Progress 04/01/08 to 03/31/13
Outputs
Target Audience: The project aided the training of numerous undergraduate and graduate students and postdoctoral scholars who conducted research on novel methods for tracking and controlling foodborne pathogens. The results of this project were also used to instruct undergraduate and graduate students in food sciences courses at Penn State University on various modern molecular methods. Results of this project were also presented to numerous national and international scientific audiences. The main objective of this project was the development of novel molecular methods for detecting and tracking epidemic clones of Salmonella and Listeria monocytogenes. Salmonella is the most frequent cause of foodborne illness in the U.S. and causes considerable morbidity in the healthy and death in infants and the elderly. Listeria monocytogenes is especially a problem for susceptible at-risk populations, including infants, the elderly and those that are immuno-compromised, such as pregnant women and their fetuses and AIDS and cancer patients. Listeria infections are especially a problem in the hispanic community due to concumption of Mexican-style cheeses, which have been frequently associated with listeriosis. The results from this project will be used to prevent contamination of these high-risk ready-to-eat foods and thus ensure the health of this miniority group. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Results of this research were presented in various short courses in the Department of Food Science related to microbial food safety. The PI met with administrators at the University of Torino to discuss research of mutual interest and also finalize a MOU between the University of Torino and Penn State University around the theme of microbial food safety. Gave an invited presenation at a workshop on molecular epidemiology of foodborne pathogens at a pre-conference workshop in Washington, DC, organized by the Grocery Manufacturers of America. Collaborated with scientists in the US, Italy and India to detect epidemic clones of Listeria monocytogenes in various ready-to-eat food products. A novel MVLST/Staphylococcal Cassette Chromosome mec (SCCmec) typing scheme and database was developed for subtyping strains of healthcare-associated and community-associated Methicillin-resistant Staphylococcus aureus (MRSA). This collaborative research project with faculty and clinicians at Penn State's Hershey Medical Center (HMC) is ongoing and is starting to focus on preventing the transmission of the pandemic clone USA300 MRSA within HMC. The evolution of epidemic clones of bacterial pathogens was presented at the Center for Infectious Disease Dynamics (CIDD). Currently working with colleagues in Italy, CDC and FDA on the US listeriosis outbreak linked to imported Italian Ricotta Cheese. Collaboration with scientists at various governmental, industrial and academic laboratories allowed my postdocs, graduate students and undergraduate students to better understand different aspects of food safety management and control from governmental, industrial and academic perspectives. It also promoted collaboration between the many different governmental agencies, food industries and academic institutions involved in these projects. The PI was invited to present the results of this research at the International Symposium on Problems of Listeriosis in Goa, India. Attendance at this conference allowed the PI to become familiar with all aspects related to detection, tracking and control of Listeria monocytogenes. How have the results been disseminated to communities of interest? The results of this research were shared each year with members of the Susquehanna Branch of the Central Atlantic States Association of Food and Drug Officials. These members are responsible for food inspection in the State of Pennsylvania. Results were also shared with members of the Eastern Meat Packers Association, so they could use the results to implement more effective prevention strategies to prevent Listeria monocytogenes contamination of meat and poultry products manufactured in the northeastern U.S. The results were also shared with participlants attending numerous short courses in the Department of Food Science at Penn State especially those that deal with food safety. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We investigated whether multi-virulence-locus sequence typing (MVLST) could detect epidemic clones of Listeria monocytogenes in outbreaks related to ready-to-eat meats in the U.S. and Canada. We determined that the 2008 and 2010 outbreaks in Toronto, Canada, along with numerous time-linked clusters of cases with similar Pulsed-Field Gel Electrophoresis profiles throughout Canada from 1988-2010 were due to a new epidemic clone, which we classified as epidemic clone five (ECV). We also analyzed numerous presumptive ECII isolates sent to us by USDA FSIS and confirmed they were ECII by MVLST. We also analyzed these ECV and ECII isolates using comK prophage junction fragment (JF) sequencing and demonstrated that the JF sequences were unique to individual meat and poultry processing plants. These findings were used by these national food safety agencies to track the epidemic clones and outbreak clones of Listeria monocytogenes to their sources within RTE meat and poultry processing plants in the U.S. and Canada. MVLST was also applied to outbreak strains from the U.S. Centers for Disease Control and Prevention that were associated with the 2011 Cantaloupe Outbreak in the U.S. These results demonstrated the existence of two additional novel epidemic clones, which we designated ECVI and ECVII. MVLST was also used to identify a novel outbreak strain associated with the 2012 outbreak associated with Ricotta Salata cheese from Italy. MVLST results from the Cantaloupe and Ricotta Salata cheese outbreaks were used by FDA and CDC to track and control these outbreaks and are also being by the food industry to prevent future outbreaks from occurring. Health agencies in Italy and India are also currently using the MVLST scheme developed in our laboratory to detect epidemic clones and outbreak clones of Listeria monocytogenes in these countries. We investigated whether highly variable genes found in Escherichia coli, collectively referred to as "rhs genes" could serve as useful sub typing markers for serotype O157:H7. Eighteen O157:H7 strains from two distinct lineages (lineages I and II) and 15 O157:H7 strains from eight previously defined clades were included. Examination of these rhs genes revealed 44 polymorphic loci (PL) and 10 sequence types (STs) among the 18 lineage strains and 280 PL and 12 STs among the 15 clade strains. Phylogenetic analysis using rhs genes generally grouped strains according to their known lineage and clade classifications. These findings also suggested that O157:H7 strains from clades 6 and 8 fall into lineage I/II and that strains of clades 1, 2, 3, and 4 fall into lineage I. Additionally, unique markers were found in rhsA and rhsJ that might be used to define clade 8 and clade 6. Therefore, rhs genes may be useful markers for phylogenetic analysis of E. coli O157:H7. Irradiated ground beef (80:20 or 93:7; lean:fat) was experimentally inoculated with ~106(6 log10) CFU/gram of various STEC (O145, O26, O45, O103, O111, O121 and O157:H7), formed into patties (~50 grams), crust-frozen with liquid nitrogen, vacuum-packaged, stored at 4°C, and subjected to HPP (QFP 2L; Avure Technologies, Franklin, Tenn.). Preliminary experiments determined that multiple, one-minute HPP cycles were more effective for reducing the STEC than single cycles for longer periods of time. Therefore, four HPP cycles (one cycle = 400 MPa for 1 minute @ 17oC) were applied to the inoculated and vacuum-packaged ground-beef patties. Ground-beef patties subjected to the four HPP cycles exhibited an internal temperature increase of ± 2°C, suggesting that short HPP cycles did not increase significantly the temperature of the final product. Yet, HPP treatments resulted in a 3 to 4 log10 CFU/gram (99.99%) reduction of the STEC in the ground-beef patties. Interestingly, the greatest reductions in the STEC were observed in 93:7 ground beef, suggesting that a higher fat content may provide a protective effect to the STEC, thereby allowing for increased survival.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Verghese, B., M. Lok, J. Wen, V. Alessandria, Y. Chen, S. Kathariou and S. J. Knabel. 2011. comK prophage junction fragments as markers for Listeria monocytogenes genotypes unique to individual meat and poultry processing plants and a model for rapid niche-specific adaptation, biofilm formation and persistence. Appl. Environ. Microbiol. 77:3279-3292.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Rocha, P. R., S. Lomonaco, M. T. Botero, A. Dalmasso, A. Dondo, C. Grattanola, F. Zuccon, B. Julini, S. J. Knabel, M. T., Caupucchio and C. Casalone. 2013. Listeria monocytogenes strains from ruminant rhombencephalitis constitute a genetically homogeneous group related to human outbreak strains. Appl. Environ. Microbiol. 79:3059-3066.
- Type:
Other
Status:
Published
Year Published:
2012
Citation:
Cutter, C., D. Depasquale, J. Hayes, C. Raines, and R. Senevirathne. 2012. Meat Science Review: HPP, ground beef, and the "Big 6" STEC. National Provisioner. Published: July 5, 2012. Accessed August 21, 2012 at: http://www.provisioneronline.com/articles/98113-meat-science-review--hpp--ground-beef-and-the--big-6--stec.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Collaborated with Dr. Edward Dudley in the Food Science Department at Penn State University, Dr. Rodolf Barrangou at Dupont and various scientists at the US Centers for Disease Control and Prevention (CDC) to develop and apply a novel Multi-Virulence-Locus Sequence Typing/Clustered Regularly Interspaced Short Palindromic Repeat (MVLST/CRISPR) scheme for subtyping the top-ten serovars of Salmonella enterica. Guest-lectured in Biochemistry, Microbiology and Molecular Biology 597A on molecular epidemiology. Co-taught Advanced Food Microbiology during Spring Semester, 2012. Initiated a research project on the control of Listeria monocytogenes in mushrooms with Dr. Luke Laborde. Presented a talk entitled, Novel DNA-sequence-based approaches for tracking and controlling bacterial pathogens at the Infectious Disease Seminar at Hershey Medical Center (HMC) and at ONIRIS in Nantes, France. Presented a talk on Advanced Molecular Methods for Pathogen Detection at the Food Microbiology Short Course. Participated in the General Mills/FDA/InnoCentive Workshop on Rapid Methods for Pathogen Detection at FDA CFSAN. Consulted with Roca Biosciences on development of optimum system for detecting foodborne pathogens. Met with administrators and faculty at the Pasteur Institute and the University of Torino to discuss research of mutual interest and also a MOU between the University of Torino and Penn State University around the theme of microbial food safety. A MVLST database was developed for assigning MVLST Sequence Types (Virulence Types) to strains of Listeria monocytogenes isolated from many different sources by governmental, industrial and academic laboratories. Collaborated with scientists at the Public Health Agency of Canada concerning the detection of a new epidemic clone of Listeria monocytogenes in Canada using MVLST. Also used this method in collaboration with scientists in the US and Italy to detect epidemic clones of this pathogen in cantaloupe and various ready-to-eat meat and cheese products. A novel MVLST/Staphylococcal Cassette Chromosome mec (SCCmec) typing scheme and database was developed for subtyping strains of healthcare-associated and community-associated Methicillin-resistant Staphylococcus aureus (MRSA). Epidemic clones of Listeria monocytogenes and their control in foodservice operations was presented at the Susquehanna Branch of the Central Atlantic States Association of Food and Drug Officials. Factors affecting Listeria monocytogenes biofilm formation in food processing plants and how to prevent it was presented at the All India Food Processors Conference in New Delhi, India on Dec. 9, 2011. HACCP, an excellent food safety risk management tool was presented at the Indian Institute of Crop Processing Technology in Tanjavour, India on Dec. 14, 2011. A talk entitled HACCP was presented at the Penn State Ice Cream Short Course. The evolution of epidemic clones of bacterial pathogens was presented at a Food Science Departmental Seminar. Worked with Dr. Sara Lomonaco and Dr. Antonio Parizi on the US listeriosis outbreak linked to imported Italian Ricotta Cheese. PARTICIPANTS: Dr. Edward Dudley and Dr. Rodolf Barrangou from the Food Science Department at Penn State University and Dr. Peter Gerner-Smidt at CDC collaborated on the Salmonella subtyping project. Dr. Bindhu Verghese and Dr. Rebecca Weinberg at Penn State University, Dr. Sara Lomonaco at the University of Torino and Dr. Antonio Parizi at the Istituto Zooprofilattico Sperimentale in Italy, Dr. Matthew Gilmour and Aleisha Reimer at the Public Health Agency of Canada, Dr. Yi Chen and Dr. Eric Brown at FDA, Dr. Mark Berrang and Dr. Richard Meinersmann at USDA, and Dr. Cheryl Tarr and Dr. Peter Gerner Smidt at CDC all collaborated on the Listeria monocytogenes MVLST subtyping project. Dr. Bindhu Verghese, Dr. Edward Dudley and Nathan Schwalm III all collaborated on the MRSA MVLST/SCCmec subtyping project. Collaboration with all of these scientists at various governmental, industrial and academic laboratories allowed my postdocs, graduate students and undergraduate students to better understand different aspects of food safety management and control from governmental, industrial and academic perspectives. It also promoted collaboration between the many different governmental agencies, food industries and academic institutions involved in these projects. TARGET AUDIENCES: These molecular subtyping research projects focused on Salmonella, Listeria monocytogenes and MRSA. All of these pathogens disportionately affect infants, the elderly and people who are immunocompromised. The results of this research was disseminated to individuals that work with such groups through scientific publications, classroom instruction, as well by numerous presentations at various informal and formal meetings, seminars and short courses. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The combined MVLST/CRISPR method we developed is currently being used by food safety experts in government, industry and academic to better track and control and ultimately prevent the many Salmonella outbreaks that have been associated with many different types of foods. The MVLST Listeria monocytogenes database was used by the US CDC, the US Food and Drug Administration (FDA), the US Department of Agriculture, The Public Health Agency of Canada (PHAC), the Pasteur Institute and colleagues in Italy to detect novel epidemic clones and outbreak clones of Listeria monocytogenes (see Knabel et al., 2012; Verghese et al., 2012 and Lomonaco et al., 2012). This allowed these governmental agencies and scientists to better understand the epidemiology of this pathogen and to suggest intervention strategies to prevent further outbreaks from occurring. Worked with clinicians and microbiologists at HMC, two postdoctoral scholars, two graduate students and a Schreyer's Honors student to subtype clinical isolates from HMC using our new MVLST/SCCmec subtyping scheme. Subtyping of MRSA isolates using our novel molecular subtyping scheme will allow healthcare and community facilities to track and control MRSA strains that are increasingly difficult to treat and are devastating in terms of their public health burden. Various scientific papers related to molecular epidemiology of foodborne pathogens and MRSA were published in collaboration with scientists from government, academia and industry (see list of publications). These publications are now allowing scientists to better detect, track and control dangerous epidemic clones and outbreak clones of numerous bacterial pathogens, thus enhancing public health.
Publications
- Chen, Y. and S. J. Knabel. 2011. Molecular Epidemiology of Foodborne Pathogens. In Genomics of Foodborne Pathogens, W. Zhang and M. Weidmann (eds.), Springer, N.Y. USA. http://link.springer.com/chapter/10.1007/978-1-4419-7686-4_12/fulltex t.html.
- Liu, F., S. Kariyawasam, B. M. Jayarao, R. Barrangou, P. Gerner-Smidt, E. M. Ribot, S. J. Knabel, and E. G. Dudley. 2011. Subtyping Salmonella serovar Enteritidis isolates from different sources using sequence typing based on virulence genes and CRISPRs. Appl. Environ. Microbiol. 77:4520-4526.
- Schwalm, N., B. Verghese, and S. J. Knabel. 2011. A novel multiplex PCR protocol for detection and differentiation of the major clonal complexes of methicillin-resistant Staphylococcus aureus. J. Microbiol. Methods 86:379-382.
- Knabel, S. J., A. Reimer, B. Verghese, M. Lok, J. Ziegler, J. Farber, F. Pagotto, M. Graham, and C. A. Nadon, the Canadian Public Health Laboratory Network (CPHLN) and M. W. Gilmour. 2012. Sequence typing confirms that a predominant Listeria monocytogenes clone caused human listeriosis cases and outbreaks in Canada from 1988-2010. J. Clin. Microbiol. 50:1748-1752.
- Verghese, B., N. D. Schwalm III, E. G. Dudley, and S. J. Knabel. 2012. A combined multi-virulence-locus sequence typing and Staphylococcus Cassette Chromosome mec typing scheme possesses enhanced discriminatory power for subtyping MRSA. Infect. Gen. Evol. 12:1816-1821.
- Lomonaco, S., R. Rosella, S. J. Knabel, and T. Civera. 2012. Detection of virulence-associated genes and epidemic clone markers in Listeria monocytogenes isolates from PDO Gorganzola cheese. Int. J. Food Microbiol. (Accepted for Publication).
- Lomonaco, S., B. Verghese, P. Gerner-Smidt, C. Tarr, L. Katz, M. Berrang, R. Meinersmann, Y. Chen, E. Brown, and S. J. Knabel. 2012. Molecular characterization of Listeria monocytogenes isolates from the recent U.S. listeriosis outbreak linked to cantaloupe provides evidence for one novel outbreak strain and two novel epidemic clones. Emerg. Infect. Dis. (Accepted for Publication).
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Results of this research were reported at the Annual Meetings of the American Society for Microbiology, The Institute of Food Technologists and the International Association for Food Protection. The results were also presented at the Annual Meeting of the Susquehanna Branch of the Central Atlantic States Association for Food and Drug officials. The results of the long-term-survival studies were published in the Penn State College of Agricultural Sciences magazine, entitled, "Penn State Ag Science". The results were shared with faculty in the College of Veterinary Science at Iowa State University and with scientists at the National Animal Disease Laboratory and Veterinary Services Laboratory in Ames, Iowa. Results were also shared with students in Dr. Knabel's and Dr. Dudley's food microbiology and molecular biology courses at Penn State University, and also presented at bi-weekly Food Microbiology Group Meetings at Penn State University. PARTICIPANTS: Along with Dr. Knabel, Dr. Edward Dudley co-advised the graduate student, Fenyun Liu, and co-authored the two Salmonella publications. Dr. Dudley and Dr. Knabel also collaborated with Dr. Rodolf Barrangou at Danisco (now Dupont) on the CRISPR portion of the Salmonella subtyping project. Dr. Dudley also collaborated with Dr. Knabel and his Ph.D. student, Jia Wen, on the L. monocytogenes gene expression publication. Dr. Anantheswaran, co-advised Jia Wen with Dr. Knabel. He served as a co-author on all of the Listeria monocytogenes publications. Dr. Knabel partnered with CDC, FDA, USDA and the National Microbiology Laboratory of Canada in conducting the molecular subtyping research described in this report. Dr. Knabel also collaborated with Dr. Sara Lomonaco from the University of Turin, Italy and Dr. Sylvain Brisse at the Pasteur Institute to determine whether or not MLST-defined clonal complexes of Listeria monocytogenes equate with epidemic clones. TARGET AUDIENCES: The research conducted in the project was especially focused on preventing foodborne illness in those populations at increased risk for foodborne illness, including infants, the elderly, cancer patients, AIS patients and patients on immuno-suppressive drug therapy. Dr. Knabel and Dr. Dudley brought the results of this molecular subtyping research into their courses on foodborne pathogens to provide the students with information on cutting edge DNA sequence-based molecular subtyping methods for tracking and controlling foodborne disease epidemics and outbreaks. They also shared this research with their professional colleagues at various scientific meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The underlying mechanisms that trigger the morphological and physiological transition of Listeria monocytogenes to the long-term-survival phase remain largely unknown. In this study, we compared the transcriptomic profiles of L. monocytogenes serotype 4b strain F2365 at different growth stages in tryptic soy broth with yeast extract (TSBYE) using a whole-genome DNA chip approach. We identified a total of 225 differentially expressed genes ( 4-fold; P < 0.05) during the transition to the LTS phase in TSBYE. Genes related to cell envelope structure, energy metabolism, and transport were most significantly upregulated in the LTS phase. The upregulation of compatible solute transporters may lead to the accumulation of cellular solutes, lowering intracellular water activity and thus increasing bacterial stress resistance during the transition to the LTS phase. The downregulation of genes associated with protein synthesis may indicate a status of metabolic dormancy of the LTS cells. Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs) which included ECII, ECIII and ECV were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30 degrees C for 48h on meat and poultry food-conditioning films, than strains lacking the comK prophage (P < 0.05). Overall, type of strain, type of conditioning film and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, a novel model is proposed in which the concept of defective comK prophage was replaced with Rapid Adaptation Island (RAI). Genes within the RAI were re-characterized as adaptons, as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety. Dr. Knabel collaborated with Dr. Sara Lomonaco to develop a multiplex SNP typing method for detecting the epidemic clones of Listeria monocytogenes. They are currently collaborating with scientists from the Pasteur Institute to apply this method to determine if various STs within clonal complexes are epidemic clones.
Publications
- Verghese, B., M. Lok, J. Wen, V. Alessandria, Y. Chen, S. Kathariou, and S. J. Knabel. 2011. comK prophage junction fragments as markers for Listeria monocytogenes genotypes unique to individual meat and poultry processing plants and a model for rapid niche-specific adaptation, biofilm formation and persistence. Appl. Environ. Microbiol. 77:3279-3292.
- Liu, F., R. Barrangou, P. Gerner-Smidt, E. M. Ribot, S. J. Knabel, and E. G. Dudley. 2011. Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica. Appl. Environ. Microbiol. 77:1946-1956.
- Liu, F., S. Kariyawasam, B. M. Jayarao, R. Barrangou, P. Gerner-Smidt, E. M. Ribot, S. J. Knabel, and E. G. Dudley. 2011. Subtyping Salmonella enterica Serovar Enteritidis isolates from different sources by using clustered regularly interspaced short palindromic repeats (CRISPRs). Appl. Environ. Microbiol. 77:4520-4526.
- Wen, J., X. Deng, Z. Li, E. G. Dudley, R. C. Anantheswaran, S. J. Knabel, and W. Zhang. 2011. Transcriptomic response of Listeria monocytogenes during transition into the long-term-survival phase. Appl. Environ. Microbiol. 77:5966-5972.
- Lomonaco, S., S. J. Knabel, A. Dalmasso, T. Civera, and M. T. Bottero. 2010. A novel multiplex SNP-based method for identifying the epidemic clones of Listeria monocytogenes. Appl. Environ. Microbiol. 77:6290-6294.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Activities during the period included conducting and analyzing experiments on the long-term-survival phase of Listeria monocytogenes and molecular subtyping of E. coli O157:H7, Listeria monocytogenes, Methicillin-Resistant Staphylococcus aureus, and the top ten serovars of Salmonella. Drs. Dudley and Knabel co-mentored a M.S. student working on molecular subtyping of E. coli O157:H7 and also a M.S. student working on molecular subtyping of Salmonella. Dr. Knabel mentored a masters degree student and a post-doctoral scholar who conducted research on molecular subtyping of Listeria monocytogenes. He also mentored an undergraduate honors student on a research project on molecular subytping of Methicillin-Resistant Staphylococcus aureus. Dr. Knabel's students presented their research results at the ABASM, IFT, ASM and IAFP conferences. Dr. Knabel collaborated with the the Food Safety and Inspection service of USDA and the Public Health Agency of Canada on research projects involving subtyping of Listeria monocytogenes. Drs. Dudley and Knabel collaborated with the Centers for Disease Control and Prevention on a research project involving subtyping clinical strains of the top ten serovars of Salmonella. Dr. Dudley presented the results of this research at the CRISPR conference in Berkeley, California and Dr. Knabel also presented this to researchers at Iowa State University and at the National Animal Disease Center/National Veterinary Services Laboratory in Ames, Iowa. Dr. Knabel shared his expertise on detection and tracking of foodborne pathogens while a member of the National Advisory Committee of Microbiological Criteria for Foods and used his experts in this area to co-author NACMCF and IFT publications. One of Dr. Knabel's graduate students took a research position on detection of foodborne pathogens with SDIX, Inc. after she graduated. PARTICIPANTS: Dr. Knabel was the PI of this project and either advised or co-advised all of the undergraduae and graduate students and the post-doctoral scholar on this project. In all cases, except for the M.S. student on the E. coli O157:H7 project, the students and post-doctoral scholar conducted their research in his laboratory. Dr. Knabel either advised or co-advised all students and the post-doctoral scholar working on this project. Dr. Dudley along with Dr. Knabel co-advised two M.S. students on this project. One student worked on the E. coli O157:H7 subtyping project and one student worked on the Salmonella subtyping project. The E. coli O157:H7 research project was conducted in Dr. Dudley's laboratory. Dr. Anantheswaran along with Dr. Knabel co-advised a Ph.D. student working on the long-term-survival phase in Listeria monocytogenes. Dr. Knabel also advised a visiting scientist from the University of Turin, Valentina Allesandria, during the duration of this project. This visiting scientist used our new molecular subtyping methods to subtype strains of Listeria monocytogenes that were isolated from various foods in Italy. Collaborators included Dr. Peter Evans at FSIS USDA, who worked on the Listeria monocytogenes subclone subtyping project; Dr. Peter Gerner-Smidt and Dr. Efrain Ribot at CDC who worked on the Salmonella subtyping project and Dr. Matthew Gilmour and Aleisha Reimer at the National Microbiology Laboratory/Public Health Agency of Canada who worked on the Listeria monocytogenes epidemic clone subtyping project. Dr. Bindhu Verghese was my post-doctoral scholar during the period and worked on all aspects of the Listeria monocytogenes subtyping projects. Nathan Scwhalm III was an undergraduate Scheyers Honors Scholar who did his honors thesis project on subytpying of Methicillin-Resistant Staphylococcus aureus in my laboratory. TARGET AUDIENCES: Research conducted during the period focused on development of novel methods for tracking and controlling foodborne pathogens such as E. coli O157:H7, Listeria monocytogenes, Salmonella and Methicillin-Resistant Staphylococcus aureus. All of these pathogens are potentially hazardous for all consumers, but are especially dangerous and life-threatening for those individuals that are immunocompromised, such as infants, the elderly, AIDS patients, cancer patients, etc. Results of this research were incorporated into various courses taught by Drs. Dudley, Anantheswaran and Knabel in the Food Science Department at Penn State University. The results of these novel methods were especially integrated into the Advanced Food Microbiology Course taught by Dr. Knabel. Results of this research were also shared at various short courses in the Department of Food Science, such as the Food Microbiology Short Course and the Penn State Ice Cream Short Course, and also with the Eastern Meat Packers Association during their annual meeting at State College, PA. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The discovery of the long-term-survival phase in Listeria monocytogenes and its subsequent increased resistance to various processes has many scientists looking at whether this could explain the persistance of Listeria monocytogenes in food processing environments and foods. This manuscript was published. The Food Safety and Inspection Service of USDA is now interested in using our novel molecular subtyping approach to track subclones of Listeria monocytogenes back to individual meat and poultry processing plants. This paper has been submitted for publication. The Public Health Agency of Canada is currently using our novel molecular subtyping methods to track the transmission of a new epidemic clone and subclone throughout Canada. The manuscript describing this advancement is currently in preparation. The Centers for Disease Control and Prevention has sent us outbreak isolates of Salmonella Enteritidis to test using our new subtyping method, because their current method (PFGE) could not differentiate these isolates. Two manuscripts describing the development and application of this new sequence-based method are currently in preparation. Presentation of our new method, which is based on Clustered Regularly Interspaced Palindromic Repeats, has been received with great interest by scientists in academia and government, who may be using this new method themselves in the near future to subtype various foodborne pathogens. It is anticipated that the above improvements in molecular subtyping will result in more accurate tracking and thus more effective control of foodborne pathogens in the near future.
Publications
- Floros, J. D., et al. 2010. Feeding the world today and tomorrow: The importance of food science and technology. Comprehensive Reviews in Food Science and Food Safety 9:573-599.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: During the Food Microbiology Short Course in May of 2008 Drs Knabel, Dudley, Cutter and Laborde presented lectures on various aspects of detection, tracking and control of foodborne pathogens. Dr. Knabel also led an activity where the participants had to interpret molecular subtyping results in a mock court case, so that they gained an appreciation for how pathogens could be traced to their company. Dr. Knabel made two presentations at the Pittsburgh CASA Conference, one on Rapid Methods for Detection of Foodborne Pathogens and one on Tracking and Controlling Listeria monocytogenes. Dr. Knabel participated in NACMCF on a subcommittee charged with reviewing issues related to novel technologies used by FSIS for detecting foodborne pathogens. Dr. Knabel also collaborated with Dr. Peter Evans at FSIS on a project to subtype L. monocytogenes ECII strains isolated by FSIS from RTE meat plants. Two of Dr. Knabel's students presented seminars at the Microbiologists at Penn State Meeting on their research projects related to molecular methods for tracking pathogens. One of Dr. Knabel's and Dr. Anantheswaran's students gave an oral presentation at the Graduate Oral Paper Competition at the annual ASM Conference on his research into the long-term-survival phase of Listeria monocytogenes and its affect on heat and pressure resistance. One of Dr. Dudley's and Dr. Knabel's students gave a poster presentation on his research on molecular subtyping of E. coli O157:H7 using rhs elements. PARTICIPANTS: Dr. Knabel served as the PI on this project and interacted closely with the co-PIs Dr. Anantheswaran and Dr. Dudley on this project. Dr. Anantheswaran and Dr. Knabel co-advised a Ph.D. student, Jia Wen, on research involving the effect of the long-term-survival phase (LTSP) in L. monocytogenes on resistance to high pressure and temperature. Dr. Anantheswaran, Dr. Dudley and Dr. Knabel are currently collaborating with Dr. Wei Zhang at the National Center for Food Science and Technology on this project to measure mRNA expression during transition to the LTSP. Dr. Dudley and Dr. Knabel co-advised a M.S. student, Kuanqing Liu, on a project involving molecular subytping of E. coli O157:H7 using rhs elements. Dr. Dudley and Dr. Knabel also mentored a postdoctoral scholar working in Dr. Knabel's laboratory on a project involving the development of novel sequence-based methods for molecular subtyping of MRSA. He also mentored a visiting scientist from the University of Turin in Italy, Dr. Sara Lomonaco, who conducted research to confirm the epidemiological relevance of MVLST, which was invented in Dr. Knabel's laboratory. Dr. Knabel also co-advised an Honors Student, Nathan Schwalm on a project related to development of a novel multiplex PCR method for differentiating clonal clusters of MRSA and collaborated with clinicians at the Milton S. Hershey Medical Center on this project. Drs. Dudley and Knabel also co-advised a M.S. student, Fenyun Liu, who is developing a sequence-based molecular subtyping method for Salmonella and are collaborating with scientists at CDC on this project. Dr. Knabel is also advising a M.S. student working on a project on molecular subtyping of L. monocytogenes and is currently collaborating with Dr. Yi Chen at FDS CFSAN and Dr. Peter Evans at USDA FSIS on this project. All of the above students are looking to pursue advanced careers in microbial food safety and/or control of infectious diseases. TARGET AUDIENCES: One of Dr. Knabel's technicians, a minority student, is currently doing an internship at FSIS in the area of policy development. Two of Dr. Knabel's current technicians are female undergraduates in the Department of Food Science at Penn State University. Research on tracking and controlling foodborne pathogens will especially enhance food safety for those individuals that are infants, the elderly and those that are immuno-compromised, as these populations are most at risk for illness and death due to a foodborne illness. Drs. Knabel, Anantheswaran and Dudley have integrated the research presented in this reported into their undergraduate and graduate courses that they teach, and into the Food Science Short Courses that they are involved in. One excellent example is the recent Food Science Camp for high school students that was held in the Department of Food Science. Participants learned how to investigate a foodborne illness outbreak and prevent future outbreaks. Participants also participated in a mock trial at the end to see how a foodborne illness outbreak can negatively impact both the effected people and food companies that are responsible. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Participants at the Penn State Food Microbiology Short Course demonstrated a significant knowledge gain in terms of how to detect, track and control foodborne pathogens. The NACMCF subcommittee that Dr. Knabel worked on got their report on new technologies for detection of foodborne pathogens by FSIS approved by the NACMCF full committee. This report will eventually be published in the Journal of Food Protection and will be available to various federal agencies and all interested scientists in the field. Dr. Knabel will share the results of a novel method that was developed in his laboratory which can very accurately differentiate clones of ECII with FSIS. This will allow FSIS to track these dangerous clones so that outbreaks can be stopped sooner and future ones prevented. This will result in a change of action on the part of federal food safety agencies and the meat industry in general, as they will be able to now "see" for the first time where these clones are located and how they are transmitted between and within RTE meat and poultry processing plants.
Publications
- Hayman, M.M., G.K. Kouassi, R.C. Anantheswaran, J.D. Floros, and S.J. Knabel. 2008. Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing. Int. J. Food Microbiol. 10:21-26.
- Hayman, M.M., R.C. Anantheswaran, and S.J. Knabel. 2008. Heat shock induces barotolerance in Listeria monocytogenes. J. Food Prot. 71:426-430.
- Wen, J., R. Anantheswaran and S.J. Knabel. 2009. Changes in barotolerance, thermotolerance and cellular morphology throughout the life cycle of Listeria monocytogenes. Appl. Environ. Microbiol. 75:1581-1588.
- Liu, K., S.J. Knabel, and E.G. Dudley. 2009. Rearrangement hot spot (rhs) genes are potential markers for multilocus sequence typing of Escherichia coli O157:H7. Appl. Environ. Microbiol. 75:5853-5862.
- Chen, Y. and S. J. Knabel. 2009. Molecular Epidemiology of Foodborne Pathogens. In, Genomics of Foodborne Pathogens, W. Zhang and M. Weidmann (eds.). Springer, NY, USA. (In Press).
- NACMCF New Technologies Subcommittee (includes S. J. Knabel). 2009. Response to the questions posed by the Food Safety and Inspection Service regarding determination of the most appropriate technologies to adopt for FSIS routine and baseline microbiological analysis. J. Food Prot. (In Press).
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Dr. Knabel is currently collaborating with Dr. Wei Zhang at the National Center for Food Safety and Technology to develop a DNA microarray approach for studying gene expression in Listeria monocytogenes in order to better understand how this organism transitions to a long-term survival stage. Dr. Knabel collaborated with Dr. Edward Dudley in the Food Science Department to apply new DNA-sequence-based subtyping technologies developed for L. monocytogenes to also track E. coli O157:H7 in dairy processing plants. Three graduate students worked on this project during this period and information on this project was also disseminated to undergraduate and graduate students taking courses on food microbiology, including Food Microbiology, Fundamentals of Food Science, Epidemiology and Molecular Epidemiology. Two visiting scientists from Italy worked on this project and one technician worked on this project in our laboratories is an African-American. Information from this project was also disseminated to participants in short courses sponsored by the Department of Food Science, including the Penn State Sanitation Short Course and the Food Microbiology Short Course. Target audiences also included food industry associations such as the Eastern Meat Packers Association, the Pennsylvania Association for Food Protection, and the Institute of Food Technologists. Additional audiences included government associations such as the Central Atlantic States Association of Food and Drug Officials and the National Advisory Committee for the Microbiological Criteria for Foods (NACMCF). PARTICIPANTS: Dr. Stephen J. Knabel, PI, was responsible for development of DNA-sequence-based molecular subtyping methods for tracking zoonotic pathogens. Dr. Edward Dudley served as a co-advisor to a M.S. student working on developing a DNA-sequence-based molecular subtyping strategy for tracking E. coli O157:H7. A visiting scientist from Italy worked on this project and one technician working on this project in our laboratories is an African-American. Dr. Sara Lomonaco, a visiting scientist from the University of Turin, Italy, conducted research on additional virulence genes in L. monocytogenes to confirm the epidemiologic relevance of MVLST. Dr. Wei Zhang at the National Center for Food Safety and Technology is collaborating with Dr. Knabel to develop a DNA microarray approach to study gene expression in L. monocytogenes during the transition to the long-term survival phase. Bindhu Verghese is a post-doctoral scholar who is attempting to develop sequence-based molecular subtyping methods for subtyping strains of Methicillin-resistant Staphylococcus aureus (MRSA). Nathen Schwalm, an undergraduate in the BMB Department is an Honor's student working on developing a multiplex PCR method for rapidly screeening MRSA. TARGET AUDIENCES: Target audiences included graduate students working on this project and undergraduate and graduate students taking courses on food microbiology, including Food Microbiology, Fundamentals of Food Science, and Advanced Food Microbiology. Target audiences also include participants in short courses sponsored by the Department of Food Science, including the Penn State Sanitation Short Course and the Food Microbiology Short Course. Target audiences also include food industry associations such as the Eastern Meat Packers Association, the Pennsylvania Association for Food Protection, and the Institute of Food Technologists. Target audiences also include government associations such as the Central Atlantic States Association of Food and Drug Officials and the National Advisory Committee for the Microbiological Criteria for Foods (NACMCF). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Dr. Knabel collaborated with Dr. Peter Evans at USDA FSIS and Dr. Todd Ward at USDA ARS on a project involving molecular subtyping of potential Epidemic Clone II Listeriais monocytogenes isolates that FSIS isolated during routine screening. Using the MVLST and multiplex PCR methods developed in his laboratory, Dr. Knabel was able to show that strains of ECII are being routinely isolated from Ready-to-Eat meat processing plants in the Northeast by FSIS. These results will help FSIS improve there risk-based inspection programs. Dr. Knabel's Visiting Scientist, Dr. Sara Lomonaco, and Ph.D. student, Dr. Yi Chen, analyzed prophages and 6 more virulence genes and 3 more virulence gene regions within L. monocytogenes using prophage sequencing and Multi-Virulence-Locus Sequence Typing (MVLST). The results identified additional single nucleotide polymorphisms (SNPs) in L. monocytogenes and confirmed that prophage sequences and MVLST possess perfect epidemiological relevance. Dr. Knabel is currently collaborating with Dr. Teresa Bottero at the University of Turin to develop a multiplex Primer Extension Reaction (PER)method to rapidly screen for epidemic clones in foods. These finding will allow scientists and food processors to use MVLST and prophage sequences and the novel SNPs within these genes. These can then be used to very accurately track the routes of transmission of epidemic clones and outbreak clones of L. monocytogenes within dairy processing plants and then implement effective intervention strategies to prevent contamination of ready-to-eat dairy and meat products. Dr. Knabel is also currently collaborating with Dr. Bottero, Sara Lomonaco, and Dr. Yi Chen, Research Microbiologist, Microbial Methods Development Branch, Division of Microbiology, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, FDA to analyze sequence data to better understand the phylogeny of Listeria monocytogenes.
Publications
- Chen, Y. and S. J. Knabel. 2008. Prophages in Listeria monocytogenes contain single nucleotide polymorphisms that differentiate outbreak clones within epidemic clones. J. Clin. Microbiol. 46:1478-1484.
- Chen, Y. and S. J. Knabel. 2008. Strain Typing. In Handbook of Listeria monocytogenes, Dongyou Liu (Ed.). CRC Press. New York. pages 203 - 240.
- Lomonaco, S., Y. Chen and S. J. Knabel. 2008. Analysis of additional virulence genes and gene regions in Listeria monocytogenes confirms the epidemiological relevance of multi-virulence-locus sequence typing. J. Food Prot. 71(12):2559-2566.
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