This study utilizes microfluidic chips to investigate the effects of Trimethoprim (TMP) on wild-type and TMP-resistant mutant E. coli strains. By observing the bacterial response to TMP, the research highlights the mechanisms of antibiotic resistance.
Take microfluidic chips with growth and drug chambers separated by a micromechanical valve.
Load wild-type and TMP-resistant mutant E. coli strains, suspended in media, into the growth chambers using pressure, where Trimethoprim (TMP) is the antibiotic tested.
Next, load the drug chambers with media containing a low concentration of TMP to induce cellular stress in bacteria.
Actuate the micromechanical valve to allow TMP diffusion into the growth chambers, enabling TMP entry into the bacteria.
Place the chips within the incubator mounted on the inverted microscope stage and initiate time-lapse imaging.
In wild-type E. coli, TMP binds to the enzyme dihydrofolate reductase, preventing dihydrofolate binding and causing stress-induced filamentation and reduced bacterial multiplication.
However, the mutant strain’s altered DHFR structure prevents TMP binding, allowing normal multiplication.
Observe the time-lapse images.
Wild-type E. coli exhibits filamentation and reduced multiplication under TMP treatment, while the mutant strain exhibits normal morphology and multiplication, indicating antibiotic resistance.
After thawing the daily frozen sample at room temperature for five minutes, inoculate a new test tube with fresh M9 medium.
Place the sample in a shaking incubator at 37 degrees Celsius overnight. The following day, use M9 medium to dilute the microbial sample tenfold and load it into the microfluidic device by pressuring the microbial sample container at five PSI. Then load the TMP containing medium into the chip by pressuring the M9 with TMP container at five PSI.
Execute the mixing between the microbe and drug by actuating the micro mechanical valve between the two chambers on the microfluidic chip for 15 minutes. Finally place the microfluidic chip in the microscope incubator mounted on the inverted microscope. With a CCD camera, acquire cell images in the growth chamber every hour for eight hours.
Calculate cell number data according to the text protocol.
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