples tested positive, and none of the produce samples had detectable Salmonella. Sixteen different serotypes were identified among the isolates, including S. enterica serotypes Give, Typhimurium, Montevideo, and Infantis. Fifty-four strains were sensitive to 12 tested antibiotics; one S. Montevideo strain was resistant to streptomycin and gentamicin. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed over 40 different pulsotypes. Several strains were isolated from water, wildlife, or soil over a period of several months, suggesting that they were persistent in this environment.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                4 December 2013
                : 8
                : 12
                : e80871
                Affiliations
                [1 ]Department of Soil and Water Science, University of Florida, Gainesville, Florida, United States of America
                [2 ]Department of Statistics, University of Florida, Gainesville, Florida, United States of America
                [3 ]Plant Pathology Department, University of Florida, Gainesville, Florida, United States of America
                Cornell University, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MT GJH JB. Performed the experiments: MM ASG JTN GJH JB. Analyzed the data: MM MCG GJH JTN JB MT. Contributed reagents/materials/analysis tools: MT GJH. Wrote the paper: MT MM MCG.

                Article
                PONE-D-13-37164
                10.1371/journal.pone.0080871
                3851777
                24324640
                a9fbc463-58ad-4118-83e7-b9402109b63f
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 11 September 2013
                : 17 October 2013
                Page count
                Pages: 11
                Funding
                This research was supported by USDA NIFA-AFRI grant # 2011-67017-30127 and by funding provided by the Center for Produce Safety through UCANR/USDA NIFA grant #2010-34608-20768 (SA7660) and the Florida Specialty Crops Foundation. A.S. George acknowledges support from the McKnight Fellowship Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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