Name: antibiotic dereplication using the antibiotic resistance platform
Uploaded: 2019-10-17T15:00:00
Description: Watch this Scientific Journal Video about Antibiotic Dereplication Using the Antibiotic Resistance Platform at JoVE.com

Research in the Wright lab pertaining to the ARP/MARP was supported by the Ontario Research Fund and Canadian Institutes of Health Research grant (FRN-148463). We would like to acknowledge Sommer Chou for assisting in the expansion and organization of the ARP library.

1. Preparation of E. coli Library Glycerol Stocks (from Agar Slants)
<ol>
	<li>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).</li>
	<li>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 &#176;C with aeration (250 rpm).</li>
	<li>Combine 800 &#956;L of cul...

<ol>
	<li>Lo Grasso, L., Chillura Martino, D., Alduina, R., Dhanasekaran, D., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Antibacterial+Compounds+from+Actinomycetes.+actinobacteria.">Production of Antibacterial Compounds from Actinomycetes. actinobacteria.</a> Basics and Biotechnological Applications. , (2016).</li><li>Thaker, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+producers+of+antibacterial+compounds+by+screening+for+antibiotic+resistance.">Identifying producers of antibacterial compounds by screening for antibiotic resistance.</a> Nature Biotechnology. 31, 922-927 (2013).</li><li>Gajd&#225;cs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Concept+of+an+Ideal+Antibiotic:+Implications+for+Drug+Design.">The Concept of an Ideal Antibiotic: Implications for Drug Design.</a> Molecules. 24, 892 (2019).</li><li>Boucher, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bad+bugs,+no+drugs:+no+ESKAPE!+An+update+from+the+Infectious+Diseases+Society+of+America.">Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America.</a> Clinical Infectious Diseases. 48, 1-12 (2009).</li><li>Gajd&#225;cs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Continuing+Threat+of+Methicillin-Resistant+Staphylococcus+aureus.">The Continuing Threat of Methicillin-Resistant Staphylococcus aureus.</a> Antibiotics. 8, 52 (2019).</li><li>Gaud&#234;ncio, S. P., Pereira, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dereplication:+Racing+to+speed+up+the+natural+products+discovery+process.">Dereplication: Racing to speed up the natural products discovery process.</a> Natural Product Reports. 32, 779-810 (2015).</li><li>Ito, T., Masubuchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dereplication+of+microbial+extracts+and+related+analytical+technologies.">Dereplication of microbial extracts and related analytical technologies.</a> The Journal of Antibiotics (Tokyo). 67, 353-360 (2014).</li><li>Van Middlesworth, F., Cannell, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dereplication+and+Partial+Identification+of+Natural+Products.">Dereplication and Partial Identification of Natural Products.</a> Methods in Biotechnology. , 279-327 (2008).</li><li>Tawfike, A. F., Viegelmann, C., Edrada-Ebel, R., Roessner, U., Dias, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+and+Dereplication+Strategies+in+Natural+Products.">Metabolomics and Dereplication Strategies in Natural Products.</a> Metabolomics Tools for Natural Product Discovery: Methods and Protocols. , 227-244 (2013).</li><li>Cox, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Common+Platform+for+Antibiotic+Dereplication+and+Adjuvant+Discovery.">A Common Platform for Antibiotic Dereplication and Adjuvant Discovery.</a> Cell Chemical Biology. 24, 98-109 (2017).</li><li>Hubert, J., Nuzillard, J. M., Renault, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dereplication+strategies+in+natural+product+research:+How+many+tools+and+methodologies+behind+the+same+concept.">Dereplication strategies in natural product research: How many tools and methodologies behind the same concept.</a> Phytochemistry Reviews. 16, 55-95 (2017).</li><li>Beutler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dereplication+of+phorbol+bioactives:+Lyngbya+majuscula+and+Croton+cuneatus.">Dereplication of phorbol bioactives: Lyngbya majuscula and Croton cuneatus.</a> Journal of Natural Products. 53, 867-874 (1990).</li><li>Mohimani, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dereplication+of+microbial+metabolites+through+database+search+of+mass+spectra.">Dereplication of microbial metabolites through database search of mass spectra.</a> Nature Communications. 9, 1-12 (2018).</li><li>Baltz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marcel+Faber+Roundtable:+Is+our+antibiotic+pipeline+unproductive+because+of+starvation,+constipation+or+lack+of+inspiration.">Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration.</a> Journal of Industrial Microbiology and Biotechnology. 33, 507-513 (2006).</li><li>Baltz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+discovery+from+actinomycetes:+Will+a+renaissance+follow+the+decline+and+fall.">Antibiotic discovery from actinomycetes: Will a renaissance follow the decline and fall.</a> Archives of Microbiology. 55, 186-196 (2005).</li></ol>

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...

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 &#946;-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 treatment3,4. These include the &#8220;ESKAPE&#8221; pathogens (i.e., vancomycin-resistant enterococci and &#946;-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 Organization3,4,5. The emergence and global spread of these MDR pathogens results in a constant need for novel antibiotics3,4,5. 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 compounds7,8. This process, referred to as dereplication, is vital to prevent and/or significantly reduce the time spent on the re-discovery of known antibiotics7,9. 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 &#8220;dereplication&#8221;, extensive efforts have been made to develop innovative strategies for the rapid identification of known antibiotics11,12. 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 methods11,13. 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 antibiotics14,15. 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 &#62;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 &#62;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 &#916;bamB&#916;tolC), 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.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Bio Shop</td>
			<td>AGR003.5</td>
			<td></td>
		</tr>
		<tr>
			<td>AlumaSeal CS Films for cold storage</td>
			<td>Sigma-Aldrich</td>
			<td>Z722642-50EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin Sodium Salt</td>
			<td>Bio Shop</td>
			<td>AMP201.100</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Mueller Hinton II Broth (Cation-Adjusted)</td>
			<td>Becton Dickinson</td>
			<td>212322</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Phytone Peptone (Soytone)</td>
			<td>Becton Dickinson</td>
			<td>211906</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Carbonate</td>
			<td>Bio Shop</td>
			<td>CAR303.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Casamino acid</td>
			<td>Bio Basic</td>
			<td>3060</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton-Tipped Applicators</td>
			<td>Fisher Scientific</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoPure Tube 1.8ml mix.colour</td>
			<td>Sarstedt</td>
			<td>72.379.992</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Bio Shop</td>
			<td>GLU501.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Culture Tube, 16x100mm</td>
			<td>Fisher Scientific</td>
			<td>14-961-29</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol Anhydrous</td>
			<td>Commercial Alcohols</td>
			<td>P016EAAN</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beads, Solid</td>
			<td>Fisher Scientific</td>
			<td>11-312C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Bio Shop</td>
			<td>GLY001.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant sealing sterilization pouch</td>
			<td>Fisher Scientific</td>
			<td>01-812-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) Sulfate Heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>F7002-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Bio Shop</td>
			<td>KAN201.50</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth Lennox</td>
			<td>Bio Shop</td>
			<td>LBL405.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MF-Millipore Membrane Filter, 0.45 &#181;m pore size</td>
			<td>Millipore-Sigma</td>
			<td>HAWP00010</td>
			<td>10 FT roll, hydrophillic, white, plain</td>
		</tr>
		<tr>
			<td>Microtest Plate 96 well, round base</td>
			<td>Sarstedt</td>
			<td>82.1582.001</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Innova&#160;44</td>
			<td>Eppendorf</td>
			<td>M1282-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc OmniTray Single-Well Plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>264728</td>
			<td>with lid, sterile, non treated</td>
		</tr>
		<tr>
			<td>Petri dish 92x16mm with cams</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pinning tools</td>
			<td>ETH Zurich</td>
			<td>-</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato starch</td>
			<td>Bulk Barn</td>
			<td>279</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Nitrate</td>
			<td>Fisher Scientific</td>
			<td>S343-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wood Applicators</td>
			<td>Dukal Corporation</td>
			<td>9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

antibiotic dereplication using the antibiotic resistance platform

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.

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...

Watch this Scientific Journal Video about Antibiotic Dereplication Using the Antibiotic Resistance Platform at JoVE.com

Antibiotic Dereplication Using the Antibiotic Resistance Platform

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.

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 ...

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