Professor of Biology
||Associate Professor, Department of Biology & Biotechnology Certificate Program, Ball State University, Muncie, IN
||Assistant Professor, Department of Biology & Biotechnology Certificate Program, Ball State University, Muncie, IN
| 8/99 – 5/03
||Assistant Professor, Microbiology & Molecular Biology, School of Biological Sciences, Louisiana Tech University, Ruston, LA (from which 2 years towards tenure were granted at BSU)
| 6/97 – 6/99
||Postdoctoral Associate, Department of Food Science & Technology, Mississippi State University, Starkville, MS
||Ph.D. Washington State University, Microbiology
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 the effect of lactoferrin and lactoperoxidase as a natural antimicrobial against bacterial pathogen growth in raw milk. Another recent project entailed optimizing the formulation of De Man, Rogosa and Sharpe (MRS) medium for recovery of selected lactic acid bacteria important to the dairy industry. This recent work has been presented at regional and national conferences including the Indiana Branch of the American Society for Microbiology (IBASM), the ASM General Meeting (ASM Microbe), the American Dairy Science Association (ADSA) Conference, 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).
Research underway with M.S. students and undergraduate students:
Project #1 – Bacillus spp. bacteriophage from dairy environments
Rachel H. Pittsley*1, Tykon Zubkov2, David A. Sanders3, Victoria M. Blake1, Katherine G. Kornilow1, Kyla L. Adamson4, & John L. McKillip1
1Department of Biology* & 2Department of Chemistry, Ball State University, Muncie, IN; 3Markey Center for Structural Biology, Department of Biology, Purdue University, West Lafayette, IN; 4Elanco Animal Health, Clinton, IN
Bacillus spp. are within a family of bacteria known to form spores for survival in extreme environments. The spores are resistant to temperature extremes, radiation, antibiotics, and many disinfectants. When ingested by or exposed to susceptible animal hosts, the spores can germinate and create biofilms or cause infections such as endophthalmitis, bacteremia, pneumonia, or gastroenteritis. The virulence potential makes these bacteria problematic in the food industry as pathogens and spoilers. Biological control of Bacillus spp. may be possible with bacteriophage. As a protective measure, dairy farmers could utilize a known bacteriophage after pasteurization to ensure dairy product quality and safety. The goal of this research is to isolate and characterize unknown bacteriophage found in dairy milk. By determining host range, DNA sequence, and morphology, phage candidates will be identified that may have promise as agents for biological control of Bacillus spp. of concern to dairy scientists and consumers. Raw milk, silage, tank water, manure, and soil samples were processed for phage extraction and enrichment. Plaques were subjected to DNA purification for sequencing, and for negative staining and transmission electron microscopy. Results revealed that the phage isolated in this study demonstrated a broad host range across several common Bacillus spp., established that levels of CaCl2 were highly influential for phage uptake, indicated the presence of shared amplicons between the unknown phage and Bacillus phage deep blue (NC_031056) and Bacillus phage BCP8-2 (NC_027355), revealed that glycerol precipitation was more efficient for DNA sequencing, and unveiled that the isolated phage had an icosahedral head and presence of a sheath and tail fibers, measuring approximately 100nm together and sharing characteristics with the well-known T4 bacteriophage known to infect Bacillus spp. Further host range studies, genomic analyses, and PCR will reveal if this phage is novel and a possible future biological control agent, and/or whether the phage harbors antibiotic resistance genes that could have food safety and public health implications.
M.S. Student Rachel Pittsley presents her poster on Bacillus spp. phage at the American Dairy Science Association (ADSA) conference in Cincinnati, OH during summer 2019.
Project #2 – Using Nitrous Acid Modified MRS Medium to Selectively Isolate and Culture Lactic Acid Bacteria from Food
Matthew A. Renschler1, Asia Wyatt1, Nnamdi Anene1, Rona Robinson-Hill1, Ethan S. Pickerill1, Nathaniel E. Fox2, Julie A. Griffith3, & John L. McKillip1*
1Department of Biology, Ball State University, Muncie, IN; 2Earl A. Chiles Research Institute, Portland, OR; 3West Virginia University, Morgantown, WV
Nitrous acid was used to modify traditional de Mann, Rogosa, Sharpe media to test our hypothesis that addition of sodium nitrite to MRS media would improve the growth rate and density of various lactic acid bacteria while preventing the same of nontarget species. Yogurt, cheese, and sauerkraut were inoculated with individual bacterial species (Bifidobacterium longum, Streptococcus salivarius, and Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Leuconostoc mesenteroides, Enterococcus faecalis and Bacillus cerus) followed by recovery and enumeration of each to compare the sensitivity between nitrous acid modified MRS (mMRS) and traditional MRS. Lactobacillus delbrueckii subsp. bulgaricus were recovered at significantly (p<0.05) higher counts from cheese in nitrous acid mMRS than MRS while there was no significant difference for other species and food systems between the two media formulatons. To augment overall comparative recovery of species on the two media types, the rate of growth of each lactic acid bacterial species in MRS -vs- mMRS broth was also measured. Growth curves were generated for the lactic acid bacteria as well as nonstarters in both mMRS and MRS to measure the selectivity of nitrous acid mMRS. Three of the tested bacterial species (Bifidobacterium longum, Streptococcus salivarius, and Lactobacillus delbrueckii subsp. bulgaricus) grew to significantly higher (p<0.05) densities more rapidly in mMRS broth than in traditional MRS during an 18h incubation regime. Conversely, two nontarget low G+C bacteria genotypically and phenotypically similar to some lactic acid bacteria, Enterococcus faecalis and Bacillus cereus, demonstrated a more prolific growth rate and significantly (p<0.05) higher OD readings in traditional MRS (15-30% higher density by 18h) compared to mMRS, in order to validate that these nontarget species would be less competitive in the improved mMRS formulation. It was determined that nitrous acid mMRS is a viable alternative medium for culturing selected lactic acid bacteria, and offers an improved formulation of MRS for use in standard methods evaluation and optimization of lactic acid bacteria in dairy and possibly other food-grade GRAS cultures.
Muncie (IN) high school junior Asia Wyatt presents her portion of the research on lactic acid bacteria recovery with the improved MRS formulation at the Indiana State Science and Engineering Fair, spring 2019. Asia received special recognition and an award from the Society of African American Women Chemists, and will be a coauthor on this manuscript, submitted summer 2019 to the Journal of Dairy Science.
Senior biology major Nnamdi Anene works on his portion of the lactic acid bacteria project during summer 2019. Nnamdi, who is an LSAMP (Louis Stokes Alliance for Minority Participation) Scholar, will be a coauthor on this manuscript, submitted summer 2019 to the Journal of Dairy Science.
Project #3 – Natural Level of Lactoferrin as an Antimicrobial against S. enterica and E. coli O157:H7
Authors: E.N. Biernbaum* and J.L. McKillip
Affiliation(s): 1Department of Biology, Ball State University, Muncie, IN 47303, USA
Keywords: lactoferrin, S. enterica, E. coli O157:H7
Improper storage conditions or processing of milk leads to potential spoilage and illness. Spoilage occurs due to shifts in temperature, which allow the bacteria in milk to reproduce at an increased rate. The bacteria break down the lactose in the milk, producing lactic acid and lowering the pH. However, proteins found in raw milk have antibacterial properties, but the natural levels have not yet been studied. One of these proteins, lactoferrin, is naturally found in mammalian body fluids and has antifungal, antibacterial, and antiviral properties. Lactoferrin binds to iron and reduces the amount available for bacteria to use for growth. We are currently investigating the effectiveness of the natural level of lactoferrin as an antimicrobial on two common milk pathogens, S. enterica and E. coli O157:H7. To test these objectives, we are using a broth system to determine the optimum protein concentration and then will move into a raw milk system. The growth rate of each bacterium will be measured for 18 hours using optical density and by using the standard plate count method. We plan to see reduced density and colony counts as a result of bacterial growth inhibition due to lactoferrin. If proven to be effective, this protein could extend the shelf-life of milk, which will also reduce spoilage and the risk of contracting foodborne illnesses.
M.S. student Erika Biernbaum, here shown teaching microbiology lab, is nearing completion of her thesis research on the lactoferring/lactoperoxidase research, which will soon be submitted as an abstract for the 2020 ASM Microbe General Meeting in Chicago.
Project #4 – Fine Focus
The most innovative 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.
Over the last few years, Fine Focus has engaged undergraduate students in multiple STEM outreach activities, all of which offer opportunities for undergraduates to learn about and become involved in publishable undergraduate research. One of these higher profile activities is the creation of a short film entitled Faces of Science.
Faces of Science is a STEM outreach collaboration between Ball State University faculty member Dr. John McKillip (Department of Biology), his Fall 2017 Virginia B. Ball Center for Creative Inquiry class, and the University of Detroit Mercy (UDM) undergraduates who are part of an NSF-funded program called ReBUILDetroit. These students at UDM are all actively engaged in various STEM undergraduate research projects, and represent academically underrepresented student groups. This film created by Ball State University students, entitled Faces of Science, features several ReBUILDetroit undergraduates telling their individual stories about how they perceive opportunities in STEM currently, and offer reflections on their role models, advice to younger students on science, society, and the individual. Early portions of this seven-minute short film were done principally by the nine BSU biology students with post-production completion by Blake Conner (BSU Digital Corps). A link to view this inspiring film will be posted in the very near future.
Fine Focus student editors Victoria Blake and Gabriella DeValeria represent Ball State University in San Francisco by promoting and marketing Fine Focus at the ASM Microbe General meeting in June 2019.
Fine Focus implemented a local STEM outreach workshop to the Muncie (IN) Unity Center, with assistance from AmeriCorps staff of the local Second Harvest Food Bank in summer 2019. Students learned basics of microbiology theory and techniques, along with professional development opportunities.
Publications (since 2010)
Adamson, K.L. & McKillip, J.L. 2019. Bacillus spp. bacteriophage and the dairy environment. J. Liberal Arts & Sciences, in press.
A. Grutsch, P. S. Nimmer, R. H. Pittsley, K. G. Kornilow, & J. L. McKillip. 2018. Molecular Pathogenesis of Bacillus spp., with Emphasis on the Dairy Industry. Fine Focus 4(2):203-222.
McKillip, J.L. 2017. Undergraduate Research and the Global Community - Partnering with students in Iceland and Continental Europe to develop a new undergraduate microbiology research journal. Council on Undergraduate Research Quarterly 37(3):27-30.
Lan, X., Zheng, N., Zhao, S., Li, S., Zhang, Y., McKillip, J., & Wang, J. 2017. Microbiological quality of raw cow’s milk and its association with herd management practices in Northern China. J. Dairy Sci. 100(6):4294-4299. doi: 10.3168/jds.2016-11631. Epub 2017 Apr 21
Rajabli, N., Williamson, L., Nimmer, P.S., Kelly-Worden, M., Bange, J.S., Ho, Y., & McKillip, J.L. 2019. The dangers of sublethal carvacrol exposure: increases in virulence of Bacillus cereus during endophthalmitis. International Journal of Biochemistry & Molecular Biology, in 9(2): 11–21.
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.