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 culture and 200 &#956;L of sterile 80% glycerol in a 1.8 mL cryovial. Mix by inverting the tubes 3&#8722;4 times, and store at -80 &#176;C.</li>
</ol>
2. ARP/MARP Frozen Stock Library Plate Preparation
<ol>
	<li>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 &#176;C.</li>
	<li>Using aseptic technique, pipette 500 &#956;L of cation adjusted Mueller Hinton broth (MHB) from a sterile reservoir into each well of a sterile 96 deep well plate.</li>
	<li>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 &#176;C&#160;(250 rpm).</li>
	<li>Ensure that there are no contaminated wells by referring to the ARP/MARP map. Repeat if contaminated. Using a multi-channel pipettor, transfer 100 &#956;L 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&#8722;2.4 in the event of frozen stock library plate contamination.</li>
	<li>Finish making the ARP/MARP frozen stock library plates by pipetting 100 &#956;L of sterile 50% glycerol into each well and mix by gently pipetting up and down.</li>
	<li>Cover the plates with sterile aluminum seals and ensure that each well is individually sealed.</li>
	<li>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.</li>
	<li>Place the plate lid on top of the aluminum seal and store at -80 &#176;C.</li>
</ol>
3. Seed Culture and Dereplication Plate Preparation
<ol>
	<li>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.</li>
	<li>Using the same wooden applicator stick, streak a sterility control on a Petri dish containing Bennett&#8217;s agar.</li>
	<li>Incubate the seed culture at 30 &#176;C with aeration for 6 days (250 rpm) and incubate the sterility control plate at 30 &#176;C for 6 days. 
	NOTE: Refer to Table 2 for SAM and Bennett&#8217;s media recipes. The above instructions are suitable for most actinomycetes. Alter growth media as necessary for other bacteria and fungi.</li>
	<li>Prepare dereplication plates by aspirating 23 mL of warm Bennett&#8217;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.</li>
	<li>Gently rotate the plate until the medium covers all areas of the plate and do not disturb it until the agar has set completely.</li>
	<li>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&#8217;s agar, the cleaner the dereplication result.</li>
	<li>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 &#956;L of seed culture onto the surface of the Bennett&#8217;s agar.</li>
	<li>Evenly spread the culture across the surface of the entire plate using a sterile cotton swab.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Put the lid back on the rectangular Petri dish and place it upside down in a sealed plastic bag. Incubate at 30 &#176;C for 6 days.</li>
</ol>
4. Dereplication Plate MHB Overlay and ARP/MARP Library Plate Preparation
<ol>
	<li>After 6 days, remove the dereplication plate from the 30 &#176;C incubator. Using sterile tweezers (autoclaved or sprayed thoroughly with 70% ethanol), carefully remove the nitrocellulose membrane from the surface of the Bennett&#8217;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.</li>
	<li>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.</li>
	<li>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 &#176;C overnight. 
	NOTE: This step allows for diffusion of secondary metabolites from the fermented Bennett&#8217;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.</li>
	<li>On the same day that the overlay is poured, inoculate a fresh ARP/MARP template by pipetting 100 &#956;L 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&#8722;3 dereplication plates. Therefore, inoculate enough 96-well ARP/MARP plates based on the number of strains that will be dereplicated.</li>
	<li>Take the frozen stock ARP/MARP library plate out of the -80 &#176;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.</li>
	<li>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.&#160;To minimize contamination during dereplication, prepare as many ARP or MARP library plates needed to only dereplicate 2&#8722;3 dereplication plates per library plate. Sterilize pinning tools between inoculating each plate.</li>
	<li>Put a new sterile aluminum seal on the frozen template once complete and return it to the -80 &#176;C freezer. Place the inoculated 96-well plates inside of a sealed plastic bag and incubate at 37 &#176;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 &#176;C as described in step 2.5.</li>
</ol>
5. Dereplicating Using the ARP/MARP
<ol>
	<li>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.</li>
	<li>Remove the dereplication plates from 4 &#176;C and allow to equilibrate to room temperature. If there is condensation, open the lids and allow to dry in a sterile environment.</li>
	<li>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.&#160;Sterilize pinning tools in between inoculating each dereplication plate.</li>
	<li>After pinning the template onto the surface of the dereplication plates, allow the template inoculum to dry for 3&#8722;5 min. Place the inoculated dereplication plates upside down in a sealed plastic bag and incubate overnight at 37 &#176;C.</li>
	<li>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).</li>
</ol>

<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 authors have nothing to disclose.

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 ...

antibiotic-dereplication-using-the-antibiotic-resistance-platform

Research

JoVE Journal

Immunology and Infection

10.4K Views.  McMaster University. 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.

Video: Antibiotic Dereplication Using the Antibiotic Resistance Platform

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms.

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.