Tiny Earth, Big Solutions

What if we could increase our chances of discovering novel antibiotics by over 10,000 every year? Tiny Earth, a Course Based Undergraduate Research Experience (CURE), offers college students around the world  – including here at NC State – the opportunity to discover antibiotics from soil bacteria right in their backyards. With the support of GOHA’s GCAP-AMR Seed Grant, NC State researcher Stephanie Mathews hoped to expand the scope of her Tiny Earth-based course by developing a research pipeline that further investigates student-identified bacteria and ultimately contributes to the fight against antimicrobial resistance.

The Global Threat of Antimicrobial Resistance

Antimicrobials, such as antibiotics, antivirals, and antifungals, are agents that help prevent and treat infectious diseases. Antimicrobial resistance occurs when the target microbes – bacteria, viruses, or fungi – are not completely killed by the drug, and the surviving individuals reproduce and pass on resistance gene(s) to the next generation. This process of antimicrobial resistance evolution can make infections difficult or impossible to treat. The process can occur naturally over time through genetic variation in pathogens, random mutations, and horizontal gene transfer, but it is also exacerbated by human activity through the overuse of antimicrobials. 

By 2050, it is projected that the growing number of antimicrobial resistant pathogens will result in the death of 10 million people worldwide. Antimicrobial resistance poses threats not only to humans, but also plants and other animals, impacting veterinary care, our food systems, and the world economy. Even with the approval of around 20 new antimicrobials since 2010, the annual rate of resistant infections keeps increasing. It is vital that new treatment methods are developed as we navigate through a post-antibiotic era, a period when existing antibiotics are no longer effective and new antibiotics are not being developed on a meaningful scale.

The natural world, and specifically our soils, contain many microorganisms with antimicrobial properties. In fact, many commercial antimicrobials are sourced from fungi and bacteria. “The ultimate goal of any bacterium is to become bacteria, just to make more of themselves,” explains Mathews. “There are lots of things that help them to survive. Producing antimicrobials and having antimicrobial resistance may be one of those things.” The soil microbiome is quite complex and relatively underexplored, therefore offering the potential for the discovery of novel compounds that could prove vital in combating antimicrobial resistance. But if antimicrobials can be found right in our backyard, why is antimicrobial resistance so difficult to treat? The answer may lie in the resources required to identify and commercialize solutions. Mathews explains, “The biggest issue in treating antimicrobial resistance is the transition from finding an antimicrobial and carrying it to market.”

Calling on Students

Tiny Earth was established in 2018 by Dr. Jo Handelsman (University of Wisconsin-Madison) and since then has inspired thousands of students to engage in scientific research. The goal of Tiny Earth is to address the diminishing supply of effective antibiotics, decline of soil health, and the need for more scientists in the workforce. Soon after it was established, NC State joined the Tiny Earth network, offering a CURE to eager undergraduate students interested in hands-on research experience.

Today, the curriculum is taught by Mathews and Co-PI Dr. Mike Taveirne in the course MB360 Scientific Inquiry in Microbiology: At the Bench. Throughout the semester, students learn how to isolate and characterize antimicrobial-producing bacteria from soil. After the chemical extraction of the bacterial secondary metabolites (small, bioactive compounds), students then test the extract for antimicrobial activity. “The goal of this class is to combat antibiotic resistance, and so we’re really looking at bacteria that could be producing a novel antibiotic compound,” says Mathews.

A Research Pipeline

Each year, NC State students are responsible for the identification of 10-20 antimicrobial-producing bacteria. While these results are promising, Mathews couldn’t help but wonder what could result from the continuation of this research. “When they finish the course, students have isolated a new bacterium, but they don’t know what it is yet,” says Mathews. With the help of GOHA funding, Mathews set out to expand the scope of the course working with colleagues and students to identify and encapsulate novel antimicrobial compounds with the ultimate goal to develop topical and oral delivery systems.

The first step in this process was sequencing the genomes of the antimicrobial-producing soil bacteria. This work was carried out by Mathews, Taveirne, and former MB360 student Anna Miller. Miller used new genome mining tools, including antiSMASH (Antibiotics and Secondary Metabolite Analysis Shell), to better understand what genes were responsible for the antimicrobial compounds. Miller explains, “That was really cool and really new. I hadn’t done that in any of my classes, so it was great to get that experience because it is very applicable to work today.” Miller also tested the animal toxicity of the antimicrobial compounds using the model organism C. elegans. “Anna completed a validated C. elegans experiment,” says Mathews. “She brought a model organism into our microbiology lab space, learned how to maintain it, created a new protocol, and synchronized their generations, which is not easy.” Miller studied the reproductive output of C. elegans, as a significant decrease in the number of eggs laid would indicate a harmful compound. “Throughout the 8 or 9 microbes that I looked at, none of them really had a significantly harmful effect, which was super promising for the future when we actually identify what the antimicrobials are,” explains Miller.

Next, Mathews set out to identify the antimicrobial compounds by exploring the chemical structure of the antimicrobial agents produced by the bacterial isolates. To help sort through the mix of bacterial compounds, Mathews recruited Adriel Guiang, NC State microbiology student with a love for chemistry, who worked with Mathews to extract the antibiotics from the compounds and test their efficacy. Extractions were performed through various methods including organic extractions, preparatory thin layer chromatography (prep-TLC), and TLC bioautography. Guiang shares, “This was very exciting for me because I never knew you could do so much with TLC and use it to test antibiotics.” Through trial and error, the preparatory TLC method proved the most effective at extracting the antibiotic. This semester Guiang will continue his research in the lab and work with broth microdilutions, testing the ability of the extract to inhibit future bacterial growth.

To meet her goal of designing controlled antimicrobial delivery methods, Mathews worked with co-PI Nathalie Lavoine and student researcher Kevin Garcia-Diaz. “When we first set out, Natalie and her team were looking to create an encapsulization method so that we could deliver the antibiotics in some type of bandage or other application,” explains Mathews. However, incompatibility between the extraction methods and the intended encapsulation methods led to some roadblocks, so this portion of the research is ongoing.

Impact and Next Steps

Through this project, Mathews worked with NC State’s Office of Technology Licensing to learn more about intellectual property and commercialization. Mathews informs her students that when discovering something novel and societally relevant, there is the opportunity for commercialization. She now includes guidelines on intellectual property in her syllabus, should the students discover a product with commercial potential when isolating and analyzing their soil bacteria.

Mathews will continue to expand this research beyond the course and is working with the NC State Department of Chemistry and Dr. Cassie Lilly to develop course modules where students can identify the chemical structures of novel antimicrobial agents identified in MB360. Currently, Guiang is developing some protocols that can be used in the CH226 Organic Chemistry Lab to replace the standard labs with inquiry-based or research-based modules.

This project not only made significant progress in isolating and investigating antimicrobial compounds, but also provided valuable research experience to the next generation of scientists. When asked about her takeaway from the project, Miller shared, “This hands-on research experience made me realize that I’m really interested in working on my own and investigating the things that interest me.” Guiang similarly shared, “As soon as research started, I knew this is exactly where I want to be.” Both students are now interested in pursuing a Ph.D., citing their research experience as one that guided them toward this goal. 

Mathews’s engaging CURE and its interdisciplinary follow-on research projects are empowering students to develop real-world solutions for the global antimicrobial resistance crisis. In wake of growing AMR and the decline of antibiotic discovery, it is vital that all resources are leveraged. Initiatives such as Tiny Earth prove that sometimes the answers can lie right in our backyards. We just need someone to look for them.