3D bioprinting of bacterial biofilms


Bacteria are microorganisms capable of growing in any ecological niche due to their adaptive and diverse metabolic activity, which is richer than in any other type of organism. They are essential engines of all ecosystems and various biogeochemical processes. These microorganisms manage to create biofilms that guarantee their survival even in hostile environments [1].


Biofilms are defined as communities of bacterial cells structured three-dimensionally in a self-produced polymeric matrix fixed to a solid surface. During the growth period of biofilms, bacteria form and degrade many compounds, which are often used to synthesize chemicals, biopolymers, enzymes, and proteins essential for biomedical applications [2].


Bacterial biofilms are responsible for establishing chronic infections, such as endocarditis and respiratory infection since they protect the microorganisms from the action of antibiotics and bactericidal agents. Microbiologists have already indicated that artificial biofilms can successfully control events such as cell-cell communication. For instance, bacterial cells within a biofilm can engage in gene transfer between member cells and demonstrate increased antimicrobial resistance. According to the World Health Organization (WHO), combating bacterial resistance is urgent action, as it is predicted to result in 10 million deaths every year worldwide by 2050 [2,3]


Figure 1: Photomicrograph of a bacterial biofilm [4].


3D bioprinting of bacterial biofilms has recently become an area of ​​great interest due to the potential of the printed constructs to resemble the in vivo bacterial growth more closely than usual 2D models. Bioprinting biofilms with controlled thickness and architecture can lead to new ways of controlling and understanding bacteria’s dynamic environment and interactions [5].


Dubbin et al. developed a bioprinting technique to pattern microbial constructs and establish geometrically defined culture conditions for bacterial biofilm growth. Using light projection stereolithography, bacteria were bioprinted within different hydrogel architectures. Distinct geometries of the bacteria-laden printed films affected nutrient flux, microbial growth within the defined 3D matrix, and as a consequence, their biocatalytic activity. The study evaluated the use of the bioprinted bacterial biofilms in applications such as uranium detection, bioremediation, and wastewater cleaning. The researchers examined the application of microbial absorption of metal ions to investigate geometric effects on metal sequestration efficiency and uranium detection capability. This work was the first demonstration of stereolithographic printing of bacteria and biofilms and opened a new perspective for future studies on the subject [6].



Figure 2: The schematic representation of the method used for the projection microstereolithographic microbial bioprinting for engineered biofilms [6].


Ning et al. fabricated 3D bioprinted bacterial biofilms for antimicrobial resistance drug testing. Solid and porous constructs containing clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa were 3D bioprinted using an alginate bioink. Bacterial viability was excellent in the bioprinted constructs and the complete five-step biofilm life cycle was observed, suggesting the formation of mature structures. The architecture of the printed biofilm influenced bacterial growth, which was faster in thinner and more porous constructs. Thicker biofilms showed greater resistance to antimicrobial therapy than thinner biofilms. Exploring complex and well-defined 3D structured biofilms is an interesting tool to aid the discovery of novel therapeutic drugs and increase the understanding of biofilm formation [2].


Figure 3: Schematic of bacterial biofilm bioprinting process [2].



REFERENCES


1. Schaffner, M., Rühs, P. A., Coulter, F., Kilcher, S. & Studart, A. R. 3D printing of bacteria into functional complex materials. Sci Adv 3, eaao6804 (2017).

2. Ning, E. et al. 3D bioprinting of mature bacterial biofilms for antimicrobial resistance drug testing. Biofabrication 11, 045018 (2019).

3. Mohammadi, Z. & Rabbani, M. Bacterial Bioprinting on a Flexible Substrate for Fabrication of a Colorimetric Temperature Indicator by Using a Commercial Inkjet Printer. J. Med. Signals Sens. 8, 170–174 (2018).

4. Maddocks, S. Biofilms – the bacterial wound communities that protect themselves from attack. The Conversation (2015).

5. McCarthy, R. R., Ullah, M. W., Booth, P., Pei, E. & Yang, G. The use of bacterial polysaccharides in bioprinting. Biotechnol. Adv. 37, 107448 (2019).

6. Dubbin, K. et al. Projection Microstereolithographic Microbial Bioprinting for Engineered Biofilms. Nano Lett. 21, 1352–1359 (2021).