Overview:
Kathryn Boor’s research program focuses on determining factors that affect the presence and persistence of pathogenic and spoilage organisms in human food products. The specific goals of her research program are to identify and characterize bacterial mechanisms that affect survival and transmission of foodborne microbes, including Listeria monocytogenes and spore-forming bacteria, throughout food systems. Her specific objectives are to integrate the tools of molecular biology and phenotypic microbiology to: (i) identify and characterize genetic factors that link the ability of bacteria to survive under various conditions, including in foods and in food processing environments, with their ability to cause human and animal disease; and (ii) rapidly identify and track spoilage and pathogenic bacteria in food systems. Research results associated with objective (i) have improved our understanding of specific cellular mechanisms that contribute to bacterial survival under widely varying environmental conditions. The latter objective directly links her research program with her extension program, in that projects undertaken are driven by current and future food industry needs. Dr. Boor’s major research accomplishments in tracking bacterial contaminants in food processing systems also have been used to generate focused educational opportunities for the dairy industry.
Background:
Pathogenesis of foodborne diseases can involve complex interactions between a bacterial pathogen, a variety of environments and one or multiple host species. As a consequence, foodborne pathogens provide ideal model systems for studying bacterial cell adaptation, survival, and maintenance of virulence characteristics under widely varying conditions, including those not directly associated with a host. Pathogenesis of bacterial diseases transmitted from animals through foods to humans may encompass all or some of the following steps: (i) bacterial survival in animal feeds or in the animal’s environment (e.g., Listeria monocytogenes and Salmonella spp. are common environmental contaminants); (ii) establishment of clinical or subclinical infections in food animals; (iii) shedding of the organism into animal products used for human consumption (e.g., milk) or contamination of the product during slaughter (e.g., meat products); (iv) bacterial survival and/or multiplication under food processing and distribution conditions; and (v) infection of human hosts, including survival of gastric passage and establishment of enteric or systemic infections. Microbial survival under rapidly changing environmental conditions thus plays a key role in the pathogenesis of many foodborne diseases. To further illustrate, microorganisms causing human disease by the oral route must survive passage through the hostile and acidic environment of the stomach. Some intracellular foodborne pathogens such as L. monocytogenes must also survive inside eukaryotic vacuoles following bacterial invasion of the host cell. Defense systems of the host cell vacuole (e.g., reactive oxygen radicals, acidic pH) pose significant stresses on bacterial cells. Most foodborne pathogens also need to survive and multiply under stressful conditions (low pH, refrigeration temperatures) in foods.
Research in Dr. Boor’s laboratory addresses the hypothesis that bacterial stress-response systems provide a crucial link between bacterial survival under various environmental conditions and the ability of the organism to cause disease. One means by which bacterial cells respond to environmental changes is through rapid changes in gene expression patterns mediated through transcriptional regulatory proteins. The stress-responsive alternative sigma factors σB and RpoS transcribe genes contributing to bacterial survival under environmental stress conditions in Gram-positive and Gram-negative bacteria, respectively, therefore, Dr. Boor selected the σB and RpoS alternative sigma factors as key target proteins for investigating the survival of bacterial pathogens under various environmental conditions. An improved understanding of the ability of bacteria to sense, respond, and survive rapid and extreme changes in environmental conditions through rapid changes in the regulation of gene expression will subsequently contribute to our understanding of factors that influence bacterial persistence and transmission in food systems.
CURRENT RESEARCH
Objective (i):
Identification and characterization of factors linking the ability of bacteria to survive under various conditions, including in foods and in food processing environments, with the ability of bacteria to cause human and animal disease.
• In collaboration with Dr. Martin Wiedmann, Dr. Boor’s research team has focused on the alternative sigma factor, σB, in Listeria monocytogenes. The collaborative team pioneered discovery of the functional characteristics of the L. monocytogenes sigma B protein; including identification of the regulon of genes controlled by sigma B; as well as some critical elements of the regulatory pathway that tightly control the activity of σB. A particularly exciting discovery was the identification of genetic linkages between the stress responsive sigma B and the ability of L. monocytogenes to invade host cells (i.e., σB contributions to inlA expression). Specifically, σB was identified as a positive direct regulator of virulence gene expression (inlA, inlB, bsh, prfA and others) in L. monocytogenes. Thus their work indicates that σB provides an important link between the ability of L. monocytogenes to respond to stressful environmental conditions and its ability to infect mammalians hosts. Their findings also suggest that multiple regulatory elements function in concert to control expression of genes contributing to transmission and virulence of foodborne bacterial pathogenes. Dr. Boor’s work currently tests the following hypotheses: (i) networks among key L. monocytogenes regulatory proteins (e.g., σB and PrfA) are necessary for appropriate expression of genes critical for pathogen transmission and virulence in different extra- and intra-host environments and (ii) multiple regulatory networks and stress response systems, including σB and the σB regulon, contribute to transmission and pathogenesis.
Objective (ii):
Rapid identification and tracking of spoilage and pathogenic bacteria in food systems.
Research in the Boor Lab has identified bacteria that can survive milk pasteurization and then increase in number under refrigeration temperatures as the biological barrier that currently limits conventional High-Temperature-Short-Time (HTST) pasteurized fluid milk product shelf-life. Results described in Huck et al. (2007) demonstrate that spore-forming bacteria, particularly Paenibacillus spp., are present in the milk processing continuum, fromthe raw milk tank truck loads that enter processingplants to the final packaged products. Their presence in both raw and processed milk means that a comprehensive farm-to-table approach will be needed to control these organisms in milk processing.
Anecdotal reports from the dairy industry initiated important research in the Boor laboratory regarding HTST fluid milk processing temperatures. Targeted research showed that the HTST pasteurization temperatures affected bacterial numbers in processed milk during refrigerated storage. Specifically – and counter-intuitively – higher bacterial numbers were found in milk that had been processed at higher temperatures. The predominant bacterial genera isolated from the milk samples did not differ by heat treatment, suggesting that the heat treatments within the range studied (72.9 to 85.2°C) do not preferentially affect a sub-population of the endospore forming bacteria present. We conclude that the endospore-forming psychrotolerant bacteria present in milk grow more effectively after a higher heat treatment. Additionally, Paenibacillus spp., which are likely present at <1 spore per mL of raw milk, are capable of growing to numbers that limit HTST pasteurized milk shelf-life, illustrating the need for a comprehensive strategy to limit the entry of endospore-forming bacteria into milk systems. Ultimately, new processing methods may need to be employed to physically remove psychrotolerant endospore forming bacteria from raw milk. Continued efforts to improve the bacteriological quality of pasteurized milk will require an increased emphasis on limiting bacterial and spore contamination along the farm-to-table continuum.
A recent study conducted by Dr. Boor’s Milk Quality Improvement Program (MQIP) at
Future Research:
Development of a single raw milk assay to predict shelf-life performance of pasteurized milk
The dairy industry has a critical need for appropriate tests that can measure raw milk quality. Our work has demonstrated that the presence of thermotolerant, cold-growing sporeforming microbes is the biological barrier currently limiting shelf-life extension of High Temperature Short Time pasteurized fluid milk. No assay currently exists to quantify the presence of these bacteria in raw milk. Our objective is to develop an assay that will quantify the presence of thermotolerant, cold-growing sporeforming microbes present in raw milk and to measure the presence of these microbes in raw and pasteurized milk samples
Locating entry points of pasteurized milk shelf-life limiting microbes in milk production systems (collaboration with
Our previous work predicts that thermotolerant, cold-growing sporeforming microbes are found in very small quantities in raw milk. Their entry points into raw milk (and into other portions of milk production systems) are currently unknown. Our objective is to identify entry points for shelf-life limiting bacteria in milk production systems.
Future Directions:
Listeria monocytogenes, a foodborne pathogen that can cause serious human disease, is estimated to cause 2,500 cases and 500 deaths annually in the
(1) Define regulons controlled by the L. monocytogenes alternative sigma factors using full genome microarrays.
(2) IdentifyL. monocytogenes proteins that co-regulate genes contributing to transmission and virulence using selected mutant bacterial strains, microarrays, qRT-PCR and bioinformatics strategies.
(3) Characterize global L. monocytogenes gene expression patterns under different environmental stress conditions and in selected intracellular and intra-host environments using microarrays and qRT-PCR.
(4) Develop a WWW-based database of L. monocytogenesmicroarray data and transcriptional profiles.
(5) Characterize stress response and virulence phenotypes of L. monocytogenes with mutations in selected genes encoding regulatory proteins and in selected genes regulated or co-regulated by alternative s factors and other transcriptional regulators.
Overall, the proposed studies will provide an improved understanding of the mechanisms used by foodborne pathogens to regulate gene expression under rapidly changing environmental stress conditions encountered during transmission and infection. This knowledge will identify mechanisms that can be targeted for development of novel and innovative bacterial control strategies including new antibacterial therapeutics.
RESEARCH ACCOMPLISHMENTS/COMPLETED PROJECTS
Vibrio parahaemlyticus characteristics and detection
Consumption of sufficiently high numbers of virulent strains of the Gram-negative Vibrio parahaemolyticus can cause gastroenteritis. Human infection with this pathogen is most frequently associated with the consumption of seafood, primarily raw or improperly cooked shellfish. Fortunately, not all strains of V. parahaemolyticus are pathogenic, however, distinguishing between virulent and non-virulent strains has been a technical challenge. In February 1996, the infection rate attributed to V. parahaemolyticus in
Mycobacterium paratuberculosis
In collaboration with scientists in the Cornell College of Veterinary Medicine, Dr. Boor’s team completed a research project to develop improved detection methods for Mycobacterium avium subsp. paratuberculosis, which is an important pathogen in dairy cattle. Although controversial, some believe this organism to be a human food-borne pathogen responsible for Crohn’s disease. Plasmid- and phage-based firefly luciferase reporter constructs were evaluated as rapid detection systems for viable Mycobacterium avium subsp. paratuberculosis (MAP). A MAP strain bearing a luciferase-encoding plasmid was detectable at 100 cells / ml in skim milk and 1000 cells / ml in whole milk. Three luciferase-encoding mycobacteriophage were evaluated for detection of wild-type MAP. The best of these, phAE85, allowed detection of > 1000 cells / ml within 24 – 48 hours. Our work clearly demonstrated that luciferase reporters show promise for rapid detection of viable MAP.
E. coli O157 susrvival in food systems and stress response
Dr. Boor’s research team determined the survival rates of E. coli O157:H7 in dairy fermentation systems, demonstrating the ability of this organism to persist as a post-pasteurization contaminant in commercial dairy products for periods of up to 35 days following initial contamination. This work scientifically disproved the commonly held, but not rigorously tested, belief in the dairy foods industry that starter cultures present in fermented dairy products would not allow the long-term persistence of pathogenic microbes in products with pH values below 4.6. This work received national attention, and was used recently by regulatory officials and other members of the dairy industry as supporting evidence for new rulings on dairy product safety in the 2005 National Conference on Interstate Milk Shipments (NCIMS) biennial meeting.
Dr. Boor’s team cloned and sequenced a key gene responsible for regulating cellular responses to changes in environmental conditions (rpoS) in the Gram negative organism, E. coli O157:H7. As described above for a wild-type E. coli O157:H7 strain, survival characteristics were measured in dairy fermentations for an E. coli rpoS mutant strain, which lacks a functional sigma-S protein. This work demonstrated that the Gram negative RpoS general stress response system (orthologous to the σB system that we identified in the gram positive L. monocytogenes, as described above) clearly plays an important survival role when E. coli O157:H7 is faced with the moderately lethal conditions encountered in the presence of dairy starter cultures. As E. coli O157:H7 (or L. monocytogenes) is exposed to similar moderately lethal conditions upon ingestion by humans, this work builds toward identification of universal characteristics of bacterial survival systems that are not only important to survival in foods, and in the environment, but may also play a role in the ability of an organism to cause disease. In addition to providing valuable information regarding survival mechanisms of foodborne pathogens in dairy products, these studies may ultimately lead to strategies for the reduction of pathogens in food animal populations. Creation of strains lacking these functional proteins, which are likely to be virulence-attenuated, ultimately may provide biological material for future vaccine development
Transmission, subtyping and characterization of spoilage Pseudomonas
Dr. Boor’s team systematically collected and characterized Pseudomonas spp. and other strains associated with dairy product spoilage from NYS dairy processing plants, with the goal of establishing a rapid bacterial contaminant tracking system designed to identify and eliminate contaminant reservoirs, and thus, reduce dairy product spoilage. The genetic “fingerprints” generated from ribotyping were assembled into a database that is accessible on the World Wide Web. Ribotyping was also applied to track and determine the cause of bacterially reduced fluid milk shelf-life in a model dairy processing system with a sole raw milk source. When recommendations for shelf-life extension developed by Dr. Boor’s team were followed, fluid product shelf-life increased from 7 days to 21 days.
L. monocytogenes transmission in different commodities and food systems
• In collaboration with Dr. Martin Wiedmann’s group, Dr. Boor’s team determined the prevalence of L. monocytogenes contamination in salmon processing plants and applied a fingerprinting system based on automated ribotyping to track the origins and the spread of L. monocytogenes strains in these operations.
Dr. Boor’s group applied information learned from the smoked salmon processing research described above to track L. monocytogenes contamination in Latin-style cheese manufacturing operations in NYS. The Hispanic cheese industry is growing rapidly, but the industry is plagued by a high prevalence of L. monocytogenes in consumer products. Our work focused on identifying bacterial contamination reservoirs and developing specific strategies for reducing the risk of contamination.
Characterization of microbial contamination patterns in raw milk and their effects on milk quality
Dr. Boor’s group completed a comprehensive characterization of the bacteriological and chemical characteristics of milk produced on approximately 10% of randomly selected farms in
Dr. Boor’s team worked with a group of NYS dairy farmers to determine the composition of bacteria present in seemingly random high bacteria counts (so-called “spikes”) in raw milk, with the goal of implementing specific control strategies targeting the sources of those bacteria. This work is of critical importance to dairy producers as farmers receive significant monetary incentives for producing milk with low bacterial counts. One “spike” can result in a severe economic penalty for a milk producer. We found that, in the absence of a significant and identifiable error in the milk handling, cooling or cleaning systems, the majority of high bacterial numbers result from the presence of “environmental” mastitis organisms such as Streptococcus uberis. Often, the animal shows no clinical symptoms of an infection, so it can be challenging to detect the presence of these organisms without standard bacterial culturing.
Shelf-life characteristics of flavored milk products, which constitute the most rapidly growing segment of the NYS fluid dairy product market were measured. This work determined that while chocolate milks spoil faster than white milks processed in the same processing plant, the major spoilage organisms in both types of products are the same on a per product/per sampling period basis. Thus, chocolate milk ingredients do not appear to uniformly introduce novel microorganisms nor do chocolate products appear to support the growth of organisms that could not grow in unflavored milks.