Our protocol is significant because it allows for both the cost effective and timely dereplication of known antibiotics without the use of specialized equipment. The main advantage to this technique is template customization. This means that an individual can tailor which resistance genes that they wish to include in their library template based on their desired level of broad or narrow substrate specificity.
For example, a beta-lactam is expressing E.coli template can be prepared in order to allow for the highly specific dereplication of beta-lactams and their various subclasses. Using a septic technique, pipette 500 microliters of cation adjusted MHB from a sterile reservoir into each well of a sterile, 96 deep well plate. With the prepared ARP or MARP strain plates, use an applicator stick to inoculate the 96 deep well plate in accordance with the ARP or MARP map.
Place a breathable sealing membrane on the surface of the deep well plate and incubate it overnight at 37 degrees Celsius, 250 RPM. Ensure that there are no contaminated wells. Using a multi-channel pipette, transfer 100 microliters from each well of the deep well plate to a sterile 96-well round bottom plate.
Add 100 microliters of sterile 50%glycerol into each well and mix by gently pipetting up and down. Prepare at least five library plates. Cover the plates with sterile aluminum seals and ensure that each well is individually sealed.
Place the plate lid on top of the aluminum seal and store at minus 80 degrees Celsius. Using a wooden applicator stick, gently scrape spores from the surface of a streptomyces colony and transfer it into a test tube containing one sterile glass bead and three milliliters of streptomyces antibiotic medium. Close the test tube loosely with a cap to allow aeration.
Using the same wooden applicator stick, streak a sterility control on a Petri dish containing Bennett's agar. Incubate the tube containing seed culture at 30 degrees Celsius with aeration for six days at 250 RPM and the sterility control plate at 30 degrees Celsius for six days. Then, on a level surface, prepare dereplication plates.
Aspirate 23 milliliters of warm Bennett's agar into a serological pipette and dispense 20 milliliters evenly across the surface of a rectangular Petri dish, leaving the remainder of the medium in the pipette to prevent air bubble formation. Cover the plate with a lid. Gently rotate the plate until the medium covers all areas of the plate and do not disturb it until the agar has set completely.
Next, prepare nitrocellulose membrane sheets by using a rectangular Petri dish lid as a tracing template so that the sheets fit the surface of the dereplication plate. Cut the sheets and autoclave them in a sterile pouch. Now, check the sterility control plate to ensure that no contaminants are present after six days of incubation.
If contamination-free, remove the lid of the rectangular Petri dish and pipette 200 microliters of the seed culture onto the surface of the Bennett's agar in the rectangular dish. Use a sterile cotton swab to evenly spread the culture across the surface of the entire plate. To position the prepared nitrocellulose membrane over top of the culture on the surface of the Petri dish, align the bottom edge of the membrane to the bottom edge of the Petri dish and slowly apply the membrane from the bottom edge to the top edge of the plate.
Use a sterile cotton swab to smooth out any air bubbles that may have formed between the membrane agar interface, ensuring that the membrane is flush to the agar. The membrane allows for organisms to sporulate on its surface while secondary metabolites may be excreted into the medium below. Put the lid back on the rectangular Petri dish and place it upside-down in a sealed plastic bag.
Incubate at 30 degrees Celsius for six days. After six days, remove the dereplication plate from the incubator. Using sterile tweezers, carefully remove the nitrocellulose membrane from the surface of the Bennett's agar.
Make sure there is only minor spore growth around the edges of the plate to decrease the chances of contaminating the overlay to be added. Ensure the work surface is level and use a serological pipette to aspirate 23 milliliters of warm cation adjusted MHB agar. Create an overlay by dispensing 20 milliliters evenly across the surface of the dereplication plate, leaving the remainder of the agar in the pipette to prevent air bubble formation.
Place the lid on the plate. Gently rotate the plate until the medium covers all areas and do not disturb it until the agar has set completely. Once cooled and solidified, return the dereplication plate to the sealed plastic bag and store it upside-down at four degrees Celsius overnight, allowing for diffusion of secondary metabolites into the MHB agar overlay.
On the same day, inoculate a fresh ARP or MARP template by first pipetting 100 microliters of cation adjusted MHB into each well of a 96-well plate. Take the frozen stock ARP or MARP library plate out of the minus 80 degrees Celsius freezer. Remove the aluminum seal before condensation begins to form on its underside.
Using sterile 96-well pinning tools, carefully pin from the frozen stock ARP or MARP library plate and inoculate the fresh MHB containing 96-well plates. To minimize contamination during dereplication, prepare as many ARP or MARP library plates needed to only dereplicate two to three dereplication plates per library plate. Once complete, put a new sterile aluminum seal on the frozen template and return it to the minus 80 degree Celsius freezer.
Place the inoculated 96-well plates inside of a loosely sealed plastic bag and incubate at 37 degrees Celsius with shaking at 250 RPM for 18 hours. Remove the ARP or MARP template from the incubator and ensure that no contaminants are present by comparing the plate to the ARP or MARP template map. Remove the dereplication plates from four degrees Celsius and allow to equilibrate to room temperature.
If there is condensation, open the lids and allow to dry in a sterile environment. Using sterile pinning tools, pin from the ARP or MARP library plate onto the surface of the MHB agar overlay of the dereplication plates. Be careful not to pierce the agar.
Allow the template inoculum to dry for three to five minutes. Place the inoculated dereplication plates upside-down in a sealed plastic bag and incubate overnight at 37 degrees Celsius. The following day, analyze dereplication results by comparing growth on the dereplication plate to wells that correspond to the ARP or MARP map.
In this ARP or MARP dereplication workflow, a positive dereplication result was achieved. Wherein the extract prepared for the strain WAC 8921 was identified as a chloramphenicol producer. The lack of ARP growth indicates the presence of either an unknown antibiotic or a less commonly found antibiotic that was not accounted for in the ARP or MARP library plate.
A growth pattern unique to the MARP was formed because of its utilization of both wild type E.coli BW25113 and a hyper-permeable and e-flux deficient mutant E.coli BW25113 bamB tolC. This result suggests the presence of a compound with antimicrobial activity that was unable to surpass an intact outer membrane. This E.coli growth patterns suggests the improper sterilization of pinning tools, resulting in the transfer of unknown E.coli strains across the overlay.
And an example of ARP or MARP frozen stock library plate contamination is shown here with three distinct E.coli colonies grown on the rectangular Petri dish surface. This result indicates that the agar overlay was pierced during dereplication. The MHB overlay related contamination can also occur during the dereplication process, showing an irregular growth pattern on the surface of the overlay.
The most important thing to remember when following this protocol is to use proper sterilization and aseptic techniques in order to ensure that you're not held back in any step due to contamination. This includes preparing the nitrocellulose membrane so that it fits the surface of the Bennett's agar plate as closely as possible in order to minimize the spreading of spores when pouring the MHB overlay. Additionally, it is also extremely important to use properly sterilized pinning equipment when inoculating the library plates and dereplicating in order to produce the cleanest result.
In addition to dereplicating known antibiotics, this method can be applied to identifying adjuvants which can be used in conjunction with existing antibiotics to rescue their activity. Although antibiotic dereplication is not a new technique, it is notorious for being resource intensive and time consuming. The ARP and MARP platform help to shed a light on how antibiotic dereplication does not need to be a large production, but rather can be completed over a two-week period using minimal equipment.
