Search

A growing world population together with a decrease in water resources have resulted in a deficit in water sources for agriculture. Nations with limited water resources are already using recycled treated water as a vital water source for irrigation. In 2019, treated wastewater made up 45% of the overall agricultural water consumption in Israel. Since recycling systems work continuously and are directly connected to irrigation reservoirs, there is a constant risk of irrigation system malfunctions in the treatment process. In cases of fecal origin, this may result in pathogens entering the irrigation water, the soil, and eventually the food chain. Early warning systems for biological water contamination are important in large-scale processing, yet less studied. This work proposes such a system, with a functional prototype for irrigation-water monitoring by measuring fluorescence spectra in the range of 300 – 520 nm at excitation wavelengths of 280 nm and 340 nm, to capture both tryptophan-like fluorescence and humic-like fluorescence. The prototype was successfully tested under simulated field conditions with treated irrigation water as the base substance, and milk injections to simulate dissolved organic matter breaches. Near real-time operation enabled recording biological and chemical dynamics within the substance for the sake of monitoring drift in process control.

Broiler litter (BL), a by-product of broiler meat production, is frequently contaminated with Salmonella and other zoonotic pathogens. To ensure the safety of crop production chains and limit pathogen spread in the environment, a pre-treatment is desired before further agricultural utilization. The objective of this study was to characterize the effect of physico-chemical properties on Salmonella persistence in BL during composting and stabilization and following soil incorporation, toward optimization of the inactivation process. Thirty-six combinations of temperature (30, 40, 50, and 60°C), water content (40, 55, and 70%; w/w), and initial pH (6, 7, and 8.5) were employed in static lab vessels to study the persistence of Salmonella enterica serovar Infantis (S. Infantis; a multidrug-resistant strain) during incubation of artificially-inoculated BL. The effect of aeration was investigated in a composting simulator, with controlled heating and flow conditions. Temperature was found to be the main factor significantly influencing Salmonella decay rates, while water content and initial pH had a secondary level of influence with significant effects mainly at 30 and 40°C. Controlled simulations showed faster decay of Salmonella under anaerobic conditions at mesophilic temperatures (3 emissions. Re-wetting the BL at mesophilic temperatures resulted in Salmonella burst, and led to a higher tolerance of the pathogen at increased temperatures. Based on the decay rates measured under all temperature, water content, and pH conditions, it was estimated that the time required to achieve a 7 log10 reduction in Salmonella concentration, ranges between 13.7–27.2, 6.5–15.6, 1.2–4.7, and 1.3–1.5 days for 30, 40, 50, and 60°C, respectively. Inactivation of BL indigenous microbial population by autoclaving or addition of antibiotics to which the S. Infantis is resistant, resulted in augmentation of Salmonella multiplication. This suggests the presence of microbial antagonists in the BL, which inhibit the growth of the pathogen. Finally, Salmonella persisted over 90 days at 30°C in a Vertisol soil amended with inoculated BL, presumably due to reduced antagonistic activity compared to the BL alone. These findings are valuable for risk assessments and the formulation of guidelines for safe utilization of BL in agriculture.

Leaf internalization is an important trait of both plant- and human-pathogens that colonize plants. Salmonella enterica was reported to efficiently internalize the leaves of multiple plants, including tomato. 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 GFP-labelled Salmonella strain in tomato, lettuce, and Arabidopsis leaves, previously shown to support different levels of Salmonella internalization. Three protocols were employed for 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 7 min. The same surface sterilization protocols resulted in different inactivation levels of Salmonella thus affecting the quantification of apparent internalization in the three tested leaves. The level of internalization varied according to the type of disinfectant and the treatment time. Treatment of tomato leaves with 70% ethanol for up to 7 min suggested possible internalization of Salmonella, while confocal microscopy showed no internalization. The lack of Salmonella internalization in tomato leaves seemed to corroborated our previous finding using a different tomato cultivar. Finally, both the plate count technique and confocal microscopy demonstrated considerable Salmonella internalization in lettuce and Arabidopsis leaves 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. Altogether, this is the first systematic study comparing the effect of the same leaf sterilization protocols on the internalization level of Salmonella in leaves of different plants, side by side with confocal microscopy analysis. 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.

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.

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.

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.

During nonventilated storage of carrots, CO(2) 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 CO(2) 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 CO(2) 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 CO(2) 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 CO(2) requires further investigation. However, this study implies an overlooked role of CO(2) 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 CO(2) 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.

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.

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.

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.