王致丞^1,2、鐘智堯²、莊漢聲^2,3

¹奇美醫學中心 外科部 泌尿科；²成功大學 生物醫學工程研究所，³醫材研發中心

Jhih-Cheng Wang^1,2, Chih-Yao Chung², Han-Sheng Chuang^2,3

Divisions of Urology, Department of Surgery, Chi Mei Medical Center¹, Tainan, Taiwan;

Department of Biomedical Engineering² and Medical Device Innovation Center³, National Cheng Kung University, Tainan, Taiwan

 

Purpose: This study combined optical diffusometry and bead-based immunoassays to develop a novel technique for quantifying the growth of specific microorganisms and achieving rapid antimicrobial susceptibility test (AST).

Materials and Methods: Optical diffusometry requires only a microscope and a camera to quantify the Brownian motion of particles. Because Brownian motion is a random and self-driven physical phenomenon, this technique can avoid the aforementioned limitations. In our concept of design, as bacteria grow and attach to particles, the measured Brownian motion tends to vary in response to the increased equivalent particle diameter. When bacteria are sensitive to an antibiotic, the change will then be halted, which can be associated with the minimum inhibitory concentration (MIC) of the drug. In an attempt with P. aeruginosa, we demonstrated that an AST process can be complete within 2 h. In addition, the minimum requirement of the sample volume is only 0.5 μL while the initial bacteria count is as low as 50 CFU per droplet (10⁵ CFU/mL).

Results: An assessment of binding specificity showed that 92 ± 2.2% of the anti-P. aeruginosa polyclonal antibody modified particles remained attached firmly to P. aeruginosa after 1 h (Fig and Video). The bacterium-particle complex was verified using a SEM. The rough surfaces of the antibody-conjugated particles were attributed to the matrix formed by the antibodies. The SEM images provided visual evidence showing the successful binding between the bacteria and the particles. The incubation time and binding efficacy here were consistent with those in previous studies based on the bead-based immunoassay

Conclusions: In summary, our findings suggest that the diffusivity of particles proportionally declines with the enlarged equivalent particle diameter because of the binding bacteria. The diffusivity of bacterium-particle complexes can be a sensitive indicator of the quantity of particular microorganisms. By analyzing the temporal diffusivity change of particles attached to bacteria, an AST assessment of the response of P. aeruginosa to gentamicin can be rapidly determined within 2 h. Our study presents a novel technique features a low sample volume (~0.5 μL), a low initial bacteria count (50 CFU per droplet ~ 10⁵ CFU/mL), high sensitivity (one bacterium on single particles), simple fabrication, and rapid AST (within 2 h). Taking advantage of the bead-based immunoassays, multiple types of bacteria can be measured simultaneously by suspending corresponding antibody-modified particles in the medium. In addition, AST evaluations for other bacterial strains can be conducted similarly. The proposed technique will provide insight into achieving rapid and sensitive AST in the near future.