Progress 07/01/15 to 06/30/20
Outputs
Target Audience:The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Changes/Problems:Final report - all objectives were accomplished as originally proposed. What opportunities for training and professional development has the project provided?This project has provided one-on-one training of a doctoral student to gain knowledge and proficiency in basic laboratory skills needed to pursue research in the field of molecular biology including qRT-PCR and RNA-seq. How have the results been disseminated to communities of interest?The doctoral student presented his findings at the American Society for Microbiology (ASM) conference and twice at annual BUILD Dairy meetings sponsored by the Western Dairy Center (housed at Utah State University) and by Dairy West. The BUILD Dairy program is a network of professors, researchers, students and dairy food industry companies and organizations in the western region of the United States. Its goal is to build university and industry linkages through learning and discovery. The work will be reported in the student's dissertation and a manuscript is being prepared. What do you plan to do during the next reporting period to accomplish the goals?N/A - Final report
Impacts
What was accomplished under these goals?
All objectives were accomplished as proposed. Objectives 1 and 2 examined the impact of habituation to lactic acid and acetic acid on expression of transcription factors and genes related to acid resistance, bile resistance and virulence in L. monocytogenes strains N1-227 and R2-499 by qRT-PCR. Objective 3 examined in vivo virulence of those same strains of L. monocytogenes using the Galleria mellonella infection model. Organic acid habituation significantly induced expression of the acid and bile stress response genes in both strains, while expression of virulence genes was strain dependent. Habituation in organic acid increased virulence of both strains as evidenced by decreased LT50 (median lethal time) of G. mellonella larvae. In addition to the original objectives, the transcriptional profile of L. monocytogenes strains N1-227 and R2-499 in the presence or absence of organic acid was additionally followed using RNA-seq. As compared to L.monoytogenes grown in standard media, more differentially expressed genes (DEGs) were identified when cells were habituated with organic acid compared to cells habituated with inorganic acid. RNA-seq data were strongly correlated with the gene expression values obtained for those genes shared in the parallel qRT-PCR analysis (R2 = 0.74 for strain N1-227 and R2 = 0.79 for strain R2-499). Other DEGs included genes involved in cell motility, membrane transport, carbohydrate and amino acid metabolism and quorum sensing. Interestingly, the DEGs involved in flagella- mediated cell motility pathways were exclusively down-regulated in both of the tested strains, and this is consistent with enhanced virulence as loss of flagella and their antigenic determinants are key to L. mono avoiding the host defense systems. The majority of the DEGs involved in amino sugar and nucleotide sugar metabolism were down-regulated under organic acid habituation for both strains, suggesting that changes in cell wall architecture is part of the L. monocytogenes response to organic acid exposure. Results from this study suggest that exposure to acetic or lactic acid can induce increased virulence in at least some L. monocytogenes strains and provide a comprehensive view of the mechanisms used by L. monocytogenes to adapt to organic acid exposure, which may provide new leads for research and help to develop better strategies to prevent L. monocytogenes contamination in food.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Luo, X., Lu, X., Cornforth, D. P., Carpenter, C. E., & Zhu, I. (2019, November). Effect of oxygen concentration in modified atmosphere packaging on color changes of the M. longissimus thoraces et lumborum from dark cutting beef carcasses. Meat Science, 161, 107999.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Changes/Problems:No changes/problems were encountered that require a deviation from the proposed objectives or schedule. What opportunities for training and professional development has the project provided?This project has provided one-on-one training of a doctoral student to gain knowledge and proficiency in basic laboratory skills needed to pursue research in the field of molecular biology. How have the results been disseminated to communities of interest?The doctoral student presented his findings at the American Society for Microbiology (ASM) conference and at the annual Build Dairy meeting sponsored by the Western Dairy Center (housed at Utah State University) and by Dairy West. The Build Dairy program is a network of professors, researchers, students and dairy food industry companies and organizations in the western region of the United States. Its goal is to build university and industry linkages through learning and discovery. What do you plan to do during the next reporting period to accomplish the goals?Objective 1,2 and 3: Data will undergo final analysis and a manuscript will be prepared and submitted for publication in a peer-reviewed journal. The doctoral student will complete writing his dissertation and defend during the spring 2020 semester.
Impacts
What was accomplished under these goals?
All experiments have been completed as proposed for the project, and the data has undergone preliminary analysis relative to the project objectives. Prior research suggested that the use of organic acids in the food industry unintentionally enhances pathogenicity of L. monocytogenes. This project employed qRT-PCR to evaluate the impact of habituation to lactic acid and acetic acid on expression of genes in Listeria monocytogenes strains N1-227 and R2-499. The genes followed included those related to general stress response (sigB), acid resistance, bile resistance (bile and bsh) and cellular infection (prfA, inlA, inlB, hly, plcA, plcB, uhpT and actA). Both strains are documented human pathogens, and their in situ virulence was evaluated using the Galleria mellonella larvae infection model. Organic acid habituation significantly induced expression of stress response genes in both strains, while expression of virulence genes was strain dependent. Habituation in acid increased virulence of both strains as evidenced by decreased LT50 of larvae.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Presentations
Li, M. (Presenter & Author), Broadbent, J. R., Carpenter, C. E., Microbe 2019, "Effect of acid exposure on virulence of Listeria mpnocytogenes," American Society for Microbiology, San Francisco. (June 21, 2019)
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Changes/Problems:No changes/problems were encountered that require a deviation from the proposed objectives or schedule. What opportunities for training and professional development has the project provided?This project has provided one-on-one training of a doctoral student to gain knowledge and proficiency in basic laboratory skills needed to pursue research in the field of molecular biology. How have the results been disseminated to communities of interest?The doctoral student presented his findings at the annual Build Dairy meeting sponsored by the Western Dairy Center (housed at Utah State University) and by Dairy West. The Build Dairy program is a network of professors, researchers, students and dairy food industry companies and organizations in the western region of the United States. It's goal is to build university and industry linkages through learning and discovery. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 and 2: Perform RNA-seq experiments on L. monocytogenes strain R2-499 and strain N1-227 after 20 minutes adaptation to acid. Objective 1,2 and 3: Data analysis, dissertation writing and manuscript submission.
Impacts
What was accomplished under these goals?
Objective 1 & 2: The quantitative PCR (qPCR) experiments for strain N1-227 and strain R2-499 were completed. Differences were observed in expression of the targeted genes in L. monocytogenes strain R2-499 and strain N1-227 after 20, 40- and 60-minutes acid-habituation. The 20-minute time will be used for the planned RNA-seq experiments. Objective 3: Determining in situ virulence using the wax worm model were completed with L. monocytogenes strain R2-499 and strain N1-227. Results were consistent with the hypothesis that adaptation of the bacteria to lactic acid or acetic acid leads increased virulence.
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Changes/Problems:No changes/problems were encountered that require a deviation from the proposed objectives or schedule. What opportunities for training and professional development has the project provided?This project has provided one-on-one training of a doctoral student to gain knowledge and proficiency in basic laboratory skills needed to pursue research in the field of molecular biology. How have the results been disseminated to communities of interest?The doctoral student presented his findings at the annual Western Dairy Center's Build Dairy meeting. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 and 2: Finish qPCR for strain R2-499 after 60 minutes acid-habituation and begin qPCR for strain N1-227. We will also perform RNAseq experiments with each strain at one time point. Objective 3: Finish the second trial using strain R2-499 and start the experiments using strain N1-277.
Impacts
What was accomplished under these goals?
Objective 1 & 2: The methodologies for RNA isolation and cDNA preparation for objectives 1 and 2 were validated and optimized and this allowed us to move into the quantitative PCR (qPCR) experiments. Differences were observed in expression of the targeted genes in L. monocytogenes strain R2-499 after 20 and 40 minutes acid-habituation, with the greater changes occurring after 20 minutes acid-habituation. Objective 3: Standard operating procedures for determining in situ virulence using the wax worm model were established and optimized. One trial with L. monocytogenes strain R2-499 was completed. Results of that single trial were consistent with the hypothesis that adaptation of the bacteria to lactic acid or acetic acid leads to increased virulence.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Target Audience The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Changes/Problems:Changes/Problems No changes/problems were encountered that require a deviation from the proposed objectives or schedule. What opportunities for training and professional development has the project provided?Opportunities This project has provided one-on-one training of a doctoral student to gain knowledge and proficiency in basic laboratory skills needed to pursue research in the field of molecular biology. How have the results been disseminated to communities of interest?Dissemination Nothing has yet been disseminated from this project. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work Objective 1 and 2: Isolate RNA and prepare cDNA from control and acid-habituated cells. Begin Q-PCR for the targeted genes. Objective 3: Establish standard operating procedures for determining in situ virulence using the wax worm model.
Impacts
What was accomplished under these goals?
Accomplishments Objective 2: Primers were designed and validated for the virulence genes targeted in objective 2 including prfA, inlA, inlB, hyl, plcA, plcB, actAm uhpT, lisR, and lisK. All primers rendered a single PCR product with no background and the products migrated at the predicted size. Sequences of the PCR products we did obtain showed 95 - 100% match with the putative gene sequences. Primers were designed and validated for the housekeeping genes to be used in the proposed studies including rpoB, gap, and 16s rRNA.
Publications
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Progress 07/01/15 to 09/30/15
Outputs
Target Audience:Target Audience The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Changes/Problems:Changes/Problems No changes/problems were encountered that require a deviation from the proposed objectives or schedule. What opportunities for training and professional development has the project provided?Opportunities This project has provided one-on-one training of a doctoral student to gain knowledge and proficiency in basic laboratory skills needed to pursue research in the field of molecular biology. How have the results been disseminated to communities of interest?Dissemination Nothing has yet been disseminated from this project. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work Objective 1: Begin Q-PCR once primer development is completed for genes targeted in both objectives 1&2. Objective 2: Primers will be developed and validated for the genes targeted in objective 2. Begin Q-PCR once primer development is completed for genes targeted in both objectives 1&2.
Impacts
What was accomplished under these goals?
Accomplishments Objective 1: Primers were designed for seven of the targeted genes identified in objective 1 including bile, bsh, sigB, gadD1,2,3 and arcA. All primers rendered a single PCR product with no background and the products migrated at the predicted size except for gadD1 gene. The basis for this observation is unknown, but one possibility is that gadD1 is absent from our test strains. Because gadD2 and gadD3 will allow us to follow the effect of our treatments on glutamate decarboxylase expression, we will not pursue gadD1 further. Sequences of the PCR products we did obtain showed 90 - 100% match with the putative gene sequences.
Publications
- Type:
Other
Status:
Published
Year Published:
2015
Citation:
Refereed Journal Articles
Zhang, Y., Carpenter, C. E., Broadbent, J. R., Luo, X. (2015). Influence of habituation to inorganic and organic acid conditions on the cytoplasmic membrane composition of Listeria monocytogenes. Food Contol, 55, 49-53.
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