Next-gen antimicrobials

August 21, 2019: EPI investigator KC Jeong aims to uncover the next generation of antimicrobials. From his microbiology lab to an experimental farm, his research explores how food animals are affected by novel and drug-resistant pathogens — and what kills them.

Next-gen antimicrobials

Drug-resistant bacteria are a metastasizing global menace. For the past seventy-five years, roughly since Franklin D. Roosevelt signed the GI Bill of Rights, society has enjoyed a wondrous time span where powerful antibiotic drugs allowed medical practitioners to aggressively fight a broad array of common bacterial infections.

But the golden years of antibiotics are waning, and many of the bugs they have reliably killed now have genes that allow them to resist our medicines unscathed. Drug-resistant tuberculosis and pneumonia, methicillin-resistant Staphylococcus aureus (MRSA), and pathogenic E. coli, Shigella, and Salmonella — these terms were once relegated to technical medical conversations but are now commonly heard on the nightly news.

UF’s Emerging Pathogens Institute investigator KC Jeong is focused on uncovering novel ways of controlling disease-causing microorganisms in farmed animals, as well as learning how these tiniest of bits of life are outwitting humanity’s best minds.

A new nano-bactericide

When Jeong arrived to the University of Florida in 2011, he had recently completed a PhD at the University of Wisconsin-Madison, where he investigated novel ways of eliminating the threat of E. coli O157:H7 — a highly virulent, and sometimes fatal, cause of food poisoning — from beef. While testing a passive antibody-based immune therapy, Jeong discovered that the substance used to deliver the antibodies, chitosan nanoparticles, harbored powerful antimicrobial activities. Chitosan is a sugar found in the hard outer shells of shrimp and other crustaceans. Nanoparticles are bits of miniscule matter that measure between 1 and 100 nanometers long.

“Chitosan nanoparticles are often used to deliver drugs to targeted sites, but before our study, nobody knew that they had antimicrobial activity by themselves,” Jeong recalls. “It was such a surprise, you don’t expect to get meaningful data from your control group.” Researchers already knew that chitosan in its raw form had antimicrobial properties under certain conditions, but no one had yet looked into its properties when it’s engineered into micro-sized particles; which is important, because microparticles will sometimes behave entirely differently from their parent material.

The chance discovery landed Jeong on EPI’s radar, and he was recruited with a position split between the EPI and UF’s Institute of Food and Agricultural Sciences animal sciences department. First on his agenda? Figuring out why chitosan nanoparticles are so good at killing pathogenic E. coli O157:H7.

KC Jeong in his laboratory

KC Jeong in his EPI microbiology lab.

Using genetics and animals, he soon discovered that the miniature chitosan bits essentially plug up “gates” in an E. coli’s bacterial cell wall that provide a portal to accept nutrients. The structure of the E. coli bacterium’s cell wall is also disrupted by the particles, causing further damage and death.

Jeong and his collaborators found that chitosan nanoparticles turned out to be broadly effective against many other disease-causing microorganisms too. Better yet, they did not produce resistance in the bacteria targeted. Jeong’s further work revealed chitosan nanoparticles reduced the presence of various strains of Salmonella enterica in agricultural water. This was eye-opening because livestock can acquire this disease-causing bacteria from drinking water, and then pass it on to people when the animals become food.

But not all particles are created equal, so Jeong studied how to best engineer chitosan nanoparticles to optimize their effectiveness against E. coli O157:H7 without producing resistance. Creating particles smaller than 300 nanometers, and adding other ingredients, turned out to be key.

“It kills bacteria very quickly, like Clorox,” says Jeong. “The beauty of this is that it does not cause antibiotic resistance. It disrupts the cell membrane so fast that the bacteria does not even have time to develop resistance mechanisms.”

It’s fair to ask how safe this new nano-bactericide is for people, given some people’s concerns about the science of nanoparticles. Chitosan is favored as a drug-delivery material specifically because of its low toxicity to humans. But because materials sometimes behave differently when reduced to a nanoscale, testing for safety is important for understanding this material’s efficacy in treating infections in animals and humans. To investigate, Jeong and colleagues performed a risk assessment using animal models. The results showed that when used at concentrations strong enough to have antimicrobial effects, chitosan nanoparticles caused no toxicity to animal and human cells.

Safeguarding food animals

After figuring out the underpinnings of how chitosan nanoparticles work against pathogenic microorganisms, Jeong set his sights on how to harness this power to benefit agricultural animals and make animal-based foods safer for people.

Metritis and mastitis are two cattle diseases that plague dairy and beef farmers, costing them millions of dollars per year. Metritis is an infection of the uterine lining caused by multiple microbes. It can strike 20 to 40 percent of postpartum cows and it directly costs the U.S. dairy industry upwards of $600 million annually. Mastitis is an infection of a cow’s mammary tissues, or udder, and it is estimated to cost farmers $110 per cow in lost income.

KC Jeong beef cattle

Beef cattle on a northcentral Florida farm participating in one of Jeong's studies on interactions between livestock, the environment, and bacteria. (Courtesy of the Jeong lab.)

But worse than the lost revenue, cattle are experiencing an increase in multi-drug resistant bacteria relevant to these infection types. This is worrisome for human health too, because bacteria are adept at sharing genes useful to their survival. Jeong has sought to characterize the specific types of bacteria and drug resistance genes involved in these infections, in order to better fight them. Often working with UF College of Veterinary Medicine associate professor Klibs Galvao, Jeong challenged dogma concerning which uterine bacteria are responsible for causing metritis.

Jeong and Galvao’s teamwork revealed that applying chitosan nanoparticles to the infected uteri of cows drastically altered the microflora to a healthier composition. The particles were effective against pathogenic Fusobacteriaceae and Bacteroidaceae, both of which are associated with acute infectious processes and are resistant to penicillin-based antibiotics.

KC Jeong and Klibs Galvao

Jeong and his collaborator, Klibs Galvao, at a dairy cow research center where the pair are investigating novel ways of treating drug-resistant infections in food animals.

 

Coupling chitosan and cephalosporin

recent $460,000 grant from the USDA National Institute for Food and Agriculture brings Jeong’s funding sum close to $1 million for investigating chitosan nanoparticles as a next-gen antimicrobial. His newest phase of inquiry will examine how to attach the best broad-spectrum antibiotics and drug extenders to chitosan nanoparticles to deliver a three-pronged infection-fighting punch directly to infection sites.

“We are very excited about testing this combination therapy,” Jeong says. “Medical doctors don’t have many antibiotics in reserve, so they are beginning to use two or three different antibiotics together in combination to fight highly resistant infections. My lab is taking this idea but repurposing it within the chitosan nanoparticle framework to bring a combination of drugs right into the uterus of cows infected with metritis.”

KC Jeong and lab assistant in laboratory

KC Jeong with Zhengxin Ma, a post-doctoral researcher, looking at a nematode, Caenorhabditis elegans, using a microscope.

The goal is to investigate how to amplify the antimicrobial properties of chitosan nanoparticles by coupling them with two other agents used to fight antibiotic resistant bacteria: third generation cephalosporin antibiotics and β-lactamase inhibitors. Cephalosporin antibiotics are one of the best broad-spectrum antibiotics on the market today, but drug-reistant bacteria threaten their usefulness. Jeong intends to test if combining them with specific nanoparticles will extend their effectiveness.

“Our work is innovative and significant because outcomes of this project are expected to lead to practical, effective new treatment options using nanotechnology,” Jeong says. “Overall, we expect that development of the new, value-added nano-antimicrobials will help improve animal and human health.”

Drug-resistant bacteria in the soil, water

While the trend amongst locavores is “farm to table,” Jeong’s research takes him on a less traveled path from laboratory to farm and back again. A significant part of his research agenda has focused on learning how beef cattle acquire pathogenic E. coli and antibiotic resistant bacteria.

“Our findings show that even animals grown without exposure to antibiotics, animals that have no history of antibiotic use in their feed or for treating infections, they have some baseline of antibiotic resistant bacteria in their gastro-intestinal tracts,” Jeong says. “Which led us to ask, ‘How did it get there?’” That seemingly simple question has led to more than $2.2 million in funding for his research between 2015 and 2020 from the U.S. Department of Agriculture.

Often working with EPI Director John G. Morris, and EPI researchers Judith Johnson and Volker Mai, Jeong’s team sampled a network of northcentral Florida farms to tease out whether antibiotic-resistant bacteria found in grass-fed cows originates in the environment (soil or water), or if it is a natural component of healthy cows’ intestinal microbiota. They identified nine bacterial isolates resistant to cefotaxime (a type of cephalosporin), six of which were identical to those found in people, and many of which also harbored high levels of multi-drug resistance.

Another of Jeong’s studies revealed that beef cattle, sampled every three months after birth, are colonized with Shiga toxin-producing E. coli within their first year of life. This is a particularly virulent strain of E. coli, and learning how it gets into cows could help mitigate its prevalence and reduce the nearly 250,000 cases of food-borne illness it causes in the U.S. alone annually. The colonization is hypothesized to occur from older cows, and the presence of this bacteria is linked with a lower diversity of gut microflora in young calves; however, their diversity then increases as they age.

“We think that the younger calves tend to have a higher burden of these disease-causing pathogens compared to older cows simply because their immune systems and GI-tract microbiota are immature,” Jeong says. “We hypothesize that as the calves age and their immune systems mature, it learns to kick out the harmful bacteria, leading to a healthier microflora composition.”

KC Jeong laboratory

KC Jeong, far right, and his laboratory research team: (from left to right) visiting scholar Haitao Yu, PhD student Peixin Fan, post-doctoral researcher Zhengxin Ma, and PhD students Lin Teng, Shinyoung Lee and Ting Lui.

Similar to the E. coli study, Jeong’s lab discovered that beef calves become colonized with cephalosporin-resistant bacteria in their GI tract even when they are raised without the use of antibiotics. Conventional dogma dictates that overuse of antibiotics in agricultural and large-animal veterinary contexts has driven the rapid increase in antibiotic resistant bacteria in farm animals through selective pressures. But Jeong’s research into animals free of antibiotic exposure acquiring resistant bacteria points to an important alternative pathway: they also get it from the environment.

Building on this study, his lab next sampled soil and agricultural water sources on the affected farms and found cefotaxime-resistant bacteria to be highly prevalent. By analyzing fecal samples from cattle, and environmental samples from 17 different farms, and combining the findings with a survey of husbandry practices, his team uncovered some fascinating trends. They found that 98.7 percent of the soil samples contained the resistant bacteria, as did 95.7 percent of the forage, and 88.6 percent of the water samples. Cefotaxime-resistant bacteria (CRB) in the cows’ feces ranged from a prevalence per farm of 21 to 87.5 percent.

Due to the wide range in occurrence of CRB from farm to farm, Jeong suspects that husbandry methods were influencing the presence of CRBs in these cattle. Further analysis revealed that when cows’ water troughs were cleaned regularly, they tended to have among the lowest CRB counts of any samples in the study, and farms with fewer than 500 cows had a far lower prevalence of CRB than farms with greater numbers of cattle.

“A lot of my research can seem very disparate at first glance,” Jeong says. “But it is all connected under the umbrella of the one health concept, the idea that animal health and people health is connected. It is my hope that we can learn how to reduce harmful bacteria in food animals, to not only increase the safety of our animal foods, but also to increase our own human public health.”


EPI Explainer

How did antibiotic-resistant bacteria get into our soil?

Consider that bacteria are the most abundant microorganisms in soil, and that a single gram of backyard dirt may contain billions of these "bugs." There are many different types of bacteria which have evolved over eons to survive against predators such as other bacteria or fungi. They produce all kinds of materials to kill the “other guys,” which is where our modern arsenal of antibiotics come from. One of the self-defense survival tools bacteria have evolved is production of the extended-spectrum beta-lactamase (ESBL) enzyme — which protects them from having their cell walls destroyed by naturally-occurring antibiotic substances produced by other bacteria. As a side effect, ESBL-producing bacteria just happen to be mucking up modern medicine, because microorganisms that can emit this substance are resistant to many different types of antibiotic drug therapies — including penicillins and cephalosporins.


Creator Credits

Written by DeLene Beeland; All photos by Tyler Jones/IFAS. Special thanks to KC Jeong for his time.

Read More

Read KC Jeong’s EPI profile here.

This article draws on the following published literature:

Reduction of Escherichia coli O157:H7 shedding in cattle by addition of chitosan microparticles to feed. Appl Environ Microbiol. 2011 Apr;77(8):2611-6. doi: 10.1128/AEM.02587-10. Epub 2011 Feb 18.

Underlying mechanism of antimicrobial activity of chitosan microparticles and implications for the treatment of infectious diseases. PLoS One. 2014 Mar 21;9(3):e92723. doi: 10.1371/journal.pone.0092723.

Chitosan Microparticles Exert Broad-Spectrum Antimicrobial Activity against Antibiotic-Resistant Micro-organisms without Increasing Resistance. ACS Appl Mater Interfaces. 2016 May 4;8(17):10700-9. doi: 10.1021/acsami.6b00894.

Application of chitosan microparticles for mitigation of Salmonella in agricultural water. J Appl Microbiol. 2017 Nov;123(5):1346-1358. doi: 10.1111/jam.13566.

Engineering of chitosan-derived nanoparticles to enhance antimicrobial activity against foodborne pathogen Escherichia coli O157:H7. Carbohydr Polym. 2018 Oct 1;197:623-630. doi: 10.1016/j.carbpol.2018.06.046.

Comprehensive in vitro and in vivo risk assessments of chitosan microparticles using human epithelial cells and Caenorhabditis elegans. J Hazard Mater. 2018 Jan 5;341:248-256. doi: 10.1016/j.jhazmat.2017.07.071. Aug 1.

Quantifying known and emerging uterine pathogens, and evaluating their association with metritis and fever in dairy cows. Theriogenology. 2018 Jul 1;114:25-33. doi: 10.1016/j.theriogenology.2018.03.016.

Application of chitosan microparticles for treatment of metritis and in vivo evaluation of broad spectrum antimicrobial activity in cow uteri. Biomaterials. 2016 Dec;110:71-80. doi: 10.1016/j.biomaterials.2016.09.016.

Identification and Characterization of Cefotaxime Resistant Bacteria in Beef Cattle. PLoS One. 2016 Sep 19;11(9):e0163279. doi: 10.1371/journal.pone.0163279.

Colonization of Beef Cattle by Shiga Toxin-Producing Escherichia coli during the First Year of Life: A Cohort Study. PLoS One. 2016 Feb 5;11(2):e0148518. doi: 10.1371/journal.pone.0148518.

Colonization Dynamics of Cefotaxime Resistant Bacteria in Beef Cattle Raised Without Cephalosporin Antibiotics. Front Microbiol. 2018 Mar 21;9:500. doi: 10.3389/fmicb.2018.00500.

High Prevalence of Cefotaxime Resistant Bacteria in Grazing Beef Cattle: A Cross Sectional Study. Front Microbiol. 2019 Feb 7;10:176. doi: 10.3389/fmicb.2019.00176.