After a day filled with exhibition booths, a keynote talk, and a bustling poster session, Nimra Khalid, D.V.M., heard her name over the loudspeaker announcing her first-place award for the Emerging Pathogens Institute Research Day 2026 graduate student poster competition.
Khalid, already with a D.V.M. in hand, is currently a Ph.D. student in the University of Florida College of Veterinary Medicine. Prior to coming to UF, she worked as a veterinarian in Pakistan, where she was struck by the widespread use of antibiotics. She was confronted with a major dilemma — antimicrobial resistance is everywhere. In 2022, UF Vet Med Assistant Professor Aria Eshraghi, Ph.D., was studying microbial virulence and antibiotic alternatives in his lab, so Khalid decided to join to begin her journey towards her Ph.D.
Initially, Khalid’s research focused on identifying new antibiotics and studying pathoblockers, a class of compounds that target bacterial disease-causing ability without inhibiting growth.
“Right now, we have five main classes of antibiotics, and some kind of resistance has been developed against all of them,” said Khalid. “Now, not every bacterium is resistant, but many, like Pseudomonas, are multidrug-resistant. The idea for our project was to identify a drug that has a mechanism that is distinct from the traditional antibiotics, like this pesticide.”
Khalid and the team shifted their focus to identify a new antimicrobial, but first, they needed to pick a bacterium as a guinea pig to test collections of different molecules. During screening of these collections, the team noted that tolfenpyrad, a pesticide currently used worldwide, stunted the growth of an infamous bacterial genus: Francisella. This genus includes F. tularensis, a pathogen that can spread rapidly via aerosol and vector-borne transmission. Interestingly, tolfenpyrad was ineffective against other tested Gram-positive and Gram-negative bacteria. As they explored further, they realized that tolfenpyrad could be enhanced for better effectiveness and lower toxicity. Its well-understood safety record across various organisms also makes it an appealing option for developing new antibiotics.
Due to its very low infectious dose and high virulence, F. tularensis is classified as a Tier 1 bioterrorism agent. The potential emergence of antibiotic resistance to this pathogen was enough to convince Khalid and her team to study the effects of this potential antimicrobial compound. A cousin of F. tularensis, F. novicida, was used as a model organism due to its lower biosafety threat and almost-perfect genome match to F. tularensis. They had found their guinea pig.
“While there are more common bacteria that have been studied in this way, like E. coli, we saw that Francisella is a larger public-health risk,” Khalid said. “It’s not common — the prevalence of the disease F. tularensis causes, known as tularemia, is low, but it’s highly pathogenic. Only 10 bacterial cells can cause the disease, which has a mortality rate up to 60%.”
Khalid and the team found that this pesticide uniquely targets bacterial energy pathways, unlike traditional pesticides. Bacteria, like human cells, need energy to survive, which is generated by a small internal “power plant.” Tolfenpyrad blocks a crucial step in this system, stopping bacteria from producing ATP, the energy molecule. Without ATP, bacteria can’t grow or survive.
This discovery could be a powerful new way to fight dangerous infections. Once Khalid completes her Ph.D., she plans to return to Pakistan to share the skills and knowledge she’s garnered from her hard work at UF.
“Techniques that I have learned here, those are replicable to other pathogens,” said Khalid. “Antibiotic resistance is a problem for every bacterial pathogen, and now I know how we can potentially translate research from one bacterium to another.”
To Khalid, one of the most exciting moments of this study was her first biochemical test with the bacteria. On her first try, the test worked, and the bacterium’s energy-generating process ceased.
“That never happens in your first attempt,” said Khalid. “You usually have to optimize the condition several times. When I told my P.I., Dr. Eshraghi, about these results, he was so excited that he printed those results and posted them on the lab fridge.”
Turning off bacterial energy: Mechanistic insights into a novel antimicrobial
Collaborators
- Nimra Khalid – College of Veterinary Medicine, University of Florida
- Chloe Van Horn – College of Veterinary Medicine, University of Florida
- Aria Eshraghi – College of Veterinary Medicine, University of Florida
Introduction
Tularemia is a highly infectious disease caused by Francisella tularensis, an intracellular pathogen capable of rapid dissemination through aerosol and vector-borne transmission. Due to its extremely low infectious dose and high pathogenicity, F. tularensis is a Tier 1 bioterrorism agent. The potential emergence of antibiotic resistance through genetic engineering or horizontal gene transfer poses a significant public health concern, highlighting the need for new antimicrobial strategies targeting previously unexplored bacterial pathways. Using its biosafety level-2 (BSL-2) lab strain as a model, we previously found that the widely used pesticide tolfenpyrad strongly inhibits Francisella growth. However, its antibacterial mechanism remains unknown. In this study, we hypothesized that tolfenpyrad suppresses Francisella growth by disrupting bacterial energy metabolism through inhibition of the electron transport chain (ETC).
Methods
To investigate the mechanism of tolfenpyrad-mediated growth inhibition, we evaluated its effects on bacterial respiration and energy production in F. novicida, a BSL-2 model organism commonly used to study the pathogenesis of F. tularensis. Biochemical and molecular assays were performed to assess ETC activity. Oxygen consumption and NADH oxidation were measured in membrane preparations of wild-type and mutant strains to evaluate respiratory activity. In addition, the effects of tolfenpyrad on the proton motive force and intracellular ATP synthesis were examined to determine its impact on cellular energy metabolism.
Results
Our findings show that tolfenpyrad disrupts cellular energy metabolism by targeting the ETC. Tolfenpyrad inhibits oxygen consumption in F. novicida membranes by blocking NADH oxidation at Complex I. This inhibition disrupts electron transport, collapses the proton motive force, and prevents ATP synthesis. As a result, bacterial energy production is impaired, leading to growth inhibition. Notably, this antibacterial activity is highly potent and specific to Francisella species.
Significance
Targeting Complex I-dependent respiration represents a promising strategy for inhibiting bacterial growth and developing new therapeutics to control antimicrobial resistance.