Salmonella enterica Serovar Typhimurium 14028s Genomic Regions Required for Colonization of Lettuce Leaves.

Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety

Salinity Stress Does Not Affect Root Uptake, Dissemination and Persistence of Salmonella in Sweet-basil (Ocimum basilicum)

Crop produce can be contaminated in the field during cultivation by bacterial human pathogens originating from contaminated soil or irrigation water. The bacterial pathogens interact with the plant, can penetrate the plant via the root system and translocate and survive in above-ground tissues. The present study is first to investigate effects of an abiotic stress, salinity, on the interaction of plants with a bacterial human pathogen. The main sources of human bacterial contamination of plants are manures and marginal irrigation waters such as treated or un-treated wastewater. These are often saline and induce morphological, chemical and physiological changes in plants that might affect the interaction between the pathogens and the plant and thereby the potential for plant contamination. This research studied effects of salinity on the internalization of the bacterial human pathogen Salmonella enterica serovar Newport via the root system of sweet-basil plants, dissemination of the bacteria in the plant, and kinetics of survival in planta. Irrigation with 30 mM NaCl-salinity induced typical salt-stress effects on the plant: growth was reduced, Na and Cl concentrations increased, K and Ca concentrations reduced, osmotic potential and anti-oxidative activity were increased by 30%, stomatal conductance was reduced, and concentrations of essential-oils in the plants increased by 26%. Despite these physical, chemical and morphological changes in the plants, root internalization of the bacteria and its translocation to the shoot were not affected, and neither was the die-off rate of Salmonella in planta. The results demonstrate that the salinity-induced changes in the sweet-basil plants did not affect the interaction between Salmonella and the plant and thereby the potential for crop contamination

Salmonella enterica Growth Conditions Influence Lettuce Leaf Internalization

Human pathogens on plants (HPOP) have evolved complex interactions with their plant host. Stomatal internalization is one such mode of interaction, where bacteria are attracted to stomata and penetrate into the substomatal cavity by a process mediated by chemotaxis. Internalization enables HPOP to evade the hostile environment of the leaf surface and find a protected, nutrient-rich niche within the leaf. Numerous studies have documented attachment and entry of the foodborne pathogens, Salmonella enterica and Escherichia coli into stomata. Internalization, however, varies considerably among different pathogens and in different plants, and both bacterial and plant’s factors were reported to influence HPOP attachment and internalization. Here we have studied the effect of laboratory growth conditions, on the internalization of Salmonella enterica serovar Typhimurium (STm) into lettuce leaf. We have further tested the potential involvement of universal stress-proteins in leaf internalization. We found that STm grown in Luria Bertani broth devoid of NaCl (LBNS), or in diluted LB (0.5×LB) internalized lettuce leaf better (62 ± 5% and 59 ± 7%, respectively) compared to bacteria grown in LB (15 ± 7%). Growth under non-aerated conditions also enhanced STm internalization compared to growth under aerated conditions. Growth temperature of 25 and 37°C did not affect STm internalization, however, growth at 42°C, significantly augmented leaf internalization. Since, the tested growth conditions represent moderate stresses, we further investigated the involvement of five universal-stress genes in STm leaf internalization following growth in LBNS medium. Knockout mutations in ydaA, yecG, ybdQ, and uspAB, but not in ynaF, significantly reduced STm internalization compared to the wild-type (wt) strain, without affecting bacterial attachment and motility. Transduction of the mutations back to the parent strain confirmed the linkage between the mutations and the internalization phenotype. These findings support a specific role of the universal-stress genes in leaf internalization. The present study highlights the complexity of bacterial internalization process and may provide partial explanation for the variable, sometimes-contrasting results reported in the literature regarding stomatal internalization by HPOP. Characterization of the regulatory networks that mediate the involvement of usp genes and the tested growth factors in STm internalization should contribute to our understanding of human pathogens-plant interactions

Bacillus strain BX77: a potential biocontrol agent for use against foodborne pathogens in alfalfa sprouts

Despite regulatory and technological measures, edible sprouts are still often involved in foodborne illness and are considered a high-risk food. The present study explored the potential of spore-forming Bacillus isolates to mitigate Salmonella and Escherichia coli contamination of alfalfa sprouts. Food-derived Bacillus strains were screened for antagonistic activity against S. enterica serovar Typhimurium SL1344 (STm) and enteropathogenic E. coli O55:H7. Over 4 days of sprouting, levels of STm and E. coli on contaminated seeds increased from 2.0 log CFU/g to 8.0 and 3.9 log CFU/g, respectively. Treatment of the contaminated seeds with the most active Bacillus isolate, strain BX77, at 7 log CFU/g seeds resulted in substantial reductions in the levels of STm (5.8 CFU/g) and E. coli (3.9 log CFU/g) in the sprouted seeds, compared to the control. Similarly, co-culturing STm and BX77 in sterilized sprout extract at the same ratio resulted in growth inhibition and killed the Salmonella. Confocal-microscopy experiments using seeds supplemented with mCherry-tagged Salmonella revealed massive colonization of the seed coat and the root tip of 4-day-old sprouted seeds. In contrast, very few Salmonella cells were observed in sprouted seeds grown with BX77. Ca-hypochlorite disinfection of seeds contaminated with a relatively high concentration of Salmonella (5.0 log CFU/g) or treated with BX77 revealed a mild inhibitory effect. However, disinfection followed by the addition of BX77 had a synergistic effect, with a substantial reduction in Salmonella counts (7.8 log CFU/g) as compared to untreated seeds. These results suggest that a combination of chemical and biological treatments warrants further study, toward its potential application as a multi-hurdle strategy to mitigate Salmonella contamination of sprouted alfalfa seeds

The response of foodborne pathogens to osmotic and desiccation stresses in the food chain

peer-reviewedIn combination with other strategies, hyperosmolarity and desiccation are frequently used by the food processing industry as a means to prevent bacterial proliferation, and particularly that of foodborne pathogens, in food products. However, it is increasingly observed that bacteria, including human pathogens, encode mechanisms to survive and withstand these stresses. This review provides an overview of the mechanisms employed by Salmonella spp., Shiga toxin producing E. coli, Cronobacter spp., Listeria monocytogenes and Campylobacter spp. to tolerate osmotic and desiccation stresses and identifies gaps in knowledge which need to be addressed to ensure the safety of low water activity and desiccated food products

Hydroponic Agriculture and Microbial Safety of Vegetables: Promises, Challenges, and Solutions

Hydroponics is a farming technique for growing plants with mineral nutrients using a soil-free medium. The plant roots are submerged in soil-free media, such as vermiculite or perlite, or just in mineral nutrient solutions. This allows for high production yields throughout the year with less water and agro-chemical inputs. Consequently, hydroponics is considered a sustainable agriculture technology. Hydroponically grown crops are usually protected from the diseases transmitted through soil or animals in open fields. Therefore, they require fewer chemicals for pest control and are safer than conventionally grown crops in terms of possible chemical contamination. Nevertheless, hydroponics guarantees neither plant health nor the microbial safety of fresh produce. In the case of microbial contamination by human pathogens, unlike soil-grown crops, the pathogens may rapidly spread through the circulating water and simultaneously infect all the plants in the facility. This review summarizes the up-to-date knowledge regarding the microbial safety of hydroponically grown crops and discusses the role of the hydroponic system in reducing the microbial hazards for leafy and fruity crops as well as the potential risks for contamination by human pathogens. Finally, it outlines the approaches and the available science-based practices to ensure produce safety. The contamination risk in hydroponic systems may be diminished by using novel planting materials and the appropriate decontamination treatment of a recirculating liquid substrate; by modulating the microbiota interactions; and by following strict phytosanitary measures and workers’ hygienic practices. There is a timely need to adopt measures, such as the Good Agricultural Practice (GAP) guidelines, to mitigate the risks and ensure safe hydroponically grown vegetables for consumers

Hydroponic Agriculture and Microbial Safety of Vegetables: Promises, Challenges, and Solutions

Hydroponics is a farming technique for growing plants with mineral nutrients using a soil-free medium. The plant roots are submerged in soil-free media, such as vermiculite or perlite, or just in mineral nutrient solutions. This allows for high production yields throughout the year with less water and agro-chemical inputs. Consequently, hydroponics is considered a sustainable agriculture technology. Hydroponically grown crops are usually protected from the diseases transmitted through soil or animals in open fields. Therefore, they require fewer chemicals for pest control and are safer than conventionally grown crops in terms of possible chemical contamination. Nevertheless, hydroponics guarantees neither plant health nor the microbial safety of fresh produce. In the case of microbial contamination by human pathogens, unlike soil-grown crops, the pathogens may rapidly spread through the circulating water and simultaneously infect all the plants in the facility. This review summarizes the up-to-date knowledge regarding the microbial safety of hydroponically grown crops and discusses the role of the hydroponic system in reducing the microbial hazards for leafy and fruity crops as well as the potential risks for contamination by human pathogens. Finally, it outlines the approaches and the available science-based practices to ensure produce safety. The contamination risk in hydroponic systems may be diminished by using novel planting materials and the appropriate decontamination treatment of a recirculating liquid substrate; by modulating the microbiota interactions; and by following strict phytosanitary measures and workers’ hygienic practices. There is a timely need to adopt measures, such as the Good Agricultural Practice (GAP) guidelines, to mitigate the risks and ensure safe hydroponically grown vegetables for consumers

Determination of Salmonella enterica Leaf Internalization Varies Substantially According to the Method and Conditions Used to Assess Bacterial Localization.

In a previous study, comparing the internalization of S. enterica serovar Typhimurium in various leaves by confocal microscopy, we have demonstrated that the pathogen failed to internalize tomato leaves. Numerous reasons may account for these findings, yet one such factor might be the methodology employed to quantify leaf internalization. To this end, we have systematically studied leaf localization of a Green-fluorescent protein-labeled Salmonella strain in tomato, lettuce, and Arabidopsis leaves by surface sterilization and enumeration of the surviving bacteria, side by side, with confocal microscopy observations. Leaf sterilization was performed using either sodium hypochlorite, silver nitrate, or ethanol for 1 to 7min. The level of internalization varied according to the type of disinfectant used for surface sterilization and the treatment time. Treatment of tomato leaves with 70% ethanol for up to 7min suggested possible internalization of Salmonella, while confocal microscopy showed no internalization. In the case of in lettuce and Arabidopsis leaves, both the plate-count technique and confocal microscopy demonstrated considerable Salmonella internalization thought different sterilization conditions resulted in variations in the internalization levels. Our findings highlighted the dependency of the internalization results on the specific disinfection protocol used to determine bacterial localization. The results underscore the importance of confocal microscopy in validating a particular surface sterilization protocol whenever a new pair of bacterial strain and plant cultivar is studied

Salmonella enterica Serovar Typhimurium 14028s Genomic Regions Required for Colonization of Lettuce Leaves.

Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety

Image_1_Bacillus strain BX77: a potential biocontrol agent for use against foodborne pathogens in alfalfa sprouts.tif

Despite regulatory and technological measures, edible sprouts are still often involved in foodborne illness and are considered a high-risk food. The present study explored the potential of spore-forming Bacillus isolates to mitigate Salmonella and Escherichia coli contamination of alfalfa sprouts. Food-derived Bacillus strains were screened for antagonistic activity against S. enterica serovar Typhimurium SL1344 (STm) and enteropathogenic E. coli O55:H7. Over 4 days of sprouting, levels of STm and E. coli on contaminated seeds increased from 2.0 log CFU/g to 8.0 and 3.9 log CFU/g, respectively. Treatment of the contaminated seeds with the most active Bacillus isolate, strain BX77, at 7 log CFU/g seeds resulted in substantial reductions in the levels of STm (5.8 CFU/g) and E. coli (3.9 log CFU/g) in the sprouted seeds, compared to the control. Similarly, co-culturing STm and BX77 in sterilized sprout extract at the same ratio resulted in growth inhibition and killed the Salmonella. Confocal-microscopy experiments using seeds supplemented with mCherry-tagged Salmonella revealed massive colonization of the seed coat and the root tip of 4-day-old sprouted seeds. In contrast, very few Salmonella cells were observed in sprouted seeds grown with BX77. Ca-hypochlorite disinfection of seeds contaminated with a relatively high concentration of Salmonella (5.0 log CFU/g) or treated with BX77 revealed a mild inhibitory effect. However, disinfection followed by the addition of BX77 had a synergistic effect, with a substantial reduction in Salmonella counts (7.8 log CFU/g) as compared to untreated seeds. These results suggest that a combination of chemical and biological treatments warrants further study, toward its potential application as a multi-hurdle strategy to mitigate Salmonella contamination of sprouted alfalfa seeds.</p