Fig 1.
The structure of the drug susceptibility testing microfluidic (DSTM) device for MIC determination of five drugs.
A) Design of the DSTM device used in the study. B) Actual image of the DSTM device. C) Precise structure of one set of fluids. D) Microscopic image of Pseudomonas aeruginosa grown in the presence of piperacillin (0, 4, 8, and 16 mg/L) for 3 h. Width of each fluid is 100 μm.
Fig 2.
The cell analyzer for analysis of images using the DSTM method.
A) Graphical interface of the software. Appropriateness of analyzed area can be checked and settled manually when they are not adequate. Calculated data in an Excel form were also available. B) Analyzed image to check bacterial cell counts. C) Image to confirm partially-overlapped long cells are counted separately.
Fig 3.
Growth of eight strains of Pseudomonas aeruginosa in the DSTM device over time.
Suspensions of McFarland 0.4–0.5 were used. Strain 2 grew too rapidly to analyze images at 3 h using the software, although it was possible to judge this visually. The images of strains 2, 3, 5, and 7 at 2 h were useful for image analysis, and all images at 3 h except for strain 2 were analyzable using the software.
Fig 4.
Effect of the inoculum size on the growth of Pseudomonas aeruginosa strain 2 in the DSTM device.
Cell suspension with an OD of 0.122 was used as McFarland 0.5. McFarland 0.1 was suitable for analysis using the software at 3 h in this strain although images from McFarland 0.5 at 3 h resulted in growth too dense to be detected using the software.
Fig 5.
Time-lapse analysis of A) Escherichia coli ATCC25922 and B) Pseudomonas aeruginosa ATCC27853 tested by piperacillin using the DSTM method. Bacterial suspension of McFarland 0.2–0.3 were used. Morphological changes were visible after 80 min in E. coli and 120 min in P. aeruginosa.
Fig 6.
Morphological changes of susceptible strains caused by the five antimicrobial agents.
Images were taken after 3 h incubation. A) Pseudomonas aeruginosa S2, B) P. aeruginosa S23. AMK: amikacin, CIP: ciprofloxacin, MPM: meropenem, CAZ: ceftazidime, PPC: piperacillin.
Fig 7.
Various morphologies caused by meropenem in a clinical isolate of Pseudomonas aeruginosa.
Cell elongation was seen at 2 mg/L, bulge formation was observed at 4 mg/L, and spheroplast formation was observed at 4 and 8 mg/L of meropenem using this strain.
Fig 8.
Different image outputs between the bottom and the middle depths of the channels.
Images from A) strain S16 and B) strain 36 at 3 h. Elongated cells were usually located on the glass surface, while spheroplast cells were floating in the fluids. The images focused on the glass surface are preferable for analysis using the software.
Table 1.
Criteria used to denote “susceptible” in the DSTM method using the software.
Table 2.
Accuracy of the DSTM method vs. the microbroth dilution method.
Fig 9.
Correlation between MICs from the DSTM method and the microbroth dilution method in Pseudomonas aeruginosa.
Domains in the bold-lines indicate sensitive or resistant (categorized by the breakpoints of CLSI) using both methods. Green shade, matching; yellow shade, 2-fold difference; orange and red shade, >4-fold difference. Red shade indicates very major errors. The matching ratios (%) shown for each drug contain 2-fold differences.
Fig 10.
Applicability of the DSTM method in clinical laboratories.
When we test positive blood culture, species name and susceptibility data of the causative organism will be available on day 3 using the ordinary method (black arrow course), and available on day 2 using the rapid methods (blue arrow course). Furthermore, those data are available on day 1 by testing blood culture directly after pre-treatment using the rapid methods (red arrow course). Consequently, we can choose the best drug for each patient on day 1, which offers significant clinical advantage.