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CL 125a Phone: 765-285-8830
Department of Biology
Ball State University
Cooper Life Science Building, CL 121
Muncie, IN 47306
The majority of the past and current research in my laboratory is focused on the development of molecular- based methods for the study of spore-forming pathogenic bacteria in food. DNA and RNA-based approaches are being employed for real-time assessment of contamination levels, bacterial stress responses, and regulation of virulence gene expression in toxigenic Bacillus spp. relevant to dairy science. In particular, current projects center around identification of novel bacterial viruses (phage) from raw milk that infect bacterial spoilers and pathogens; we are also interested in production and prevention of bacterial biofilms by spore-forming bacteria in fluid dairy products, and are just completing a study investigating the presence and expression of regulator and toxin genes in a novel species of Bacillus from ultra-high temperature (UHT) pasteurized milk. This recent work has already been presented at regional and national conferences including the Indiana Branch of the American Society for Microbiology (IBASM), the ASM General Meeting, as well as an invited talk at the 4th International Symposium on Dairy Cow Nutrition and Milk Quality, Beijing, China in May of 2015 (and again invited for the May 2017 Symposium in Beijing, as well).
Earlier in my career at BSU (~2005-2009), I explored various research collaborations with the Departments of Physiology & Health Science, Physics and Astronomy, Mathematical Sciences, Computer Science, Criminal Justice & Criminology, Hamilton County (IN) Sheriff’s Office, Anderson (IN) Sexual Assault Treatment Center, and the Indiana Chapter of the International Association of Forensic Nurses, all of which fostered interactions among my students and expanded my network on and off campus, producing multiple presentations at conferences, an Emmy Award-winning documentary that aired on PBS, grant submissions, and two peer- reviewed publications from these collaborative efforts in forensic science.
I feel that my past and current research objectives are consistent with the mission of our department and the university. That is, I believe in teaching through research, exposing students to relevant literature, opportunities to present their data, write grant proposals, and mentor new students as they come into the lab. This is true for both the more traditional bench research in my laboratory, as well as for students engaged in Fine Focus. As such, I have submitted four substantially large (>$20,000 TDC) research proposals over the
past two years, reflecting current research and teaching interests. I have two publications in press at the present time (early-to-mid 2017 publication date) – a pedagogical perspective article, and a book chapter. Additionally, each of the projects described in the first paragraph above will result in a manuscript for submission to the Journal of Dairy Science by May or June 2017. The undergraduate and M.S. students involved in each specific project will be lead authors.
Work in progress (research underway with M.S. students; undergraduate students also involved to varying degrees):
Project #1 – Discovery of new viruses that infect dairy spoilage bacteria. M.S. student research project with expected completion date 5/2017. Current status = ~ 1⁄2 complete as of 5.20.2016.
Many Bacillus species are able to form spores, and therefore can survive some forms of pasteurization. In this study, soil samples around campus will be screened for novel bacterial-specific viruses, termed phages, which are specific to toxigenic strains of Bacillus. Once phages have been detected, they will be characterized using both SEM and TEM microscopy. They will also be DNA sequenced. These new phages could be used for detection and control of toxigenic strains of Bacillus in dairy foods, where they pose many costly problems during yogurt and cheese production. Using phages as a method for controlling spoilage causing and pathogenic bacteria is an idea that has recently been reintroduced in the dairy field. Furthermore, because of the vast number of unique phages (10!") globally, it is likely that novel phages will be discovered.
Project #2 – Effects of Subinhibitory Carvacrol Levels on Bacillus cereus Virulence During Endophthalmitis In Retinal Pigmented Epithelial Cells (ARPE-19). M.S. student research project with expected completion date 12/2016. Current status = ~90% complete as of 5.20.2016.
Bacillus cereus is one of the major pathogens causing posttraumatic endophthalmitis in humans. B. cereus can result in permanent eye damage within 24 hours after initial infection via production of multiple extracelluar virulence factors. Carvacrol, an antimicrobial essential oil, may be a promising treatment for B. cereus endophthalmitis. Specific events of transcriptional regulation of virulence genes in B. cereus during infection of human retinal pigment epithelial (RPE) cells are not well understood. Our aim is to measure relative levels of regulator and virulence gene expression in SIC carvacrol-stressed B. cereus during infection of laboratory-cultured ARPE-19 cells. Results of this work will elucidate mechanisms of virulence regulation in this increasingly important food and clinical pathogen. ARPE-19 cells were treated with 105 B. cereus alone, and B. cereus + SIC (1mM) of carvacrol. Relative differences in expression of plcR, nprC, hblC, and nheA regulator and virulence gene expression in B. cereus treated with SIC of carvacrol, and untreated B. cereus was quantified by mean cycle threshold (CT) value comparisons using real-time PCR, and statistically analyzed using one-way ANOVA in Minitab17. Furthermore, plcR gene expression was measured through detection of the green fluorescent protein (GFP) production using transformed B. cereus ATCC 14579 with a promoter trapping expression vector (pBAD:plcR:GFP) treated with and without the SIC of carvacrol (1mM). The bacteria were inoculated in ARPE-19 cells and visualized using fluorescence microscopy and results quantified by enumeration of fluorescing bacteria within ARPE-19 cells. Results reveal that Untreated B. cereus invaded ARPE-19 cells significantly less compared to SIC carvacrol-treated bacteria, which revealed full internalization within 48h. GFP (plcR)- expressing B. cereus were detected as fluorescing significantly more under carvacrol conditions compared to control replicates. These data indicate that sublethal chemical stress by carvacrol significantly increases potential virulence of this pathogen, via an increase in the global regulator (PlcR) production.
Project #3 – Bacillus amyloliquefaciens biofilm activity and composition in Ultra-high temperature (UHT) organic milk. M.S. student research project with expected completion date 5/2017. Current status = ~10% complete as of 5.20.2016.
Bacillus spp. are Gram positive rod-shaped bacteria that are common instigators of food-associated illness. Bacillus amyloliquefaciens is known to survive ultra-high temperature (UHT) pasteurization in milk, and may form biofilms on the surfaces in which the milk is stored and/or transported. The UHT process is intended to make transportation of milk easier, as shipment and retail storage can be done without refrigeration. B. amyloliquefaciens may grow and produce biofilm in the storage container and can cause spoilage, reducing shelf life of milk. Once established, a biofilm can produce an extracellular polymeric substance (EPS), which serves as a protective shield against mechanical disruption and direct contact from harmful agents. This makes the biofilm resilient against chemical agents, further contributing to the spoilage issue. The combination of these factors that B. amyloliquefaciens exhibits in the presence of organic milk make it potentially harmful to the dairy industry.
Project #4 – Bacillus amyloliquefaciens biofilm activity and composition in Ultra-high temperature (UHT) organic milk. M.S. student research project with expected completion date 5/2017. Current status = ~10% complete as of 5.20.2016.
Lactic acid bacteria require a modified medium for culture and characterization. This modified medium can be used for hygiene monitoring of starter culture batch fermentations as well as recovering lactic acid bacteria from food samples. Lactic acid bacteria are commonly used in the dairy industry to set up fermentations for dairy products, such as milk, cheese, and yogurt, as well as other fermented foods, including sauerkraut. Some lactic acid bacteria commonly used in these fermentations include Lactobacillus bulgaricus, Streptococcus thermophilus, Lactococcus lactis, Lactobacillus helveticus, and Leuconostoc mesenteroides. The lowering of pH and increased rate of acidification are beneficial to the culture in preventing contamination by Streptococcus pyogenes and other contaminants. The medium used for culturing is also critical for establishing starter cultures for fermentations. While MRS medium is used today for general culturing of lactic acid bacteria, Davidson and Cronin developed a nitrous acid- based medium to selectively isolate lactic acid bacteria from food systems . Nitrous acid with MRS medium (pH to 5.5) may be able to selectively isolate lactic acid bacteria from food samples, with nitrous acid being the selective agent. This modified MRS medium (mMRS) could increase hygiene monitoring of starter cultures on a variety of lactic acid bacteria. The increased hygiene monitoring of starter cultures is needed by the industry due to an increase in governmental regulations. We hypothesize that a nigrous acid-modified MRS medium may be developed as an improved means of quality control and hygiene monitoring for lactic acid bacteria starter cultures in the dairy industry. We propose to utilize a cocktail of known type strain lactic acid bacteria in a series of artificially-inoculated dairy food systems to develop such a medium.
The most unusual teaching platform I have developed is implementation of immersive learning in several courses since my last promotion. First, in 2008, with the BSU Virginia B. Ball Center for Creative Inquiry seminar entitled State of Assault, a student-produced documentary on sexual assault that won an Emmy Award in 2009, and more recently Fine Focus. Since 2013, Fine Focus has been implemented as a product-based course at Ball State University, comprised of 12-24 undergraduates each term from a variety of disciplines. Fine Focus students have developed and manage the first (and only) international digital and print journal for undergraduate microbiology research, with the American Society for Microbiology (ASM) as a community partner. The student review team in this course is responsible for making important decisions that direct the future of the journal, including learning the process of manuscript management through our double-blind peer review system using experts from our external Editorial Board. The marketing/design team is responsible for determining how to best target Fine Focus to our global audience, soliciting manuscripts, promoting Fine Focus through our website and social media, and creating an Executive Committee. This faculty-led, student-driven “immersive learning” model allows the students to be the authors of, and authorities on, their own education.
Full text access: https://cardinalscholar.bsu.edu/handle/123456789/199369
Submission Website: finefocussubmissions.org
Undergraduate students Rachel Pittsley (senior, microbiology) and Natasha Rollings (senior, wildlife biology) present at the Ball State University Student Symposium spring, 2016.
Cassandra Bormann (senior, biology/theatre) presents a Fine Focus polo shirt to guest speaker Dr. Roger Shanks, Emeritus Professor of Animal Science & Editor-in-Chief, Journal of Dairy Science, during a visit to our class last year.
University of Akureyri (Iceland) Fine Focus biotechnology students University of Akureyri (Iceland) Fine Focus biotechnology student Sigþóra Brynja Kristjánsdóttir and others work on various aspects of anaerobic bacteriology as part of their undergraduate research. Fine Focus was managed during Fall 2015 semester in Iceland by a dynamic group of students from five countries and four disciplines.
Fine Focus students lead a discussion on scientific writing and peer review in a biotechnology class at Indiana University recently.
Publications (since 2010)
Williamson, L.M., Nimmer, P.S., Kelly-Worden, M., Bange, J.S., Ho, Y., & McKillip, J.L. 2016. Subinhibitory carvacrol exposure increases virulence potential of Bacillus cereus in vitro and during endophthalmitis. Int’l J. Med. Microbiol., in press.
Turner, D.A., Pichtel, J., Rodenas, Y., McKillip, J.L., & Goodpaster, J.V. 2015. Microbial degradation of gasoline in soil: effect of season of sampling. Forensic Sci. Intl.251:69-76.
Turner, D.A., Pichtel, J., Rodenas, Y., McKillip, J.L., & Goodpaster, J.V. 2014. Microbial degradation of ignitable liquids in soil: comparison by soil type. J. Bioremed. Biodeg. 5(2):1-7. http://dx.doi.org/10.4172/2155-6199.1000216
Counsil, T.I., & McKillip, J.L. 2013. Molecular forensic analysis of ribonucleic acid (RNA)-based approaches. J. Liberal Arts Sci. 18(2):107-125.
Nimmer, P.S., Beer, M.R., & McKillip, J.L. 2013. Bacillus cereus: a bacterial species of environmental and clinical significance. J. Liberal Arts Sci. 18(2):21-32.
Smith, A.R., Ellison, A.L., Robinson, A.L., Drake, M.A., McDowell, S.A., Mitchell, J.K., Gerard, P.D., Heckler, R.A., & McKillip, J.L. 2013. Enumeration of sublethally injured Escherichia coli O157:H7 and E. coli B-41560 using selective agar overlays versus commercial methods. J. Food Prot.76:674-679.
Gracias, K.S., & McKillip, J.L. 2011. Triplex PCR-based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk. J. Basic Microbiol. 51:147-152.
Robinson, A.L. & McKillip, J.L. 2010. Biology of Escherichia coli O157:H7 in human health and food safety with emphasis on sublethal injury and detection. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. A. Méndez-Vilas (Ed.), vol. 2. Formatex, Badajoz, Spain.
Troxell, B., Volk, C., McKillip, J.L., & Warnes, C.L. 2010. Legionella pneumophila occurrence in waters of East Central Indiana. Proc. Indiana Acad. Sci. 119:205-211.
Counsil, T.I., & McKillip, J.L. 2010. Forensic blood evidence analysis using RNA targets and novel molecular tools. Biologia 65:175-182.
McKillip, J.L. 2010. Transformative undergraduate research: Students as the authors of and authorities on their own education. Council on Undergraduate Research Quarterly 30:10-15.
Work submitted but not yet accepted for publication:
Williamson, L., Nimmer, P.S., Kelly-Worden, M., Bange, J.S., Ho, M., Rajabli, N., & J. L. McKillip. 2017. Sublethal carvacrol exposure increases virulence of Bacillus cereus. Int’l J. Mol. Biol., submitted.
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