High Levels of CO2 Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes.

During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8 h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.

Dynamics of bacterial communities in alfalfa and mung bean sprouts during refrigerated conditions.

Sprouts are considered a healthy ready-to-eat food and has gained popularity in recent years. The objective of the present study was to determine the dynamics of sprouts' microbiome during cold storage to the end of their shelf-life at home. The microbiological quality of fresh alfalfa (Medicago sativa) and mung bean (Vigna radiata) sprouts from two commercial brands was tested and the number of APC ranges from 5.0 to 8.7 log CFU/g in alfalfa and 6.7 to 9.3 log CFU/g in mung bean sprouts. In the case of alfalfa, but not mung beans, there were differences in the mean numbers of APC between the two brands. The number of coliform bacteria ranges from 4.3 to 7.7 log CFU/g in alfalfa and 4.1 to 8.1 log CFU/g in mung bean sprouts. Four independent batches of sprouts were used for DNA preparation and were sampled immediately after purchase and once a week during subsequent storage in refrigerator until the end of their shelf-life. Microbial population of the sprouts was determined using next generation sequencing of 16S rRNA amplicons. Alfalfa sprouts were dominated by Pseudomonas throughout the storage time with relative abundance of >60% at 3 weeks. Fresh mung bean sprouts were dominated by both Pseudomonas and Pantoea, but Pantoea became the dominant taxa after 2 weeks of storage, with >46% of relative abundance. The bacterial communities associated with sprouts were largely dependent on the sprout type, and less dependent on the brand. The species richness and diversity declined during storage and the development of spoilage. Among the 160 genera identified on sprouts, 23 were reported to contain known spoilage-associated species and 30 genera comprise potential human pathogenic species. This study provides new insight into the microbiome dynamics of alfalfa and mung bean sprouts during cold storage.

Microbiome dynamics during ensiling of corn with and without Lactobacillus plantarum inoculant.

Microbial population dynamics associated with corn silage, with and without Lactobacillus plantarum treatment, was studied. Whole crop corn was ensiled using laboratory silos and sampled at different times, up to 3 months. The dominant bacteria, before ensiling, were Acinetobacter (38.5%) and Klebsiella (16.3%), while the dominant fungi were Meyerozyma (53.5%) and Candida (27.7%). During ensiling, the microbial population shifted considerably, and Lactobacillus (> 94%) and Candida (> 74%) became the most dominant microbial genera in both treated and untreated silages. Yet, lactic acid content was higher in the treated silage, while the microbial diversity was lower than in the untreated silage. Upon aerobic exposure, spoilage occurred more rapidly in the treated silage, possibly due to the higher abundance of lactic acid-assimilating fungi, such as Candida. Our study is the first to describe microbial population dynamics during whole-crop corn ensiling and the results indicate that microbial diversity may be an indicator of aerobic stability.

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.

Persistence of Salmonella enterica during dehydration and subsequent cold storage.

Despite the fact that Salmonella enterica serotype Typhimurium SL 1344 has served as a model pathogen in many studies, information regarding its desiccation response is still scarce. In this study, we investigated environmental conditions that affect Salmonella survival following dehydration and subsequent cold storage, using a 96-well polystyrene plate model. The SL 1344 strain exhibited high survival compared with other Typhimurium isolates and S. enterica serotypes. Further characterization of desiccation tolerance in this strain revealed that temperature, stationary-phase of growth, solid medium, and the presence of increasing NaCl concentrations (0.5-5.0%) in the growth medium enhanced desiccation tolerance. Dehydration at basic pHs (8-10), or in trehalose, sucrose, but not in glycine-betaine, improved bacterial persistence. Dehydrated Salmonella survived over 100 weeks at 4 °C with a ∼5-log reduction in numbers. However, viability staining revealed only a ∼50% reduction in viable cells, suggesting bacterial transition into a viable-but-not-cultivable state (VBNC). Addition of chloramphenicol reduced bacterial survival implying that adaptation to desiccation stress requires de-novo protein synthesis. Consistent with this finding, shortening the dehydration time resulted in lower survival. This study emphasizes the impact of environmental conditions on the fate of dried Salmonella in the food chain and highlights the potential transition of the pathogen to the VBNC state.

Air-ozonolysis activation of polyolefins versus use of laden finishing to form contact-active nonwoven materials.

Two synthetic approaches were explored for modification of the polyolefins polyethylene/polypropylene (PE/PP) to form contact-active nonwoven materials. In the first approach, polymer surfaces were activated by O2-free air-ozonolysis, and then the active agent (trimethoxysilyl) propyl-octadecyl-dimethyl-ammonium chloride (C18-TSA) was covalently bound. In the second approach, the active agent was directly conjugated to the commercial 'finishing' that was then applied to the polymer. The chemical, physical and microscopic properties of the modified polymers were comprehensively studied, and their active site density was quantified by fluorescein sodium salt-cetyltrimethylammonium chloride reaction. The antimicrobial activity of the prepared nonwovens against Bacillus subtilis (Gram-positive) and Salmonella enterica (Gram-negative), and their stability at various pHs and temperatures were examined. The two approaches conferred antimicrobial properties to the modified polymers and demonstrated stable linkage of C18-TSA. However, the performance of the nonwovens formed by the first approach was superior. The study suggests two feasible and safe pathways for the modification of polyolefins to form contact-active nonwoven materials that can be further applied in various fields, such as hygiene products, medical fabrics, sanitizing wipes, and more.

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.

Quantification of bacteria in water using PLS analysis of emission spectra of fluorescence and excitation-emission matrices.

Bacterial contamination of drinking water is a considerable concern for public health. Tryptophan-like fluorescence (TLF) has been widely suggested to enable fast and inexpensive monitoring and quantification of bacterial contamination of water. Typically, TLF is determined at a certain excitation (ex)/emission (em) wavelengths pair. The aim of this study was to assess fluorescence spectroscopy supported with partial least squares (PLS) algorithms as a tool for a rapid evaluation of the microbial quality of water, by comparing the use of a single ex/em wavelengths pair, of the spectrum of emission obtained at a single excitation wavelength to that of whole excitation-emission matrices (EEMs). For that, laboratory-grown Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa were studied as the model systems, as well as 90 groundwater samples from 6 different wells in Israel. The groundwater samples were characterized for fluorescence emission, coliforms, fecal coliforms, fecal streptococci and heterotrophic plate counts. The PLS analysis of emission spectra and, especially, of EEMs was capable of meaningfully reducing the detection limit of microorganisms in model systems, as compared with the single ex/em wavelengths pair-based determination commonly used, reaching a detection threshold as low as 10 CFU/ml. Use of PLS-analyzed EEMs becomes beneficial also in terms of correlation and similarity between the actual and predicted bacterial concentrations. Similarly, improved detection of bacteria was also achieved in groundwater samples. Furthermore, at a level of >104 CFU/ml, use of EEMs coupled with PLS enabled discrimination between E. coli, B. subtilis and P. aeruginosa.

Bacterial Dynamics of Wheat Silage.

Knowledge regarding bacterial dynamics during crop ensiling is important for understanding of the fermentation process and may facilitate the production of nutritious and stable silage. The objective of this study was to analyze the bacterial dynamics associated with whole crop wheat silage with and without inoculants. Whole crop wheat was ensiled in laboratory silos, with and without Lactobacillus inoculants (L. plantarum, L. buchneri), for 3 months. Untreated and L. plantarum-treated silages were sampled at several times during ensiling, while L. buchneri-treated silage was sampled only at 3 months. Bacterial composition was studied using next generation sequencing approach. Dominant bacteria, before ensiling, were Pantoea (34.7%), Weissella (28.4%) and Pseudomonas (10.4%), Exiguobacterium (7.8%), and Paenibacillus (3.4%). Exogenous inoculants significantly affected bacterial composition and dynamics during ensiling. At 3 months of ensiling, Lactobacillus dominated the silage bacterial population and reached an abundance of 59.5, 92.5, and 98.2% in untreated, L. plantarum- and L. buchneri-treated silages, respectively. The bacterial diversity of the mature silage was lower in both treated silages compared to untreated silage. Functional profiling of the bacterial communities associated with the wheat ensiling demonstrated that the abundant pathways of membrane transporters, carbohydrate and amino acids metabolisms followed different pattern of relative abundance in untreated and L. plantarum-treated silages. Only three pathways, namely base-excision repair, pyruvate metabolism and transcription machinery, were significantly different between untreated and L. buchneri-treated silages upon maturation. Lactic acid content was higher in L. plantarum-treated silage compared to untreated and L. buchneri-treated silage. Still, the pH of both treated silages was lower in the two Lactobacillus-treated silages compared to untreated silage. Aerobic stability test demonstrated that L. plantarum-, but not L. buchneri-supplement, facilitated silage deterioration. The lower aerobic stability of the L. plantarum-treated silage may be attributed to lower content of acetic acid and other volatile fatty acids which inhibit aerobic yeasts and molds. Indeed, high yeast count was recorded, following exposure to air, only in L. plantarum-treated silage, supporting this notion. Analysis of bacterial community of crop silage can be used for optimization of the ensiling process and the selection of appropriate inoculants for improving aerobic stability.

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.

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.

Emergence of Leuconostoc mesenteroides as a causative agent of oozing in carrots stored under non-ventilated conditions.

Long-term storage and transport of post-harvest carrots (Daucus carota L.) require a low-temperature, high-relative-humidity environment, usually with low ventilation. Following long-term storage, a slimy exudate (oozing) often appears on the carrots, leading to severe spoilage. We characterized the environmental conditions leading to these symptoms and identified the causative agent. Simulation of non-ventilated storage conditions revealed accumulation of CO2 (to 80%) and ethanol (to 1000 ppm); then, a transparent exudate appeared on the carrot surface which, upon ventilation, developed into tissue browning and soft rot. Peels from oozing carrots contained over 10-fold the total bacterial counts of healthy carrots. The total peel microbiome was determined by 16S rDNA sequencing. During oozing stage, the surface of carrots incubated in a CO2 -rich (98%) environment harboured a bacterial population dominated by Lactobacillales and Enterobacteriales, differing markedly from those incubated in air. Three prevalent bacterial isolates from the oozing carrots were identified as Pantoea agglomerans, Rahnella aquatilis and Leuconostoc mesenteroides. Inoculation of carrot discs with L. mesenteroides, but not the others, induced oozing under high CO2 , suggesting that this bacterium is responsible for oozing of stored carrots. These findings should enable development of approaches to preventing carrot spoilage during long-term storage.

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

In 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.

Survival of Salmonella enterica serovar infantis on and within stored table eggs.

Contaminated table eggs are considered a primary source of foodborne salmonellosis globally. Recently, a single clone of Salmonella enterica serovar Infantis emerged in Israel and became the predominant serovar isolated in poultry. This clone is currently the most prevalent strain in poultry and is the leading cause of salmonellosis in humans. Because little is known regarding the potential transmission of this strain from contaminated eggs to humans, the objective of this study was to evaluate the ability of Salmonella Infantis to survive on the eggshell or within the egg during cold storage or at room temperature. Salmonella cells (5.7 log CFU per egg) were inoculated on the surface of 120 intact eggs or injected into the egg yolk (3.7 log CFU per egg) of another 120 eggs. Half of the eggs were stored at 5.5 ± 0.3°C and half at room temperature (25.5 ± 0.1°C) for up to 10 weeks. At both temperatures, the number of Salmonella cells on the shell declined by 2 log up to 4 weeks and remained constant thereafter. Yolk-inoculated Salmonella counts at cold storage declined by 1 log up to 4 weeks and remained constant, while room-temperature storage supported the growth of the pathogen to a level of 8 log CFU/ml of total egg content, as early as 4 weeks postinoculation. Examination of egg content following surface inoculation revealed the presence of Salmonella in a portion of the eggs at both temperatures up to 10 weeks, suggesting that this strain can also penetrate through the shell and survive within the egg. These findings imply that Salmonella enterica serovar Infantis is capable of survival both on the exterior and interior of table eggs and even multiply inside the egg at room temperature. Our findings support the need for prompt refrigeration to prevent Salmonella multiplication during storage of eggs at room temperature.

Air-ozonolysis to generate contact active antimicrobial surfaces: activation of polyethylene and polystyrene followed by covalent graft of quaternary ammonium salts.

Air-ozonolysis was revealed as an accessible and effective approach for surface activation and further functionalization of hydrocarbon polymers. Antimicrobial contact active polyethylene (PE) and polystyrene (PS) were designed by generation on their surfaces OH-functional groups and covalent graft of dimethyloctadecyl [3-(trimethoxysilyl) propyl] ammonium chloride (C18-TSA) quaternary ammonium salt. The shortened analog, trimethyl [3-(trimethoxysilyl) propyl] ammonium chloride (C1-TSA), was also covalently attached to the activated PE and PS surfaces. X-ray photoelectron spectroscopy (XPS) and FTIR confirmed the surface modifications. Scanning electron (SEM) and confocal microscopy were utilized to monitor surface morphology and bacteria interactions. The antimicrobial effect of the C18-TSA grafted polymer surfaces was demonstrated on Gram-negative and Gram-positive bacteria species including human pathogen, Salmonella enterica. The shorter C1-TSA grafted polymers did not demonstrate bactericidal activity, suggesting the critical role of the alkyl chain length. The described strategy may establish a new general and safe platform for future development and application of contact active antimicrobial polymers.

Root internalization, transport and in-planta survival of Salmonella enterica serovar Newport in sweet basil.

It is now acknowledged that food-borne pathogens present in the irrigation water or soil can become associated with crop plants in the field, penetrate internal plant tissues via the root, translocate and survive inside plants. Only little information is available concerning interaction between enteric pathogens and plants. The present study evaluated the potential for contamination of the aromatic plant, sweet basil during cultivation, by Salmonella enterica serovar Newport. Root internalization was plant-age-dependent, with the highest susceptibility occurring at the beginning of the rapid growth phase of the root. Higher incidence of internalization was detected in vegetative than reproductive plant organs, pointing at bacterial transport in the transpiration stream. Internalized Salmonella survived only < 30 h in the phyllosphere. In contrast, survival of Salmonella on the leaf surface was much pronounced (at least 8 days), and the initial decay rate was lower at the abaxial (lower) compared with the adaxial (upper) side of the leaf. Although the experiments were conducted with high concentration of Salmonella unlikely to happen in the field, internalization occurred at a low frequency and in-planta survival was limited to less than 30 h. These findings imply that leaf surface contamination, rather than root internalization, may pose higher risk for human infection following consumption of contaminated basil.