1a. Objectives (from AD-416):
1. Develop and evaluate novel antagonists (e.g. Bacteriovorax, Bdellovibrio and non-pectolytic Pseudomonas) for biological-based intervention strategies, and identify means of combining pathogen microbial ecology with effective chemical and physical interventions. 2. Develop and optimize chemical decontamination interventions (e.g. novel sanitizer formulations and advanced gas-phase antimicrobial treatments), making use of pathogen microbial ecology information generated under Objective 1. 3. Develop nonthermal technologies (e.g. cold plasma, high-intensity monochromatic light and irradiation) as effective, waterless physical treatments, and establish protocols for combination treatments with interventions developed in the first two objectives.
1b. Approach (from AD-416):
A holistic approach to the development of new effective intervention technologies will be followed. In an iterative process of technology development, increased knowledge of pathogen ecology on produce surfaces will be used to optimize biological, chemical and physical control strategies; combinatorial use of intervention technologies will be examined for additive and synergistic effects while maintaining product quality. More rapid and successful commercialization will be fostered by determining the equipment and infrastructure required for large-scale implementation, as well as economic costs and benefits expected from the use of the new technologies. A key aspect of this effort to facilitate commercialization will be collaboration and information sharing with industry, including direct contact with potential end-users of technologies. Stakeholders will be updated on research goals and objectives of the project, and input will be sought from them to identify key problems to be solved. By proactively fostering these interactions in conjunction with site visits, annual scientific meetings, industry trade shows and similar venues, there will be opportunities in the early, mid-phase and in late stages of research will allow for the practical needs of industry to be addressed as the research is formulated and conducted.
3. Progress Report:
Objective 1. Bdellovibrio like organisms are predatory bacteria. (Deltaproteobacterium) that resides in soil and fresh-water ecosystems, and are obligate predators of Gram negative bacteria. We assessed the predation of E. coli O157:H7 and S. enterica by B. bacteriovorus 109J in co-cultures assays. The predatory cultures of host-dependent B. bacteriovorus 109J was cultured at 30°C on E. coli ML35 prey cells (a surrogate and non-pathogenic strain) on HEPES buffer (pH 7.6) containing CaCl2 and MgSO4 for 24-48h on an orbital shaker. The liquid suspension of predatory bacteria, B. bacteriovorus 109J (1 mL) were pipetted in separate 50 ml sterile conical flasks containing 3 mL each, of E. coli O157 strains 43894, 43895, 35150 and S. enterica 2380 plus 20 mL of HM buffer and incubated at 30°C for 24 and 48 hrs. Pathogen survival was quantified by dilution plating on McConkey Agar and XLT-4 for E. coli O157:H7 and S. enterica, respectively. A control in which pathogen strains were incubated in HM buffer without the predator at the same temperature was used. The results of co-culture study showed that relative to the un-treated control, reductions of E. coli O157:H7 strains were 2.6+0.5 Logs (43894), 1.5+0.2 Logs (43895), and 3.8+0.0 Log CFU/ml (35150), whereas for S. enterica 2380, the pathogen reduction was 1.0+0.4 Log CFU/ml of HM buffer substrate. The research findings of this study indicate that B. bacteriovorus 109J can inactivate and inhibit E. coli O157:H7 and S. enterica in liquid substrates in vitro. The predators were recovered and enumerated by double plaque assay on dilute nutrient broth (DNB) agar (1.5% agar for bottom layer and 0.6% agar for top layer) with Bdellovibrio populations (plaque forming units) in the range of 4.8 X 108 to 5.1 x 108 PFU/mL of liquid substrate. Our SEM images also confirmed the predation by Bdellovibrio as attack-phase predator cells were abundant. The result showed that B. bacteriovorus 109J is capable of suppressing populations of E. coli O157:H7 and Salmonella in-vitro. Objective 2. Control of Salmonella Typhimurium on sprouts and minimally processed produce is crucial for food and consumer safety. The aim of this research was to assess natural microflora populations on soybean and evaluate the effects of gaseous chlorine dioxide (ClO2) and biocontrol Pseudomonas on the survival of S. Typhimurium on soybean sprouts. Aerobic mesophilic bacteria, lactic acid bacteria, yeast and molds, and Pseudomonas sp. were enumerated on seeds using total plate count agar (TPC), de Man, Rogosa, and Sharpe agar (MRS), potato dextrose agar (PDA), and Pseudomonas Agar F (PAF), respectively. Soybean sprouts were dip-inoculated with S. Typhimurium prior to the application of biocontrol microbes (P. chlororaphis and P. fluorescens). After inoculation with S. Typhimurium, sprouts were treated with ClO2 at 0.4 mg/L for 1 h (90% R.H., 13 deg C). For pathogen and biocontrol recovery, Pseudomonas strains and Salmonella were assessed on PAF and XLT-4 media, respectively. Natural microflora populations (aerobic mesophilic bacteria, lactic acid bacteria, Pseudomonas sp., yeasts and molds) were low and ranged from 0.93-2.91 log CFU/g of soybean seed. Pseudomonas strains reduced Salmonella by less than 1 log CFU/g of sprouts. The reduction of S. Typhimurium on soybean sprouts by ClO2 ranged from 2.55-5.35 log CFU/g. Gaseous ClO2 treatment reduced S. Typhimurium by 3.90 (0 h), 4.47 (24 h), and 3.61 log CFU/g (168 h). When S. Typhimurium was co-inoculated with Pc or Pf, pathogen reductions on sprouts were similar. Treatments of soybean sprouts with ClO2 and biocontrol Pseudomonas can enhance the safety of soybean sprouts at post-harvest. Objective 3. There is a potential for combination treatments of biocontrol organisms with conventional or nonthermal antimicrobial interventions. However, while research has been undertaken to establish the response of human pathogens (the “bad bugs”) to these interventions, we lack a clear understanding of the sensitivity of the biocontrol organisms (the “good bugs”). Particularly for combination treatments, this knowledge is essential. Bdellovibrio bacteriovorus 109J is a biocontrol strain which attacks and kills Escherichia coli. In this study, cultures of B. bacteriovorus 109J were inoculated into a liquid buffer substrate, or onto butter leaf lettuce. The samples were irradiated with gamma radiation at doses up to 1.0 kGy and the survivors enumerated. The non-pathogen E. coli ML 35 was similarly inoculated, treated, recovered, and counted. Based on the survivorship at each dose, a survivor curve was calculated, and a D10 value determined. (The D10 is the radiation dose required to reduce bacterial population by 1 log, or 90%.). E. coli ML35 had a D10 of 0.24 kGy when suspended in buffer, and 0.367 on butter leaf lettuce. These values are comparable to those obtained for pathogenic E. coli O157:H7 strains, which confirms that this surrogate is a valid choice for these predation studies. The biocontrol organism B. bacteriovorus 109J was considerably more sensitive to irradiation, showing D10 values of 0.08 kGy on both buffer and butter leaf lettuce. This suggests that sequential treatments of biocontrol-plus-irradiation will follow an optimized design of co-inoculation of the biocontrol organism first, with irradiation used as a terminal processing step. These results also confirm that conventionally efficacious irradiation doses will inactivate residual biocontrol populations in the distribution chain.
1. Inactivation of foodborne pathogens by predatory bacteria in co-cultures. Fresh produce can be contaminated by bacterial pathogens, putting consumers at risk. Certain “good” bacteria can block, inactivate, or even attack the pathogens, acting as a biocontrol system to improve food safety. In this study, ARS scientists at Wyndmoor, Pennsylvania applied a biocontrol bacterium (Bdellovibrio bacteriovorus 109J) against strains of the human pathogens Escherichia coli O157:H7 and Salmonella enterica in a liquid culture, hoping to better understand exactly how the “good” bacteria are stopping the “bad” bacteria. After 24 h, E. coli O157:H7 was reduced by 97 - 99.99%, Salmonella was reduced by 90%, and a non-pathogenic strain of E. coli was reduced by 99%. Examination of the bacteria with a scanning electron microscope confirmed that the “good” biocontrol bacteria were actively attacking the “bad” pathogenic bacteria. These results will be useful for identifying and evaluating other biocontrol organisms, and in optimizing the way they are applied and used on foods. Ultimately, these biocontrol organisms can help to inactivate human pathogens on foods, making food safer for consumers without chemical sanitizers.
2. Enhancement of microbial safety of butter lettuce by post-harvest treatment with Bdellovibrio bacteriovorus 109J. The human pathogens Escherichia coli O157:H7 and Salmonella on leafy greens continue to cause illnesses and put consumers at risk. Certain “good” bacteria can block, inactivate, or even attack the pathogens, acting as a biocontrol system to improve food safety. In this study, ARS researchers at Wyndmoor, Pennsylvania inoculated leafy greens with E. coli O157:H7, then treated the leaves with Bdellovibrio bacteriovorus, a “good” bacterium that seeks out and attacks this pathogen. After 24 h of storage at 26 C (a mildly abusive temperature), E. coli O157:H7 was reduced by 99%. Reductions after storage at 4 C (proper refrigeration) was 86%, and 53% after storage at 30 C (a very abusive temperature). Direct counts of the “good” bacteria showed that it grew less effectively at higher temperatures. These results help to determine the best ways to use biocontrol organisms to block or eliminate pathogens like E. coli O157:H7 on leafy greens. Reducing pathogens will benefit consumers by making food safer.
3. Reduction of bacterial contamination on post-harvest produce by predatory Halobacteriovorax. Fresh produce can be contaminated by bacterial pathogens, putting consumers at risk. Certain “good” bacteria can block, inactivate, or even attack the pathogens, acting as a biocontrol system to improve food safety. In this study, ARS scientists at Wyndmoor, Pennsylvania, inoculated grape tomatoes, lettuce, and clover sprouts with Salmonella, then applied Halobacteriovorax, a naturally-occurring biocontrol organism that is known to attack these kind of pathogens. On tomatoes, Halobacteriovorax reduced Salmonella by 93% after 1 h, and by 99% after 24 h of storage. Halobacteriavorax reduced Salmonella by 94-99% on clover sprouts and by 69-97% on butter lettuce, depending on how the biocontrol organism was applied. These results help to determine the best ways to use biocontrol organisms to block or eliminate pathogens like Salmonella on a variety of fresh produce commodities. Reducing pathogens will benefit consumers by making food safer.
4. Cold plasma inactivation of Salmonella on packaged salad mixes. Fresh produce, including fresh and fresh-cut, pre-packaged salad mixes, can be contaminated by bacterial pathogens, putting consumers at risk. New ways to inactivate these pathogens are needed. In this study, ARS scientists at Wyndmoor, Pennsylvania used cold plasma (a form of electrically energized gas) to inactivate Salmonella on mixed tomato-and-lettuce salads. Two different inoculation methods were evaluated to address cross-contamination of Salmonella from cherry tomatoes to lettuce and vice versa. In separate studies, either cherry tomatoes (55g) or romaine lettuce (10g) were inoculated with a Salmonella cocktail and placed into a commercial plastic container and treated with high-voltage cold plasma corona discharge for 3 min. When lettuce was the inoculated counterpart, the kill level of Salmonella was significantly greater on tomatoes (83% reduction) compared to lettuce (55% reduction). Salmonella was significantly reduced on mixed salad only when lettuce was the inoculated counterpart (49% reduction). It was determined that cold plasma can kill Salmonella in a pre-packaged, mixed salad, with efficacy dependent on the nature of contamination, direction of transfer, and on the surface topography of the contaminated commodity. This information will help design even better cold plasma treatments, and facilitate the development of this novel technology as a tool to make food safer, thereby protecting the public.
5. Gas composition influences cold plasma inactivation of E. coli. Cold plasma is a novel method of killing human pathogens on fresh and fresh-cut produce. Since cold plasma kills pathogens through the production of free reactive chemical species, ARS scientists at Wyndmoor, Pennsylvania sought to enhance the antimicrobial efficacy by augmenting the cold plasma with either water vapor (no carbon atoms), carbon dioxide (one carbon atom), or ethanol vapor (two carbon atoms). Blueberries were inoculated with a non-pathogenic bacteria, then exposed to the augmented cold plasma in a vacuum chamber for 0, 1, 5 or 10 min. With only air and electricity, cold plasma reduced the bacteria by 53%. Adding water vapor boosted that reduction to 99%. Carbon dioxide and ethanol vapor were not as effective. These results show that the chemical composition of the cold plasma can be “tweaked” to greatly enhance kill of bacteria on foods. This information will guide further development of cold plasma, facilitating the development of this novel technology into an efficient tool to make food safer, thereby protecting the public.
6. Irradiation inactivation of antibiotic resistant bacteria. Irradiation effectively reduces foodborne pathogens and spoilage microorganisms on fresh produce. However, limited research is available regarding its effect on antibiotic-resistant bacteria, or on the genes that convey that resistance. In collaboration with Virginia Tech, ARS scientists in Wyndmoor, Pennsylvania, inoculated lettuce leaves with a compost slurry containing multi-drug resistant Escherichia coli O157:H7 and Pseudomonas aeruginosa. The lettuce was washed with a mild sanitizer, irradiated, and stored for 14 days at 4 C. A 1.0kGy dose of irradiation reduced both pathogens by 99.9%, and no significant regrowth was seen during storage. No differences in abundance or distribution of the antibiotic resistance genes were observed between irradiated and control lettuce over time. These results show that irradiation effectively reduces antibiotic resistant bacteria on romaine lettuce. This information can be used to help design use protocols for irradiation as it is applied to fresh and fresh-cut produce, improving food safety and protecting the public.
7. Cold plasma inactivates Salmonella on Valencia oranges. Because of past outbreaks associated with orange juice, and the risk of cross-contamination during peeling and processing, there is a need for antimicrobial interventions which can effectively eliminate pathogens from fruit surfaces. ARS scientists at Wyndmoor, Pennsylvania, inoculated Valencia oranges with Salmonella Anatum on the peel, in the stem scar, or in the blossom end, then exposed the oranges to cold plasma, a novel type of sanitizer created with high-voltage electrical discharge. The oranges were treated for 1, 3, or 5 minutes, at distances of 0 cm or 7.5 cm from the cold plasma emitter head. All treatments significantly reduced Salmonella on oranges, on all surfaces tested. The 0 cm treatments yielded log reductions ranging from 89 – 99% (stem scar), 98 – 99.98% (blossom end), and 99.7 – 99.992% (peel), with longer treatment times yielding greater reductions. The 0 cm were uniformly more effective than the 7.5 cm treatments, which yielded log reductions ranging from 30 – 98% (stem scar), 91 – 98% (blossom end), and 58 – 94% (peel). These results suggest cold plasma could be an effective, waterless, chemical-free sanitation step for peel-on fruits such as Valencia oranges. Reducing human pathogens on whole fruits will improve food safety and protect the public.
8. Organic sanitizers effectively inactivate Salmonella on tomatoes. Fresh produce can be contaminated by bacterial pathogens, putting consumers at risk. ARS scientists at Wyndmoor, Pennsylvania, inoculated Salmonella enterica onto tomato stem scars, then applied an organic acid wash followed by a newly formulated bioactive coating, each for various times. One minute each of acid wash and bioactive coating reduced Salmonella by 99.8%. Extending the time beyond one minute reduced the pathogens to undetectable levels, at least a 99.999% reduction. On the untreated tomatoes, the pathogen persisted during 21 days of storage; on the treated tomatoes, in contrast, pathogen levels fell to undetectable. The acid wash plus bioactive coating also completely inactivated mold and yeast. These results indicate that the integrated treatment can provide a safe and effective means of improving the safety of tomatoes, thereby protecting the public.
9. Chlorine dioxide gas improves safety of freshly grown sprouts. Fresh sprouts can be contaminated by bacterial pathogens, putting consumers at risk. ARS scientists at Wyndmoor, Pennsylvania, inoculated soybean sprouts with Salmonella Typhimurium, then treated them with chlorine dioxide (a gaseous antimicrobial sanitizer) and biocontrol microbes (“good” bacteria that block and attack “bad” bacteria). Alone, the biocontrol microbes reduced Salmonella by 90%. Chlorine dioxide alone reduced Salmonella by 99.8 – 99.999%, depending on the concentration used and the duration of treatment. The two treatments applied together gave similar results. These results are being used to further optimize combination treatments, paving the way for more rapid, less intensive chemical treatments that still deliver the required reductions of pathogens and spoilage microorganisms. By reducing harmful pathogens on sprouts, the safety of this product is enhanced, protecting the consumer.
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