Prior work with regard to susceptibility testing of dalbavancin has been valuable in development of a standardized methods and acknowledgement for this work is given to JMI Laboratories, Robert Rennie and Beth Goldstein.

Note: Refer to CLSI documents M7-A10 and M100-S25 and/or ISO/FDIS 20776-1 for full details of the reference broth microdilution method for antimicrobial susceptibility2, 3, 4. The reference methods allow for options in some of the procedures specific to inoculum preparation and MIC panel production to achieve the same end result. The method detailed here relates to one antimicrobial agent, dalbavancin, and some of the steps represent one of potentially several ways the reference procedure can be done. Appropriate safety precautions (consistent with biosafety level 2) should be utilized10. The MIC panel format used for the purpose of this video publication is shown in Table 1.
1. Store Dalbavancin Powder
<ol>
	<li>Upon receipt of diagnostic grade dalbavancin powder, store at -20 oC in a desiccated environment in a non-defrosting freezer. Prior to use, the powder should equilibrate to reach RT before opening.</li>
</ol>
2. Prepare MIC Panel Dilutions
<ol>
	<li>Prepare a stock solution no higher than 1,600 &#181;g/ml of dalbavancin in neat (pure) DMSO in sterile glass or plastic tubes, and use on the same day of preparation or store at -20 to -60 oC or below for future use in a non-defrosting freezer. Take into consideration the potency of dalbavancin as provided on the documentation received with the powder, when weighing the powder (see Equation 1 for example 800 &#181;g/ml stock preparation).</li>
	<li>Dilute the stock dilution similar to the scheme as is shown in column 1 (&#8220;Source Concentration&#8221;) in Table 2 with neat DMSO in sterile glass or plastic tubes. Use one pipet for measuring diluent and another pipet for adding the initial dalbavancin stock to the first tube. For each subsequent dalbavancin stock concentration use a new pipet.</li>
	<li>Prepare 100X final MIC panel concentration dilutions (intermediate concentrations) with neat DMSO. As is shown in columns 2-4 of Table 2, combine appropriate volumes of source and DMSO to achieve desired intermediate concentration (volumes to be used will depend on number of MIC panels to be made).</li>
	<li>Prepare 0.004% P80: Prepare a fresh working stock solution of 2% P80 by adding 0.1 ml P80 to 4.9 ml dH2O. Sterilize by passing through a 0.22 micron filter and use solution on the same day of preparation. Prepare 0.004% P80 diluent by making a 1:500 dilution of 2% P80 (e.g., 0.3 ml of 2% P80 to 149.7 ml of CAMHB).</li>
	<li>Further dilute the intermediate concentrations prepared in Step 2.2 1:100 in cation adjusted Mueller Hinton broth (CAMHB) supplemented with 0.004% (v/v) polysorbate-80 (P-80) P-80 and/or LHB (for streptococci) added at double the final concentration because addition of inoculum (step 4.3) will result in a 1:2 dilution. See columns 6 and 7 in Table 2.</li>
</ol>
3. Prepare MIC Panels
<ol>
	<li>Dispense 50 &#181;l of each dalbavancin solution prepared in step 2.3 into appropriate wells of the MIC panel and include media only in one well (growth control well). A multi-channel pipet with sterile tips can be used for this step.</li>
	<li>Use panels immediately or seal with plastic film, place in plastic bags and immediately place in a non-defrosting freezer at &#8804;-20 oC (preferably at &#8804;-60 oC) until needed. If frozen panels are used, remove seals and place individual panels on lab bench for 15-30 min&#160;(until well contents are thawed) before proceeding to panel inoculation.</li>
</ol>
4. Inoculate MIC Panels, Perform Purity and Setup Colony Count
<ol>
	<li>Select several well-isolated colonies from an 18-24 hr blood agar or other non-selective agar plate. Touch the top of each colony with a sterile loop or swab and transfer to 1-5 ml CAMHB or saline until turbidity is equivalent to a 0.5 McFarland standard. Assess turbidity by visual comparison to the 0.5 McFarland or with a photometric device.</li>
	<li>Within 15-30 min&#160;of preparation, dilute the inoculum 1:100 in CAMHB (100 &#181;l into 10 ml CAMHB). For most bacteria tested against dalbavancin, with the exception of S. pneumoniae, this dilution will provide a final well concentration of 5 x 105 CFU/ml (acceptable range is 2-8 x 105 CFU/ml). For S. pneumoniae, bacterial concentration based on comparison of turbidity to a 0.5 McFarland is typically considerably less, therefore, dilute the inoculum 1:25 (400 &#181;l into 10 ml CAMHB+10% +LHB).</li>
	<li>Within 15 min after inoculum preparation, transfer 50 &#181;l of the final inoculum to each well (with exception of the sterility control well) of the MIC panel prepared in step 3. A multi-channel pipet using sterile tips can be used for this step.</li>
	<li>Perform a purity check by transferring and spreading a 1-10 &#181;l aliquot from the positive growth control well using a sterile loop to a nonselective agar (e.g., trypticase soy agar with 5% sheep blood).</li>
	<li>Setup colony count by removing 10 &#181;l from the growth control well with a single channel pipet and sterile tip and transfer to 10 ml of saline (1:1,000 dilution). Mix and transfer 100 &#181;l with a single channel pipet and sterile tip to a suitable, nonselective agar medium (e.g., trypticase soy agar with 5% sheep blood) and spread over the entire agar surface with a sterile loop, repeating two times in different directions to assure even distribution of the inoculum (1:10 dilution).</li>
</ol>
5. Incubate MIC Panels, Colony Count and Purity Plates
<ol>
	<li>Seal each MIC panel or stack of no more than 4 panels in a plastic bag, with plastic tape or with a tight-fitting plastic cover before incubating. Alternatively, place empty MIC panel on the top of stack of no more than 4 MIC panels, place a damp paper towel in a plastic container, place MIC panels in the plastic container and close container securely with lid.</li>
	<li>Incubate MIC panels in an ambient air incubator at 35 &#176;C &#177; 2 &#176;C for 16-20 hr (staphylococci and enterococci) and 20-24 hr (streptococci) within 30 min&#160;of inoculation. Incubate the colony count and purity plates under same conditions except incubate streptococci in a 5% CO2 incubator.</li>
</ol>
6. Read the MIC and Colony Count Plates; Check Purity Plate
<ol>
	<li>Read the MIC as the lowest concentration that completely inhibits bacterial growth in the wells as detected by the unaided eye.</li>
	<li>Count colonies on the colony count plate. Multiply each colony by dilution factor (1:10,000) (e.g., 50 colonies is equivalent to 5 x 105 CFU/ml). An acceptable range is 20-80 colonies (2-8 x 105 CFU/ml) and is used as an approximate guideline.</li>
	<li>Check purity plate. If all colonies are similar to the colonies used in step 4.1, then the inoculum can be considered pure. If there are any other colonies present, then there is potential for a contaminant to be present in the MIC panel and the test should be repeated.</li>
</ol>
7. Check Quality Control Results.
<ol>
	<li>Refer to Table 3 for expected ranges of CLSI quality control strains. Only report results of test isolates if dalbavancin MIC results for quality control organism(s) are within the expected range.</li>
	<li>If an MIC of &#8805;0.25 &#181;g/ml is obtained for any clinical S. aureus isolate, repeat the dalbavancin MIC testing and/or send to a reference laboratory for verification.</li>
</ol>

<ol><li>Boucher, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Once-weekly+dalbavancin+versus+daily+conventional+therapy+for+skin+infection%5BTitle%5D)+AND+%22The+New+England+journal+of+medicine%22%5BJournal%5D)">Once-weekly dalbavancin versus daily conventional therapy for skin infection.</a> The New England journal of medicine. 370, 2169-2179 (2014).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22M07-A10.+Methods+for+Dilution+Antimicrobial+Tests+for+Bacteria+that+Grow+Aerobically.%3A+Approved+Standard%22">M07-A10. Methods for Dilution Antimicrobial Tests for Bacteria that Grow Aerobically.: Approved Standard</a>. , Tenth Edition, Clinical and Laboratory Standards Institute. Wayne, PA. (2015).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22M100-S25.+Performance+Standards+for+Antimicrobial+Susceptibility+Testing%3B+Twenty-Fifth+Informational+Supplement%22">M100-S25. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement</a>. , Clinical and Laboratory Standards Institute. Wayne, PA. (2015).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+laboratory+testing+and+in+vitro+diagnostic+test+systems+--+Susceptibility+testing+of+infectious+agents+and+evaluation+of+performance+of+antimicrobial+susceptibility+test+devices+--+Part+1%3A+Reference+method+for+testing+the+in+vitro+activity+of+antimicrobial+agents+against+rapidly+growing+aerobic+bacteria+involved+in+infectious+diseases%5BTitle%5D)+AND+%22ISO%2FFDIS%22%5BJournal%5D)">Clinical laboratory testing and in vitro diagnostic test systems -- Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices -- Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases.</a> ISO/FDIS. 20776-1, International Organization for Standardization. (2006).</li>
<li>Jones, R. N., Farrell, D. J., Flamm, R. K., Sader, H. S., Dunne, M. W., Mendes, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surrogate+analysis+of+vancomycin+to+predict+susceptible+categorization+of+dalbavancin%5BTitle%5D)+AND+%22Diagn+Microbiol+Infect+Dis%22%5BJournal%5D)">Surrogate analysis of vancomycin to predict susceptible categorization of dalbavancin.</a> Diagn Microbiol Infect Dis. , (2015).</li>
<li>Turnidge, J., Paterson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Setting+and+revising+antibacterial+susceptibility+breakpoints%5BTitle%5D)+AND+%22Clinical+microbiology+reviews%22%5BJournal%5D)">Setting and revising antibacterial susceptibility breakpoints.</a> Clinical microbiology reviews. 20, 391-408 (2007).</li>
<li>Rennie, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+influencing+broth+microdilution+antimicrobial+susceptibility+test+results+for+dalbavancin%2C+a+new+glycopeptide+agent%5BTitle%5D)+AND+%22Journal+of+clinical+microbiology%22%5BJournal%5D)">Factors influencing broth microdilution antimicrobial susceptibility test results for dalbavancin, a new glycopeptide agent.</a> Journal of clinical microbiology. 45, 3151-3154 (2007).</li>
<li>Arhin, F. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+polysorbate+80+on+oritavancin+binding+to+plastic+surfaces%3A+implications+for+susceptibility+testing%5BTitle%5D)+AND+%22Antimicrobial+agents+and+chemotherapy%22%5BJournal%5D)">Effect of polysorbate 80 on oritavancin binding to plastic surfaces: implications for susceptibility testing.</a> Antimicrobial agents and chemotherapy. 52, 1597-1603 (2008).</li>
<li>Farrell, D. J., Mendes, R. E., Rhomberg, P. R., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revised+reference+broth+microdilution+method+for+testing+telavancin%3A+effect+on+MIC+results+and+correlation+with+other+testing+methodologies%5BTitle%5D)+AND+%22Antimicrobial+agents+and+chemotherapy%22%5BJournal%5D)">Revised reference broth microdilution method for testing telavancin: effect on MIC results and correlation with other testing methodologies.</a> Antimicrobial agents and chemotherapy. 58, 5547-5551 (2014).</li>
<li>Ribbon Panel, B. lue <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+for+Safe+Work+Practices+in+Human+and+Animal+Medical+Diagnostic+Laboratories%5BTitle%5D)+AND+%22MMWR+Supplements%22%5BJournal%5D)">Guidelines for Safe Work Practices in Human and Animal Medical Diagnostic Laboratories.</a> MMWR Supplements. 61 (01), 1-101 (2012).</li>
<li>Anderegg, T. R., Biedenbach, D. J., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initial+quality+control+evaluations+for+susceptibility+testing+of+Dalbavancin+%28BI397%29%2C+an+investigational+glycopeptide+with+potent+gram-positive+activity%5BTitle%5D)+AND+%22Journal+of+clinical+microbiology%22%5BJournal%5D)">Initial quality control evaluations for susceptibility testing of Dalbavancin (BI397), an investigational glycopeptide with potent gram-positive activity.</a> Journal of clinical microbiology. 41, 2795-2796 (2003).</li>
<li>Koeth, L. M., Windau, A. R., Goldstein, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+Broth+Microdilution+and+Agar+Dilution+Dalbavancin+and+Vancomycin+MIC+Results+for+42+Aerobic+Gram-positive+Bacteria.+Poster+132%5BTitle%5D)+AND+%22ASM%22%5BJournal%5D)">Comparison of Broth Microdilution and Agar Dilution Dalbavancin and Vancomycin MIC Results for 42 Aerobic Gram-positive Bacteria. Poster 132.</a> ASM. , San Francisco, CA. (2012).</li>
<li>Fritsche, T. R., Rennie, R. P., Goldstein, B. P., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+dalbavancin+MIC+values+determined+by+Etest+%28AB+BIODISK%29+and+reference+dilution+methods+using+gram-positive+organisms%5BTitle%5D)+AND+%22Journal+of+clinical+microbiology%22%5BJournal%5D)">Comparison of dalbavancin MIC values determined by Etest (AB BIODISK) and reference dilution methods using gram-positive organisms.</a> Journal of clinical microbiology. 44, 2988-2990 (2006).</li>
<li>Mushtaq, S., Warner, M., Johnson, A. P., Livermore, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+dalbavancin+against+staphylococci+and+streptococci%2C+assessed+by+BSAC+and+NCCLS+agar+dilution+methods%5BTitle%5D)+AND+%22The+Journal+of+antimicrobial+chemotherapy%22%5BJournal%5D)">Activity of dalbavancin against staphylococci and streptococci, assessed by BSAC and NCCLS agar dilution methods.</a> The Journal of antimicrobial chemotherapy. 54, 617-620 (2004).</li>
<li>Jones, R. N., Flamm, R. K., Sader, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surveillance+of+dalbavancin+potency+and+spectrum+in+the+United+States+%282012%29%5BTitle%5D)+AND+%22Diagnostic+microbiology+and+infectious+disease%22%5BJournal%5D)">Surveillance of dalbavancin potency and spectrum in the United States (2012).</a> Diagnostic microbiology and infectious disease. 76, 122-123 (2013).</li>
</ol>

Sandra McCurdy is an employee of Durata Therapeutics; Laboratory Specialists, Inc., employer of Laura Koeth and Jeanna Fisher, was contracted by Durata Therapeutics to prepare the manuscript. Publication fees for this article were paid by Laboratory Specialists, Inc.

The following method details are presented for additional considerations when preparing for dalbavancin MIC testing. The design of the MIC panel will depend on the number of antimicrobial agents to be tested (i.e., dalbavancin alone or multiple agents) and the species of bacteria to be tested. The dalbavancin concentrations should encompass the interpretive breakpoints for all bacterial species to be tested and the entire range of dilutions expected for at least one QC strain. The antimicrobial dilution series can be organized either in rows or columns depending on the number of antimicrobial agents and/or isolates/plate and method of inoculation. The volume of dalbavancin powder and broth should be calculated to assure that optimal amounts are produced and waste is minimal. For the example used in this publication, volumes are based on preparation of two MIC panels. Cation adjusted Mueller Hinton broth should be prepared and autoclaved according to manufacturer&#8217;s instructions and sterility of the media verified. MIC panels can be made and used on same day of preparation or they can be tested with appropriate quality control organisms and the validated panels stored frozen for future use. It is important to use sterile techniques throughout the procedure.
There is no strict criteria for verification of inoculum concentration with colony counts. Colony counts may be checked initially for each species tested and/or periodically to validate the dilution step performed in step 4.2. It is important that the validity of the 0.5 McFarland standard is also checked routinely with E. coli ATCC 25922 using a procedure similar to step 4.5. If results are not equivalent to 1-2 x 108 CFU/ml, a new 0.5 McFarland standard should be obtained. If the colony counts from MIC wells are outside of the acceptable range (2-8 x 105 CFU/ml and MIC results for the clinical isolate(s) vary from the expected normal distribution (eg. 0.03-0.12 ug/ml for S. aureus) and/or MIC results for quality control isolate(s) are outside of expected ranges, validation testing is necessary and method modification may be warranted. It is suggested that a validated 0.5 McFarland standard is used to repeat the colony count(s) and if results are again outside of expected results, the inoculum dilution as performed in step 4.2 should be adjusted accordingly and the MIC repeated. Note that a purity check is necessary even if a colony count is performed because the colony count procedure uses a diluted volume that may not necessarily detect lower concentrations of contaminating organisms.
For a MIC test to be considered valid, acceptable growth must occur in the growth control well. For most bacteria that will be tested against dalbavancin (i.e., staphylococci, streptococci and enterococci, growth will be seen as a button (typically &#62;2 mm) and less frequently, growth may appear as turbidity in the well. Unlike some antimicrobial agents that may exhibit trailing effect as concentrations increase, dalbavancin endpoints are typically well defined. There are various viewing devices intended to facilitate reading microdilution tests, which may be used to read dalbavancin containing plates as long as there is no compromise in the ability to discern growth in the wells.
The critical steps within the protocol are related to the parts of the procedure that are related to the solubility of dalbavancin and addition of P80. It is important that the maximum concentration of dalbavancin used is no greater than 1,600 &#181;g/ml, that DMSO is used as a solvent and that intermediate concentrations are prepared in DMSO prior to making the broth dilutions so that the final concentration of DMSO in the MIC panel wells is no greater than 1%. DMSO is hygroscopic and therefore, the use of older bottles of DMSO was a consideration with regard to dalbavancin solubility. However, while a small BMD study with quality control strains has shown this not to be an important variable (see results section), it is good practice to use new or recently opened bottles of DMSO. It is also important that P80 is added to the broth in the final step of drug dilution preparation and is at a final concentration of 0.002% in the MIC well.
The BMD procedure included here utilizes preparation of MIC panels using 50 &#181;l/well at dalbavancin and P-80 concentrations two times the final concentration and addition of inoculum with 50 &#181;l/well so that final volume/well is 100 &#181;l. This method allows for one MIC panel format to be produced for the testing of both S. aureus and S. pneumoniae because the lysed horse blood can be added to the inoculum. Alternative methods for preparation of MIC panels using 100 &#181;l/well with addition of up to 10 &#181;l/well are described in the CLSI and ISO procedures.
The dalbavancin susceptible breakpoint for S. aureus is &#8804;0.12 &#181;g/ml (FDA package insert). As is shown in recent surveillance, the majority of isolates are inhibited by dalbavancin at levels of 0.06 &#181;g/ml15. With a susceptible breakpoint close to the modal MIC, any variation in MIC as a result of method differences can affect susceptibility rates. Because the BMD procedure for dalbavancin includes some unique steps (specifically, the use of DMSO in preparation of stock and intermediate drug dilutions and 0.002% P80 in the MIC well, it is imperative that the method is followed exactly. The S. aureus QC organism (ATCC 29213) with a very clear dalbavancin modal MIC of 0.06 &#181;g/ml, has been a good indicator of method variation and should be used routinely to validate the BMD method. If MIC results are with the expected range of 0.03-0.12 &#181;g/ml, but are trending to either the low or high side of this range, further validation of the method is warranted.
The addition of 0.002% P80 is recommended for all relevant Gram-positive organisms (staphylococcci, enterococci, and streptococci.) However, it is noteworthy that it has been shown that lysed horse blood acts similar to P-80 relative to its anti-binding capability and that the addition of P-80 to CAMHB+LHB has no additional effect on dalbavancin streptococci MIC results.
There is currently no disk diffusion method for testing dalbavancin and because of the poor correlation of BMD and AD results, susceptibility testing of dalbavancin using the AD method is not recommended12, 14. The BMD method should be used for the future assessment of dalbavancin susceptibility and as a gold standard for the development of commercial antimicrobial susceptibility tests.

Dalbavancin is a semi-synthetic lipoglycopeptide that was approved by the FDA in May 2014 for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms1. The primary goal of detailing this method in a video format is to provide clear guidance to the clinical laboratory and research scientists for testing and reporting of accurate and reproducible dalbavancin susceptibility results.
The determination of an antimicrobial agent MIC by commercial methods is routinely performed in clinical laboratories for assessment of an agent&#8217;s in vitro activity against pathogenic bacteria. The method described here is the broth microdilution method as recommended by CLSI and ISO 2,3,4. Since dalbavancin commercial susceptibility methods are not currently available (at the time of this publication) and disk diffusion and agar dilution methods are not currently recommended for dalbavancin, reference broth dilution methods are the current options available 12,14. As this reference method is not a commercial and FDA cleared in vitro diagnostic method, clinical laboratories in the USA that use this procedure for patient testing must abide by regulatory requirements for a laboratory developed test (LDT). It is anticipated that as the dalbavancin use increases, more laboratories will have a need to test this compound. Vancomycin testing as a surrogate for determination of dalbavancin susceptibility of S. aureus has been validated5. However, dalbavancin may be active against vancomycin intermediate S. aureus (VISA) and therefore, clinicians may request dalbavancin MIC results. In addition, MIC results for other bacterial species may be requested.
The FDA susceptible breakpoint for dalbavancin against S. aureus has been established at &#8804;0.12 &#181;g/ml. FDA assigned only a susceptible category because of the lack of data on resistant isolates. The normal MIC distribution among wild type S. aureus isolates is between 0.03-0.12 &#181;g/ml, with a clear modal MIC of 0.06 &#181;g/ml. A slight variation in MIC results due to method difference, in addition to the inherent BMD variation of &#177; one dilution, could potentially have a significant impact on the susceptibility rates.6 Similar to other lipoglycopeptide agents, dalbavancin susceptibility testing can be challenging as a result of solubility and the relatively large size of the molecule and its binding properties7,8,9. The reference BMD method described here includes two specific steps that are unique to insoluble agents and/or lipoglycopeptide agents: (1) the use of DMSO for preparation of stock solutions and (2) addition of 0.002% P-80 in the final MIC panel dilutions. The objective of this video publication is to provide clarity to the dalbavancin BMD method and to specifically focus on these key steps, to assure accurate and reproducible MIC results.

<table><tbody><tr><td>Dalbavancin</td><td>Metrics, Inc.</td><td>Greenville, NC</td><td>BI-397</td></tr><tr><td>Vancomycin</td><td>Sigma-Aldrich</td><td>St. Louis, MO</td><td>C5793</td></tr><tr><td>Cation Adjusted Mueller Hinton Broth</td><td>Becton Dickinson</td><td>Sparks, MD</td><td>212322</td></tr><tr><td>Lysed horse blood</td><td>Cleveland Scientific</td><td>Bath, OH</td><td></td></tr><tr><td>Dose-it peristaltic pump</td><td>Integra Biosciences</td><td>Hudson, NH</td><td></td></tr><tr><td>Multi-channel pipet (Viaflo II)</td><td>Integra</td><td>Hudson, NH</td><td>4164</td></tr><tr><td>12 channel pipet (Matrix)</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>Single channelpPipets (Ovation 10 &amp;micro;l - 100 &amp;micro;l)</td><td>VistaLab</td><td>Brewster, NY</td><td></td></tr><tr><td>96 well microplate</td><td>Greiner Bio-one</td><td>Monroe, NC</td><td>650161</td></tr><tr><td>50 ml flat cap conical bottom centrifuge tubes</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>16x125 mm disposable glass tubes</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>12x17 mm snap cap, polystyrene tubes</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>16 ml centrifuge tubes</td><td>BioExpress</td><td>Kaysville, UT</td><td>C-3394-2</td></tr><tr><td>Reagent reservoirs</td><td>Integra Biosciences</td><td>Hudson, NH</td><td>4312</td></tr><tr><td>Disposable sterilelLoops (1 &amp;micro;l &amp;amp; 10 &amp;micro;l)</td><td>Biologix</td><td>Lenexa, KA</td><td>65-0001,65-0010</td></tr><tr><td>Tryptic soy agar + 5% sheep blood</td><td>Becton Dickinson</td><td>Sparks, MD</td><td></td></tr><tr><td>Incubator (ambient)</td><td>Sanyo</td><td></td><td></td></tr><tr><td>Incubator (5% CO2)</td><td>Sanyo</td><td></td><td></td></tr><tr><td>Analytical balance&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>Waltham, MA</td><td></td></tr><tr><td>Gloves</td><td>Kimberly-Clark</td><td></td><td>52817</td></tr><tr><td>Lab coats</td><td></td><td></td><td></td></tr></tbody></table>

a reference broth microdilution method for dalbavancin in vitro susceptibility testing of bacteria that grow aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

Watch this Scientific Journal Video about A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically at JoVE.com

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

28.9K Views.  Laboratory Specialists, Inc. Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology. 

Video: A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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 ...

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 ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

Biology

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

Source: de-Souza-Silva, C. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57127">Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds and their minimum inhibitory concentration (MIC).

1. Solutions and Media

	Prepare 2X Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), Sabouraud dextrose broth, and ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth

Source: Zheng, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55273">System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an in-broth assay to determine antibiotic efficacy against Mycobacterium tuberculosis. The bacterial culture is incubated with different concentrations of a bactericidal antibiotic. The bactericidal efficacy is measured by assessing the presence of viable bacteria using a resazurin assay.

1. Bacterial Strain and Growth Medium

	Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

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