Of all the problems facing healthcare systems, chronic wounds are one of the most challenging to address. Characterised as any wound that fails to heal in a timely manner – commonly diabetic foot ulcers or venous leg ulcers – they are costly to treat, distressing for patients, and marked by high recurrence rates. They are also becoming more prevalent as the population ages, affecting around 1.5-2 million people in Europe and 2.4-4.5 million in the US.

While the causes vary – no two chronic wounds are the same – we can surmise that something has happened to interrupt the healing process. This might be a circulatory problem, a systemic illness, immunosuppression or something else. On top of that, these wounds are prone to bacterial colonisation of the most pernicious kind – the formation of a biofilm.

“It hasn’t necessarily been proven that it’s the presence of a biofilm that causes an acute infection to develop into a chronic infection, but they certainly do worsen wound healing,” says Barbara Conway, head of pharmacy at the University of Huddersfield, UK, and co-director of the Institute of Skin Integrity and Infection Prevention. “It’s that combination of a chronic wound in a non-healing state, and the presence of the biofilm that together provides a real problem.”

Why are biofilms such a problem?

In their free-living, ‘planktonic’ state, bacteria are easy enough for the body to deal with. Rampaging phagocytes (a type of white blood cell) engulf the bacteria and chew them up. However, bacteria have found a way to defend themselves within hostile environments: they adhere to surfaces, where they organise themselves into fortress-like structures known as biofilms.

“The bacteria tend to grow together in large groups and surround themselves with a sticky, slimy matrix,” says Sarah Rowe-Conlon, a research associate professor in the Department of Microbiology and Immunology at the University of North Carolina. “This matrix is the biofilm, and it acts like a forcefield protecting the encased bacteria from our immune systems and other assaults, such as antibiotics.”

Sometimes described as ‘cities for microorganisms’, biofilms are complex communities in which members work together to fend off attacks. Like any other city, they are cosmopolitan in nature – composed not just of bacteria, but of a mixture of different entities, including polysaccharides, proteins and DNA.

“You get them on all sorts of surfaces,” says Conway. “You get them on medical devices, you get them in natural environments like rocks and rivers, and you also get them in other places within the human body. We’re disturbing and removing a biofilm every time we brush our teeth.”

Unfortunately, the biofilms that form in chronic wounds aren’t as easy to dispel as dental plaque. Even once the wound has been debrided (physically scraped out), cells can remain, and the bacterial ‘city’ can start to rebuild its walls. Forty percent of diabetic foot ulcers will have reappeared within a year, rising to 65% within five years.

“I often compare the current therapeutic strategy to removing a weed in your garden by cutting the stem off using scissors,” says Rowe-Conlon. “You can cut it off, but you're going expect to it to grow back, and it might grow back in the same place or it might proliferate somewhere else.”

While healthcare professionals do administer antibiotics, this tends to bring mixed success. For one thing, certain classes of drugs can struggle to penetrate the biofilm. For another, sluggishly growing ‘persister cells’ within the biofilm can tolerate very high concentrations of antibiotics. Biofilms are even known to contribute to antibiotic resistance, which can happen when bacteria of different species swap genetic material.

“For example, the first reported incidence of a strain of Staphylococcus aureus acquiring vancomycin resistance from Enterococcus was from a patient who had a diabetic foot ulcer and was co-infected with both pathogens,” says Rowe-Conlon “This is an example of bacteria sharing resistance mechanisms within biofilms.”

The research community is responding

Evidently, we need approaches that get rid of the biofilm altogether. Some researchers are looking at strategies that disperse biofilm communities, while others are more interested in stopping the biofilm from forming in the first place.

“It’s important to understand the biofilm lifecycle, from early colonisation through to maturity, because the approach that is needed will be determined by the stage it has reached,” says Conway. “You can either try to stop the adhesion and the formation of the biofilm, or you can try to destroy it after it’s already in existence. People are working on targeting entities within the biofilm like the polysaccharides and proteins.”

The percentage reduction in MRSA infection achieved in the wounds of diabetic mice using the sonotherapy approach.
Huddersfield University

Conway herself works within pharmaceutical formulation, designing drug delivery systems that target drugs towards the sites where they’re needed. In the context of wound care, she is particularly interested in strategies involving nanomaterials, in which the microbial agent is encased in tiny particles and (to continue the city analogy) smuggled through the city walls.

“The idea is to develop nanoparticles that can penetrate biofilms,” she says. “These would be smart release carriers, which are triggered to release the antimicrobial when they reach the bacteria. That could be used either for preventing biofilm formation, or for disrupting current biofilms.”

While she is hopeful about the wave of research being conducted in this area, she points out it can be tricky to turn lab studies into real-world applications. What’s more, the disparate healthcare professionals involved in wound care tend to work in siloes. More translational research is needed, and the field needs to become more joined up.

“The idea is to develop nanoparticles that can penetrate biofilms. These would be smart release carriers, which are triggered to release the antimicrobial when they reach the bacteria. That could be used either for preventing biofilm formation, or for disrupting current biofilms.”
Barbara Conway

“You can grow biofilms in the lab, but that biofilm won’t be the same as it’s going to be in a wound,” she says. “People are different, the types of constituents in each wound are different, it changes over time, and you’ve got that complex ongoing healing process as well. That’s why a one-size-fits-all approach doesn’t work. We’re always after realistic models to use to test our formulations.”

A promising approach

Across the Atlantic, Rowe-Conlon’s team have also been looking for ways to deliver antimicrobials through biofilms. Their approach combines a topical antibiotic (gentamicin), an antibiotic adjuvant (palmitoleic acid) and non-invasive ultrasound. It was used successfully in an animal model, reducing MRSA infection by 94% in the wounds of diabetic mice.

“Our method, which we call sonotherapy, involves adding topical antibiotics on top of the wound, along with these nanoscale droplets,” says Rowe-Conlon. “Once the nanoscale droplets have penetrated inside the biofilm, we add an ultrasound wave. This causes the little droplets to expand into much larger microbubbles, which oscillate in response to the ultrasound pressure and causes gaps to form. The fluid movement around the bubble helps the drugs penetrate inside the biofilm and kill the embedded bacteria.”

Ultrasound-responsive microbubbles have been used for decades within diagnostic imaging, but they’ve only recently been tested within therapeutic applications. According to Virginie Papadopoulou, a research assistant professor in the UNC-NCSU Joint Department of Biomedical Engineering, and the ultrasound lead of the study, this is the first time they have been used topically rather than intravenously in vivo.

“We just pipette them on top of the wound, to see if we can use them for drug delivery,” she says. “That's one aspect that's new – the nanoscale droplet formulation variant we’ve used allows for increased stability and penetration into the biofilm. We are also using a slightly different type of bubble. When we start our treatment, they’re tiny liquid droplets in a lipid shell. Once we apply the ultrasound, we can convert them back into a gas bubble about the size of a red blood cell.”

What’s notable about this research is that the antibiotic chosen (gentamicin) is typically ineffective against MRSA. That’s because it performs badly against the persister cells. In this case, the palmitoleic acid works as a novel, non-toxic antibiotic adjuvant – helping the antibiotic wipe out the persister cells and even reversing antibiotic resistance.

“Gentamicin is great at stopping the spread of the infection, but it's not very good at actually removing a biofilm infection,” says Rowe-Conlon. “Using our approach we can reduce the biofilm by 94%, and three of our eight mice had no detectable bacteria left in their wounds. We're very excited that this topical only, non-invasive approach is so effective at reducing the biofilm, compared to the standard of care.”

The team at UNC are now looking to scale up their research into a larger animal model, and, if that works, a safety study in humans. While their technique is still a long way from the clinic, it could one day be used as an add-on therapy in multiple contexts. The ultrasound is portable (meaning it could be used in outpatient settings) and potentially compatible with any antibiotic.

“There was a recent study that suggested by 2050, more people will succumb to antibiotic-resistant infections than currently succumb to cancer,” says Rowe-Conlon. “With the void in the drug development pipeline, we need strategies to make our current arsenal of antibiotics work better, rather than relying on the development of new molecules.”

For anyone involved in chronic wound care – or in treating infections more generally – biofilms represent a challenging battlefront. Understanding their complexities will be key if we want to use antibiotics effectively and improve patients’ quality of life.