Progress 12/15/15 to 12/14/18
Outputs
Target Audience:Academic and governmental researchers and food safety professionals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided scientific training to 3 graduate students and 1 post-doctoral research scientist. How have the results been disseminated to communities of interest?Results of this project have been and will continue to be disseminated to the scientific community through peer-reviewed publications and presentations at professional conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
It is known that foodborne bacterial pathogens such as Salmonella enterica and Listeria monocytogenes can contaminate fresh-cut fruits, which has led to a number of multi-state foodborne disease outbreaks in the U.S. However, the conditions and underlying mechanisms that allow bacterial transfer and survival in fresh-cut fruits are much less known. This is extremely important when trying to validate decontamination regimes, particularly when contaminated fresh-cut fruits are used as ingredients into a food process or a finished product. In this project, conventional microbiological methods and high-throughput transcriptome sequencing tools were used to study the transfer and survival mechanisms of S. enterica and L. monocytogenes in common fresh-cut fruits. Findings from this project provide important scientific data on specific bacterial determinants, food attributes and processing conditions that have a collective impact the transfer and survival of bacterial pathogens in fresh-cut produce. In the Objective #1 of this project, we investigated two major physical mechanisms of bacterial transfer and internalization into fresh-cut fruits, i.e. via cutting knives and disposable gloves. Specifically, we assessed the transfer rates of S. enterica Newport (strain 36796) on fresh-cut tomatoes and cantaloupes via cutting knives at different inoculation levels (i.e. low 103-4 MPN/ml or high 107-8 CFU/ml). Our results showed that cutting knives transferred ~20 MPN/cm2 to >105CFU/cm2 S. enterica cells from an inoculated fruit to an un-inoculated one. Cucumbers and apples displayed higher bacterial transfers than tomatoes and cantaloupes and bacterial transfer was most significant between the initial two cuts of an un-inoculated fruit (p < 0.05). We used fresh-cut cantaloupe as a food model to study glove-mediated transfer of pathogens. A four-strain Listeria monocytogenes cocktail (F8027 serovar 4b, F8369 serovar 1/2a, F8255 serovar 1/2b, ATCC 51779 serovar 1/2c) and two different inoculation levels (high 106-7 CFU/ml or low 104-5 CFU/ml) were used for inoculation. Fruit-to-fruit and surface-to-fruit transfer via gloves were investigated, and 3 different types of single-use gloves (nitrile, polyvinyl chloride, polyethylene) were tested with different contact pressures (0.05, 0.18, 0.37 psi) and time (2, 5,10 s). Predictive modeling and validation analysis suggested that glove-mediated L. monocytogenes transfer can last for more than 85 touches of cantaloupe; however, glove materials, contact time, and contact pressures did not result in significant difference in L. monocytogenes transfer on fresh-cut cantaloupe. We evaluated the survival rates of S. enterica in fresh-cut produce under commercial storage conditions. The fresh-cut fruits were individually inoculated with ~107 CFU/ml of S. enterica Newport strain 36796 or Typhimurium strain LT2 19585, and stored in the forms of slices or blended juice at 4oC for up to 7 days. S. enterica population remained unchanged in tomatoes and cantaloupes but declined approximately 0.5 log in apples and cucumber after 7 days. A cocktail of four L. monocytogenes strains (F8027, F8255, F8369, ATCC51779) was used to inoculate in filter-sterilized apple, tomato, cucumber, celery, honeydew, cantaloupe, corn, spinach juice and stored at 4for 7 days. Results showed that bacterial population displayed a direct correlation with the pH of fresh produce juice (R2=0.86049 at 105 CFU/ml and R2=0.69452 at 109 CFU/ml). In the Objective #2 of this project, we used the high-throughput transcriptome sequencing method to investigate molecular mechanisms of bacterial survival in fresh-cut fruits. Specifically, RNA-seq was used to identify differentially-expressed genes in S. enterica inoculated on fresh-cut tomatoes and cantaloupes after 1h storage at 4oC, in comparison to that in 0.1% buffered peptone water as a control. A total of 325 and 358 genes inSalmonellaNewport (FDR <0.05,log2 fold_change ≥1.5) were found to be differentially expressed in fresh-cut tomatoes and cantaloupes, respectively. A total of 57 and 114 genes were commonly up- and down-regulated in both fresh-cut tomatoes and cantaloupes. The major functional categories of commonly up-regulated genes included carbohydrate utilization (n=6), amino acid utilization (n=8), transporter (n=8) and virulence factor (n=8). The major functional categories of commonly down-regulated genes included citrate cycle (TCA) (n=6), pentose and glucuronate interconversions (n=5), arginine and proline metabolism (n=5), and phosphotransferase system (PTS) (n=7). Gene set enrichment analysis (GSEA) identified 7 biological processes that were commonly overrepresented (p <0.05, FDR <0.1), including amino acid metabolism, carboxylic acid metabolism, multi-organism process (virulence), ribosome biogenesis, ion transport, carbohydrate derivative metabolism and carbon utilization. Global transcriptomic responses of L. monocytogenes during its residence on fresh-cut produce were also evaluated by using RNA-seq. Fresh-cut apple, cantaloupe, cucumber, celery and tomato samples were inoculated with 109 CFU of L. monocytogenes strain F8027 that was previously isolated from celery. Samples were stored at 4°C for 48 hours. A reference sample was prepared by subjecting the same strain in phosphate buffered saline (PBS) under the same storage condition. Total RNA of L. monocytogenes was extracted, purified, reverse transcribed to cDNA, and sequenced on an Illumina MiSeq platform. A total of 110 and 158 genes were significantly up- and down-regulated in all the five fresh-cut produce samples (FDR <0.05,log2 fold_change ≥3.0). Major functional categories of commonly up-regulated genes included membrane transport (n=8), transcriptional regulation (n=10), and amino acid metabolism (n=12). Major functional categories of commonly down-regulated genes included phosphotransferase system (PTS) (n=12) and carbohydrate metabolism (n=38). The transcriptome in response to apple was distinct from those in other produce samples by featuring the largest numbers of genes that were uniquely up- or down-regulated in this fruit. GSEA analysis indicated that genes (p <0.05, FDR <0.25) related to amino acid metabolism were overrepresented, whereas genes related to carbon metabolism and phosphotransferase system for transporting monosaccharide or polysaccharide were underrepresented. Collectively, our study demonstrated that the fresh-cut process can effectively facilitate bacterial pathogens to transfer from contaminated food contact surface such as cutting knives and gloves to the finished product. Considering the low infectious dose of these pathogens in susceptible populations, in which a few hundred pathogen cells can lead to an infection, such high transfer rates through cutting knives and disposable gloves are particularly alarming. In addition, our transcriptomic analyses revealed novel molecular mechanisms used by S. enterica and L. monocytogenes outbreak strains to adapt to and persist in fresh-cut fruits under storage conditions relevant to common industry and retail practices. These new findings fill knowledge gaps of the underlying mechanisms of cross-contamination and persistence of these foodborne bacterial pathogens in fresh-cut fruits, which may contribute to developing more effective mitigation and intervention strategies to ensure the safety of fresh-cut produce.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Qi Y, Li S, He Y, Mann DA, Zhang W, Deng X. 2018. Transcriptomic analysis of Listeria monocytogenes adaptation on fresh-cut produce. The 2018 International Association for Food Protection Annual Meeting, Salt Lake City, Utah.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
He Y, Chen R, Zhang S, Qi Y, Deng X, Zhang W. 2018. Salmonella transfer and survival on fresh-cut fruits. The 2018 International Association for Food Protection Annual Meeting, Salt Lake City, Utah.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Qi Y, He Y, Zhang W, Deng X. 2019. Glove-mediated transfer of Listeria monocytogenes to fresh-cut cantaloupe under laboratory conditions. Food Microbiol
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
Qi Y, Li S, He Y, Mann DA, Zhang W, Deng X. 2019. Transcriptomic analysis of Listeria monocytogenes adaptation on fresh-cut produce. Appl Environ Microbiol
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
He Y, Chen R, Zhang S, Qi Y, Deng X, Zhang W. 2019. Salmonella transfer and survival on fresh-cut fruits. Food Microbiol
- Type:
Journal Articles
Status:
Submitted
Year Published:
2019
Citation:
He Y, Chen R, Zhang S, Qi Y, Deng X, Zhang W. 2019. Transcriptomic analysis of Salmonella enterica survival and adaptation on fresh-cut fruits. Appl Environ Microbiol
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Progress 12/15/16 to 12/14/17
Outputs
Target Audience:Academic and governmental researchers and food safety professionals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Research training was provided to one graduate student and one postdoctoral scientist in this project. How have the results been disseminated to communities of interest?Results of this project will be disseminated to the scientific community through peer-reviewed publications and presentations at professional conferences. What do you plan to do during the next reporting period to accomplish the goals?We plan to evaluate the impact of lower initial inoculum on the survival and transfer of S. enterica and L. monocytogenes in various fresh-cut fruits. Validation of RNA-seq results will be carried out by qRT-PCR.
Impacts
What was accomplished under these goals?
In the second year of this project, we evaluated the survival rates of S. enterica in fresh-cut produce under commercial storage conditions. The fresh-cut fruits were inoculated with ~107 CFU/ml of S. enterica Newport strain 36796 or Typhimurium strain LT2 19585, and stored in the forms of slices or blended juice at 4oC for up to 7 days. S. enterica population remained unchanged in tomatoes and cantaloupes but declined approximately 0.5 log in apples and cucumber after 7 days. RNA-seq was used to identify differentially-expressed genes in S. enterica strain 36796 on fresh-cut tomatoes and cantaloupes after 1h storage at 4oC, in comparison to that in 0.1% buffered peptone water as a control. A total of 95 and 106 genes showed significant up- and down-regulation (fold change ≥4.0, p <0.05, FDR <0.05), respectively, in both tomatoes and cantaloupes. The up-regulated genes included genes in biosynthesis of antibiotics (7 genes), cationic antimicrobial peptide resistance (4 genes), carbon metabolism (6 genes), ABC transporters (14 genes), quorum sensing (4 genes), amino acid biosynthesis (5 genes) and metabolism (7 genes). The down-regulated genes included those involved in citrate cycle (TCA) (6 genes), ascorbate and aldarate metabolism (6 genes), arginine and proline metabolism (6 genes), biosynthesis of antibiotics (10 genes), carbon metabolism (9 genes), fructose and mannose metabolism (4 genes) and phosphotransferase system (PTS) (7 genes). Gene set enrichment analysis (GSEA) identified 12 commonly up-regulated pathways (p <0.05, FDR <0.1) including amino acids biosynthesis and metabolism, methane metabolism, pantothenate and CoA biosynthesis, aminoacyl-tRNA biosynthesis and antibiotics biosynthesis. Two commonly down-regulated pathways (p <0.05, FDR <0.1) were related to citrate cycle and butanoate metabolism. Global transcriptomic responses of L. monocytogenes during its residence on fresh-cut produce were also evaluated by using RNA-seq. Fresh-cut apple, cantaloupe, cucumber, celery and tomato samples were inoculated with 109 CFU of L. monocytogenes strain F8027 that was previously isolated from celery. Samples were stored at 4°C for 48 hours. A reference sample was prepared by subjecting the same strain in phosphate buffered saline (PBS) under the same storage condition. We identified a total of 118 and 78 genes were commonly up- and down-regulated in all five fresh-cut produce samples. Major functional categories of commonly up-regulated genes included membrane transport (n=18), transcriptional regulation (n=10), and amino acid metabolism (n=11) such as that of arginine, lysine, cysteine, methionine and histidine. Major functional categories of commonly down-regulated genes were phosphotransferase system (PTS) (n=12) and carbohydrate metabolism (n=33) including that of fructose, mannose, starch, sucrose, pentose phosphate, pentose and glucuronate. The transcriptome in apple appeared to be different from other produce samples by featuring the largest numbers of genes uniquely up- or down regulated. We visitedlocal stores of two major retail chains in Griffin, GA regarding in-store preparation of fresh cut produce. Use of gloves is a common practice and a potential route of food borne pathogen transfer onto fresh cut produce. We investigated the transfer of L. monocytogenes on fresh-cut cantaloupes via single-use gloves of three different materials including nitrile, polyvinyl, and polyethylene. Fresh-cut cantaloupes (11-13g /slice, ~3.5×4.5×0.75 cm) were inoculated with 106-7 CFU/ml of a cocktail of four strains (F8027, serotype 4b, from celery; F8255, serotype 1/2b, from peach; F8369, serotype 1/2a, from corn; and ATCC51779, serotype 1/2c, from a dairy product). An un-inoculated sample was first hand pressed with a glove for 10 s, followed by similar press of an inoculated sample and a series of consecutive uninoculated samples at 10°C. The samples were stomached for 1 min and plated onto MOX for enumeration or stored for 2 days for enrichment analysis. L. monocytogenes was detectable by culture enrichment after 41 times of transfers by all three types of gloves. Logarithmic model was developed to quantitatively assess glove-mediated transfer of L. monocytogenes between fresh-cut cantaloupe samples. We estimated that with the initial inoculum at 105-6 CFU/piece, transfer can last for ~100 consecutive presses by all three types of cloves. No significant difference was predicted among transfer rates of three types of gloves (p=0.47). These results suggest that all the commonly used gloves for retail preparation of fresh-cut fruits can lead to sustained transfer of L. monocytogenes cells during the fresh-cutting.
Publications
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2018
Citation:
Qi Y, Li S, He Y, Mann DA, Zhang W, Deng X. 2018. Transcriptomic analysis of Listeria monocytogenes adaptation on fresh-cut produce. The 2018 International Association for Food Protection Annual Meeting, Salt Lake City, Arizona.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2018
Citation:
He Y, Chen R, Zhang S, Deng X, Zhang W. 2018. Salmonella transfer and survival on fresh-cut fruits. The 2018 International Association for Food Protection Annual Meeting, Salt Lake City, Arizona.
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Progress 12/15/15 to 12/14/16
Outputs
Target Audience:Academic and governmental researchers and food safety professionals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Research training was provided toone graduate student and one postdoctoral scientist in this project. How have the results been disseminated to communities of interest?Results of this project will be disseminated to the scientific community through peer-reviewed publications and presentations at professional conferences. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we willtest additional bacterial outbreak strains and serotypesduring infiltration into fresh-cut fruits. We will also focus on exploring the in-depth molecular mechanisms of bacterial stress response and adapation in fresh-cut produce.
Impacts
What was accomplished under these goals?
In the first year of this project, we evaluated 2 Salmonella enterica strains representing serovars Newport and Typhimurium for the bacterial transfer rates during fresh-cutting of 4 different types of fresh produce including cantaloupes, apples, tomatoes and cucumbers. Produce samples were inoculated with bacteria on the surface and incubated at 25°C and 90% humidity for 0 d and 1 d. A single un-inoculated sample was cut (negative control) with a sterilized knife, followed by cutting an inoculated sample with the same knife. The artificially contaminated knife was then used to cut 4 additional un-inoculated samples in a sequence. Fresh-cut samples were homogenized for 2 minutes in a sterile blender and serially diluted for plate counting on XLD agar plates. An average of 0.42 to 1.81 log reductions of Salmonella was found after the first cut of an un-inoculated produce with a contaminated knife. Cucumbers showed the highest bacterial transfer; whereas tomatoes showed the lowest bacterial transfer among the 4 types of produce tested. Between samples analyzed after 0 d and 1 d of incubation, we detected significant differences (p<0.005) among the transfer rates of S. Newport. Specifically, bacterial transfer rates to the first un-inoculated samples were 0.15%±0.02%, 0.17%±0.05%, 0.20%±0.07%, 0.08%±0.03% at 0 d; and 0.35%±0.07%, 0.38%±0.09%, 0.31%±0.14%, 0.17%±0.06% at 1 d, in cantaloupes, apples, tomatoes and cucumbers, respectively. In contrast, S. Typhimurium did not display any significant differences between 0 d and 1 d, suggesting that length of storage of contaminated produce and Salmonella serotype both played roles in determining the bacterial transfer dynamics in fresh-cut produce. The fate of Listeria monocytogenes in fresh-cut produce under refrigerated storage was also evaluated. A cocktail of four strains (F8027, serotype 4b, from celery; F8255, serotype 1/2b, from peach; F8369, serotype 1/2a, from corn; and ATCC51779, serotype 1/2c, from a dairy product) was inoculated in filter-sterilized juice of tomato, cucumber, celery, honeydew, spinach, cantaloupe and corn, and stored at 4°C for 7 days. We found that the changes of bacterial population displayed a correlation with the pH of the produce (R2=0.50 at medium inoculum level of 105 CFU/ml; R2=0.60 at high inoculum level of 109 CFU/ml). Significant (p<0.05) reduction of L. monocytogenes was observed in apple juice at pH 3.61 (2.36 to 6.4 log). Minor population fluctuations (< 0.5 log) of the pathogen were observed in tomato juice at pH 4.36 and cucumber juice at pH 5.9, in contrast to more than 4 log increase in corn juice at pH 7.41 and cantaloupe juice at pH 7.43. To characterize transcriptomic profiles of L. monocytogenes cells transferred or internalized into fresh or fresh-cut produce, we developed a method for collecting listerial total RNA from fresh-cut produce. Specifically, L. monocytogenes cells (109 CFU) were inoculated onto a nitrocellulose membrane (0.45 µm) and the membrane was sandwiched by two halves of fresh-cut apple, cucumber and cantaloupe. This configuration allows listeria cells to come in direct contact with fresh-cut produce flesh while facilitating the separation and harvesting of the bacterial cells. Using this method, we were able to collect high quality and quantity of listerial total RNA after up to 7 d of storage under 4°C, which will allow us to carry out RNA-Seq studies in the next phase of this project.
Publications
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