(188x) A 3D Printed, Two-Compartment Model for Antibiotic Susceptibility Testing

Antibiotic resistance is a natural phenomenon that necessitates the creation of new antibiotics and faster diagnostic methods. Increasing resources needed to progress from development to FDA approval and the threat of bacteria evolving resistance has led to a reduction in the number of new antibiotics. As drug development progresses, the cost associated with testing increases due to growing complexity and safety of the methods involved. Determining the likelihood of failure of an antibiotic earlier in the drug development process could help reduce costs. Here we present a 3D printed, two compartment, in vitro device to determine antibiotic susceptibility of a bacterial strain under pharmacokinetic profiles that more closely mimic in vivo environments. This 3D printed device utilizes a 3D printed insert, both printed using polyjet printing. This insert is created using a print-pause-print method that allows for integration of porous membranes to mimic diffusion barriers found in vivo. The purpose of the device is to mimic a drug’s pharmacokinetics in the body by flowing antibiotic growth media under the central compartment allowing diffusion into the compartment (absorption) and subsequent diffusion into the secondary compartment where bacteria would reside (distribution). The gradient is then reversed by flowing growth media without antibiotic (excretion). Utilizing a ratio of extracellular ATP released by the bacterial culture compared to the turbidity of the culture (OD600) allows for the rapid evaluation of antibiotic susceptibility within 1 hour of reaching the C_max of the antibiotic. Testing Levofloxacin (C_max = 17.4 ± 3.2 µM) against Kanamycin-resistant E. coli yielded a ratio of 1109.0 ± 196.0 nM/OD600 when compared to a control (27.9 ± 16.3 nM/OD600; p < 0.05). The difference is measurable due to extracellular ATP being degraded or hydrolyzed by live bacteria during later stages of the growth phase (lower ATP/OD600) as well as an increase in extracellular ATP caused by dying bacterial cells (higher ATP/OD600).