Antimicrobial resistance is an increasing problem worldwide, with the World Health Organization calling for urgent action to avoid a “post-antibiotic era” in which common infections and minor injuries can once again kill.

Bacteria in acute infections usually exist in a free-swimming state and can be treated with antimicrobials. Chronic infections, however, can lead to the development of bacterial biofilms – 3D structures of bacterial cells – which are up to 1000 times more resistant and can render standard antimicrobial therapies ineffective. Unfortunately, most methods used to study biofilm resistance are 2D in nature, and thus unable to reflect complex 3D infection processes seen in vivo.

As such, there’s an urgent need for 3D biofilm models that realistically represent clinical infection. With this aim, a team headed up at the University of Strathclyde has developed a way to 3D print bacteria biofilms for antimicrobial resistance testing and screening of new drugs (Biofabrication 10.1088/1758-5090/ab37a0).

“Our aim is to create clinically relevant 3D bacteria biofilms that can potentially mirror in vivo bacterial growth and behave more closely than traditional 2D models, for example, in their responses to drug treatment,” says senior author Wenmiao Shu.

3D bioprinting

Shu and colleagues created a bacteria-laden bioink by mixing live bacteria into a partially-crosslinked hydrogel. They employed a custom-built bioprinter to extrude this bioink into constructs with precisely controlled thickness and design. After printing, the researchers immersed the structures in barium chloride solution to increase hydrogel cross-linking and boost stability.

Over time, as the bacteria grew, 3D biofilms developed on the constructs. The researchers observed excellent bacterial viability in the constructs, with production and maturation of biofilms possible for at least 28 days. This long-term stability more closely mirrors clinical biofilms than previous biofilm models.

To determine the ideal construct design, the researchers bioprinted solid and porous constructs with thicknesses from 0.25 to 4 mm. Using E. coli, they saw that biofilm formation was greater in thinner (0.25–1 mm) than thicker (4 mm) constructs, attributed to restricted diffusion of nutrients and oxygen. However, the thinner constructs were not robust enough for physical manipulation and analysis after 14 days.

Growth in solid constructs was slower than in porous constructs for all thicknesses, likely due to the porous design facilitating fluid transport and nutrient and oxygen diffusion. The optimal structure for an E.coli biofilm was a 1 mm porous construct.

Whilst aerobic E. coli grew less in thicker constructs, anaerobic bacteria can thrive in oxygen-depleted conditions. To test this, the researchers grew films of the anaerobic P. aeruginosa in non-porous 2- and 4-mm thick constructs. They found that P. aeruginosa formed an extremely dense biofilm layer in these structures, and note that the 4 mm biofilm is likely the thickest 3D bioprinted biofilm construct reported to date.

“The key challenge was the development of a stable hydrogel system that would allow 3D biofilm formation over a longer period of time,” explains Shu. “In this study, we successfully developed a stable hydrogel system that will last over four weeks and allows us to observe the full life-cycle of 3D biofilm development: formation, growth, maturation and finally dispersal, which is migration to a new site for infection.”

Resistance testing

To assess the susceptibility of 2D and 3D bacterial cultures to treatment, the researchers compared 3D printed biofilms with 2D bacterial cultures, using S. aureus. Most strains of this pathogen, including methicillin-susceptible S. aureus (MSSA), are sensitive to antibiotics. However, there is a growing worldwide prevalence of methicillin-resistant S. aureus (MRSA) infections.

The team determined the minimum concentrations of methicillin that prevented growth of MRSA and MSSA in 2D culture and  in 3D biofilms. As expected, MRSA required higher drug concentrations than MSSA, indicating greater resistance to antimicrobials. However, for both strains, far higher doses were required to treat the biofilm than corresponding 2D cultures.

The researchers also compared 1- and 2-mm thick bioprinted E. coli constructs exposed to tetracycline. Thicker biofilms showed greater resistance to the antimicrobial drug, with more bacteria surviving in the 2 mm than the 1 mm constructs after seven days of treatment. They surmised that the thicker and longer-lasting biofilms behaved more closely to biofilms in in vivo infection.

This demonstrated ability to design and control the thickness of biofilms will also allow the team to study drug penetration in 3D, which is impossible for 2D biofilms. A more immediate benefit could be testing the impact of drugs on biofilm infections for an individual patient. More accurate bacterial biofilm models may also aid future development of novel drug compounds and therapies for tackling the disease.

“We are already working with the clinicians who are very excited about the new possibilities enabled by the new research,” Shu tells Physics World. “Our next focus is on applying the new bioprinting technology for studying and developing more effective drug treatment for specific biofilm-associated infections.”