Antibiotic Dereplication Using the Antibiotic Resistance Platform

Summary

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

Abstract

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

Introduction

Since the discovery of penicillin in 1928, natural products derived from environmental microorganisms have proven to be a rich source of antimicrobial compounds¹. Approximately 80% of natural product antibiotics are derived from bacteria of the genus Streptomyces and other actinomycetes, while the remaining 20% is produced by fungal species¹. Some of the most common antibiotic scaffolds used in the clinic such as the β-lactams, tetracyclines, rifamycins, and aminoglycosides, were originally isolated from microbes². However, due to the rise of multidrug resistant (MDR) bacteria, our current panel of antibiotics has become less effective in treatment^3,4. These include the “ESKAPE” pathogens (i.e., vancomycin-resistant enterococci and β-lactam-resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter sp.), which are a subset of bacteria deemed to be associated with the highest risk by a number of major public health authorities such as the World Health Organization^3,4,5. The emergence and global spread of these MDR pathogens results in a constant need for novel antibiotics^3,4,5. Regrettably, the past two decades have demonstrated that the discovery of novel antibiotics from microbial sources is increasingly difficult⁶. Current approaches to drug discovery include the high-throughput screening of bioactive compounds, including natural product extract libraries, allowing for thousands of extracts to be tested at a given time². However, once antimicrobial activity is detected, the next step is to analyze the contents of the crude extract to identify the active component and eliminate those containing known or redundant compounds^7,8. This process, referred to as dereplication, is vital to prevent and/or significantly reduce the time spent on the re-discovery of known antibiotics^7,9. Although a necessary step in natural product drug discovery, dereplication is notoriously laborious and resource-intensive¹⁰.

Ever since Beutler et al. first coined the term “dereplication”, extensive efforts have been made to develop innovative strategies for the rapid identification of known antibiotics^11,12. Today the most common tools used for dereplication include analytical chromatographic systems such as high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance-based detection methods^11,13. Unfortunately, each of these methods requires the use of expensive analytical equipment and sophisticated data interpretation.

In an attempt to develop a dereplication method that can be rapidly performed without specialized equipment, we established the antibiotic resistance platform (ARP)¹⁰. The ARP can be used for the discovery of antibiotic adjuvants, the profiling of new antibiotic compounds against known resistance mechanisms, and the dereplication of known antibiotics in extracts derived from actinobacteria and other microbes. Here, we focus on its application in antibiotic dereplication. The ARP utilizes a library of isogenic Escherichia coli strains expressing individual resistance genes that are effective against the most commonly re-discovered antibiotics^14,15. When the E. coli library is grown in the presence of a secondary metabolite-producing organism, the identity of the compound can be deduced by the growth of E. coli strains that express its associated resistance gene¹⁰. When the ARP was first reported, the library consisted of >40 genes conferring resistance to 16 antibiotic classes. The original dereplication template was designed to encompass a subset of resistance genes per antibiotic class to provide information regarding antibiotic subclass during the dereplication process. Today, the ARP is comprised of >90 genes that confer resistance to 18 antibiotic classes. Using our extensive collection of resistance genes, a secondary dereplication template has been developed and is known as the minimal antibiotic resistance platform (MARP). This template was created to eliminate gene redundancy and to simply provide information regarding the general antibiotic class that a dereplicated metabolite is related to. Additionally, the MARP template possesses both wildtype and a hyperpermeable/efflux deficient strain of E. coli BW25113 (E. coli BW25113 ΔbamBΔtolC), compared to the original incarnation of the ARP, which only utilizes the hyperpermeable strain. This unique aspect creates additional phenotypes during dereplication, indicating a compounds ability to cross the outer membrane of Gram-negative bacteria. Here, we describe a robust protocol to be followed when dereplicating with either the ARP and/or MARP, highlight the most critical steps to be followed, and discuss the various possible outcomes.

Protocol

1. Preparation of E. coli Library Glycerol Stocks (from Agar Slants)

1. Streak the ARP/MARP E. coli strains from lysogeny broth (LB) agar slants onto Petri dishes containing LB agar and the appropriate selectable marker (Table 1).
2. Prepare cultures for each of the E. coli strains by inoculating 3 mL of LB containing the appropriate selectable marker with a single colony. Grow overnight at 37 °C with aeration (250 rpm).
3. Combine 800 μL of culture and 200 μL of sterile 80% glycerol in a 1.8 mL cryovial. Mix by inverting the tubes 3−4 times, and store at -80 °C.

2. ARP/MARP Frozen Stock Library Plate Preparation

1. Streak the ARP/MARP strains from the glycerol stocks prepared in section 1 onto a new set of Petri dishes containing LB agar and the appropriate selectable marker. Grow overnight at 37 °C.
2. Using aseptic technique, pipette 500 μL of cation adjusted Mueller Hinton broth (MHB) from a sterile reservoir into each well of a sterile 96 deep well plate.
3. With the plates prepared in step 2.1, use an applicator stick to inoculate the 96 deep well plate in accordance with the ARP/MARP map (Supplemental Figure 1 and Supplemental Figure 2). Ensure that the appropriate selectable marker is added to each well. Place a breathable sealing membrane over the surface of the deep well plate and incubate overnight at 37 °C (250 rpm).
4. Ensure that there are no contaminated wells by referring to the ARP/MARP map. Repeat if contaminated. Using a multi-channel pipettor, transfer 100 μL from each well of the deep well plate to a sterile 96-well round bottom plate. Repeat this step to create multiple frozen stock library plates.
   NOTE: It is best to prepare at least 5 library plates at a time to keep from repeating steps 2.1−2.4 in the event of frozen stock library plate contamination.
5. Finish making the ARP/MARP frozen stock library plates by pipetting 100 μL of sterile 50% glycerol into each well and mix by gently pipetting up and down.
6. Cover the plates with sterile aluminum seals and ensure that each well is individually sealed.
7. Number the plates and dedicate only one frozen stock library plate for inoculating new templates at a given time. Keep the remainder as back-ups in the event of frozen stock library plate contamination.
8. Place the plate lid on top of the aluminum seal and store at -80 °C.

3. Seed Culture and Dereplication Plate Preparation

1. Using an applicator stick, inoculate 3 mL of Streptomyces antibiotic medium (SAM) (or other appropriate medium for the organism being tested) in a test tube containing one sterile glass bead (to break-up the mycelium) with the producing strain that is to be dereplicated. For Streptomyces, gently scrape spores from the surface of a colony.
2. Using the same wooden applicator stick, streak a sterility control on a Petri dish containing Bennett’s agar.
3. Incubate the seed culture at 30 °C with aeration for 6 days (250 rpm) and incubate the sterility control plate at 30 °C for 6 days.
   NOTE: Refer to Table 2 for SAM and Bennett’s media recipes. The above instructions are suitable for most actinomycetes. Alter growth media as necessary for other bacteria and fungi.
4. Prepare dereplication plates by aspirating 23 mL of warm Bennett’s agar into a serological pipette and dispense 20 mL evenly across the surface of a rectangular Petri dish (Table of Materials), leaving the remainder of the medium in the pipette to prevent air bubble formation.
   NOTE: Ensure that the surface being used to pour plates is level and perform this step before the agar has cooled too much; a flat surface is imperative for library pinning in the next stages.
5. Gently rotate the plate until the medium covers all areas of the plate and do not disturb it until the agar has set completely.
6. Prepare nitrocellulose membrane sheets (Table of Materials) 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.
   NOTE: This membrane allows for organisms to sporulate on its surface, while secondary metabolites may be excreted into the medium below. Once grown, the membrane is removed to provide a clean surface for dereplication. The closer fit that the membrane paper has on the surface of the Bennett’s agar, the cleaner the dereplication result.
7. Check the sterility control plate to ensure that no contaminants are present after 6 days of incubation. If contamination-free, remove the lid of the rectangular Petri dish and pipette 200 μL of seed culture onto the surface of the Bennett’s agar.
8. Evenly spread the culture across the surface of the entire plate using a sterile cotton swab.
9. Place the nitrocellulose membrane prepared in step 3.6 over top of the culture on the surface of the Petri dish. Begin by aligning 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.
10. 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.
11. Put the lid back on the rectangular Petri dish and place it upside down in a sealed plastic bag. Incubate at 30 °C for 6 days.

4. Dereplication Plate MHB Overlay and ARP/MARP Library Plate Preparation

1. After 6 days, remove the dereplication plate from the 30 °C incubator. Using sterile tweezers (autoclaved or sprayed thoroughly with 70% ethanol), carefully remove the nitrocellulose membrane from the surface of the Bennett’s agar.
   NOTE: This step will remove the hydrophobic spores and mycelia grown on the surface of the membrane to provide a clean surface for dereplication, facilitating step 4.2.
2. As described for step 3.4, ensure the work surface is level and use a serological pipette to aspirate 23 mL of warm cation adjusted MHB agar. Create an overlay by dispensing 20 mL evenly across the surface of the dereplication plate, leaving the remainder of the medium in the pipette to prevent air bubble formation.
3. 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 at 4 °C overnight.
   NOTE: This step allows for diffusion of secondary metabolites from the fermented Bennett’s medium into the MHB agar overlay. If the nitrocellulose membrane was not prepared properly there will be spore growth around the edges of the plate, which have hydrophobic properties that repel the MHB agar. Do not pour the overlay on top of these spores because it can result in contamination of the overlay.
4. On the same day that the overlay is poured, inoculate a fresh ARP/MARP template by pipetting 100 μL of cation adjusted MHB into each well of a 96-well plate.
   NOTE: To reduce the chance of spreading contamination during dereplication, use a single ARP/MARP library plate to only dereplicate 2−3 dereplication plates. Therefore, inoculate enough 96-well ARP/MARP plates based on the number of strains that will be dereplicated.
5. Take the frozen stock ARP/MARP library plate out of the -80 °C freezer. Remove the aluminum seal before condensation begins to form on its underside, thereby decreasing the chance of contaminating neighboring wells in the library plate.
6. Using sterile 96-well pinning tools (or other forms of inoculation equipment), carefully pin from the frozen stock ARP/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 2−3 dereplication plates per library plate. Sterilize pinning tools between inoculating each plate.
7. Put a new sterile aluminum seal on the frozen template once complete and return it to the -80 °C freezer. Place the inoculated 96-well plates inside of a sealed plastic bag and incubate at 37 °C with aeration (250 rpm) for 18 h.
   NOTE: New frozen stock library plates can be prepared from this step after ensuring that no contamination is present. Add glycerol to the plate before storing at -80 °C as described in step 2.5.

5. Dereplicating Using the ARP/MARP

1. Remove the ARP/MARP template from the incubator and ensure that no contaminants are present. Always dereplicate using a template that is freshly prepared and not directly from the frozen stock.
2. Remove the dereplication plates from 4 °C and allow to equilibrate to room temperature. If there is condensation, open the lids and allow to dry in a sterile environment.
3. Using sterile pinning tools (or other inoculation equipment), pin from the ARP/MARP library plate onto the surface of the MHB agar overlay of the dereplication plates. Be careful not to pierce the agar. Sterilize pinning tools in between inoculating each dereplication plate.
4. After pinning the template onto the surface of the dereplication plates, allow the template inoculum to dry for 3−5 min. Place the inoculated dereplication plates upside down in a sealed plastic bag and incubate overnight at 37 °C.
5. Analyze dereplication results the following day by comparing growth on the dereplication plate to wells that correspond to the ARP/MARP map (Table 3 and Table 4).

Results

The following results were obtained when a collection of antibiotic-producing strains of interest were dereplicated using the ARP and/or MARP.

A diagram of the ARP/MARP dereplication workflow is depicted in Figure 1, and library plate maps are shown in Supplemental Figure 1 and Supplemental Figure 2. Figure 2 demonstrates a positive dereplication result wherein the environmental extract WAC 8921 is id...

Discussion

The protocol described above can be applied to both the discovery of novel antimicrobial compounds and adjuvants that can be used in conjunction with existing antibiotics to rescue their activity. The platform takes advantage of the high substrate specificity of resistance mechanisms and their cognate antibiotics, to dereplicate compounds within crude natural product extracts. Although the time required for dereplication plates to be prepared is lengthy (~2 weeks), the dereplication process itself is complete after a sin...

Materials

Name	Company	Catalog Number	Comments
Agar	Bio Shop	AGR003.5
AlumaSeal CS Films for cold storage	Sigma-Aldrich	Z722642-50EA
Ampicillin Sodium Salt	Bio Shop	AMP201.100
BBL Mueller Hinton II Broth (Cation-Adjusted)	Becton Dickinson	212322
BBL Phytone Peptone (Soytone)	Becton Dickinson	211906
Calcium Carbonate	Bio Shop	CAR303.500
Casamino acid	Bio Basic	3060
Cotton-Tipped Applicators	Fisher Scientific	23-400-101
CryoPure Tube 1.8 mL mix.colour	Sarstedt	72.379.992
D-glucose	Bio Shop	GLU501.5
Disposable Culture Tube, 16 mm x 100 mm	Fisher Scientific	14-961-29
Ethyl Alcohol Anhydrous	Commercial Alcohols	P016EAAN
Glass Beads, Solid	Fisher Scientific	11-312C
Glycerol	Bio Shop	GLY001.4
Hydrochloric Acid	Fisher Scientific	A144-212
Instant sealing sterilization pouch	Fisher Scientific	01-812-54
Iron (II) Sulfate Heptahydrate	Sigma-Aldrich	F7002-250G
Kanamycin Sulfate	Bio Shop	KAN201.50
LB Broth Lennox	Bio Shop	LBL405.500
Magnesium Sulfate Heptahydrate	Fisher Scientific	M63-500
MF-Millipore Membrane Filter, 0.45 µm pore size	Millipore-Sigma	HAWP00010	10 FT roll, hydrophillic, white, plain
Microtest Plate 96 well, round base	Sarstedt	82.1582.001
New Brunswick Innova 44	Eppendorf	M1282-0000
Nunc OmniTray Single-Well Plate	Thermo Fisher Scientific	264728	with lid, sterile, non treated
Petri dish 92 mm x 16 mm with cams	Sarstedt	82.1473.001
Pinning tools	ETH Zurich	-	Custom order
Potassium Chloride	Fisher Scientific	P217-500
Potato starch	Bulk Barn	279
Sodium Chloride	Fisher Scientific	BP358-10
Sodium Nitrate	Fisher Scientific	S343-500
Wood Applicators	Dukal Corporation	9000
Yeast Extract	Fisher Scientific	BP1422-2