Progress 04/01/03 to 03/31/08
Outputs
OUTPUTS: Results of research on high pressure processing and molecular subtyping were presented at the Annual Meetings of the Institute of Food Technologists and the American Society for Microbiology. Results of multi-virulence-locus sequence typing were shared with scientists from USDA FSIS and ARS. As a result, we are now collaborating with FSIS scientists to analyze suspect L. monocytogenes isolates in our laboratory using multiplex PCR, MVLST and prophage sequencing to determine if the isolates are epidemic clones and outbreak clones of this pathogen. A lecture on MVLST was presented by one of my Ph.D. students in my Microbial Diversity course at Penn State. A seminar on the mechanism of inactivation by high pressure processing was presented by another of my Ph.D. students at the Microbiologists at Penn State Meeting. Both of these Ph.D. students graduated from Penn State in 2007. The results of our molecular subtyping project were disseminated to the Eastern Meat Packer
Association. The attendees were cautioned about the potential hazard of Epidemic Clone II Listeria monocytogenes in meat plants in the Northeastern U.S.
PARTICIPANTS: Partner Organization: Scientists at the USDA Food Safety Inspection Service and Agricultural Research Service are currently collaborating with me on the analysis of Listeria monocytogenes isolates that have been collected from Northeast U.S. meat plants during normal surveillance testing. Collaborators and contacts: My collaborators and contacts (above) are Dr. Peter Evans (FSIS) and Dr. Todd Ward (ARS). Dr. Edward Dudley, a new Assistant Professor in the Food Science Department, is now collaborating with me on the new Milk Safety Project to develop a similar DNA-based subtyping scheme for tracking E. coli O157:H7. We are currently co-advising a graduate student on this project. Dr. Cynthia Whitener, Co-Chair of Clinical Affairs at Hershey Medical Center, is currently collaborating with me to develop a similar subtyping scheme for Methicillin-Resistant Staphylococcus aureus. I am currently co-advising (with Dr. Phillip Mohr in the Biochemistry and Molecular Biology
Department) an honors student on this project. Dr. Peter Hudson, Director of the Huck Institute is currently seeking funds for Dr. Whitener and I to support a postdoctoral scholar on this project. Training or professional development: A visiting scientist from the University of Turin, Italy is currently working in my laboratory to learn about our new DNA-sequence-based subtyping methods.
TARGET AUDIENCES: This project supported 4 graduate students during the period, two of which were females. The project also supported a technician in my laboratory who is an African-American. The project also supported an honors student in my laboratory who was from an economically disadvantaged family in the coal-mining region of Pennsylvania. Efforts. The results of our HPP research were presented to members of the Center for Food Manufacturing and to the Microbiologists at Penn State. The results of our molecular subtyping research were presented to the Eastern Meat Packers Association and to my Microbial Diversity class.
Impacts
Multi-virulence-locus sequence typing and sequencing of prophage genes has revolutionized molecular subtyping of Listeria monocytogenes. These DNA-sequence-based methods yield almost perfect discriminatory power and epidemiological concordance, the two most important criteria in the field of molecular epidemiology. FSIS scientists have acknowledged the superiority of these novel methods, which is why they are now sending us isolates to analyze to see if they are epidemic clones and outbreak clones of this important pathogen. These new techniques will allow food industries and governmental agencies to track L. monocytogenes both between and within food processing plants. This will clarify the routes by which these dangerous clones are transmitted to foods, which will then allow application of more effective intervention strategies to prevent contamination of foods and thus prevent foodborne disease. MVLST data were utilized in a major court case to demonstrate that
epidemic clone II L. monocytogenes was present in two different processing plants associated with a major outbreak of listeriosis and thus both plants were held responsible. Research demonstrated that growth temperature, growth phase, heat shock and water activity all significantly affect the destruction of L. monocytogenes during high pressure processing. Food industries that utilize this new non-thermal technology need to take these factors into account to ensure destruction of this pathogen during high pressure processing.
Publications
- Sawant, A. A., N. V. Hegde, B. A. Straley, S. C. Donaldson, B. C. Love, S. J. Knabel, and B. M. Jayarao. 2007. Antimicrobial-resistant enteric bacteria from dairy cattle. Appl. Environ. Microbiology 73:156-163.
- Hayman, M. M., R. C. Anantheswaran and S. J. Knabel. 2007. The effects of growth temperature and growth phase on the inactivation of Listeria monocytogenes in whole milk subject to high pressure processing. Inter. J. Food Microbiol. 115(2):220-226.
- Chen, Y., W. Zhang, and S. J. Knabel. 2007. Multi-Virulence-Locus Sequence Typing identifies single nucleotide polymorphisms that differentiate epidemic clones and outbreak strains of Listeria monocytogenes. J. Clin. Microbiol. 45:835-846.
- Chen, Y and S. J. Knabel. 2007. Multiplex PCR for simultaneous detection of bacteria of the genus Listeria, Listeria monocytogenes, and major serotypes and epidemic clones of L. monocytogenes. Appl. Environ. Microbiol. 73:6299-6304.
- Hayman, M. M., R. C. Anantheswaran and S. J. Knabel. 2007. Heat shock induces barotolerance in Listeria monocytogenes. J. Food Prot. (In Press)
- Hayman, M. M., G. K. Kouassi, R. C. Anantheswaran, J. D. Floros and S. J. Knabel. 2007. Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing. Inter. J. Food Microbiol. (Pending)
- Chen, Y. and S. J. Knabel 2007. Prophages in Listeria monocytogenes contain single nucleotide polymorphisms that differentiate outbreak clones within epidemic clones. J. Clin. Microbiol. (Pending)
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Progress 01/01/06 to 12/31/06
Outputs
A recently developed multi-virulence-locus sequence typing (MVLST) method showed improved discriminatory power for subtyping genetically diverse Listeria monocytogenes isolates and allowed identification of an epidemic clone II among isolates associated with two recent U.S. multistate listeriosis outbreaks. To evaluate the ability of MVLST to distinguish other epidemic clones and outbreak strains of L. monocytogenes a total of 58 isolates from 14 outbreaks and 49 non-outbreak isolates were analyzed. Results showed that MVLST provided very high discriminatory power (D = 0.99), epidemiological concordance (E = 1.0), stability and typeability. MVLST allowed accurate identification of three previously known epidemic clones (epidemic clone I, II and III) and suggested another epidemic clone (epidemic clone IV) in serotype 4b of L. monocytogenes. A set of 28 single nucleotide polymorphisms (SNPs) allowed satisfactory differentiation of epidemiologically unrelated isolates.
A subset of 16 SNPs differentiated all 4 epidemic clones and outbreak strains. Phylogenetic analysis showed congruence between MVLST clusters, serotypes and previously defined genetic lineages of L. monocytogenes. Work is in progress to identify SNPs in other hypervariable genes that can differentiate outbreak strains within epidemic clones. SNPs in virulence genes appear to be excellent molecular markers for the epidemiological investigation of epidemics and outbreaks caused by L. monocytogenes. A multiplex PCR scheme was developed which can differentiate all 4 epidemic clones. Another multiplex PCR scheme is being developed to detect Listeria spp, L. monocytogenes, serotypes 4b and 1/2a. The end goal is to develop methods that can first screen for the presence of L. monocytogenes and determine its serotype and whether it is an epidemic clone. If multiplex PCR indicates it is an epidemic clone or outbreak strain then SNP typing will be used to confirm this. The combination of
multiplex PCR screening and confirmation via SNP typing will provide food, government and academic laboratories with a comprehensive molecular strategy for detection and control of L. monocytogenes.
Impacts
Listeriosis due to contamination of foods with Listeria monocytogenes is a significant health risk to humans and a significant financial risk to food companies. Most cases of listeriosis are caused by a small subset of L. monocytogenes strains known as epidemic clones. Listeria monocytogenes contamination in food plants is very widespread and includes numerous strains that are not epidemic clones. Therefore, molecular methods that can identify and track the epidemic clones are needed. Results from this research have shown that single mutations (SNPs) within virulence genes can be used to very accurately identify and track epidemic clones of Listeria monocytogenes. Also, amplification of other genes, which was also developed in our lab, can also be used to screen food samples rapidly to determine if L. monocytogenes is present. The combination of rapid screening of L. monocytogenes and SNP typing will allow food, government and academic laboratories to accurately track
and control these dangerous epidemic strains of L. monocytogenes.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Multi-virulence-locus sequence typing (MVLST) was applied to isolates from 14 different major listeriosis outbreaks. MVLST results were validated by sequencing two additional virulence genes (actA and inlA). MVLST was able to very accurately identify four epidemic clones: ECI (Canada coleslaw, LA soft cheese, and Switzerland soft cheese), ECII (1998 hot dog and 2002 turkey deli) and ECIII (1989 hot dog and 2000 turkey deli), which had previously been described by Kathariou et al. and a new epidemic clone, ECIV (1979 Boston vegetable and 1989 UK pate). ECI, ECII and ECIV were all serotype 4b in Lineage I and ECIII was serotype 1/2a in lineage II. In all cases the virulence gene sequences were identical within each epidemic clone. Within all outbreaks inlA (2.18%) had the highest percentage of polymorphic sites, followed by actA (2.06%), dal (1.13%), inlC (0.72%), inlB (0.69%), prfA (0.64%) and clpP (0%). In conclusion, MVLST can be combined with PFGE to detect
epidemics and outbreaks and thus can help clarify the epidemiology of listeriosis in the future.
Impacts
The new Multi-Locus Sequence Typing methods that were developed and optimized will help food processors track Listeria monocytogenes epidemic clones of Listeria monocytogenes in food processing plants and thus will allow them to implement effective intervention strategies that prevent contamination of Ready-to-Eat (RTE) foods and reduce the burden of foodborne listeriosis.
Publications
- Chen, Y., Zhang W. and Knable, S. J. 2005. Multi-virulence-locus sequence typing clarifies the epidemiology of two recent listeriosis outbreaks. J. Clin. Microbiol. 43:5291-5294.
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Progress 01/01/04 to 12/31/04
Outputs
A Multi-Virulence-Locus Sequence Typing (MVLST) method was developed, which targeted six virulence genes in Listeria monocytogenes. This molecular subtyping method yielded higher discriminatory power than standard Pulsed-Field Gel Electrophoresis when 28 selected strains of L. monocytogenes were analyzed. The MVLST method is currently being optimized to reduce time and cost of analysis, while maintaining high dicriminatory power. The BAX for Screening Listeria monocytogenes PCR product was cloned and sequenced. The 423 bp product was found to be a partial putative gene Imo2234, which was most closely related to a gene encoding a sugar phosphate isomerase/epimerase. Studies are ongoing to develop a MSLT strategy for subtyping all Listeria species, which will allow processors to track the transmission of Listeria species in their processing plants. High pressure treatment of Listeria monocytogenes in milk indicated that cells recovered in optimized Penn State University
broth, but not in U.S. Food and Drug Administrations Listeria Enrichment Broth immediately after high pressure processing. In addition, injured cells recovered rapidly in milk when stored at 4C and subsequently grew rapidly during temperature abuse. Further studies are underway to understand the mechanisms by which Listeria monocytogenes are injured during high pressure processing and their kinetics and mechanisms of recovery. Studies are also being conducted to develop a multi-hurdle strategy for destroying or inhibiting Listeria monocytogenes in ready-to-eat meats and dairy foods.
Impacts
The new Multi-Locus Sequence Typing methods that are being developed and optimized will help food processors track both Listeria species and Listeria monocytogenes in food processing plants and thus will allow them to implement effective intervention strategies that prevent contamination of Ready-to-Eat (RTE) foods. The multi-hurdle strategy being developed will destroy or prevent the growth of any Listeria monocytogenes that may contaminate ready-to-eat meats during packaging. Both of these intervention strategies are expected to reduce the incidence of listeriosis in ready-to-eat foods, including dairy and meat products.
Publications
- Zhang, W., Jayarao, B.M., and Knabel, S. J. 2004. Multi-virulence-locus sequence typing of Listeria monocytogenes. Appl. Environ. Microbiol. 70:913-920.
- Zhang, W., Hughes, A., Wilt, G., and Knabel, S. J. 2004. The BAX PCR assay for screening Listeria monocytogenes targets a partial putative gene Imo 2234. Journal of Food Prof. 57:1507-1511.
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Progress 01/01/03 to 12/31/03
Outputs
A multi-virulence-locus sequence typing (MVLST) method was developed for subtyping Listeria monocytogenes by analyzing six virulence or virulence-associated genes (prfA, inlB, inlC, dal, lisR, and clpP). This method was optimized by developing a multiplex PCR assay. Comparison of MVLST to pulsed-field gel electrophoresis (PFGE), ribo typing (RT) and multilocus sequence typing (MLST) methods demonstrated that MVLST provided the highest discriminatory power for differentiating L. monocytogenes strains. Using a modified drill press and a cutting mechanism to prevent cross contamination, Escherichia coli O157:H7 cells were found to penetrate up to 3,800 um into apple tissue immediately after a 5,000 um puncture, but no penetration was observed if E. coli cells were added 48 h after puncture. Cells remained within the boundaries of the puncture. Population of the cells increased by approximately 3 log cfu/g within a fresh puncture after 48-h incubation at 21oC. This is
likely due to the release of moisture and nutrients within punctures and cuts, but not large amounts of inhibitory malic acid, which is located in vacuoles within the apple tissues. E. coli O157:H7 penetrated 8,400 um into the open calyx of apples, but only 5,600 um into apples with a closed calyx. Scanning electron microscopy revealed that E. coli O157:H7 cells preferentially associated themselves within the grooves on the stem and on the flower remnants on the calyx region. It was observed that over a 3-h period, E. coli O157:H7 cells continued to accumulate on the stem after which, they reached a maximum level and did not increase significantly. Significant number of cells was recovered from the stem after only 30 sec inoculation. This indicated that cells are immediately drawn toward the stem upon submersion in an inoculum. Rinsing the stem in deionized water (control) resulted in approximately 0.2 log cfu reduction whereas; treatment with Trisodium Phosphate (pH 12.3) resulted in
additional 0.6 log reduction of the cells. It was also observed that efficiency of recovery of E. coli O157:H7 from the stem was reduced from 94% after 30 sec inoculation to almost 70% after 3h. It was hypothesized that firm attachment of the cells to the stem over time, may result in lower efficiency of recovery during sample preparation. This firm attachment may also contribute to resistance of the cells to treatment. Lower efficiency of recovery may result in underestimating the extent of bacterial contamination over time. Since the stem remained in the inoculum solution throughout the study and was removed only during sampling times, desiccation of the bacterial cells due to drying was ruled out. Hydrophobic or hydrophilic polystyrene beads that are either positively or negatively charged were allowed to associate themselves with the apple parts. Preliminary data indicated that hydrophobic-negatively charged beads preferentially associate with discontinuities or cut surfaces of
apple.
Impacts
The new method developed (Multi-locus Sequence Typing) will help food processors track Listeria monocytogenes in food processing plants and thus implement intervention strategies to prevent contamination of Ready-to-Eat (RTE) foods and prevent listeriosis. Understanding how foodborne pathogens contaminate fruit and vegetable products will help food scientists develop methods to prevent contamination of these RTE foods and increase the efficiency of decontamination treatments.
Publications
- No publications reported this period
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