Source: UNIV OF WISCONSIN submitted to
CONTAMINATION OF FRUITS, NUTS, AND VEGETABLES BY FILAMENTOUS SALMONELLA; PERSISTENCE AND VIRULENCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0220799
Grant No.
2010-65201-20563
Project No.
WIS01472
Proposal No.
2009-04336
Multistate No.
(N/A)
Program Code
93231
Project Start Date
Feb 1, 2010
Project End Date
Jul 31, 2013
Grant Year
2010
Project Director
Kaspar, C. W.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Bacteriology
Non Technical Summary
Recent outbreaks associated with nuts, peanut butter, toasted oats, and vegetables in the U. S. have increased concerns of Salmonella contamination, survival, and infectivity in or on products with reduced water activity. These products may become contaminated with Salmonella at one or more stages of pre-harvest, harvest, processing, or storage. The organism does not normally grow in low-moisture environments but some serovars can survive for prolonged periods. Salmonella encounters a myriad of stressful conditions in the food production chain that can trigger specific and general stress-protection systems that render cells more tolerant to stress and promote persistence in the environment or food. One interesting response to stress is the formation of filaments that can reach >100 I?m in length. Much of the basic biology of filamentation is unknown including triggers, production on foods, survival characteristics, and virulence. The formation of Salmonella filaments is significant to food safety because under favorable conditions the filaments will form septa and divide the filament into multiple, typical cells that can impact estimations of pathogen numbers, the effectiveness of intervention practices (i.e., critical control points), and risk assessments. This proposal will study Stress-Induced Filamentous Salmonella (SIFS) on fruits, nuts, and vegetables and specifically determine there: 1.) pathogen load assessments and persistence on foods, 2) formation and characterization, 3) mitigation measures, 4) infectious dose and virulence. This project will provide fundamental information on stress response in Salmonella that impacts persistence on fruits, nuts, and vegetables and define effective harvest and/or production practices that prevent formation or inactivate these stress-induced cells.
Animal Health Component
(N/A)
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124010110050%
7125010110050%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria; 5010 - Food;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The goal of this project is to study the formation and persistence of Stress-Induced Filamentous Salmonella (SIFS) on fruits, nuts, and vegetables. The specific objectives are to determine the impact of SIFS on: (1) pathogen load assessments and persistence in or on foods, (2) growth and survival properties of the pathogen, (3) mitigation pratices, and (4) infectious dose and virulence. This project will provide fundamental information on the formation and survival properties of SIFS that can influence persistence on fruits, nuts, and vegetables and virulence of this pathogen. Additionally, we will attempt to define effective production and/or storage practices that prevent formation or inactivate SIFS.
Project Methods
Salmonella present on the surface of fruits, nuts, and vegetables will likely encounter one or more of the stress conditions identified in our preliminary studies trigger formation of SIFS. Five serovars of Salmonella enterica will be studied for filament formation. Filaments will also be used to inoculate tomato, peanuts, lettuce and stainless steel surfaces and cell morphology and viable numbers monitored over time. The growth and survival properties of filaments will be compared with normal cells in five different Salmonella serovars. Microarray comparisons of filaments and normal cells will be conducted with three strains; Typhimurium, Typhi, and Agona. The mean hybridization signal intensities for each gene will be calculated from the multiple probe targets per gene. Genes will be considered differentially expressed when the signal-to-noise ratios exceed 3, the paired Student's t test of the average RNA levels are significantly different from controls (P<0.05), and the mean RNA level changes are at least twofold. The results from this objective will determine whether filamentous cells are more or less robust than normal cells and provide insights into their formation, survival, and virulence. The preceding objectives will characterize the formation and persistence of SIFS. Results from these experiments will be used to set the specific parameters of storage and the basis for formulating control strategies by modifying storage or process interventions. This objective will evaluate conditions that failed to support or repressed filament formation (i.e., storage temperature, storage humidity, time) as an intervention step during the storage of in-shell peanuts, tomatoes, and lettuce to determine if these conditions will render Salmonella more sensitive to subsequent roasting (peanuts) and sanitizing (tomatoes and lettuce). The roasting process will be modified (i.e., time and temperature of roast, blanch-roast) as needed to determine the necessary time and temperature to achieve a significant reduction of Salmonella numbers. The free chlorine concentration, temperature, and exposure time will be tested to determine the most effective combination for Salmonella reduction on tomatoes and lettuce. To assess virulence, the ability of SIFS and normal cells of Salmonella to attach to, invade and multiply within intestinal epithelial cells, using the Caco-2 human intestinal epithelial cell line, will be compared. The virulence of SIFS will be tested in a mouse model of gastrointestinal bacterial infection using the genetically susceptible C57Bl/6 strain of mice. Salmonella cells will be inoculated directly into the gastrointestinal tract to mimic the natural route of infection. Results from this project will disseminated in peer-reviewed publications, presentations at scientific meetings, and through extension and outreach activities.

Progress 02/01/10 to 07/31/13

Outputs
Target Audience: The target audiences of our efforts were industry, government, public health, and food safety professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided excellent training opportunities and professional development. With oversight, mentoring, and direction of the research coordinated by the Principal Investigators (Kaspar, Wong, and Czuprynski), six undergraduate students, four Graduate Students, one Postdoctoral Research Associate, and one senior technician were involved in this project. The four Graduate students (Shiroda, Stackhouse, Stasic, and Tsarouha) were supported in total or in part by this project and all completed a Master of Science degree in Bacteriology. Dr. Pratt was the postdoctorate supported by this project and is now employed as a faculty member at St. Norbert College. Six undergraduate students received training and helped generate data for this project; T. Borchert, B. Chen, B. Gietman, A. King, J. Leshmore, and K. Yang. All of the participants involved in this project presented findings of their research at local and national meetings. How have the results been disseminated to communities of interest? The findings from this project were disseminated to communities of interest (industry, government and academic food safety professionals) at local and national conferences including: the Food Research Institute, the International Association of Food Protection, the American Society for Microbiology, and the Raper Symposium coordinated by the Department of Bacteriology at UW-Madison. Ten talks or posters on research findings were presented during this project. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (1.) SIFS had a significant impact on quantitative assessments of microbial numbers (CFU/ml or g). A number of stress conditions result in the formation of SIFS. In our studies, reduced water activity (aw) was used to generate aseptate filaments with multiple nucleoids that formed single colonies on solid culture media even though they are comprised of multiple cells (10-100 cells). Thus, samples containing SIFS can yield misleading numbers of CFU and presents challenges to retrospective determinations of salmonellae numbers in foods. The protein and DNA levels in SIFS were similar to control cells when cell populations were standardized by biomass rather than CFU. However, SIFS contained more ATP than control cells despite reduced levels of two FoF1-ATP synthase subunits. Based upon these findings, a plating method was developed to enumerate the number of SIFS that was consistent with microscopic enumerations. (2.) The growth and survival of SIFS were compared to non-filamentous Salmonella control cells. The transfer of salmonellae cultures containing SIFS from stress conditions (i.e., hyperosmotic conditions) to fresh growth media resulted in septa formation and the formation of normal-sized cells along with a corresponding rapid increase in CFU. Cultures containing SIFS had a more rapid increase in CFU/ml than control cells when beginning with the same number CFU. However, outgrowth in conditions of stress (i.e., osmotic stress) was similar or slower than control cells. The survival of SIFS was evaluated in a number of conditions including: high temperature, low pH, and hypertonic conditions. The survival of SIFS was not significantly different from control cells when inoculum was standardized by biomass rather than CFU. These results indicate that SIFS are not more resistant than control cells to the intrinsic and extrinsic factors tested. (3.) To define effective mitigation practices, results from objectives 1 and 2 were necessary. Results found that the general stress protection system of salmonellae, regulated by the alternative sigma factor RpoS, is essential to survival and persistence in detrimental conditions such as those in low-moisture foods. The formation of SIFS was not directly linked to RpoS but was necessary for the growth and survival of salmonellae in stress conditions that permitted the formation of SIFS. Thus, conditions that repress RpoS synthesis or decrease stability would be effective mitigations strategies. Anaerobic conditions was one factor identified as a possible intervention strategy that resulted in stress sensitive salmonellae. (4.) SIFS were found to be virulent in both in vitro and in vivo methods. SIFS invaded and multiplied in Caco-2 human intestinal cells to a similar degree as control cells when comparable CFU of filament and control cells were tested. SIFS successfully infected mice following intragastric inoculation. The SIFS colonized the gastrointestinal tract and disseminated to the spleen and liver at levels comparable to those attained with control cells even when animals were inoculated with 10- to 100-fold fewer CFU.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Stackhouse, R., N. Faith, C. W. Kaspar, C. Czuprynski, and A. Wong. 2012. Formation, survival, and virulence of stress-induced filamentous Salmonella. Appl. Environ.Microbiol. 78:2213-2220.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Pratt, Z., B. Chen, C. J. Czuprynski, A. C. L. Wong, and C. W. Kaspar. 2012. Characterization of osmotic-induced filaments of Salmonella enterica. Appl. Environ. Microbiol. 78:6704-6713.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Stasic, A. J., A. C. L. Wong, and C. W. Kaspar. 2012. Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires rpoS. Current Microbiol. 65:660-665.
  • Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Shiroda, M., Z. Pratt, D. Dopfer, A. C. L. Wong, and C. W. Kaspar. 2013. Survival and growth of Salmonella enterica during osmotic stress is dependent upon RpoS. J. Appl. Microbiol.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Stackhouse, R., N. Faith, C. W. Kaspar, C. Czuprynski, and A. Wong. 2010. Filamentous Salmonella: formation, survival, and virulence. International Association of Food Protection. Anaheim, CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Pratt, Z., B. Chen, B. Gietman, C. W. Kaspar, and A. C. L. Wong. 2011. Molecular characterization of filaments of Salmonella formed during growth on media with reduced water activity. Kenneth B. Raper Symposium, Univ. Wisc.-Madison
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Pratt, Z., B. Chen, B. T. Gietman, A. C. L. Wong, and C. W. Kaspar. 2012. Characterization of osmotic-induced filaments of Salmonella enterica. Am. Soc. for Microbiol., San Francisco, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Gietman, B., Z. Pratt, A. C. L. Wong, and C. W. Kaspar. 2012. Filamentous Salmonella enterica formed during osmotic stress undergo changes to nucleoid morphology and ATP generation over time. Food Res. Institute Meeting, Madison, WI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Chen, B., Z. Pratt, C. W. Kaspar, and A. C. L. Wong. 2012. Levels of ATP in osmotic-induced filaments of Salmonella enterica are greater than in non-filaments despite reduced levels of succinate dehydrogenase. Food Research Institute Meeting, Madison, WI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Tsarouha, J., N. Faith, C. W. Kaspar, A. Wong, and C. Czuprynski. 2012. Motility of filamentous cells of Salmonella enterica serovar Enteritidis E40. Food Research Institute Meeting, Madison, WI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Faith, N., J. Tsarouha, C. W. Kaspar, A. Wong, and C. Czuprynski. 2012. Motility of filamentous cells of Salmonella enteritidis E40. Ann. Meeting of the Intern. Assoc. Food Prot., Providence, RI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Borchardt, T., Z. Pratt, C. W. Kaspar, and A. Wong. 2013. Oxidative stress observed in Salmonella enterica grown in hyperosmotic conditions. Food Research Institute Meeting, Madison, WI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Lensmire, J., Z. Pratt, and C. W. Kaspar. 2013. Involvement of ROS scavenging enzymes in Salmonella enterica survival in hyperosmotic conditions. 2013. Kenneth B. Raper Symposium, Madison, WI


Progress 02/01/12 to 01/31/13

Outputs
OUTPUTS: OUTPUTS: The purpose of this study is to characterize the basic biology of filamentation and the impact of filamentous salmonellae on transmission and virulence. The outputs for this study have been produced by five undergraduate students, four graduate students, one postdoctorate, and a senior technician. Oversight, mentoring, and the direction of the research was provided by the project principal investigators. The findings for this study were disseminated at conferences, including the Food Research Institute annual meeting that is attended by industry, government and academic food safety professionals, the International Association of Food Protection annual meeting, the American Society for Microbiology annual meeting, and the Raper Symposium which is coordinated by the Department of Bacteriology at UW-Madison. PARTICIPANTS: PIs: Charles W. Kaspar, A.C.L. Wong, and C.J. Czuprynski; postdoctorate, Z. Pratt; technician, N. Faith; graduate students: Megan Shiroda, A.J. Stasic, B. Stackhouse, and J. Tsarouha; undergraduate students: A. King, B. Gietman, T. Borchert, K. Yang, and B. Chen. TARGET AUDIENCES: The target audiences are industry, government and academic food safety professionals as well as students in these areas of study. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The removal of water from food items (desiccation) is an effective method of food preservation. Salmonella enterica form smooth, aseptate filaments with multiple nucleoids by an unknown mechanism when cultured in hyperosmotic conditions (desiccation). Filaments are comprised of multiple cells (10-50); yet, form single colonies on growth media that results in an underestimation in the number of viable cells present within a sample. These findings have importance to retrospective assessments of the infectious dose of salmonellae determined from contaminated food samples. Results have determined that the general stress protection system of salmonellae, regulated by RpoS, is essential to persistence of salmonellae in low-water environments and is indirectly involved in filamentation by enabling the cell to survive and elongate. It was determined that during desiccation, Salmonella generate reactive oxygen species that contribute to the lethality of low-water conditions. The results generated from this study will be useful in making informed recommendations for intervention practices and risk assessments of Salmonella-contaminated foods.

Publications

  • Chen, B., Z. Pratt, C. W. Kaspar, and A. C. L. Wong. 2012. Detection of filamentous Salmonella on low-aw food items and enumeration method of filamentous Salmonella. UW-Madison Undergraduate Research Symposium, Madison, WI.
  • Wong, A. C. L. Salmonella in low moisture foods. Food Research Institute Spring Meeting. May 22, 2012
  • Pratt, Z. L., B. Chen, C. J. Czuprynski, A. C. Wong, and C. W. Kaspar. 2012. Characterization of osmotically induced filaments of Salmonella enterica. Applied and Environmental Microbiology. 78:6704-6713.
  • Stackhouse, R., N. Faith, C. W. Kaspar, C. Czuprynski, and A. Wong. 2012. Formation, survival, and virulence of stress-induced filamentous Salmonella. Applied and Environmental Microbiology 78:2213-2220.
  • Stasic, A. J., A. C. L. Wong, and C. W. Kaspar. 2012. Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires rpoS (σ38). Current Microbiology 65:660-665.
  • Gietman, B., Z.L. Pratt, A.C.L. Wong, and C.W. Kaspar. 2012. Filamentous Salmonella enterica formed during osmotic stress undergo changes to nucleoid morphology and ATP generation over time. Kenneth B. Raper Symposium, Madison, WI.
  • Stasic, A.J., A.C.L. Wong, and C. W. Kaspar. 2012. Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires rpoS (σ38). Kenneth B. Raper Symposium, Madison, WI.
  • Faith, M., J. Tsarouha, C. W. Kaspar, A. Wong, and C. Czuprynski. 2012. Motility of filamentous cells of Salmonella enteritidis E40. Annual Meeting of the International Association of Food Protection, Providence, RI.
  • Pratt, Z.L., B. Chen*, B.T. Gietman*, A.C.L. Wong, and C.W. Kaspar. 2012. Characterization of Osmotic-induced Filaments of Salmonella enterica. American Society for Microbiology General Meeting, San Francisco, CA.
  • Shiroda, M., Z.L. Pratt, and C.W. Kaspar. 2012. Contribution of RpoS to Growth and Survival of salmonellae during Osmotic Stress. American Society for Microbiology General Meeting, San Francisco, CA.
  • Gietman, B., Z.L. Pratt, A.C.L. Wong, and C.W. Kaspar. 2012. Filamentous Salmonella enterica formed during osmotic stress undergo changes to nucleoid morphology and ATP generation over time. Food Research Institute Spring Meeting, Madison, WI.
  • Chen, B., Z.L. Pratt, C.W. Kaspar, and A.C.L. Wong. 2012. Levels of ATP in Osmotic-induced Filaments of Salmonella enterica are Greater than in Non-filaments Despite Reduced Levels of Succinate Dehydrogenase. Food Research Institute Spring Meeting, Madison, WI.
  • Tsarouha, J., N. Faith, C. W. Kaspar, A. Wong, and C. Czuprynski. 2012. Motility of filamentous cells of Salmonella enterica serovar Enteritidis E40. Food Research Institute Spring Meeting, Madison, WI.


Progress 02/01/11 to 01/31/12

Outputs
OUTPUTS: Experiments were conducted and results analyzed to better understand and characterize the basic biology of filamentation and the impact of filamentous salmonellae on food safety. Methods and techniques have been modified or developed to facilitate these studies. These outputs have been generated by four undergraduate students, one postdoctorate research associate, and one senior technician that mandates mentoring and coordination by the project PIs. The findings from these studies have been disseminated at conferences including the Food Research Institute annual meeting that is attended by government, industry and academic food safety professionals, the International Association of Food Protection annual meeting, the annual conference of North Central Association of Food and Drug Officials, and the Raper Symposium which is coordinated by the Department of Bacteriology at UW-Madison. PARTICIPANTS: PI: Charles W. Kaspar; Co-PIs: Drs. A.C.L. Wong and C. Czuprynski; Postdoctorate: Dr. Z. Pratt; Senior technician: Nan Faith; Undergraduate researchers: Brad Gietman, KaHoua Yang, Jon Szewczk, and Alisa King TARGET AUDIENCES: The target audiences are industry, government, and academic food safety and public health professionals as well as students in these areas of study. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Reducing the water activity (aw) of food items is an effective method of food preservation and inhibiting microbial growth. Salmonella enterica form smooth, aseptate filaments with multiple nucleoids by an unknown mechanism when cultured on reduced-aw media (achieved by supplementing media with sodium chloride) or foods. Multi-chromosomal filaments are viable and form single colonies on agar plates even though they are actually comprised of multiple cells (10-50). When encountering favorable growth conditions, filaments quickly form septa and separate into individual cells that results in a shorter lag phase and more rapid exponential growth phase than non-filamentous control cells. Thus, samples containing filamentous salmonellae can yield misleading numbers of CFU and presents challenges to retrospective determinations of infectious dose and risk assessments.

Publications

  • Kaspar, C.W. 2011. Desiccation tolerance in Salmonella serovars. Food Research Institute annual meeting. Pyle Center, Madison, WI
  • Shiroda, M., C. W. Kaspar, and Z. Pratt. 2011. RpoS contribution to growth and survival of Salmonella serovars under desiccation conditions. Food Research Institute annual meeting. Pyle Center, Madison, WI
  • Stackhouse, R., N. Faith, C. W. Kaspar, C. Czuprynski, and A. Wong. 2011. Formation, survival and virulence of stress-induced filamentous Salmonella. Applied and Environmental Microbiology (submitted)


Progress 02/01/10 to 01/31/11

Outputs
OUTPUTS: Experiments were conducted to understand and characterize the basic biology of filamentation in Salmonella. Methods and techniques have been modified or developed to facilitate these studies. These outputs have been generated primarily by several undergraduate students and a postdoctorate research which involves mentoring by the project PIs. The results from these studies have been disseminated at conferences (one local and one national meeting) and as part of a workshop. PARTICIPANTS: C. W. Kaspar, project PI. A. C. L. Wong, Co-PI. C. Czuprynski, Co-PI. N. Faith, technician, partial support. Z. Pratt, post-doctorate fellow. Contributing undergraduate researchers, 4 (Megan Shiroda, Aaron Gnas, Matt Bozile, and Sarah Meyer) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Salmonella encounters a myriad of stressful conditions in the food production chain that can trigger specific and general stress-protection systems that render cells more tolerant to stress and promote persistence in the environment or food. One interesting response to stress is the formation of filaments (elongated cells that have not formed septa) that can reach >100 &#956;m in length. Much of the basic biology of filamentation is unknown including triggers, production on foods, survival characteristics, and virulence. The formation of Salmonella filaments is significant to food safety because under favorable conditions the filaments will form septa and divide the filament into multiple, typical cells that will impact estimations of pathogen numbers, the effectiveness of intervention practices (i.e., critical control points), and risk assessments. Outcomes: S. Enteritidis E40 developed filaments when exposed to reduced aw. In addition, filamentation occurred when the organism was exposed to ultra-violet light. When the filaments were resuspended in fresh TSB at 25 or 37C, cells formed septa and divided into regular-sized single cells, resulting in a more rapid increase in viable counts than a control population. Control cells and filaments survived all stress conditions tested; generally higher decreases in filament viable numbers were observed with the exception of pH 2, where the reverse was observed. Filamentous cells are virulent; they invaded and grew in Caco-2 cells, infected multiple organs in mice, and appeared to persist longer than control cells in the gastrointestinal tract. Impacts: Results from these studies will impact risk assessments and determinations of the infectious dose of pathogens.

Publications

  • Stackhouse, R., N. Faith, C. W. Kaspar, C. Czuprynski, and A. Wong. 2010. Formation, survival and virulence of stress-induced filamnetous Salmonella. International Association of Food Protection, P2-134. Anaheim, CA.
  • Whitemarsh, R., A. Gnas, and C. W. Kaspar. 2010. Desiccation tolerance in Salmonella. International Association of Food Protection, T2-01. Anaheim, CA.