The fate of salmonellae applied to tomato plants was investigated. Five Salmonella serotypes were used to inoculate tomato plants before and after fruits set, either by injecting stems with inoculum or brushing flowers with it. Ripe tomato fruits were subjected to microbiological analysis. Peptone wash water, homogenates of stem scar tissues, and homogenates of fruit pulp were serially diluted and plated on bismuth sulfite agar before and after enrichment. Presumptive Salmonella colonies were confirmed by serological tests, PCR assay using HILA2 primers, and enterobacterial repetitive intergenic consensus PCR. Of 30 tomatoes harvested from inoculated plants, 11 (37%) were positive for Salmonella. Of the Salmonella-positive tomatoes, 43 and 40%, respectively, were from plants receiving stem inoculation before and after flower set. Two of eight tomatoes produced from inoculated flowers contained Salmonella. Higher percentages of surface (82%) and stem scar tissue (73%) samples, compared to pulp of Salmonella-positive tomatoes (55%), harbored the pathogen. Of the five serotypes in the inoculum, Montevideo was the most persistent, being isolated from tomatoes 49 days after inoculation, and Poona was the most dominant, being present in 5 of 11 Salmonella-positive tomatoes. Results suggest that Salmonella cells survive in or on tomato fruits from the time of inoculation at flowering through fruit ripening. Tomato stems and flowers are possible sites at which Salmonella may attach and remain viable during fruit development, thus serving as routes or reservoirs for contaminating ripened fruit.

The possibility of uptake of salmonellae by roots of hydroponically grown tomato plants was investigated. Within 1 day of exposure of plant roots to Hoagland nutrient solution containing 4.46 to 4.65 log 10 CFU of salmonellae/ml, the sizes of the pathogen populations were 3.01 CFU/g of hypocotyls and cotyledons and 3.40 log 10 CFU/g of stems for plants with intact root systems (control) and 2.55 log 10 CFU/g of hypocotyls and cotyledons for plants from which portions of the roots had been removed. A population of >3.38 log 10 CFU/g of hypocotyls-cotyledons, stems, and leaves of plants grown for 9 days was detected regardless of the root condition. Additional studies need to be done to unequivocally demonstrate that salmonellae can exist as endophytes in tomato plants grown under conditions that simulate commonly used agronomic practices.Tomatoes, seed sprouts, and melons are among the raw produce repeatedly identified as potential vehicles of human salmonellosis (9,20,21,29). The inner tissue of sound produce is generally considered to be sterile. However, endophytic bacteria are known to reside in a wide range of plant tissues (7,16,28), including those consumed as raw vegetables (11,12,19,23,25,27,31). Samish et al. (23) studied 10 fruits and vegetables and found that bacteria, mostly members of two families, the Pseudomonadaceae and the Enterobacteriaceae, were present in sound, raw cucumber and tomato fruits. Ralstonia solanacearum, which causes bacterial wilt of tomatoes, is known to enter the plant through the root, to penetrate the xylem, and to systemically colonize the stem (18,22,30). Pseudomonas syringae pathovar tomato, the causative agent of bacterial speck of tomato, inhabits leaf trichomes (24).None of the chemical or physical treatments currently authorized by regulatory agencies in the United States for use as disinfectants for raw produce can be relied on to eliminate all types of pathogens (2). Devising successful intervention steps to reduce populations of human pathogens on and in fruits and vegetables eaten raw should be aided by information concerning the sources of contamination and the ecology of pathogens affected by agronomic and minimal processing practices (2, 3).In a previous study (6), we observed that salmonellae inoculated onto flowers and into stems of tomatoes survived for at least 49 days and were recovered from ripened fruits. Contact of tomato fruits with soil containing salmonellae can result in infiltration of the pathogens into subsurface tissues (5). The objectives of the study reported here were to investigate the possibility of association of salmonellae with hypocotyls, cotyledons, stems, and leaves of young plants grown in a hydroponic nutrient solution inoculated with the pathogen. Bacterial cultures.Five serotypes of Salmonella enterica obtained from the Centers for Disease Control and Prevention, Atlanta, Ga., were used. S. enterica serotype Montevideo (serogroup C 1 ) was isolated from a patient in an outbreak of salmonellosis associated with consumption of raw t...

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Salmonellosis has been linked to the consumption of several types of raw fruits and vegetables, some of which may have been contaminated with Salmonella before harvesting. The objectives of this study were to investigate water and soil as reservoirs of Salmonella for the contamination of mature green tomato fruits. Salmonella survived for at least 45 days in inoculated moist soil. The population of Salmonella on tomatoes in contact with soil increased by 2.5 log10 CFU per tomato during storage for 4 days at 20 degrees C and remained constant for an additional 10 days. The number of cells inoculated on tomatoes decreased by approximately 4 log10 CFU per tomato during storage for 14 days at 20 degrees C and 70% relative humidity. Fruits in contact with inoculated soil for 1 day at 20 degrees C harbored Salmonella only near or on the skin surface. More Salmonella cells were observed in stem scar and subsurface areas of tomatoes as the time of storage increased. PCR fingerprinting revealed that among five Salmonella serotypes in the inoculum, Salmonella Montevideo was the most persistent on tomatoes in contact with inoculated soil and on spot-inoculated tomatoes, followed by Salmonella Poona and Salmonella Michigan. The results of this study demonstrate that an enhanced green fluorescent protein marker can be used to detect cells and monitor the growth of Salmonella in the presence of other microorganisms. Observations on the infiltration of Salmonella into tomato tissues support the contention that preharvest contact of produce with contaminated water or soil exacerbates problems associated with the postharvest removal of pathogens or their accessibility to treatment with sanitizers.

Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of l-proline, an amino acid with medicine, feed, and food applications. To facilitate l-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards l-proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an l-proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces l-proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing.