Progress 01/01/21 to 12/31/24
Outputs
Target Audience:Students who worked on this project presented results from each year of field work as posters at the main US food safety conference: the Annual Meeting of the International Association for Food Protection (see other products). This provided an opportunity for food safety professionals to discuss the results with them. Attendees included staff from major US leafy greens growers. We have also published 3 peer-reviewed papers on this work, and all open access to encourage dissemination. Changes/Problems:As described in earlier sections, we had to adapt our field-work protocols for maximum industry relevance, and also real world conditions. Concerning relevance, the first aim intended to use entirely inoculated field trials. While we did successfully complete an inoculated field-trial in Year 1, at pilot-scale, we realized that these types of experiments would not be the best approach to answering the most important sampling questions related to large-scale production. So, instead, from the second half of the first year, we pivoted to conducting large-scale sampling experiments in commercial fields to recover only naturally occurring microbes. This would ensure that whatever data we collected would represent real world contamination profiles, making the work directly relevant to real world sampling. While this was broadly a gain for the project, it did mean that we were at the mercy of real world conditions for conducting follow up work in fields with pathogen positive tests. Throughout the course of the project, our industry partners only identified one field with a pathogen positive test. We were able to mount a large-scale sampling response to that event, as intended, but unfortunately (for our project, not for the partner), none of those samples recovered a pathogen. This confirms that real-world contamination is rare, and low-level - great news for the produce industry - but also meant we were not able to directly validate the power of our new methods with pathogen positive tests. We did also take advantage of large-scale flooding in Salinas to additionaly test our soil sampling methods to recover bacteria in response to this real world event. What opportunities for training and professional development has the project provided?This project has provided projects for 2 PhD students and 1 MS student. Students were trained to do Biosafety Level 1 and 2 microbiology work, to keep digital laboratory notebooks, to analyze samples collected by field work with industry collaborators. Concerning professional development, the lead PhD student on the project had extensive opportunities to coordinate fieldwork and sample collection in commercial leafy greens fields. This involved frequent e-mails and update meetings with our industry collaborator, arranging travel logistics, designing protocols, adapting protocols to day-of-study developments, and then presenting the results of these analyses back to the collaborators. All students on the project also assisted the PI in developing the posters for presentation at the yearly project directors meeting. Each student on the project has also published 1 lead-author peer-reviewed paper on their main contributions to this work. How have the results been disseminated to communities of interest?This project has provided research training to the 3 graduate students working on the project. In addition, the 3 students who worked on this project presented results from the field work every year from 2021-2024 at the main US food safety conference: the Annual Meeting of the International Association for Food Protection (see other products). This provided an opportunity for food safety professionals to discuss the results with them. Attendees included staff from major US leafy greens growers. We have also published 3 peer-reviewed papers on this work. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Develop aggregative sampling for preharvest food safety testing. We developed an aggregative soil sampling for preharvest product testing. In year 1, aggregative swabbing in commercial fields at pilot testing scale showed comparable, if not better, quality indicator results than grab samples. Then, follow up aggregative swabbing in commercial fields at large scale showed promise from quality indicator results including reduced variance compared to grab sampling.. Critically, swabs show reduced variance in APC and Coliform counts. No generic E. coli were detected in any samples. As year 1 research demonstrated that manual aggregative sampling using dry cloths performs no worse than composite produce sampling in the recovery of quality indicators, but underperforms recovering safety indicators. Year 2 aimed to improve the aggregative sampling technique by testing pressure and hydration as factors affecting the recovery of quality and safety indicators to justify future, more expensive, work on pathogen recovery. In the main experimental work for the project, we found pre-hydrated cloth sampling performs better than dry cloths as an aggregative sampling technique. Hydrated cloth also performs no worse than composite tissue sampling in recovering quality and safety indicator bacteria from commercial romaine lettuce fields. As year 2 concluded that pre-hydrated cloths attached to the manual sampling device was considered the best technique, additional field work in Year 3 found that aggregative sampling on romaine lettuce using pre-hydrated cloth performs similarly as composite romaine tissue samples recovering quality and safety indicator bacteria from commercial romaine lettuce fields. Sampling was performed during the shoulder season in commercial fields planted with romaine lettuce in Salinas, California. The improved technique recovered similar APC and similar total coliforms as romaine samples. Only aggregative samples recovered generic E. coli. This year's work suggested that improved methods using pre-hydrated cloths in aggregative sampling improves the recovery of APC and coliforms and performs no worse in the recovery of generic E. coli when compared to current composite produce grab sampling. It justified future work on pathogen detection. We also developed an aggregative boot cover sampling method. In year 1, the team started considering that aggregative boot cover sampling may be a more representative, practical, and powerful method for preharvest produce soil testing than grab sampling because boot covers aggregate soil from larger areas. We tested if boot cover sampling results reflect quality and safety indicator organisms and community diversity of grab sampling. Overall, aggregative boot cover sampling was similar to both grab methods for recovering quality and safety indicator organisms and representative microbiomes. This justified future work testing aggregative soil sampling for foodborne pathogen detection. In year 2, the team considered that hydration could be a factor which influenced the performance of aggregative sampling methods. The team compared hydrated bootie and hydrated drag sampling methods with composite grab soil sampling. This data suggested that aggregative testing performs similar to, at least not worse, than composite grab for indicator organism testing in soil from common produce fields. Objective 2.Validate the power of aggregative sampling in commercial fields with previous pathogen positive test results. In year 2, we did perform sampling in response to one event where a commercial field had a presumptive positive Salmonella test result; Unfortunately, none of the subsequent testing in this field confirmed as Salmonella positive, neither in the testing done by our lab or by our industry partner as part of their independent follow-up activities. Still, our work is briefly described here to show good faith effort to complete this objective as written. Response to the presumptive positive involved receiving notification of the presumptive positive test results on June/27/2022, spending 1 day adapting our pre-planned experimental protocols to the realities of the field and staffing situation, and then remotely coordinating 2 interns (1 for produce sampling, 1 for soil sampling) working with our industry partner to gather ship back to our lab a total of 105 samples. These included 25 aggregative swab samples, 26 tissue samples, 18 aggregative soil samples, 18 drag swabs, and 18 bootie swabs. All samples were shipped as tissue, soil, or swabs, for dilution and lab testing at Illinois. We recovered Salmonella-typical colonies on XLD from a total of 54 samples across all types. Unfortunately, all but one of these did not show typical Salmonella results based on invA PCR (typical meaning single band of 284 bp size). One thing that we learned in this process is that the classic invA PCR for Salmonella confirmation is not ideal when one expects a background of other Gram - organisms. Many of our samples did amplify multiple PCR bands, consistent with the initial paper reporting this assay. While we were not able to validate our aggregative sampling method in year 2, this exercise allowed us to further strengthen our working relationships with that group, get additional feedback on our sampling methods, and continue to stress-test our protocols for collecting samples, shipping them to Illinois, and processing them in the lab. In year 3, we did not have the opportunity to respond to any commercial field pathogen positive test results. But, we were able to positively respond to the large-scale flooding in the Salinas valley by working to test our aggregative soil sampling methods in previously flooded fields in year 3. We continued our extension of the scope of the project to aggregative soil sampling in the same commercial fields, using single-use booties of similar materials to produce samples. While composite grabs did occasionally recover generic E. coli, it might be due to grabs being designed to pick up soil 2 inches deeper than the surface while boot covers only swabbed the surface. Also, high total coliform levels in boot covers might affect the enumeration of generic E. coli. This justified future work for soil testing in response to flooding or other events.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Gathman, R. J., J. Quintanilla Portillo, G. A. Reyes, G. Sullivan, and M. J. Stasiewicz. 2024. Aggregative swab sampling method for romaine lettuce show similar quality and safety indicators and microbial profiles compared to composite produce leaf samples in a pilot study. Foods. 13(19)2080. https://doi.org/10.3390/foods13193080.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Quintanilla Portillo, J,. R.J. Gathman, J. Wu, E. Wilhelmsen, M.J. Stasiewicz. 2025. Aggregative sampling performs similar to composite produce samples to recover quality and safety indicators throughout romaine lettuce production. Journal of Food Protection. 88(5)100481. https://doi.org/10.1016/j.jfp.2025.100481.
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:The 2 students who worked on this project presented interim results from the third year of field work as posters at the main US food safety conference: the 2023 Annual Meeting of the International Association for Food Protection (see other products). This provided an opportunity for food safety professionals to discuss the results with them. Attendees included staff from major US leafy greens growers. Changes/Problems:One challenge was the lack of opportunity to respond to a pathogen positive field test this year. But, we were able to positively respond to the large-scale flooding in the Salinas valley by working to test our aggregative soil sampling methods in previously flooded fields. What opportunities for training and professional development has the project provided?This project period has provided projects for 2 PhD students. Students continue to train to do Biosafety Level 1 and 2 microbiology work, to keep digital laboratory notebooks, to analyze samples collected by field work with industry collaborators. Concerning professional development, the lead PhD student on the project had extensive opportunities to coordinate field-work and sample collection in commercial leafy greens fields. This involved frequent e-mails and update meetings with our industry collaborator, arranging travel logistics, designing protocols, adapting protocols to day-of-study developments, and then presenting the results of these analyses back to the collaborators. All students on the project also assisted the PI in developing the poster for presentation at the yearly project directors meeting. How have the results been disseminated to communities of interest?This project has provided research training to the 2 graduate students working on the project. In addition, the 2 students who worked on this project in this period presented interim results from the third year of field work as posters at the main US food safety conference: the 2023 Annual Meeting of the International Association for Food Protection (see other products). This provided an opportunity for food safety professionals to discuss the results with them. Attendees included staff from major US leafy greens growers. What do you plan to do during the next reporting period to accomplish the goals?During this NCE we will write up the remaining manuscripts for the project and take them through the peer review and publication process.
Impacts
What was accomplished under these goals?
Produce Tissues Focused This year's work built upon previous work which demonstrated that manual aggregative sampling using prehydrated cloths performs no worse than composite produce sampling in the recovery of quality indicators but underperforms recovering safety indicators. Work in Y2 concluded that pre-hydrated cloths attached to the manual sampling device was considered the best technique. Henceforth, all aggregative samples collected in Y3 were collected following this technique. At the end of Y3 work, we found that aggregative sampling on romaine lettuce using pre-hydrated cloth performs similarly as composite romaine tissue samples recovering quality and safety indicator bacteria from commercial romaine lettuce fields. Sampling was performed during the shoulder season in commercial fields planted with romaine lettuce in Salinas, California. Sampling consisted of collecting: (1) Aggregative samples using pre-hydrated cloths collected by using a manual sampling device (n=20), and (2) Composite romaine lettuce samples (n=20), compositing 60 produce grabs for a total of 375g composite per sample, as comparison data. Samples were tested for quality metrics - aerobic plate counts (APC) - and safety indicators - Coliform and generic Escherichia coli counts. Means of the counts were analyzed in ANOVA and differences in generic E. coli detection using Chi-square test. Aggregative samples showed no significant different APC 6.1 ± 0.55 Log(CFU/g sample) than produce samples that recovered 5.7 ± 0.7 Log(CFU/g) (p=0.07). Coliform recoveries from aggregative samples were 5.8 ± 0.78 Log(CFU/g of swab), while produce samples were 5.4 ± 0.87 Log(CFU/g). These recoveries were not significantly different (p=0.15). Generic E. coli was only found in 2/20 aggregative samples, and 0/20 in produce samples, this was insignificantly different (p=0.49). The improved technique recovered similar APC and similar total coliforms as romaine samples. Only aggregative samples recovered generic E. coli. Soil Focused This year's work suggests that improved methods using pre-hydrated cloths in aggregative sampling improves the recovery of APC and coliforms and performs no worse in the recovery of generic E. coli when compared to current composite produce grab sampling. It justifies future work on pathogen detection. We continued our extension of the scope of the project to aggregative soil sampling in the same commercial fields, using single-use booties of similar materials to produce samples. Two sampling trials were performed in February and March 2023 in response to a large-scale flooding in Salinas, California. For each sampling, 20 hydrated boot covers were used to collect soil samples in pairing with 20 composite grabs. Twenty-five grams of well-mixed soil sample, or the whole bootie, was processed to enumerate generic E. coli and total coliforms. Wilcoxon tests were conducted to compare statistically significant differences in means of plate counts. In February, there was no generic E. coli detected in the soil collected using boot covers, while 4 composite grabs had countable generic E. coli ranging from 1.0 to 1.2 log(CFU/g). Total coliforms recovery on boot covers and composite grabs were 6.1 ± 0.4 and 3.5 ± 1.0 log(CFU/g), respectively. A significant difference (P<0.001) of 2.6 log(CFU/g) was observed. In March, there was no generic E. coli detected in the soil collected using boot covers, while 1 composite grabs had 0.7 log(CFU/g) generic E. coli recovered. Total coliforms recovery on boot covers and composite grabs were 7.2 ± 0.4 and 3.6 ± 0.9 log(CFU/g), respectively. A significant difference (P<0.001) of 3.6 log(CFU/g) was observed. Across two sampling trials, soil collected using boot covers recovered 2-3 log(CFU/g) more total coliforms than composite grabs, but 5 out of 40 composite grabs did generate countable generic E. coli results while boot covers did not. When testing in response to this flooding event, boot covers showed promising ability to recover total coliforms. While composite grabs did occasionally recover generic E. coli, it might be due to grabs being designed to pick up soil 2 inches deeper than the surface while boot covers only swabbed the surface. Also, high total coliform levels in boot covers might affect the enumeration of generic E. coli. This justified future work for soil testing in response to flooding or other events. We did not have the opportunity to respond to any commercial field pathogen positive test results this year. But, we were able to positively respond to the large-scale flooding in the Salinas valley by working to test our aggregative soil sampling methods in previously flooded fields. Here we continued to develop the methods using quality indicators to demonstrate their utility.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Wu, J., R. J. Gathman, J. Quintanilla Portillo, C. Gaulke, M. Kim, and M. J. Stasiewicz. 2023. Aggregative soil sampling using boot covers compared to soil grabs from commercial romaine fields shows similar indicator organism and microbial community recoveries. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2023.100177:100177.
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:The threestudents who worked on this project presented interim results from the second year of field work as posters at the 2022 Annual Meeting of the International Association for Food Protection. This provided an opportunity for food safety professionals to discuss the results with them. Attendees included staff from major US leafy greens growers. Changes/Problems:One positive change was the easy addition of aggregative soil sampling to the field work. A suite of problems involved our testing in response to a presumptive positive field not confirming as pathogen positive (either by our work, or our partner's) and our planned response being overly burdensome for the on-site interns who helped us collect samples. Given the rapid nature of the response that is required to respond to a presumptive positive, there is nothing we can do about the risk of not confirming positive. We cannot wait for industry partner confirmation. We will also consider assaying the collected opportunistic samples for quality and safety indicators to get additional information out of their results. Based on the overly burdensome field work we continue to refine our sampling plan, have improved our staging of materials with our partner so there is less work to do be done to prepare for the tests, and our graduate students will work hard to travel at short notice to assist the interns with the sample collection. What opportunities for training and professional development has the project provided?This project period has provided projects for twoPhD students and oneMS student. Students have learned to perform Biosafety Level 1 and 2 microbiology work to keep digital laboratory notebooks, and to analyze samples collected by field work with industry collaborators. Concerning professional development, the lead PhD student on the project had extensive opportunities to coordinate field-work and sample collection in commercial leafy greens fields. This involved frequent e-mails and update meetings with our industry collaborator, arranging travel logistics, designing protocols, adapting protocols to day-of-study developments, and then presenting the results of these analyses back to the collaborators. All students on the project also assisted the PI in developing the presentation and extended abstract for presentation at the yearly project directors meeting. How have the results been disseminated to communities of interest?This project has provided research training to the threegraduate students working on the project. In addition, the threestudents who worked on this project presented interim results from the second year of field work as posters at the 2022 Annual Meeting of the International Association for Food Protection. This provided an opportunity for food safety professionals to discuss the results with them. Attendees included staff from major US leafy greens growers. What do you plan to do during the next reporting period to accomplish the goals?We will continue to be ready to respond to additional presumptive positive events for validation sampling in Year 3. This time with further improved protocols. We are ready to respond to up to twoevents. For example, we are currently discussing sampling in response to recent flooding events in central CA as a potential opportunity to recover pathogens in soil pre-planting, and then would follow up with sampling in fields subsequently planted for romaine.
Impacts
What was accomplished under these goals?
In the second year of this project (Jan 22 - Dec 22) we made progress developing the aggregative sampling technique using testing for quality indicators in commercial fields. We continue to plan validation testing for pathogen testing in Year 3. Objective 1. Develop aggregative sampling for preharvest food safety testing. Year 1 research demonstrated that manual aggregative sampling using dry cloths performs no worse than composite produce sampling in the recovery of quality indicators, but underperforms recovering safety indicators. This year aimed to improve the aggregative sampling technique by testing pressure and hydration as factors affecting the recovery of quality and safety indicators to justify future, more expensive, work on pathogen recovery. In the main experimental work for the project, we found pre-hydrated cloth sampling performs better than dry cloths as an aggregative sampling technique. Hydrated cloth also performs no worse than composite tissue sampling in recovering quality and safety indicator bacteria from commercial romaine lettuce fields Sampling was performed in commercial fields planted with romaine lettuce in Salinas, California. Sampling consisted of collecting: (1) Aggregative samples: either as (i) Dry or pre-hydrated cloths (testing for hydration) and (ii) collected by hand or using manual sampling device (testing for pressure) (n=31), and (2) Composite produce samples (n=21) as comparison data using sixtyproduce grabs for a total of 375g composite per sample. Samples were tested for quality metrics - aerobic plate counts (APC) - and safety indicators - Coliform and generic Escherichia coli counts. Means of the counts were analyzed in ANOVA and differences in generic E. coli detection using Chi-square test. Pre-hydrated cloths samples showed significantly higher mean APC 6.67±0.94 log(CFU)/g (p<0.001), significantly higher mean Coliform counts (6.48±0.94 log(CFU)/g) (p<0.05) compared to dry cloths that showed APC of 5.08±0.3 log(CFU)/g and Coliform counts of 2.03±1.29 log(CFU)/g. Pressure showed no significant effect (p=0.13) on bacterial recovery. Pre-hydrated cloths attached to the manual sampling device was considered the best technique moving forward. Comparing the best aggregative technique to the reference composite samples, aggregative sampling showed significantly higher (p<0.001) mean APC (6.67±0.94 log(CFU/g)), compared to reference composite samples that showed 5.28±0.36 log(CFU/g). For total coliform counts, the improved aggregative sampling showed significantly higher (p<0.001) total coliform counts (6.48±0.94 log(CFU/g)), compared to reference composite samples that showed 4.91±0.8 log(CFU/g). Generic E. coli was found in 9/13 produce samples, while 6/15 improved aggregative swabs, this was insignificantly different (p=0.24). This work to date suggests that using prehydrated cloths in aggregative sampling improves the recovery of quality and safety indicators when compared to current tissue grab sampling. It justifies future work on pathogen detection for tissue aggregative samples. As a relatively low-cost, but highly-informative extension of the scope of the project, we were able to have the students working on this project also evaluate aggregative soil sampling in the same commercial fields, using single-use booties of similar materials to produce samples, taken on the same sampling trips. Here we found that aggregative soil sampling shows similar indicator bacteria recovery ability compared to grab soil sampling from commercial romaine fields To compare aggregative booties and drag sampling methods with composite grab sampling, sampling was performed in six beds of commercial romaine lettuce fields in Salinas, California. Three sampling methods were used to take soil samples: booties (walking in the fields with wearing sterilized, perforated, and hydrated bootie covers), drags (dragging a piece of hydrated gauze in the fields), and grabs (grabbing composite soil samples by using shovels). Twenty samples were taken for each of them. Booties covering approx. 333 ft, drags covering approx. 333 ft, in pair with 25 grams of well-mixed grab samples, were processed to enumerate generic E. coli, total coliforms, and aerobic plate counts. ANOVA were used to compare means. There was no generic E. coli detected in any sample. Total coliforms recovery on booties, drags, and grabs were 2.50 ± 0.89, 2.91 ± 1.02, and 1.94 ± 0.48 log CFU/g, respectively. Grabs showed a lower (p = 0.0013) mean than drags, whereas booties showed no significant difference from either grabs (p = 0.088) or drags (p = 0.27). Aerobic plate counts recovery on booties, drags, and grabs were 7.03 ± 0.30, 7.16 ± 0.11, 6.98 ± 0.11 log CFU/g, respectively. The means of booties (p = 0.024) and grabs (p = <0.001) were lower than drags. There is no significant difference (p = 0.44) between grabs and booties. These data suggest that aggregative testing performs similar to, at least not worse, than composite grab for indicator organism testing in soil from common produce fields. This justifies future work on pathogen detection for soil bootie sample. Objective 2.Validate the power of aggregative sampling in commercial fields with previous pathogen positive test results. We did perform sampling in response to one event where a commercial field had a presumptive positive Salmonella test result; Unfortunately, none of the subsequent testing in this field confirmed as Salmonella positive, neither in the testing done by our lab or by our industry partner as part of their independent follow-up activities. Still, our work is briefly described here to show how we are better prepared for testing next year. Response to the presumptive positive involved receiving notification of the presumptive positive test results on June/27/2022, spending oneday adapting our pre-planned experimental protocols to the realities of the field and staffing situation, and then remotely coordinating twointerns (onefor produce sampling, onefor soil sampling) working with our industry partner to gather ship back to our lab a total of 105 samples. These included 25 aggregative swab samples, 26 tissue samples, 18 aggregative soil samples, 18 drag swabs, and 18 bootie swabs. All samples were shipped as tissue, soil, or swabs, for dilution and lab testing at Illinois. We recovered Salmonella-typical colonies on XLD from a total of 54 samples across all types. Unfortunately, all but one of these did not show typical Salmonella results based on invA PCR (typical meaning single band of 284 bp size). One thing that we learned in this process is that the classic invA PCR for Salmonella confirmation is not ideal when one expects a background of other Gram - organisms. Many of our samples did amplify multiple PCR bands, consistent with the initial paper reporting this assay. While we were not able to validate our aggregative sampling method in Year 2, this exercise allowed us to further strengthen our working relationships with that group, get additional feedback on our sampling methods, and continue to stress-test our protocols for collecting samples, shipping them to Illinois, and processing them in the lab.
Publications
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Progress 01/01/21 to 12/31/21
Outputs
Target Audience:The lead PhD student on the project presented interim results from the first year of field work as a poster session at the main U.S. food safety conference: The 2021 Annual Meeting of the International Association for Food Protection (see Other Products). Changes/Problems:Illinois inoculated field trials were planned for Year One, to provide additional data comparing the power to recover rE. coli from leafy greens. Due to COVID-related planting delays and weather, these trials were not successful. They may be re-attempted in Year Two or Year Threeif needed, such as if the data in commercial fields are insufficient to prove the technique. Still, we feel our pivot to conducting the pilot scale experiments in commercial fields with our industry partner was, ultimately, more useful. What opportunities for training and professional development has the project provided?This project period has provided projects for twoPhD students and oneMS student. Students have learned to perform Biosafety Level 1 and 2 microbiology work to keep digital laboratory notebooks, to analyze samples collected by field work with industry collaborators. Concerning professional development, the lead PhD student on the project had extensive opportunities to coordinate field-work and sample collection in commercial leafy greens fields. This involved frequent e-mails and update meetings with our industry collaborator, arranging travel logistics, designing protocols, adapting protocols to day-of-study developments, and then presenting the results of these analyses back to the collaborators. All students on the project also assisted the PI in developing the presentation and extended abstract for presentation at the yearly project directors meeting. How have the results been disseminated to communities of interest?The lead PhD student on the project presented interim results from the first year of field work as a poster session at the main U.S. food safety conference: The 2021 Annual Meeting of the International Association for Food Protection (see Other Products). What do you plan to do during the next reporting period to accomplish the goals?Future work in Year Twowill demonstrate and validate the use of these techniques for detecting foodborne pathogens. Specifically, we intend to iterate with our industry partner the exact method to apply the aggregative swab to the leafy green fields to make the process easier, more reproducible, and more powerful. Then, in Fall 2023, we hope to return to the commercial fields to conduct at least one sampling in a field with a previous pathogen positive test.
Impacts
What was accomplished under these goals?
In the first year of this project (January 21 - December 21) we made progress developing the aggregative sampling technique using both inoculated field trials and initial testing for quality indicators in commercial fields. We continue to plan validation testing for pathogen testing in Years 2-3. Objective 1. Aggregative swabbing in commercial fields at pilot testing scale shows comparable, if not better, quality indicator results than grab samples. 26 total aggregative swabs and tissue composites were collected from commercial Romaine fields in Salinas, California with some areas of the field being inoculated with rifampicin-resistant Escherichia. coli. Aerobic plate counts (APC), coliform concentrations, and presence of generic E. coli from grabs and swabs were determined using 3M Petrifilms. Further identification of the bacteria, their abundances, and the microbial diversity of the samples were determined from microbial community profiling using PacBio full length 16S DNA sequencing including store-bought tissue samples as an outgroup. APC and coliform counts revealed that aggregative swabs could pick up similar, if not higher, concentrations of bacteria compared to grabs. Recovery of APC were 9.17±0.43 and 9.21±0.42 log(CFU/g) for grabs and swabs, respectively (p=0.38 by Mann-Whitney-Wilcoxon test). Coliform recoveries were 3.52±1.17 and 4.01±1.46 log(CFU/g) for grabs and swabs, respectively (p=0.36 by non-paired t-test). Presence of generic E. coli showed that swabs could detect E. coli more often than grab samples with 8/12 (67%) of swabs detecting E. coli compared to 3/14 (38%) of grabs. Microbial profiling revealed that swab and grab samples have similar alpha diversities and the most prevalent bacterial taxa in similar abundances. Inoculation did not have a significant effect in any tests. Aggregative swabbing in commercial fields at large scale shows promise from quality indicator results including reduced variance compared to grab sampling Aggregative swabs and N60 tissue composites were collected from commercial Romaine fields in Salinas, California. Two trials collected: (i) Aggregative Swab Samples (at least two per bed), (ii) Composite Tissue Samples (at least two samples of 60 tissue grabs for a total of 150g per sample), and (ii) High Resolution Composite Tissue Samples (12 composite samples of 25 g per sample) each from three areas: Center, Road Edge, Field Border. Swabs showed higher APC 5.58±0.54 log(CFU/g), compared to composite tissue 4.48±0.27 log(CFU/g), across all locations. Coliform detection was higher in tissue samples, where composite tissue recovered 2.45±1.29 log(CFU/g), compared to 1.23±0.36 log(CFU/g) recovered by swabs. Critically, swabs show reduced variance in APC and Coliform counts. No generic E. coli were detected in any samples. Objective 2. The specific work for Objective 2 was originally planned as a Year Two or Year Threetask. But in Year Onewe did lay the foundation for validation in commercial fields. Specifically, we did have a sampling trip to our industry partner to pilot test our methods in commercial fields while analyzing for quality indicators. This allowed us to strengthen our working relationships with that group, get feedback on our sampling methods, and stress-test our protocols for collecting samples, shipping them to Illinois, and processing them in the lab.
Publications
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