Photodynamic patterning of living bacterial biofilms with high resolutions for information encryption and antibiotic screening

Controlling the growth of bacterial biofilms in a specific pattern greatly enhances the study of cell-to-cell interactions and paves the way for expanding their biolog-ical applications.However,the development of simple,cost-effective,and highly resolved biopatterning approaches remains a persistent challenge.Herein,a pio-neering photodynamic biopatterning technique for the creation of living bacterial biofilms with customized geometries at high resolutions is presented.First of all,an outstanding aggregation-induced emission photosensitizer is synthesized to enable efficient photodynamic bacterial killing at a low concentration.By combining with custom-designed photomasks featuring both opaque and transparent patterns,the viability of photosensitizer-coated bacteria is successfully manipulated by control-ling the degree of light transmittance.This process leads to the formation of living bacterial biofilms with specific patterns replicated from the photomask.Such an innovative strategy can be employed to generate living bacterial biofilms composed of either mono-or multispecies,with a spatial resolution of approximately 24µm.Furthermore,its potential applications in information storage/encryption and antibi-otic screening are explored.This study provides an alternative way to understand and investigate the intricate interactions among bacteria within 3D biofilms,hold-ing great promise in the controlled fabrication of dynamic biological systems for advanced applications.

Automated and miniaturized screening of antibiotic combinations via robotic-printed combinatorial droplet platform

Antimicrobial resistance(AMR)has become a global health crisis in need of novel solutions.To this end,antibiotic combination therapies,which combine multiple antibiotics for treatment,have at-tracted significant attention as a potential approach for combating AMR.To facilitate advances in anti-biotic combination therapies,most notably in investigating antibiotic interactions and identifying synergistic antibiotic combinations however,there remains a need for automated high-throughput plat-forms that can create and examine antibiotic combinations on-demand,at scale,and with minimal reagent consumption.To address these challenges,we have developed a Robotic-Printed Combinatorial Droplet(RoboDrop)platform by integrating a programmable droplet microfuidic device that generates antibiotic combinations in nanoliter droplets in automation,a robotic arm that arranges the droplets in an array,and a camera that images the array of thousands of droplets in parallel.We further implement a resazurin-based bacterial viability assay to accelerate our antibiotic combination testing.As a demonstration,we use RoboDrop to corroborate two pairs of antibiotics with known interactions and subsequently identify a new synergistic combination of cefsulodin,penicillin,and oxacillin against a model E.coli strain.We therefore envision RoboDrop becoming a useful tool to efficiently identify new synergistic antibiotic combinations toward combating AMR.

Logic-signal output of fluorescent proteins for screening antibiotic combinations

A new method to screen antibiotic combinations is demonstrated,which takes advantage of the logic-signal output of genetically engineered drug-resistant E.coli strains expressing different fluorescent proteins.Thirty-six antibiotic combinations for nine antibiotics were investigated.The operation of different logic gates can reveal the susceptibility,resistance,or synergistic effect of the antibiotic combinations in a rapid(7–8 h versus 24–28 h for typical growth-based assays),simple,quantitative and high-throughput manner.This logic-signal-based output patterns provide the basis for novel and reliable screening of antibiotic combinations and help us to both gain insight into the mechanisms of multi-drug action.