Progress 07/01/09 to 06/30/14
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:
Nothing Reported
What opportunities for training and professional development has the project provided? Opportunities The accomplishments for involved a visiting scientist from Shandong Agricultural University, China. Training for the visiting scientist largely involved one-on-one work with the project's principal investigators. The accomplishments involved a visiting scientist from Shandong Agricultural University (SAU), China. Training for the visiting scientist largely involved one-on-one work with the project's principal investigators. This scientist was simultaneously a grad student at SAU, and she has since graduated with her doctoral degree. She has most recently has accepted a faculty position and intends to continue this line of research owing to its international interest for food safety. We have obtained extramural support for a doctoral graduate student to continue this line of research. How have the results been disseminated to communities of interest? Dissemination The results from this year's accomplishments were published in the refereed scientific journal as included in the publications list. We opted to publish in Meat Science, a journal with readership targeted to those responsible for food safety in the meat industry. Our hope is to maximize the direct impact of our research by putting it in front of the meat industry that widely employs organic acids as washes to decontaminate meat surfaces and the addition of organic acid salts salts to prevent bacterial growth in processed meats. Results from this project have been published in three refereed journal articles as previously reported. A fourth article is currently being prepared for submission. What do you plan to do during the next reporting period to accomplish the goals? Plan of Work The objective of our project is to characterize the relevant physiology of L. monocytogenes to growth in the presence of different organic acid anions. We have completed studies that followed the inducible resistance to acid and bile in response to habituation in various organic acids. The results of those studies were described in the Accomplishments section of our AD421 annual report, and reports of those studies will be submitted for publication in 2013. We will implement parallel studies during 2014 to determine the effects of organic acids on membrane composition because it is a putative effector of resistance to acid and bile. We hope to submit a proposal for a new UAES project to look at how habituation to organic acids may induce virulence of Listeria monocytogenes.
Impacts
What was accomplished under these goals?
Accomplishments 1) Major activities completed We evaluated the intrinsic and inducible resistance of four human pathogenic strains of Listeria monocytogenes to acid and bile, factors associated with virulence. Cells were grown in media at pH 7.4, or in media at pH 6.0 containing 0 (HCl control) or 4.75 mM of different organic acids, harvested at stationary or mid log phase, and challenged for 1 h in in acid or bile. Stationary phase cells were intrinsically more resistant to either challenge than log phase cells, and large differences between strains were evident among the latter. Compared to the HCl control, habituation to log phase with organic acids induced significant (p < 0.05) and meaningful (≥1 log) increases in acid resistance of three of four strains tested, and in bile resistance of two strains 2) Specific objectives met The activities completed objective 1 to characterize the relevant physiology of Listeria monocytogenes in response to growth in the presence of various organic acids. 3) Significant results achieved The results demonstrate that habitual exposure of L. monocytogenes to organic acid anions under only mildly acid conditions can induce acid and bile resistance, factors known to enhance virulence in this important food-borne pathogen. 4) Key impacts or other accomplishments realized. Organic acids are widely employed as antimicrobials by the food industry, and this research supports the possibility that such widespread use of organic acids may inadvertently increase virulence in L. monocytogenes and should be considered in the design of food safety programs and protocols to ensure public health and safety. 1) Major activities completed In addition to what has been previously reported for this project, we completed parallel studies to determine the effects of organic acids on membrane composition of Listeria monocytogenes. We are currently performing final analysis of the data and preparing a paper for submission. Our results indicate that organic acids do not effect membrane composition in a manner that is consistent with their capacity to induce resistance to acid and bile. This suggests that the organic acids are influencing expression of genes that impart resistance to acid and bile independent of changes in membrane composition. 2) Specific objectives met Objective 1 was completed to characterize the relevant physiology of L. monocytogenes in response to growth in the presence of various organic acids. Objective 2 was not completed during the duration of this project because the needed external funding was not realized. However, the results from completing objective 1 have provided the the information needed to refine and target objective 2 as a separate proposal for outside funding. 3) Significant results achieved The results from this project demonstrate that habitual exposure of L. monocytogenes to organic acid anions under only mildly acid conditions can induce resistance to acid and bile. Those changes will promote survival of Listeria as it travels through the gastrointestinal tract, and are also tied to expression of virulence factors related to colonization of the intestine, septicemia, and neuropathic disease. 4) Key impacts or other accomplishments realized. Organic acids are widely employed as antimicrobials by the food industry, and this research supports the possibility that such widespread use of organic acids may inadvertently increase virulence in L. monocytogenes and should be considered in the design of food safety programs and protocols to ensure public health and safety.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zhang, Y., Carpenter, C. E., Broadbent, J. R. (2014). Habituation to organic acid anions induces resistance to acid and bile in Listeria monocytogenes. Meat Science, 96(3), 1152 - 1157
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Progress 01/01/13 to 09/30/13
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:
Nothing Reported
What opportunities for training and professional development has the project provided? The accomplishments for involved a visiting scientist from Shandong Agricultural University, China. Training for the visiting scientist largely involved one-on-one work with the project's principal investigators. How have the results been disseminated to communities of interest? The results from this year's accomplishments were published in the refereed scientific journal as included in the publications list. We opted to publish in Meat Science, a journal with readership targeted to those responsible for food safety in the meat industry. Our hope is to maximize the direct impact of our research by putting it in front of the meat industry that widely employs organic acids as washes to decontaminate meat surfaces and the addition of organic acid salts to prevent bacterial growth in processed meats. What do you plan to do during the next reporting period to accomplish the goals? The objective of our project is to characterize the relevant physiology of L. monocytogenes to growth in the presence of different organic acid anions. We have completed studies that followed the inducible resistance to acid and bile in response to habituation in various organic acids. The results of those studies were described in the Accomplishments section of our AD421 annual report, and reports of those studies will be submitted for publication in 2013. We will implement parallel studies during 2014 to determine the effects of organic acids on membrane composition because it is a putative effector of resistance to acid and bile.
Impacts
What was accomplished under these goals?
1) Major activities completed We evaluated the intrinsic and inducible resistance of four human pathogenic strains of Listeria monocytogenes to acid and bile, factors associated with virulence. Cells were grown in media at pH 7.4, or in media at pH 6.0 containing 0 (HCl control) or 4.75 mM of different organic acids, harvested at stationary or mid log phase, and challenged for 1 h in in acid or bile. Stationary phase cells were intrinsically more resistant to either challenge than log phase cells, and large differences between strains were evident among the latter. Compared to the HCl control, habituation to log phase with organic acids induced significant (p < 0.05) and meaningful (≥1 log) increases in acid resistance of three of four strains tested, and in bile resistance of two strains 2) Specific objectives met The activities completed objective 1 to characterize the relevant physiology of Listeria monocytogenes in response to growth in the presence of various organic acids. 3) Significant results achieved The results demonstrate that habitual exposure of L. monocytogenes to organic acid anions under only mildly acid conditions can induce acid and bile resistance, factors known to enhance virulence in this important food-borne pathogen. 4) Key impacts or other accomplishments realized. Organic acids are widely employed as antimicrobials by the food industry, and this research supports the possibility that such widespread use of organic acids may inadvertently increase virulence in L. monocytogenes and should be considered in the design of food safety programs and protocols to ensure public health and safety. Opportunities The accomplishments involved a visiting scientist from Shandong Agricultural University, China. Training for the visiting scientist largely involved one-on-one work with the project's principal investigators. Dissemination The proposed studies have been completed, and we will publish our results in 2014 as described in the plan of work for 2014.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Resistance to acid and bile is associated with virulence of Listreria monocytogenes, and we investigated the ability of human pathogenic strains of L. monocytogenes to resist challenges from acid and from bile. Two serotype 1/2a strains (R2-499 and C1-056) and two serotype 4b strains (N1-227 and N3-013) of L. monocytogenes were obtained from the International Life Sciences Institute (ILSI) North American Database at Cornell University. Cells were grown in tryptic soy broth (TSB) at 37C and harvested 4 hours after inoculation during log phase growth. The harvested cells were suspended in peptone and inoculated into TSB (without dextrose) at pH = 3.0, 2.5, 2.0, or 1.5 or into TSB containing 0.1, 0.2, 0.3, 0.4, and 0.5% bile salts (w/v) (1:1 sodium cholate:sodium deoxycholate). The challenges were maintained at 37C for one hour and samples plated on tryptic soy agar to determine numbers of viable cells. The pH and bile concentration at which surviving cells were not detectable varied among the strains: R2-499, pH = 2.0 and 0.3% bile; C1-056, pH = 2.5 and 0.4% bile; N1-227, pH = 2.0 and 0.3% bile; N3-013, pH = 1.5 and 0.4% bile. The challenges at pH = 3 or at bile concentration of 0.2% resulted in 2 to 5 log decreases in cell numbers across all strains, thus exhibiting substantial, but not complete, lethality. Those conditions will be employed in future studies to determine whether organic acids affect changes in acid and bile tolerance that contribute to virulence in L. monocytogenes. PARTICIPANTS: Yimin Zhang, Visiting Scholar, Shandong Agricultural University, China TARGET AUDIENCES: The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Information will be disseminated to those audiences via publication of results in relevant peer reviewed journals and by presentations at affiliated scientific and industry venues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Resistance to acid and bile is associated with ability of food-born pathogens to survive transport though the stomach and upper intestine thereby allowing the pathogen to colonize the intestine and cause illness. The results from our research to date add understanding about the extent to which various human pathogenic strains of Listeria monocytogenes differ in their capacity to resist acid and bile. The next step in our research will be to study how various organic acids modify acid and bile resistance, possibly leading to increased virulence in L. monocytognes. The meat industry widely employs organic acids as antimicrobials, and our research represents the first attempt to establish the extent of bacterial adaptation that might be introduced as a consequence of the widespread addition of organic acid salts to ready-to-eat meats and enhanced fresh meats. In the most practical sense, the information generated from the proposed research will facilitate selection of organic acid for addition to processed meats based on both their efficacy to inhibit pathogen growth and their likelihood to inadvertently enhance virulence. The information learned from our research will be used to design the most effective total food safety programs to ensure public health and safety.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: A mathematical model of intracellular anion accumulation into bacteria was developed based on fundamental principles of chemistry. This model predicts that for any conditions where the pH outside the cell (pHout) is less than the pH inside the cell (pHin) (which would be the case in mildly acidic foods), acid anions will accumulate within the cell ([A-]in). Intracellular accumulation of anions was described mathematically as follows: i) Protonated organic acid freely transverses the cell membrane to establish equilibrium: [HA]out = [HA]in (Equation 1) ii) Once inside the cell, protonated acid dissociates according to the Henderson-Hasselbach equation: [A-]in = antilog(pHin - pKa) x [HA]in (Equation 2) iii) Substituting equation 1 into equation 2: [A-]in = antilog(pHin - pKa) x [HA]out (Equation 3) iv) Protonated acid outside the cell is also defined by the Henderson-Hasselbach equation: [A-]in = antilog(pHin - pKa) x ([A-]out/antilog(pHout - pKa)) (Equation 4) v) Applying algebra: [A-]in = [A-]out x antilog(pHin - pHout) (Equation 5) vi) Protons and anions are released equally from acid dissociation: [A-]in = [H+]released = [A-]out x antilog(pHin - pHout) (Equation 6) The model predicts that the driving forces for cellular accumulation of anion and cellular acidification are two fold: external anion concentration and the internal:external pH gradient. The model also predicts that while the extent of dissociation of a given acid within and outside the cell depends on its pKa (equations 2 and 4), the accumulation of anion and protons is independent of pKa (equation 6). This novel principle will be employed in our studies to elucidate the antimicrobial effects of organic acids. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Information will be disseminated to those audiences via publication of results in relevant peer reviewed journals and by presentations at affiliated scientific and industry venues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Knowledge gained from these studies has direct application for controlling important food-borne pathogens thereby leading to a safer food supply. Food poisoning costs the United States $152 billion dollars annually, $505 for every American. Food safety is the highest rated non-economic issue for United States consumers, and they indicate a willingness to pay up to 30 percent more for products with a safety certification label. That translates into an economic impact of over $170 billion based on yearly supermarket food sales alone.
Publications
- UTAO+267 Carpenter, C. E., Smith, J. V., & Broadbent, J. R., (2011). Efficacy of washing meat surfaces with 2% levulinic, acetic, or lactic acid for pathogen decontamination and residual growth inhibition: Meat Science, 88: 256-260. (Published).
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Meat samples were spray washed with water (control), 2% lactic acid, 2% acetic acid, or 2% levulinic acid at 130F. After drying, the meat samples were inoculated with pathogenic bacteria and vacuum packaged. The experiment was replicated in four model systems including: Esherichia coli O157:H7 inoculated onto pieces of beef carcass tissue; Salmonella spp. inoculated onto pork carcass tissue; Salmonella spp. inoculated onto chicken carcass tissue, and; Listeria monocytogenes inoculated onto slices of ready-to-eat turkey roll. Turkey slices were stored at 4 deg. C and L. monocytogenes counts determined at 0, 2, 4, 8, 12, and 16 weeks. All other samples were stored at 8 deg. C and pathogen counts determined at 0, 2, 4, 6, and 8 weeks. In the decontamination studies, acid washes lowered recoverable numbers of pathogens by only 0.6 to 1 log/sq cm as compared to controls that did not receive any wash treatment, and only lactic acid lowered the number of pathogens recovered as compared to the water wash. Washing with levulinic acid at higher temperatures (68.3 or 76.7 deg. C) did not result in additional decontamination of Escherichia coli O157:H7 from beef plate. Only acetic acid prevented growth of E. coli O157:H7 on beef plate and Listeria monocytogenes on turkey roll, and this treatment also imparted a residual effect that reduced numbers of Salmonella on chicken skin to below recoverable levels. In conclusion, washing with levulinic acid displayed no advantages for reducing pathogens on meat surfaces as compared to the current industry standards of lactic and acetic acids. Only lactic acid washes added small decontaminating capacity as compared to water, and only acetic acid displayed residual activity to prevent growth of pathogens. This highlights the need for the meat industry to reconsider the effectiveness of organic acid washes relative to their combined ability to decontaminate meat tissues and subsequently inhibit growth of pathogens, and especially the extent to which washing with organic acids contributes to a total food safety program. PARTICIPANTS: American Meat Institute Foundation TARGET AUDIENCES: The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Information will be disseminated to those audiences via publication of results in relevant peer reviewed journals and by presentations at affiliated scientific and industry venues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Knowledge gained from these studies has direct application for controlling important food-borne pathogens thereby leading to a safer food supply. Food poisoning costs the United States $152 billion dollars annually, $505 for every American. Food safety is the highest rated non-economic issue for United States consumers, and they indicate a willingness to pay up to 30 percent more for products with a safety certification label. That translates into an economic impact of over $170 billion based on yearly supermarket food sales alone.
Publications
- Ward, R. E., & Carpenter, C. E., 2010. Food Analysis (4th edition), In Neilsen, S.S. (Ed.), Ch. 12: Traditional methods for mineral analysis. Springer, New York.
- Carpenter, C. E., & Ward, R. E., 2010. Food Analysis Laboratory Manual (2nd edition), In Neilsen, S.S. (Ed.), Ch. 15: Enzyme analysis to determine glucose content. Springer, New York.
- Carpenter, C. E., 2010. Food Analysis Laboratory Manual (2nd edition), In Nielsen, S.S. (Ed.), Ch. 4: Determination of fat content. Springer, New York.
- Carpenter, C. E., & Ward, R. E., 2010. Food Analysis Laboratory Manual (2nd edition), In Nielsen, S.S. (Ed.), Ch. 9: Iron determination in meat using Ferrozine assay. Springer, New York.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We established a model system to study the effects of organic acids on growth of bacteria. Specifically, we investigated the effects of levulinate, acetate, D- or L-lactate, and HCl (organic anion-free control acid) on growth of Listeria innocua in a laboratory medium (Brain-Heart Infusion; BHI) at pH 5.8 and 6.5, values typical of many RTE foods such as processed meats. Organic acid concentrations were adjusted considering the pKa of each acid to achieve external anion concentrations of 3 mM or 15 mM at pH 5.8 or 6.5, respectively. Those conditions were designed to attain a targeted internal anion concentration of 475 mM. The treatment media were inoculated from a stock culture of L. innocua, and bacterial growth at 37C monitored by measuring absorbance at 600 nm. The various organic anions generally inhibited growth more than HCl, but specific effects were clearly dependent on factors such as isomer, pKa, and external acidity. For example, both lactate isomers similarly inhibited growth at external pH = 5.8 as compared to HCL, but D-lactate was much more inhibitory to growth at external pH = 6.5 than was L-lactate. This suggests that the bacterial cell is better able to control the intracellular levels of L-lactate depending on external pH, and this could have important food safety implications for selecting lactic acid additives. As another example, acetic acid inhibited growth at external pH = 6.5 more than HCl, but acetic acid inhibited growth at external pH = 5.8 less than HCl. This observation suggested that acetic acid provides a protective effect from external acidity in the more acid environment. We speculate that anion exchange of acetate for expelled glutamate contributes to the recovery of alkaline internal pH, because the pKa for acetate is greater than that of glutamate, and that this phenomenon provides an increasingly beneficial effect to the cell as external pH becomes more acidic. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The food industry and government regulatory agencies are the primary target audience, especially those segments dealing with food safety issues. Information will be disseminated to those audiences via publication of results in relevant peer reviewed journals and by presentations at affiliated scientific and industry venues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The model system we established advocates an approach for directing internal anion levels by controlling external concentration of anions and pH. This approach overcomes critical flaws in contemporary experimental design that invariably target concentration of either protonated acid or total acid in the growth media, thereby leaving anion concentration to vary depending on the pKa of the acids involved. Failure to control external concentration of anions has undoubtedly confounded results, and it has likely led to misleading conclusions regarding the antimicrobial action of organic acids. Our model system effectively separates the general effects of acidity from the specific effects of organic acids and their anions, thereby offering additional opportunity to study the mechanisms by which organic acids inhibit bacterial growth. Knowledge gained from such studies will have application in the control of important food-borne pathogens such as Listeria monocytogenes and may also facilitate efforts to promote the survival in foods or beverages of desirable probiotic bacteria.
Publications
- Carpenter, C.E., Broadbent, J.R. 2009. External concentration of organic acid anions and pH: Key independent variables for studying how organic acids inhibit growth of bacteria in mildly acidic foods. J. Food Sci. 74:R12-15.
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