Non-Tuberculous Mycobacteria abundance and diversity in response to climate change
Carolina Mehaffy, Colorado State University
Location: Colorado
Abstract
This CURE engages student in hypothesis-driven research to investigate how seasonal climate affects microbial diversity, particularly Non-Tuberculous Mycobacteria (NTM) communities found in environmental samples such as soil and water. The course focuses on NTM diversity and physiology and its relationship with climate. Students are also introduced to climate change and how it may affect microbial diversity and abundance with implications to human health.
Student Goals
- Apply the scientific method to formulate and test a hypothesis for a specific research question.
- Demonstrate reasoning and troubleshooting skills when conducting experiments and analyzing data.
- Synthesize scientific literature and write protocols to apply to your research
Research Goals
- Characterize water and soil microbial communities by analyzing their diversity and abundance during two different climate seasons.
- Compare and contrast abundance and diversity of culturable mycobacteria vs 16S metagenomics.
Context
This CURE was designed in a large research university to introduce microbiology students to research experiences. The course is designed for upper level division students (juniors and seniors). The course is intended to be delivered as a 15 week, two times a week, 3 hours per session course. Students are expected to have a good knowledge of microbiology and microbiology related laboratory skills such as pipetting, culturing, serial dilutions, etc. Students should have completed a course on Introduction to Research.
Target Audience:Major
CURE Duration:A full term
CURE Design
The premise of this CURE is the rise in NTM infections worldwide. Authors have hypothesized that this may be due to an increase in NTM abundance in the environment due to climate change. Students are introduced to current concepts in climate change and how it may affect microbial diversity. Students are also introduced to current literature on NTMs in the environment. Students work first individually and then as a larger team to identify potential research questions associated with the topic. Because climate change cannot be observed in one semester, we use climate seasons as a proxy. Students sample water and soil samples during two different seasons (early on and mid semester). Formative assignments are designed to identify gaps before experiments are carried on, and also offer the opportunity for the instructor to provide feedback to all students individually.
The stakeholders are scientists as well as the general public who want to know more about how climate affects microbial diversity and human health. More specifically, NTM researchers and clinicians are also interested in understanding the relationship between NTMs and climate and its association with human health.
Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Planning and carrying out investigations
Nature of Research:Basic Research
Tasks that Align Student and Research Goals
Student Goals ↓
Measure environmental variables associated with water and soil samples during two climate seasons. Variables include pH, sample temperature, air temperature, GPS coordinates and soil type.
Extract and sequence environmental DNA from soil and water samples to conduct 16S metagenomic analysis.
Culture water and soil samples during two different climate seasons. Identify colonies with potential to be NTMs by 16S and rpoB sequencing; and by MALDI-ToF biotyper.
Determine the microbial composition of the samples by analyzing the sequence data through a bioinformatic pipeline and krona charts.
Correlate environmental variables with microbiological findings.
Compare and contrast the microbiological data (culturable bacteria) with the sequencing data (culturable and unculturable bacteria). In particular students focus on diversity and abundance of NTMs in each sample.
Instructional Materials
For the 16S microbiome analysis students follow the tutorial: https://training.galaxyproject.org/training-material/topics/metagenomics/tutorials/mothur-miseq-sop/tutorial.html
A modified version that facilitates implementation in the classroom can be provided upon contact.
Protocols for soil and water decontamination are provided, however, instructors are encouraged to have students draft their own protocol after reviewing the literature.
PPT Introduction (PowerPoint 2007 (.pptx) 62kB Jul29 22)
Protocols (Acrobat (PDF) 415kB Jul29 22)
Syllabus and schedule example (Acrobat (PDF) 219kB Jul29 22)
Reagents and Equipment NTM CURE.docx (Microsoft Word 2007 (.docx) 15kB Jul29 22)
Assessment
Assessments (Microsoft Word 2007 (.docx) 23kB Jul29 22)
Rubrics (Acrobat (PDF) 208kB Jul29 22)
Instructional Staffing
This CURE can be taught by one instructor. It is expected students will be in charge of most (if not all) needed preparation such as making buffers and media, autoclaving trash and cleaning the lab after each session.
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Author Experience
Carolina Mehaffy, Colorado State University
I have developed and taught the CURE on Non-Tuberculous Mycobacteria and climate. This submission includes the final version of protocols and assessments that increase success of students obtaining meaningful results as it relates to the research questions and learning goals. I am interested in identifying opportunities to create a national network for this CURE.
Advice for Implementation
Ideally this CURE should be taught twice a week, 3 hours each lab session. Some of the protocols (i.e. decontamination and plating) will take more than 2 hours of hands-on involvement, specially the first time the students perform it.
Iteration
Students are involved in all aspects of trouble-shooting and problem solving. When an experiment does not work, students are asked to provide ideas and with guidance from the instructor develop a plan. Most common "failures" are: lack of NTM colonies, and lack of band in PCR. Because some samples will not contain culturable NTMs, students can change the location for the second round of sampling. However, since the protocol includes culturing of samples with and without decontamination, the chance of culturing increases. There are multiple opportunities built in the schedule (as floating or catch up days) for students to troubleshoot and repeat experiments including repeat PCR or DNA extractions. In addition, this CURE is iterative in nature since students perform two rounds of sampling in the semester. The second time of sampling most students feel very confident on their skills and knowledge of the protocols and they complete these much faster than the first time. This also allows time for discussion and review of the literature.
Using CURE Data
Students contribute their findings to a google sheet database. This is used to share with future students and long term can be analyzed for a future publications. All students share their individual work during the poster presentations.
Resources
A document with a list of manuscripts will be provided List of literature.pdf (Acrobat (PDF) 165kB Jul29 22). Faculty are encouraged to read all manuscripts on the list. Students are encouraged during the beginning of the semester to choose one or two to read from the list. A few other can be incorporated as assignments through the semester.
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