Source: NORTH DAKOTA STATE UNIV submitted to
ENHANCING MICROBIAL FOOD SAFETY BY RISK ANALYSIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1003967
Grant No.
(N/A)
Project No.
ND02426
Proposal No.
(N/A)
Multistate No.
S-1056
Program Code
(N/A)
Project Start Date
Oct 1, 2014
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Project Director
Bergholz, TE.
Recipient Organization
NORTH DAKOTA STATE UNIV
(N/A)
FARGO,ND 58105
Performing Department
Microbiological Sciences
Non Technical Summary
Foodborne illness is a significant public health concern, with an estimated 48 million cases of foodborne illness each year in the U.S. alone. In addition to health care costs, the presence of human pathogens in the food supply has a substantial economic effect on the food industry in the U.S. due to product recalls and loss of consumer confidence. Pathogens such as E. coli, Salmonella,andListeria monocytogenesare responsible for the majority of severe foodborne illness. These pathogens are capable of surviving in a number of different food products, including fresh produce and low-moisture foods. Aclearer understanding of how these pathogens are capable of surviving on foods during handling and processing will allow development of new control strategies to reduce pathogens in the food supply.To determine how bacterial pathogens are able to survive different stresses present on foods, we will expose them to different stresses and measure changes in gene expression and quantify the number of bacteria that survive. Changes in gene expression are one indication of how a bacterium is reacting to a stress in order to protect itself from further damage. In some cases, the genes induced by one stress can lead to increased resistance to a subsequent different stress. Using this information against the bacteria, such as combining stresses or processing steps in a different way, could lead to sensitization of the bacteria to the stress, making them easier to elminate from the food.
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124010110060%
7125010104040%
Keywords
Goals / Objectives
Risk Management: Develop science-based interventions to prevent and mitigate food safety threats
Project Methods
To characterize molecular mechanisms that foodborne pathogens use to survive stresses that are present in foods or encountered during handling and processing of foods, we will first evaluate bacterial survival under single stresses that bacteria can encounter in foods. These stresses will include typical intrinsic and extrinsic properties of foods, such as pH and osmolarity, as well as bacteriostatic and bactericidal compounds. In conjunction with publically available gene expression data, this will allow us to construct deletion mutations in specific genes to determine their role in survival under these stress conditions. Molecular mechanisms shown to be important under an single stress will then be evaluated on a food product that has the same intrinsic/extrinsic property.Gene content can also play a role in the stress resistance properties of a foodborne pathogen. We will assess the impact of the presence or absence of specific sets of genes that have previously been shown to contribute to stress resistance in a population of genetically diverse bacterial strains representative of the population ofListeria monocytogenesknown to cause human disease. All quantitative data will be evaluated for statistical significance using an appropriate test for each specific experiment. Signficant differences in survival phenotypes between mutants and wild type strains will be indicative of the importance of a specific gene product in the survival phenotype, and will warrant further investigation into the regulation of expression of that protein, and the stimuli that can alter its expression.

Progress 10/01/14 to 09/30/18

Outputs
Target Audience:For this reporting period, the target audience was food safety and veterinary researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided a research training opportunity for one undergraduate researcher. Ryan Callahan worked on genomic characterization of the ruminant listeriosis strains, focusing specifically on the Listeria pathogenicity island 3.This project has also provided a research training opportunity for one graduate student, Maria Cardenas Alvarez, who worked on the Listeria in ruminants project. This research was published this year. How have the results been disseminated to communities of interest?Results have been disseminated through peer-reviewed publications and presentations at regional and national microbiology meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Listeria monocytogenes (Lm) is an intracellular pathogen that causes disease in humans and animals. It is frequently found as contaminant in food and animal feed, and has been associated with different clinical manifestations such as central nervous system (CNS) infections, bacteremia (BAC) and maternal-neonatal (MN) infections. Variation in virulence has been reported among strains. Data on clinical isolates from ruminants in Europe have provided evidence of hypervirulent clones causing rhombencephalitis in lineage I (LI) and have described sequence type (ST) 1 (from clonal complex -CC 1) as predominant, suggesting increased neurotropism of ST1. However, there is also evidence of hypervirulent clones in lineage II (LII), indicating that this feature is not restricted to strains from LI. A key remaining gap in knowledge is whether the association of CCs with a higher frequency in listeriosis cases, and with a specific clinical outcome could be attributed to higher invasiveness and increased virulence of a group of strains. Our working hypothesis was that different CCs of Lm will show distinct mortality rates in G. mellonella, an insect model for measuring virulence among L. monocytogenes. Strains from CC14 (LII) showed lower LD50 values than strains from CC1, CC7, CC37 and CC204 at all inoculum concentrations, and was significantly lower when compared to CC7. When comparing outcomes, strains causing MN showed LD50 significantly lower than strains related to CNS and BAC. These results indicate that strains associated with MN infections have a higher virulence potential and need a lower number of bacterial cells to cause the disease.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Steckler, A. J., M. X. Cardenas-Alvarez, M. K. Townsend Ramsett, N. Dyer, and T. M. Bergholz. Genetic characterization of Listeria monocytogenes from ruminant listeriosis from different geographical regions in the U. S. 2018. Veterinary Microbiology 215:93-97
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Bergholz, T. M., M. K. Shah, L. S. Burall, M. Rakic Martinez, and A. Datta. Genomic and phenotypic diversity of Listeria monocytogenes clonal complexes associated with human listeriosis. 2018. Applied Microbiology and Biotechnology 102:3475-3485.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:For this reporting period, the target audience was food safety and veterinary researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided a research training opportunity for two undergraduate researchers. Abbey Steckler worked on the sequence typing of ruminant listeriosis strains. She was able to present her research at a national meeting. Ryan Callahan worked on genomic characterization of the ruminant listeriosis strains, focusing specifically on the Listeria pathogenicity island 3. He was able to present his research at a regional meeting. This project has also provided a research training opportunity for one graduate student, Sahar Malekmohammadi, who worked on the nisin resistance experiments. This research was published this year. How have the results been disseminated to communities of interest?Results have been disseminated through peer-reviewed publications and presentations at regional and national microbiology meetings. What do you plan to do during the next reporting period to accomplish the goals?We will continue typing ruminant listeriosis isolates, and we have obtained whole genome sequences for these isolates and will start performing comparative genomic analyses.

Impacts
What was accomplished under these goals? Listeria monocytogenes infections are an important disease of ruminants worldwide, causing encephalitis, septicemia, and abortions . Ruminant listeriosis can also pose a food safety risk due to the potential for L. monocytogenes to enter the food supply via the farm environment. Data on the genetic diversity of L. monocytogenes from ruminant clinical cases in the United States is limited. Our goal was to assess the genetic diversity of clinical listeriosis isolates from ruminants in the Upper Great Plains states, a population not well-studied, and compare this population to isolates from ruminants in New York State. Multi-locus sequence typing (MLST) was used to classify and compare the genetic diversity of the isolates from the two regions. Loci sequences were compared to all known sequence types using the Pasteur Institute L. monocytogenes MLST database. Four novel sequence types (ST) were identified among the Upper Great Plains isolates, and four new STs were classified in the New York collection. Five STs were found to be common across the 2 geographical regions; ST 1, 7, 191, and 204. Strains of ST 7 were most frequently isolated (7/46 isolates). Strains of ST 91 were all associated with fetal infections from the Upper Great Plains. Our results demonstrate that while there are some subtypes commonly found between the two geographic regions, there are also subtypes distinct to each region. L. monocytogenes possesses a number of virulence factors, one of which is Listeria Pathogenicity Island 3 (LIPI-3). The eight genes of LIPI-3 are responsible for the production and post-translational modification of Listeriolysin S (LLS), a bacteriocin that helps establish infection in the intestinal tract. Previous studies on LIPI-3 have focused primarily on gene function using reference strain F2365 and little work has been done on the prevalence of LIPI-3 in L. monocytogenes isolates from the United States, specifically the Upper Midwest. In this study, L. monocytogenes isolates from the Upper Midwest as well as a handful of reference genomes from around the world were screened for the presence of LIPI-3. Isolates containing LIPI-3 were further categorized using multi-locus sequence typing (MLST) methods. Using this information, phylogenetic trees were constructed to show the divergence of LIPI-3 genes based on MLST subtype. Comparison of the LIPI-3 gene diversity to MLST data showed that the LIPI-3 genes exhibit a higher degree of diversity than MLST genes. More so, diversity of LIPI-3 genes exists even within clonal complexes established by MLST.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Malekmohammadi, S., K. K. Kodjovi, J. Sherwood, and T. M. Bergholz. 2017. Genetic and environmental factors influence Listeria monocytogenes nisin resistance. Journal of Applied Microbiology 123:262-270.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Steckler, A. J., M. Cardenas Alvarez, N. Dyer, and T. M. Bergholz. Comparison of Listeria monocytogenes from ruminant listeriosis isolated in 2 geographical regions of the U.S. American Society for Microbiology Microbe 2017 meeting, New Orleans, Louisiana. 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Callahan, R. C. and T. M. Bergholz. Characterization of LIPI-3 Positive Listeria monocytogenes in the Upper Midwest. North Central Branch American Society for Microbiology Annual Meeting, De Pere, Wisconsin. 2017


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:For this reporting period, the target audience was food safety and veterinary researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided a research training opportunity for one undergraduate researcher, Abbey Steckler, who worked on the sequence typing of ruminant listeriosis strains. She was able to present her research at a national meeting. This project has also provided a research training opportunity for one graduate student, Sahar Malekmohammadi, who worked on the nisin resistance experiments. She was able to present her research at a regional conference. How have the results been disseminated to communities of interest?Through presentations at research conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue typing ruminant listeriosis isolates, and will also begin whole genome sequencing of those isolates so we can perform comparative genomic analyses.

Impacts
What was accomplished under these goals? We have determined that inducible nisin resistance in L. monocytogenes can be influenced both by environmental conditions as well as the genetic composition of the strain. This information is important for food processors and food safety professionals when considering antimicrobial additives to control L. monocytogenes on ready to eat foods. We have also assessed the genetic diversity of L. monocytogenes strains isolated from cases of ruminant listeriosis in the upper great plains region. Our results indicate that the isolates causing listeriosis in ruminants in the upper Midwest are genetically diverse, with new sequence types of L. monocytogenes still being discovered. Our results also demonstrate that isolates from ruminants in this region belong to the same genetic subtypes as isolates found in the food supply and human cases of listeriosis, and further studies are warranted to understand the ecology of this pathogen in the farm environment.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Steckler, A. J., M. Cardenas Alvarez, N. Dyer, and T. M. Bergholz. Genetic characterization of Listeria monocytogenes isolated from cases of Listeriosis in Ruminants in the Upper-Midwest. American Society for Microbiology Microbe 2016 Meeting, Boston, Massachusetts.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Malekmohammadi, S., K. K. Kodjovi, J. Sherwood, and T. M. Bergholz. Influence of genetic and environmental factors on antimicrobial peptide resistance in L. monocytogenes. Frontiers in Biomedical Research Symposium, Fargo, North Dakota. June 4, 2016.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:For this reporting period, the target audience was food safety researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided a research training opportunity for an undergraduate student, Kouevi Kodjovi. He was able to present this research at local and national conferences. How have the results been disseminated to communities of interest?Through presentations at research conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to evaluate the genetic basis of inducible nisin resistance inL. monocytogenes, and also begin evaluation of other antimicrobials that are approved for use on foods to determine if genetic and environmental factors may contribute to resistance to these compounds.

Impacts
What was accomplished under these goals? We have determined thatinducible nisin resistance in L. monocytogenes can be influenced both by environmental conditions as well as the genetic composition of the strain. This information is important for food processors and food safety professionals when considering antimicrobial additives to controlL. monocytogeneson ready to eat foods.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Influence of the 5 gene stress survival islet on salt-induced nisin resistance of Listeria monocytogenes Kouevi Kodjovi, Julie Sherwood, and Teresa M. Bergholz NDSU Explore conference, November 4, 2014