Patterned Biofilm Formation to Investigate Biofilm-Associated Antibiotic Resistance

Biofilm-related high-level tolerance to antimicrobials is a leading cause of chronic infections in humans and persistent biofouling in industry. However, the mechanism of such tolerance is still not fully understood due to the intrinsic heterogeneity in biofilm structure and associated spatial variation in cell physiology. Previously, we demonstrated that patterned biofilms with well-defined size and shape of micron-scale cell clusters and distance between adjacent cell clusters can be obtained using gold surfaces with patterned functional alkanthiols to control structural heterogeneity. Using this system, we found that multicellular connections can be formed between cell clusters depending on the size of interacting clusters and the distance between them.

These results led to our hypothesis that antibiotic tolerance of biofilm cells may change during biofilm growth and the cells involved in inter-cluster interactions are more sensitive to antibiotics. To test this hypothesis, antibiotic treatment of Escherichia coli biofilm cells during biofilm formation was monitored using live/dead staining and colony forming units (CFU) viability assays. The results revealed that the antibiotic susceptibility of biofilm cells increased in the first two hours after incubation and then gradually decreased to the level of overnight planktonic cultures. In addition, E. coli RP437 cells attached between micron-scale square patterns were found more sensitive to ampicillin compared to the cells within the square-shaped cell clusters. These findings are consistent with our earlier report that active interaction between all clusters is involved during biofilm formation, and provide new information for maximizing the outcome of antibiotic treatment.