(330g) Antifouling Topographies to Combat Biofilm-Associated Antibiotic Tolerance

Huan Gu^1,2, Sang Won Lee^1,2, and Dacheng Ren^1,2,3,4*

¹Department of Biomedical and Chemical Engineering, ²Syracuse Biomaterials Institute, ³Department of Civil and Environmental Engineering, ⁴Department of Biology, Syracuse University, Syracuse, NY 13244, USA

Bacterial attachment to indwelling medical devices and the subsequent formation of multicellular biofilms, are the primary causes of hospital-acquired infections. Biofilms are composed of microbes encased in self-produced extracellular polymeric substrates (EPS). Due to the protection of EPS, a hallmark characteristic of mature biofilms is their high-level tolerance (up to 1,000 times) to conventional antibiotics compared to planktonic cells. Such multidrug tolerance makes it essentially impossible to eradicate mature biofilms, leading to persistent infections with high morbidity and mortality, and posing a heavy burden on the healthcare system.

To effectively control biofilms, it is important to understand the dynamic change in antibiotic susceptibility during biofilm formation and associated mechanisms. By tracking the susceptibility of Escherichia coli biofilm cells to antibiotics during the first 24 h biofilm formation, we found that biofilm cells were not always tolerant to antibiotics. Specifically, E. coli biofilms were formed in Lysogeny Broth (LB) on glass surfaces and treated with 200 µg/mL ampicillin (Amp) or 5 µg/mL ofloxacin (Ofx) for 1 h at 37 °C. The results show that the susceptibility of E. coli biofilm cells to both Amp and Ofx increased in the first 2.5 h after the inoculation. After this time point, the susceptibility to Amp decreased with the start of EPS production, while the susceptibility to Ofx did not decrease until 16 h after inoculation. Interestingly, the embedded biofilm cells remained active and thus susceptible to antibiotics if dispersed from the surface. Similar phenomena were observed during the biofilm formation of Pseudomonas aeruginosa PAO1 and Uropathogenic E. coli (UPEC) cells. These results indicate that physiological changes during early-stage biofilm formation can sensitize bacterial cells to conventional antibiotics. Thus, materials engineering to create dynamic and active topographies may render bacteria cells stay in an active stage and thus susceptible to antibiotics. We will present two examples of material design for such effects.

The first example is biofilm control using dynamic topographies generated by shape recovery of t-butyl acrylate (tBA) based shape memory polymer (SMP). This polymer can achieve one-way shape recovery from the temporary to permanent shape when the temperature increases from 25 to 40°C. We demonstrate that such on-demand actuation of shape recovery causes effective removal of both bacterial and fungal biofilms, e.g., 99.9% (3 logs) removal of P. aeruginosa biofilms. Along with biofilm removal, the detached cells were sensitized to antibiotics. For example, the susceptibility of P. aeruginosa to 50 µg/mL tobramycin increased by 2,479-times compared to the static flat control. This was attributed to both the disruption of biofilm structure and the increase in cellular activities as evidenced by the increase in intracellular level of ATP and expression of the rrnB gene.

Encouraged by the effects of dynamic topographies, we further developed a biofilm control strategy based on active topographies with magnetically driven micron-sized pillars. By turning the electromagnetic field on and off at a tunable frequency, active pillars can beat periodically, and thus, achieve long-term control of biofilm formation by inhibiting bacterial attachment and removing mature biofilms on demand (with stronger actuation). Active topographies with the optimized design prevented biofilm formation and removed established biofilms of UPEC, P. aeruginosa, and Staphylococcus aureus, with up to 3.7 logs of biomass reduction compared to the flat and static controls. In addition, the detached biofilm cells were sensitized to bactericidal antibiotics to the level comparable to exponential-phase planktonic cells. Such effects demonstrate a synergy between smart materials and antibiotics, which may help design safer medical devices.