Jeri D. Barak

Role of soil, crop debris, and a plant pathogen in Salmonella enterica contamination of tomato plants.

Background In the U.S., tomatoes have become the most implicated vehicle for produce-associated Salmonellosis with 12 outbreaks since 1998. Although unconfirmed, trace backs suggest pre-harvest contamination with Salmonella enterica. Routes of tomato crop contamination by S. enterica in the absence of direct artificial inoculation have not been investigated. Methodology/Principal Findings This work examined the role of contaminated soil, the potential for crop debris to act as inoculum from one crop to the next, and any interaction between the seedbourne plant pathogen Xanthomonas campestris pv. vesicatoria and S. enterica on tomato plants. Our results show S. enterica can survive for up to six weeks in fallow soil with the ability to contaminate tomato plants. We found S. enterica can contaminate a subsequent crop via crop debris; however a fallow period between crop incorporation and subsequent seeding can affect contamination patterns. Throughout these studies, populations of S. enterica declined over time and there was no bacterial growth in either the phyllosphere or rhizoplane. The presence of X. campestris pv. vesicatoria on co-colonized tomato plants had no effect on the incidence of S. enterica tomato phyllosphere contamination. However, growth of S. enterica in the tomato phyllosphere occurred on co-colonized plants in the absence of plant disease. Conclusions/Significance S. enterica contaminated soil can lead to contamination of the tomato phyllosphere. A six week lag period between soil contamination and tomato seeding did not deter subsequent crop contamination. In the absence of plant disease, presence of the bacterial plant pathogen, X. campestris pv. vesicatoria was beneficial to S. enterica allowing multiplication of the human pathogen population. Any event leading to soil contamination with S. enterica could pose a public health risk with subsequent tomato production, especially in areas prone to bacterial spot disease.

Human enteric pathogens in produce: un-answered ecological questions with direct implications for food safety.

Recent outbreaks of gastroenteritis linked to the consumption of fresh produce raise questions about the mechanisms by which human pathogens colonize plants and persist within marketable produce. Neither Salmonella nor Escherichia coli appear to produce enzymes that degrade plant cell walls, therefore it is not yet certain how these bacteria enter plant tissues and spread within them. Similar to plant-associated bacteria, enterics use cellulose and aggregative fimbriae for their attachment to plant surfaces. Salmonella can be an effective plant endophyte, even though it is capable of triggering plant defenses. Plant-associated microbiota contributes to the fitness and translocation of these human pathogens within plant hosts, although interactions and mechanisms of communication between plant-associated microbiota and enteric pathogens are not yet characterized.

Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection.

A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng microl(-1) and 0.45 ng microl(-1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.

Recovery of Surface Bacteria from and Surface Sanitization of Cantaloupes

Practical, effective methods that could be implemented in a food service establishment (restaurant or delicatessen) for the surface sanitization of cantaloupes were microbiologically evaluated. Cantaloupes (Cucumis melo L. var. reticulates) were immersed in an inoculum containing Salmonella enterica serovar Poona or Pantoea agglomerans at ca. 10(4) to 10(5) CFU/ml. An efficient method for the recovery of bacteria from the cantaloupe surface was developed and validated. The method consisted of washing the entire melon with Butterfields buffer containing 1% Tween 80 in a plastic bag placed inside a plastic pail affixed to an orbital shaker. Levels of S. enterica Poona recovered by washing the entire melon were significantly higher than those recovered by the more common laboratory method of blending the rind. P. agglomerans can be used as a non-pathogenic proxy for S. enterica Poona. A three-compartment surface sanitization method consisting of washing with an antimicrobial soap solution, scrubbing with a brush in tap water, and immersion in 150 ppm of sodium hypochlorite reduced the initial level of recoverable viable bacteria by 99.8%. When examined separately, scrubbing with a vegetable brush in tap water, washing with soap, and dipping in chlorine were found to reduce the bacterial load by 70, 80, and 90%, respectively.

Nutrient limitation of phytoplankton growth in Waquoit Bay, Massachusetts, USA: a nutrient enrichment study

We conducted nutrient enrichment experiments and field sampling to address three questions: (1) is there nutrient limitation of phytoplankton accumulation within an estuary whose waters are exposed to relatively high nitrogen loading rates, (2) where in the salinity gradient from fresh to seawater (0 to 32‰) is there a shift from phosphorus to nitrogen limitation of phytoplankton accumulation, and (3) is there a seasonal shift in limiting function of phosphorus and nitrogen anywhere in the estuarine gradient. Nitrogen and phosphorus enrichment experiments in the Childs River, an estuary of Waquoit Bay, Massachusetts, USA, showed that the accumulation of phytoplankton biomass in brackish and saline water was limited by supply of nitrate during warm months. The effects of enrichment were less evident in fresh water, with short-lived responses to phosphate enrichment. There was no specific point along the salinity gradient where there was a shift from phosphorus- to nitrogen-limited phytoplankton accumulation; rather, the relative importance of nitrogen and phosphorus changed along the salinity gradient in the estuary and with season of the year. There was no response to nutrient additions during the colder months, suggesting that some seasonally-varying factor, such as light, temperature or a physiological mechanism, restricted phytoplankton accumulation during months other than May-Aug. There was only slight evidence of a seasonal shift between nitrogen- and phosphorus-limitation of chlorophyll accumulation. Phytoplankton populations in nutrient-rich estuaries with short flushing times grow fast, but at the same time the cells may be advected out of the estuaries while still rapidly dividing, thereby providing an important subsidy to production in nearby deeper waters.

Potential Interactions between Salmonella enterica and Ralstonia solanacearum in tomato plants.

Over the past decade, the Eastern Shore of Virginia (ESV) has been implicated in at least four outbreaks of salmonellosis associated with tomato, all originating from the same serovar, Salmonella enterica serovar Newport. In addition to Salmonella Newport contamination, the devastating plant disease bacterial wilt, caused by the phytopathogen Ralstonia solanacearum, threatens the sustainability of ESV tomato production. Bacterial wilt is present in most ESV tomato fields and causes devastating yield losses each year. Although the connection between bacterial wilt and tomato-related salmonellosis outbreaks in ESV is of interest, the relationship between the two pathogens has never been investigated. In this study, tomato plants were root dip inoculated with one of four treatments: (i) 8 log CFU of Salmonella Newport per ml, (ii) 5 log CFU of R. solanacearum per ml, (iii) a coinoculation of 8 log CFU of Salmonella Newport per ml plus 5 log CFU of R. solanacearum per ml, and (iv) sterile water as control. Leaf, stem, and fruit samples were collected at the early-green-fruit stage, and S. enterica contamination in the internal tissues was detected. S. enterica was recovered in 1.4 and 2.9% of leaf samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. S. enterica was recovered from 1.7 and 3.5% of fruit samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. There were significantly more stem samples from plants coinoculated with Salmonella Newport plus R. solanacearum that were positive for S. enterica (18.6%) than stem samples collected from plants inoculated with Salmonella Newport only (5.7%). Results suggested that R. solanacearum could influence S. enterica survival and transportation throughout the internal tissues of tomato plants.

Automated Real-Time PCR Biosensor for the Detection of Pathogens in Produce Irrigation Water

A flow-through real-time PCR system has been developed. PCR reagents were pumped ninto a borosilicate reaction chamber and sealed by the pump and a valve prior to thermal cycling. nThe system successfully reached temperature setpoints within 2°C and was able to measure nfluorescence as a function of cycle number. The flow-through PCR system results were compared nwith results from a commercial real-time PCR machine. In both systems, primer dimers were formed nin negative control reactions. The commercial instrument formed a product with the expected melting ntemperature with extracted Salmonella enterica DNA as the target while the flow-through PCR nsystem formed a primer dimer with the extracted DNA sample. The cause of the unexpected primer ndimer formation is being investigated.

The Biggest Food Safety Threat from the Tiniest of Crops

Sprouted seeds are the most common vehicle of pathogenic Escherichia coli and salmonellosis outbreaks. Sprouted seed contamination by enteric human pathogens poses a unique challenge. Bacterial pathogens that cause food-borne illnesses replicate exponentially as seeds sprout, forming biofilms that adhere to sprouted seeds and cannot be cleaned off. The long-term survival of these pathogens on seeds presents a serious obstacle for sprout producers and food safety risk for consumers. Attempts to decontaminate seeds have failed, and testing seed or sprout lots for pathogens has proven unreliable. Eliminating seed contamination remains the only viable option. However, avoiding risk factors for seed contamination by enteric human pathogens may not be feasible. Once contamination occurs, seed represents the number one risk factor for sprout contamination.

Effect of plant surface microstructure and material on attachment of pathogenic bacteria.

Consumption of fresh vegetables contaminated with human pathogenic bacteria has caused many recent outbreaks of foodborne diseases. To avoid further outbreaks, the nature of the bacterial attachment to vegetable surface has been extensively studied in hope of finding a way to prevent the attachment. Nevertheless, the studied natural plant surfaces contain uncontrolled variables of both materials and microstructures, which result in doubtful deductions of the attachment nature. Our goal is to understand how plant surface material and microstructure affect bacterial attachment by using artificial plant surfaces, which contain various microstructures and is coated with selected organic materials, to mimic natural plant surfaces. The microstructures with desired dimensions and shapes require the microfabrication technology. Paraffin wax and pectin are the selected organic materials. The fabricated surfaces attached by the bacteria will also be washed by water to further examine the bacteria’s attachment strength. Finally, selected strains of the bacteria with different biomolecular attachment factors will be allowed to attach on the artificial plant surfaces. Then their different attachment capabilities under different surface conditions will be evaluated.

Attachment of Escherichia coli to Artificial Plant Surfaces

Consumption of fresh vegetables contaminated with pathogenic Escherichia coli has caused several recent outbreaks of foodborne disease. To avoid further outbreaks, the nature of the bacterial attachment to vegetable surface has been extensively studied in hope of finding a way to prevent the attachment. Nevertheless, the studied natural plant surfaces contain uncontrolled variables of both chemistry and topography, which result in doubtful deductions of the attachment nature. Our research is to study the E. coli attachment to the artificial plant surfaces, microfabricated by a replica molding method, one of soft lithography techniques, to have desired topography. The natural plant surface chemistry is also imitated by creating thin films of paraffin wax and cellulose acetate. In future work, the attachment strength examined by water flow shear stress testing will be performed. Also selected strains of E. coli with different extracellular characteristics, related to the attachment ability, will be studied. This research can help us understand the obscure nature of the E. coli attachment.