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 c....

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 cul.......

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 1.8ml mix.colour	Sarstedt	72.379.992
D-glucose	Bio Shop	GLU501.5
Disposable Culture Tube, 16x100mm	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 92x16mm 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