The preparation of fluorescent antibiotics allows for assessment of the localization of these therapeutic reagents within bacteria through convenient analytical techniques such as spectrophotometry and microscopy. The main advantage of this technique is the ease with which the antibiotic localization can be determined. This localization is relevant to a number of phenomena including efflux.
To perform the click A reaction procedure, place the azide antibiotic of interest in a round bottom flask and add 25 milliliters of tert-butanol and 25 milliliters of water per millimole of azide to the flask. Add the prepared fluorophore alkyne to the solution and heat the reaction to 50 degrees Celsius. Next, add 0.6 equivalents of copper sulfate and 2.4 equivalents of ascorbic acid to the flask.
Stir the reaction for one hour or until analysis by LCMS indicates reaction completion. Then cool and purify the reaction as appropriate for the antibiotic scaffold. Here, the key key click chemistry reaction for the preparation of fluorescent antibiotics with examples of the structure synthesized from the corresponding antibiotics via an azide intermediate based on ciprofloxacin, linezolid, and trimethoprim are shown.
In these liquid chromatography mass spectrometry traces from a ciprofloxacin azide and an NBD alkyne click reaction, the azide was eluted at 3.2 minutes and the product was eluted at 3.8 minutes. The progress of the click reaction can be followed by the disappearance of the azide peak. In these spectra, the impact of the purification can be visualized with erroneous peaks disappearing.
To assess the antimicrobial activity of the synthesized antibiotic, streak glycerol stocks of bacterial strains appropriate for the antibiotic scaffold onto LB agar plates and grow the cultures overnight at 37 degrees Celsius. The next morning, pick a single colony from each plate and culture the colonies overnight in five milliliters of CAMHB per culture at 37 degrees Celsius. The next day, dilute the cultures approximately 40-fold in fresh CAMHB and grow the bacteria to mid log phase with an optical density at 600 nanometers between 0.4 and 0.8.
Next, prepare stock solutions of each fluorescent antibiotic at 1.28 milligrams per milliliter in 20%dimethyl sulfoxide in sterile water and add 10 microliters of antibiotic to each well of the first column of a 96-well plate. Add 90 microliters of CAMHB to each well of the first column and 50 microliters to all of the other wells. Then perform a serial two-fold dilution across the plate.
After thoroughly mixing, dilute the mid log phase cultures to approximately one times 10 to the sixth colony forming units per milliliter and add 50 microliters of each culture to the dilution wells to obtain a final concentration of approximately five times 10 to the fifth colony forming units per milliliter. When all of the bacteria have been plated, place lids onto the plates and incubate the cultures for 18 to 24 hours at 37 degrees Celsius without shaking. The next day, visually inspect the plates.
The minimum inhibition concentration will be the lowest concentration well with no visible growth. For probe accumulation analysis, streak glycerol stocks of the bacterial strains onto LB agar plates for an overnight incubation at 37 degrees Celsius. The next morning, pick a single colony from the plate for overnight culture in lysogeny broth at 37 degrees Celsius.
The next morning, dilute the overnight culture approximately 50-fold in fresh medium. When the culture reaches mid log phase, pellet the bacteria by centrifugation and decant the medium. Resuspend the bacteria in one milliliter of PBS and centrifuge the bacteria again.
Decant the supernatant and resuspend the washed pellet in PBS to an optical density at 600 nanometers of two. Add 10.1 microliters of 10 millimolar CCCP in PBS to one milliliter of bacteria and incubate the bacteria at 37 degrees Celsius for 10 minutes. At the end of the incubation, collect the bacteria by centrifugation and resuspend the pellet in one milliliter of 10 to 100 micromolar fluorescent antibiotic solution in PBS.
After a 30 minute incubation at 37 degrees Celsius, wash the cells by centrifugation four times in one milliliter of cold PBS per wash. After the wash, lyse the bacteria with 180 microliters of lysis buffer and 70 microliters of lysozyme. After 30 minutes at 37 degrees Celsius, freeze-thaw the bacteria three times at minus 78 degrees Celsius for five minutes and 34 degrees Celsius for 15 minutes respectively.
After the last round of freeze-thawing, sonicate the sample for 20 minutes followed by a 30 minute incubation at 65 degrees Celsius. At the end of the incubation, collect the lysed sample by centrifugation and strain the tube contents through a 10 kilodalton filter membrane. Wash the filter four times with 100 microliters of water per wash and aliquot each wash into individual wells of a black flat bottom 96-well plate.
Then measure the fluorescence intensity on a plate reader with excitation and emission wavelengths appropriate to the fluorophore. These typical results from the assessment of intracellular accumulation by fluorescence spectroscopy in the presence and absence of efflux show that the intracellular fluorescence of the bacteria is significantly higher after pre-treatment with CCCP indicating that efflux reduces the accumulation within the bacteria. In these representative confocal microscopy images of Gram positive and Gram negative bacteria, the localization of the antibiotic within the bacteria can be visualized after CCCP treatment.
This phenomenon is not observed when no CCCP is added. When using fluorescent antibiotics, be mindful of what information you are aiming to gather and be sure to consider which protocol will be most useful when obtaining this data. Following their synthesis, fluorescent antibiotics can be used to study a number of bacterial processes including drug target interactions and resistance modifications.
