Rapid Bacterial Motility Monitoring Using Inexpensive 3D-Printed OpenFlexure Microscopy Allows Microfluidic Antibiotic Susceptibility Testing

Abstract

Antibiotic susceptibility testing is vital to tackle the emergence and spread of antimicrobial resistance. Inexpensive digital CMOS cameras can be converted into portable digital microscopes using 3D printed x-y-z stages. Microscopic examination of bacterial motility can rapidly detect the response of microbes to antibiotics to determine susceptibility. Here, we present a new simple microdevice-miniature microscope cell measurement system for multiplexed antibiotic susceptibility testing. The microdevice is made using melt-extruded plastic film strips containing ten parallel 0.2 mm diameter microcapillaries. Two different antibiotics, ceftazidime and gentamicin, were prepared in Mueller-Hinton agar (0.4%) to produce an antibiotic-loaded microdevice for simple sample addition. This combination was selected to closely match current standard methods for both antibiotic susceptibility testing and motility testing. Use of low agar concentration permits observation of motile bacteria responding to antibiotic exposure as they enter capillaries. This device fits onto the OpenFlexure 3D-printed digital microscope using a Raspberry Pi computer and v2 camera, avoiding need for expensive laboratory microscopes. This inexpensive and portable digital microscope platform had sufficient magnification to detect motile bacteria, yet wide enough field of view to monitor bacteria behavior as they entered antibiotic-loaded microcapillaries. The image quality was sufficient to detect how bacterial motility was inhibited by different concentrations of antibiotic. We conclude that a 3D-printed Raspberry Pi-based microscope combined with disposable microfluidic test strips permit rapid, easy-to-use bacterial motility detection, with potential for aiding detection of antibiotic resistance.

Current and Future Flow Cytometry Applications Contributing to Antimicrobial Resistance Control

“…However, they take several days to provide a result because they require the isolation of bacteria isolated from patients who were then exposed to the antibiotics. To overcome this delay in conventional AST assays, new phenotypic strategies have been proposed for the rapid measurement of various phenotypic characteristics of bacteria, such as morphology, metabolism, biochemical composition, and growth after exposure to antibiotics, including isothermal microcalorimetry [ 28 , 29 ], electrochemical impedance spectroscopy [ 30 , 31 ], microscopy [ 32 , 33 ], electrochemical ASTs [ 34 , 35 ], spectroscopy [ 36 ], flow cytometry [ 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ], and spectrometry [ 46 ]. Rapid phenotypic AST methods have detected microbial growth and/or metabolism as well as morphological changes in biological samples with very low microbial loads.…”

Preparation, Stimulus–Response Mechanisms and Applications of Micro/Nanorobots

“…In terms of scalability, more stress design preparation methods for more material systems need to be investigated to meet different application requirements. At the micro-and nanoscale, 3D direct laser writing (DLW) allows the precise batch preparation of complex polymer structures with high resolution [161][162][163]. Typically, we use DLW techniques to fabricate helically structured micromotors (Figure 6A) [9,20,31,65].…”

“…Typically, we use DLW techniques to fabricate helically structured micromotors (Figure 6A) [9,20,31,65]. At the micro-and nanoscale, 3D direct laser writing (DLW) allows the precise batch preparation of complex polymer structures with high resolution [161][162][163]. Typically, we use DLW techniques to fabricate helically structured micromotors (Figure 6A) [9,20,31,65].…”

Mask-Moving-Lithography-Based High-Precision Surface Fabrication Method for Microlens Arrays

“…As micro/nano structural components with continuous surfaces, fabrication technologies for microlens arrays include laser direct writing [4][5][6], multi-layer etching [7,8], grayscale lithography [9][10][11], 3D printing technology [12][13][14], and mask-moving technologies [15]. Direct writing technology offers a high precision and resolution but is costly and less efficient, so it is unsuitable for mass fabrication or fabrication of large-sized micro-optical elements.…”