A patient with long-term cardiac issues makes the decision to get a pacemaker. The surgery goes well, and they’re excited to feel strong and confident in their heart again. But after the surgery, they start feeling chest pain, fatigue, and a creeping fever. Doctors say the implant is infected, and course after course of strong antibiotics isn’t helping. The patient goes back into surgery again to remove the infected pacemaker, but doctors say they can’t guarantee they’ll be able to remove all of the bacteria from the heart tissue.

If this sounds like a clear-cut case of antibiotic resistance, think again. The World Health Organization (WHO) recognizes antimicrobial resistance — when microbes like bacteria evolve immunities to antibiotics — as “one of the top global public health and development threats,” but it’s not the only reason that antibiotics stop working (1). 

Antibiotic tolerance is another major driver of chronic infections. It occurs when bacteria are still genetically susceptible to antibiotics, but external factors prevent the drugs from working. One of the biggest causes of antibiotic tolerance are biofilms — thick, slimy groups of bacteria that quarantine themselves from the outside environment. Biofilms happen when bacteria find themselves in a hostile environment like the human body, causing them to slow down their metabolisms like a hibernating bear and secrete a viscous, extracellular matrix to block would-be attackers.

“It basically is like a force field,” said Sarah Rowe-Conlon, a microbiologist at the University of North Carolina at Chapel Hill. 

Biofilms can form on medical implants, in the wounds of diabetic patients, and across organs of the body, leading to long-term conditions like chronic urinary tract infections or pneumonia. They’re found in 60 percent of chronic wounds, and they slow healing while threatening complications like sepsis, amputation, and death (2,3).

Antibiotic tolerance due to biofilms is a separate phenomenon from antibiotic resistance, but the two are linked. Environmental stress — such as exposure to antibiotics — drives bacterial evolution, and while biofilms help keep bacteria safe from outside forces, they also stress the bacteria with low oxygen and limited nutrients. As doctors throw long courses of antibiotics at chronic infections, stress and prolonged exposure to antibiotics create the perfect conditions for bacteria to evolve new resistance mechanisms. So, though not all biofilms are associated with antibiotic-resistance, the two can and do occur together. 

It’s like a weed in your yard. If you go into your yard, and you cut the top off a weed with the scissors but the root is still there, it's obviously going to grow back. 
- Sarah Rowe-Conlon, University of North Carolina at Chapel Hill

Biofilms are especially difficult to treat because the sticky extracellular matrix they secrete is dense and negatively charged, which prevents most antibiotics from getting in. If a drug can penetrate the matrix, it faces another challenge: To cope with low oxygen and limited nutrients, some bacteria slow their metabolisms into a dormant state. These are called persister cells. Because most antibiotics block processes like DNA replication or cell wall synthesis — regular steps in a fast-growing cell — they lose potency against these slow-growing persister cells. 

Rowe-Conlon compared the problem to throwing a wrench into a machine full of cogs. If the cogs are moving quickly, a wrench will jam the whole system, but if the cogs are barely moving, the wrench will bounce off and do little damage.

There are currently no reliable cures for biofilm-associated infections. Patients may need to visit a clinic for uncomfortable weekly cleanings or put up with the side effects and risks of long-term antibiotic regimens. Some physicians may physically cut or scrape off infected tissue to remove a biofilm, but Rowe-Conlon said this technique isn’t foolproof.

“It’s like a weed in your yard,” said Rowe-Conlon. “If you go into your yard, and you cut the top off a weed with the scissors but the root is still there, it's obviously going to grow back.”

The promise of ultrasound

To eliminate biofilms, roots and all, researchers like Rowe-Conlon think one answer is a tool already in many physicians’ arsenals: ultrasound. 

Ultrasound has been a staple in medical imaging since the 1960s. These devices sense and emit high-frequency sound waves that bounce and scatter in predictable ways on different types of tissue, allowing healthcare providers to visualize structures inside the body. Ultrasound imaging is enhanced with the help of microbubbles, tiny gas-filled bubbles about the size of a red blood cell that amplify the contrast between different tissue types. Under the right conditions, ultrasound waves can push these microbubbles around to generate disturbances at the cellular level — just enough to break up the viscous matrix of a bacterial biofilm. 

Rowe-Conlon and Virginie Papadopoulou, a biomedical engineer at the University of North Carolina at Chapel Hill, employ ultrasound to eradicate biofilms in diabetic chronic wounds. Using a combination of phase-shift microbubbles and gentamicin, a broad-spectrum antibiotic, the researchers shared promising results in a mouse model, published in the journals Biofilm and Cell Chemical Biology (4,5).

The specially engineered phase-shift microbubbles begin as a liquid, but they transform into a gas at a specific ultrasound pressure. Then, as the ultrasound waves ebb and flow, the gaseous microbubbles rhythmically expand and contract. When the microbubbles' size fluctuates, tiny, Swiss-cheese holes momentarily form in the biofilm, allowing the antibiotic molecules to rush in.

“We're kind of creating these silos that help antibiotics penetrate deep into the biofilm,” explained Papadopoulou. 

But just getting the antibiotic into the biofilm isn’t always enough; they also needed to enhance the antibiotic so that it’s effective against persister cells. To do this, Rowe-Conlon and Papadopoulou incorporated an adjuvant that helped the gentamicin latch onto persister cells.

I think of it as a little pin that inserts itself into the membrane of the bacteria. It will allow the drug to get through, even in those subsets that are very metabolically inactive. 
- Virginie Papadopoulou, University of North Carolina at Chapel Hill

“I think of it as a little pin that inserts itself into the membrane of the bacteria,” said Papadopoulou.  “It will allow the drug to get through, even in those subsets that are very metabolically inactive.”

When Rowe-Conlon and Papadopoulou used this technique, called sonotherapy, to treat diabetic mice with chronic wounds infected by methicillin-resistant Staphylococcus aureus (MRSA), they found the ultrasound combination effectively killed biofilm-associated bacteria. Mice that underwent sonotherapy presented with 99 percent less bacteria than those that received standard diabetic chronic wound care. Because mice were euthanized to quantify bacterial levels, the researchers were not able to observe how the mice’s wounds continued to heal. 

Rowe-Conlon and Papadopoulou’s research is promising for patients with external biofilm-associated infections, but for biofilms that form inside the body, research is still in its early stages. Luckily, ultrasound is already used for internal imaging, so its ability to penetrate deep into tissues is well-characterized.

At the University of Oxford, mechanical engineer Eleanor Stride is beginning to use ultrasound to target internal biofilms in chronic bladder and urinary tract infections. Using an organoid model, she’s found that injecting a microbubble-antibiotic combination and applying ultrasound improved antibiotic penetration 16-fold (6). Further in vitro  studies showed that the technique curbed bacteria levels by 99 percent (7).

From in vitro models to human infections

So far, research on ultrasound’s effects on biofilm-associated infections has been limited to in vitro  studies, animal models, and organoids, but researchers said they’re hopeful the translation process will move quickly. 

Ultrasound has the support of decades of safety research, and it’s already established for certain therapeutic uses including uterine fibroid treatment and cataract removal (8). Rowe-Conlon and Papadopoulou said their next concern is adapting the method to larger animals — though one group has already found success treating septic arthritis in a pig model (9).

Because Rowe-Conlon, Papadopoulou, and Stride’s drug and microbubble formulas feature minor tweaks to established products, the researchers are currently embarking on the regulatory approval process.

“Our biggest challenge is probably getting regulatory approval for the bubbles themselves,” said Stride. “We're actually sticking the drug molecules onto the bubble, so we're really getting very targeted delivery. Unfortunately, that means from a regulatory perspective, we've invented a new drug.”

Stride said once her team passes those hurdles, she’s confident they can quickly translate the technique into humans. 

Rowe-Conlon, Papadopoulou, and Stride all expressed concerns about ultrasound potentially dislodging bacteria and sending it into the bloodstream — risking systemic infection — but they’ve seen little evidence of this being a hazard during their trials.

“Talking to the clinicians we work with, they said, ‘Look, compared to the damage we do when we actually mechanically debride infected tissue, this is not a risk,’” said Stride.

Stride added that her main concern was that patients’ immune systems won’t be strong enough to clear an infection, even with the help of ultrasound. Many patients with chronic wounds or infections have poor circulation and dampened immune systems, and breaking down the biofilm is only the first step in the healing process.

Ultrasound isn’t the only technique in development for biofilm-associated infection treatment. Some researchers are engineering nanoparticle-based drugs, which are small enough to slip through the sticky biofilm matrix. Others are using enzymes to digest that extracellular matrix. Also in the running are chemical agents that disrupt extracellular communication between bacteria needed to build and maintain a biofilm.

While she’s hopeful for the future of ultrasound-mediated therapy, Stride said it’s likely that to successfully manage biofilms, physicians will need to use all the tools in their arsenal. 

“The general agreement was we need everything,” said Stride, speaking about discussions at a conference on biofilms and microbial resistance. “It's not that one is going to win. We need all these techniques for different infections because the problem is so urgent.”

References

1. World Health Organization. Antimicrobial resistance (2023).
2. James, G.A. et al. Biofilms in chronic wounds. Wound Repair Regen  16, 37-44 (2008).
3. Schultz, G. et al. Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds. Wound Repair Regen  25, 744-757 (2017).
4. Durham, P.G. et al. Harnessing ultrasound-stimulated phase change contrast agents to improve antibiotic efficacy against methicillin-resistant Staphylococcus aureus biofilms. Biofilm  3, 100049 (2021).
5. Papadopoulou, V. et al. Overcoming biological barriers to improve treatment of a Staphylococcus aureus wound infection. Cell Chem Biol  30, 513-526 (2023). 
6. Horsley, H. et al. Ultrasound-activated microbubbles as a novel intracellular drug delivery system for urinary tract infection. J Control Release  301, 166-175 (2019).
7. LuTheryn, G. et al. Bactericidal and anti-biofilm effects of uncharged and cationic ultrasound-responsive nitric oxide microbubbles on Pseudomonas aeruginosa biofilms. Front Cell Infect Microbiol  12, 956808 (2022).
8. Miller, D.L. et al. Overview of therapeutic ultrasound applications and safety considerations. J Ultrasound Med  31, 623-634 (2012). 
9. Zhao, N. et al. Microbubble cavitation restores Staphylococcus aureus antibiotic susceptibility in vitro and in a septic arthritis model. Commun Biol  6, 425 (2023).