Antibiotic Properties of Spices and the Evolution of Antibiotic Resistance,
OR: "The Spice of Life"
Proposal for HHMI - CRE course module development by Drs. Kathy Winnett-Murray (Biology)
and Maria Burnatowska-Hledin (Chemistry and Biology).
Requested for period 1 June 2014 -1 June 2015. Amount requested: $2,500
Abstract – An existing lab module developed for GEMS 161 (Biotechnology and You) will be
re-structured and augmented to emphasize research-like experiences for both non-science majors
and science majors, and be disseminated to a broader community of undergraduate science
educators. The current lab, “Microbiology and Antibiotic Resistance” will be re-written in a
format to allow instructor choice of new components on the evolution of resistance in microbes
as well as different quantitative analysis choices for student data, allowing close matching to
student level and course goals. In addition, we will enhance the relevant themes in human
culture and biology that richly derive from an exploration of the historical and modern use of
spices to reduce food-borne pathogens. The re-worked module will emphasize the following
CRE elements: 1) outcome unknown to students (and instructor), 2) students have some or all
input in creating the experimental design, 3) students become responsible for the project, 4)
students analyze data, and 5) students critique the work of other students. An appropriate
assessment plan to gauge student response to these course elements will be developed, and the
module will be disseminated on campus and off campus, most likely as a major workshop at the
2015 meeting of the Association for Biology Laboratory Education (ABLE).
Project Description
We propose to modify an existing laboratory used in GEMS 161, "Microbiology and
Antibiotic Resistance", currently developed and taught by Dr. Maria Burnatowska-Hledin, with
specific goals to enhance the research-like experiences of this module, using the specific criteria
established by CURE findings. These goals are to: 1) provide a more research-based
experience for GEMS students, 2) to increase research-based components, quantitative elements,
and direct coverage of evolutionary concepts, so that the module is also suitable for potential
implementation in other science courses, such as Biology 106, which includes an Evolution
component, and 3) disseminate this research-based module to other undergraduate institutions.
The scientific literature is rich with examples of research on antibiotic resistance (e.g.
Neu 1992, Williams and Heymann 1998, Falkow and Kennedy 2001) and this is reflected in a
growing interest among the general public in the problem of resistant strains of bacteria and
other human pathogens (e.g. Radetsky 1998). While the widespread use of antibiotics has saved
millions of lives, the effectiveness of antibiotics in controlling certain human pathogens (e.g.
Staphylococcus aureus, one of the bacterial species that Dr. Hledin's GEMS 161 lab currently
investigates), is declining due to the evolution of bacterial resistance. At the same time,
misconceptions about the nature of evolution, a foundational principle of biology, continue to
hamper public understanding of bacterial resistance and ultimately limit understanding of
medically and biologically important processes in undergraduate science courses (Nesse and
Williams 1994). Naturally occurring spices, which have been used for centuries to enhance
1
flavors and fragrances of food, and for medicinal purposes (Lai and Roy, 2004, Rahman et al.
2011), provide an intriguing context in which to investigate antimicrobial aspects of food
preparation in the context of human adaptation. Volatile oils of some spices have antibacterial
effects against some food-borne pathogens (e.g. Dorman and Deans 2000), stimulating a
resurgence in scientific hypotheses that propose a synergistic relationship between human
evolutionary history with pathogens, and the cultural development of spice usage as a protection
against food-borne illnesses and food poisoning (Sherman and Flaxman 2001). In the last few
decades, there has been a very strong research interest in testing ultimate explanations for the
adaptive value of spices used in human foods. A wealth of diverse literature exists on the topic,
and there are even several K-12 and undergraduate laboratories that have been developed to
explore the "spices as potential antibiotics" theme. However, the array of spice attributes, along
with the taxonomic variety of food-borne pathogens, continue to render this field imminently
promising for open-ended investigations since responses by microbes to particular compounds
may vary significantly by species, concentration, or even by the anatomical part of the plant
(spice) used, and/or its method of preparation. The possibilities for various combinations of
spice/microbe variables in experimental research and the variety of environmental factors
mediating the outcome of those interactions are virtually inexhaustible. As a potential
framework for possible student-generated hypotheses, a framework proposed by Sherman and
Flaxman (2001) is of particular interest, since they incorporate themes of geographic and cultural
diversity in the use of spices. They propose four predictions:
1) spices used in cooking should exhibit antimicrobial activity. This prediction forms the basis
of the main component of Dr. Hledin's GEMS 161 lab: students prepare extracts of a chosen
spice in both water and in methanol, and test growth of bacteria (E. coli and S. aureus) on agar
plates containing sterile paper discs inoculated with various concentrations of the spice
extract(s). 2) use of spices should be greatest in hot climates, where unrefrigerated foods spoil
quickly.3) the spices used in each country should be particularly effective against the local
bacteria. 4) within a country, meat recipes should be spicier than vegetable recipes. 5) within a
country, recipes from lower latitudes and altitudes should be spicier because of the presumably
greater microbial diversity and growth rates in those regions
These general predictions form the basis of a wealth of potential student-generated
hypotheses that can integrate the principles of antibiotic resistance, evolution of antibiotic
resistance, and how human cultural attributes may reflect the environmental conditions (e.g.
climate, latitude) that mediate these relationships. For example, in a simple, but elegant
laboratory developed by Sousa and Waldman (2013), students make qualitative comparisons of
microbial growth on Brazilian rice pudding "cultures", prepared with and without ground
cinnamon or ground cloves. The novelty of what we propose to do with the existing GEMS 161
Antibiotic Resistance lab derives from our deliberate combining of the relevance and inherent
real-world interest of spice use by human cultures with the issue of evolution of antibiotic
resistance in microbes. We have good resources from which to draw established protocols for
testing antibiotic resistance (e.g. Dr. Hledin's lab attached as Appendix A with this proposal,
Lessem 2007, Marion and Preszler 2010, Joseph et al. 2013). Although other laboratory outlines
explore antibiotic effects of spices, as previously described, to our knowledge, no other
undergraduate laboratory specifically combines effects of spices with antibiotic resistance in an
evolutionary context.
2
We have identified five course elements from the CURE attributes of research-like
experiences that will characterize this lab module. Of these five, one of them (outcome unknown
to students (and instructor) characterizes Dr. Hledin's existing lab; the other four will be
strengthened as outlined in Table 1, and include deliberate focus on: "students have some or all
input in creating the experimental design", "students become responsible for the project",
"students analyze data", and "students critique the work of other students". In addition, we have
included in Table 1 a goal of making this lab module more interdisciplinary by inclusion of an
evolutionary context. The methods for achieving these changes are outlined in Table 1. We
desire to develop particular elements in alternative forms such that different instructors teaching
different courses (e.g. GEMS vs. Biology 106 (introductory level) vs. advanced biology
electives) could select elements appropriate to that class level. For example, the degree of
quantitative rigor in the data analysis course element will be presented in 3alternative forms: 1)
a basic graphical comparison of zones of inhibition (of bacterial growth) similar to Dr. Hledin's
current lab, 2) an analytical component that introduces ANOVA as an inferential statistical tool
to test student-generated hypotheses, and 3) a more advanced introduction to two-way ANOVA
to test student-generated hypotheses involving the potential synergistic, interactive effects of two
independent variables (e.g. microbe species and spice species, or spice species and mode of its
preparation). The second course element that will assume variety in "packaging" is the extent to
which evolutionary concepts are incorporated into the lab material. While it is critical for all
students, including those at the introductory level, to understand bacterial resistance as an
evolutionary response to changing environments, it is desirable for more advanced students in
biology to also begin to explore concepts such as genetic variation, plant anatomy, chemical
composition of plant compounds and their evolution, calculation of relative biological fitness,
and differential responses of diverse microbial taxa to various plant compounds. To that end, we
will package background information, imbedded assessment questions, and lab report criteria in
such a way as to facilitate the ability of instructors to "pick and choose" appropriate extensions
for their particular courses.
Dissemination - Our goal is to disseminate this research-based lab both on and off campus. On
campus, we will first implement the lab in GEMS 161 and then take advantages of opportunities
to potentially rotate this lab with other previously developed evolution modules in Biology 106,
a decision that would rest primarily with the Biology faculty collectively, and with the Bio 106
instructors, in particular. Since components of several introductory biology modules will
probably transition and alternate among years, we anticipate that this is a strong possibility. It is
also possible that an advanced version of this lab module could be used in upper level biology
courses such as Evolutionary Biology, Ethnobotany or Plant Physiology. Dissemination offcampus will begin with the anticipated major workshop at ABLE 2015 and will include the
HHMI group institutional consortium.
Assessment - We plan to use the CURE assessment instrument as our primary summative
evaluation tool. We will develop a complete assessment plan in conjunction with the HHMI
CRE summer workshop. Additional summative assessment will be accomplished in the form of
the instructor- and peer-evaluated final report (either oral or written; Table 1). Formative
assessments will include both instructor- and peer-evaluation of the Guided Inquiry Proposal,
and "understanding checkpoint" questions imbedded in the lab instructions and analysis
instructions. We may also include a background preparation assignment as a formative
assessment that students complete prior to the lab that will help them focus on generating
3
appropriate hypotheses for their planned experiments, and to help them in completion of the
Guided Inquiry
Brief Timeline and Staffing Resources Needed
June 1-15 2014: KW-M and MB-H will assemble protocols to be tested by student assistant.
June 15-30 2014: Student assistant works with KW-M for 2 weeks (consulting MB-H) to test
protocol variations, analyze results, assemble resources on the following themes:
cultural/historical use of spices, scientific research on antimicrobial properties of spices,
environmental variance in response of microbes to antibiotics, and the evolution of antibiotic
resistance. Student assistant and KW-M develop Guided Inquiry Proposal format, and three
alternative versions of quantitative analysis (basic, ANOVA, and two-way ANOVA) to be
reviewed by MB-H
Summer 2014: KW-M will spend additional portions of summer 2014 finalizing write-ups, with
review by MB-H.
September 2014: 1) Research-enhanced antibiotic resistance lab to be implemented in GEMS
161, including a to-be-developed assessment of student learning for this lab in Dr. Hledin's fall
2014 class. 2) Research-enhanced lab to be submitted for consideration as a major workshop at
the 2015 ABLE (Association for Biology Laboratory Education) meeting.
Fall 2014: MB-H will have one or two undergraduate T.A.s assigned to GEMS 161, as is
customary. KW-M will participate in at least one of the weekly labs in which this lab is
implemented. KW-M and MB-H meet to evaluate outcomes of the modifications and assessment
results; make further modifications as necessary. KW-M and MB-H share results with the
biology department faculty and discuss outcomes and potential for use as a possible future
alternative exercise in Biology 106 evolution module. Preliminary report submitted September
2014 and presentation to HHMI lunch bunch during the fall.
June 2015: Anticipated presentation as a major workshop at the 2015 annual meeting of ABLE
(Association for Biology Laboratory Education), and subsequent peer-reviewed publication at
ABLE's on-line website for proceedings. Final report to HHMI - CRE program.
Bibliography
Ceylan, E. 2003. Antimicrobial activity of spices against Escherichia coli O157:H7 and their
application in solid and liquid foods. M.S. Dissertation, Kansas State University.
Dorman,, H.J. and S.G. Deans. 2000. Antimicrobial agents from plants: antibacterial activity of
plant volatile oils. J. Appl. Microbiology 88: 308-316.
Falkow, S. and D. Kennedy. 2001. Animals, Antibiotics, and People - Again! Science 291: 397.
Joseph, M., V. Pierre-Louis, H. Singer, A. Wachs and K. Nolan. 2013. Student-designed
projects that ascertain antibiotic properties of natural substances. Pages 443-448 in Tested
Studies for Laboratory Teaching. Volume 34 (K. McMahon, Editor). Proceedings of the 34th
Conference of the Association for Biology Laboratory Education (ABLE), 499 pages.
http://www.ableweb.org/volumes/vol-34/v34reprint.php?ch=53.
4
Lai, P.K. and J. Roy. 2004. Antimicrobial and chemopreventative properties of herbs and
spices. Current Medical Chemistry 11: 1451-1460.
Lessem, P. B. 2008. The antibiotic resistance phenomenon: use of minimal inhibitory
concentration (MIC) determination for inquiry based experimentation. Pages 357-362, in Tested
Studies for Laboratory Teaching, Volume 29 (K.L. Clase, Editor). Proceedings of the 29th
Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 433 pages.
Marion, A.L. and R.W. Preszler. 2010. Evolution of bacterial resistance to antibiotics. Pages
216-225, in Tested Studies for Laboratory Teaching, Volume 31 (K.L. Clase, Editor).
Proceedings of the 31st Workshop/Conference of the Association for Biology Laboratory
Education (ABLE), 534 pages.
Nesse, R.M. and G.C. Williams. 1994. Why we Get Sick: The New Science of Darwinian
Medicine. Vintage Books, New York.
Neu, H.C. 1992. The crisis in antibiotic resistance. Science 257: 1064-1073.
Ozcan, N.M., O. Sagdic, and G. Ozkan. 2006. Inhibitory effects of spice essential oils on the
growth of Bacillus species. J. Med. Food. 9: 418-421.
Radetsky, P. 1998. Last days of the wonder drugs. Discover, November, 1998.
Rahman, S., A. Khasru Parvez, R. Islam, and M. Hossain Khan. 2011. Antibacterial activity of
natural spices on multiple drug resistant Escherichia coli isolated from drinking water,
Bangladesh. Annals of Clinical Microbiology and Antimicrobials 10: 10.
Sherman, P. and S.M. Flaxman. 2001. Protecting ourselves from food. American Scientist 89:
142-151.
Sousa, A.M. and W.R. Waldman. 2013. Antimicrobial properties of spices: an activity for high
school or introductory chemistry or biology. J. Chemical Education 91: 103-106.
Takikawa, A., K. Abe, M. yamamoto, S. Ishimaru, M. Yasui, Y. Okubo, and K. Yakoigawa.
2002. Antimicrobial activity of nutmeg against Escherichia coli O157. J. Biosci. Bioeng. 94:
315-320.
Williams, R.J. and D.L. Heymann. 1998. Containment of antibiotic resistance. Science 279:
1153-1157.
5
Table 1. Course elements identified for transition to greater research-like experiences in GEMS 161
Lab: Microbiology and Antibiotic Resistance
Identified Course
Element
outcome unknown
to students (and
instructor);
research-like in
being "discovery"
based
Students have some
or all input into
creating the
experimental design
Students become
responsible for
project
Current
Attributes
Unknown
outcome;
instructor familiar
with outcomes
but results vary
with type of
spices chosen, etc
Students have
minimal control of
choices, but some
(they choose
which spices to
test)
Proposed Attributes
Stronger studentcentered approach with
a greater element in
choice of independent
variables, while retaining
essential elements of a
common protocol so that
results across student
groups are comparable
1) build background content and
readings to assist students in
constructing rationale for
"guided proposals"
Students are
responsible for
carrying out the
experiment and
for a formal
written lab report
Build on existing
"ownership" aspects to
strengthen other aspects
of student ownership in
the investigation
1) Develop "guided inquiry
proposals" with students able to
identify some variables, as
above.
2) oral presentation of group
results, with peer evaluation of
presentations OR peer
evaluation of written lab report
requirement; either will involve
development of a student
scoring rubrics.
3) Participation in "debate";
construct an experience in
which students can use their
own data in the debate; develop
a debate evaluation rubric.
Retain variety in choices
of independent
variables; retain openended "discovery"
nature
Proposed Method/Grant
Activity
Essentially no modification of
this element; currently this is
one of the strongest researchlike elements of the
investigation
2) Create streamlined "guided
inquiry proposals" - students
identify variables, propose a
rationale for their hypotheses,
and identify the relationship
between predictions that derive
from their hypothesis and the
experimental design. These may
consist of a one-page "form" so
instructor/TA can grade quickly
during class.
6
Analyze data
Basic data analysis
in qualitatively
and graphically
comparing zones
of inhibition
Strengthen possibilities
for data analysis,
retaining the current
comparisons of zones of
inhibition as the main
dependent variable, but
including 2 quantitative
alternatives (choices for
GEMS courses vs. majors
courses)
1) Create background
information and statistical
instructions with worked
examples for 3 analysis choices:
a) "basic"- as used currently, b)
descriptive statistics with
ANOVA, and C) descriptive
statistics with two-way ANOVA,
enabling instructors to choose
the best "fit" for individual
courses
Critique the work of
other students
Currently, Dr.
Hledin has
students engaged
in various forms
of peer
evaluation, but
not in this
particular lab
Microbiology and
some chemistry
Include opportunities to
critique the work of
other students
Possibilities include:
1) peer evaluation of guided
inquiry proposals
2) peer evaluation of debate,
with a focus on evaluating how
well other groups make use of
scientific evidence in their
argument(s)
1) build background aimed at
connecting with evolutionary
principles, using antibiotic
resistance as the theme
2) develop question-based
elements th/out the lab handout
encouraging students to connect
results with concepts such as
genetic variation, relative
fitness, natural selection,
"artificial" selection, etc.
3) include resources and library
research assignment(s) related
to both a) the evolution of
antibiotic resistance, and b)
theories on use of spices in
human cultures
4) package new elements so
that instructors can choose their
own emphasis
Interdisciplinarity
(added by the
authors)
Expand conceptual
framework to include
evolution:
1) biological evolution as
evidenced by antibiotic
resistance
2) cultural evolution as
exemplified by historical
and current use of spices
in human cultures
7
8