Name: A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically (Video) | JoVE Journal
Uploaded: 2015-09-09T14:00:00.000+00:00
Duration: 11 min 28 s
Description: Learn the broth microdilution method for dalbavancin susceptibility testing in Gram-positive bacteria. This protocol ensures accurate MIC determination using DMSO stock solutions and polysorbate 80.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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

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.

Standardized MIC methods are necessary in order to monitor resistance development, an important goal of various antimicrobial surveillance programs. Dalbavancin MIC results for isolates collected in Europe and USA in 2011-2013 were &lt;0.25 µg/ml for 99.9% of S.aureus and 100% of streptococci (Table 4).
To assess the importance of using fresh (sealed) DMSO, triplicate testing of dalbavancin BMD MICs for QC strains S. aureus ATCC 29213, and E. faecalis ATCC 29212 and duplicate testing for S. pneumoniae ATCC 49619 was performed using (1) DMSO from sealed ampules, (2) DMSO from a 1 L bottle that was within the expiration date and had been used previously for 2 years and (3) an aliquot of DMSO from the same 1 L bottle (sample 2) that was allowed to sit in an open beaker for 72 hr prior to use. Dalbavancin MIC results using panels made with all three DMSO samples were similar and within the acceptable QC ranges (Table 5).
The loss of dalbavancin activity following exposure of the stock solution to plastic and without the addition of P-80 has been well documented7. In 2 additional studies, the addition of P-80 to the initial and intermediate stock solutions (Table 6) and the use of glass or plastic tubes for the preparation of the initial and intermediate stock solutions (Table 7) did not have an effect on the dalbavancin MIC results. There was also no effect on dalbavancin MIC results when polypropylene and polystyrene plates from three manufacturers were used (Table 7).
The presence of lysed horse blood in the broth media for testing of streptococci has been shown to have a surfactant-like activity, comparable to P-807, 8. The dalbavancin reference method for streptococcus does include both P-80 and lysed horse blood, however, unlike staphylococci and enterococci, dalbavancin MIC results for streptococci are similar (within +/- 1 dilution) with and without the addition of P-80.
As has been previously presented and shown in Figure 1, dalbavancin agar dilution (AD) MICs are typically 2-8 fold higher compared to the current BMD reference12, 13, 14. Unlike BMD, the addition of 0.002% P-80 does not affect dalbavancin AD MIC results12.
<img alt="Preparation of 800 μg/mL stock solution, equation showing calculation of 21.5 mg dalbavancin." src="/files/ftp_upload/53028/53028eq1.jpg" data-altupdatedat="1787630408000" data-alt="Equation 1"> 
Equation 1: Example of Dalbavancin Stock Solution Preparation. Preparation steps for 25 ml of 800 µg/ml dalbavancin solution.
<img alt="Dalbavancin vs Vancomycin comparative bar chart; mean MIC analysis of bacterial strains." src="/files/ftp_upload/53028/53028fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Comparison of Dalbavancin and Vancomycin BMD to AD.  Results of study that compared dalbavancin and vancomycin BMD and AD MIC results for select Gram-positive bacterial isolates. Average (A) Dalbavancin and (B) Vancomycin Broth Microdilution and Agar Dilution MIC Results in µg/ml for 15 Staphylococcus aureus, 8 Coagulase Negative Staphylococci (CNS), 5 Enterococcus faecalis and 14 Streptococci (Groups A, B, and C). <a href="https://www.jove.com/files/ftp_upload/53028/53028fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>7</td>
			<td>8</td>
			<td>9</td>
			<td>10</td>
			<td>11</td>
			<td>12</td>
		</tr>
		<tr>
			<td>A</td>
			<td>DAL 0.002</td>
			<td>DAL 0.004</td>
			<td>DAL 0.008</td>
			<td>DAL 0.015</td>
			<td>Dal     0.03</td>
			<td>DAL    0.06</td>
			<td>DAL   0.12</td>
			<td>DAL    0.25</td>
			<td>DAL      0.5</td>
			<td>DAL         1</td>
			<td>DAL         2</td>
			<td>Pos Ctrl.</td>
		</tr>
		<tr>
			<td>B</td>
			<td>DAL 0.002</td>
			<td>DAL 0.004</td>
			<td>DAL 0.008</td>
			<td>DAL 0.015</td>
			<td>Dal     0.03</td>
			<td>DAL    0.06</td>
			<td>DAL   0.12</td>
			<td>DAL    0.25</td>
			<td>DAL      0.5</td>
			<td>DAL         1</td>
			<td>DAL         2</td>
			<td>Pos Ctrl.</td>
		</tr>
		<tr>
			<td>C</td>
			<td>DAL 0.002</td>
			<td>DAL 0.004</td>
			<td>DAL 0.008</td>
			<td>DAL 0.015</td>
			<td>Dal     0.03</td>
			<td>DAL    0.06</td>
			<td>DAL   0.12</td>
			<td>DAL    0.25</td>
			<td>DAL      0.5</td>
			<td>DAL         1</td>
			<td>DAL         2</td>
			<td>Pos Ctrl.</td>
		</tr>
		<tr>
			<td>D</td>
			<td>DAL 0.002</td>
			<td>DAL 0.004</td>
			<td>DAL 0.008</td>
			<td>DAL 0.015</td>
			<td>Dal     0.03</td>
			<td>DAL    0.06</td>
			<td>DAL   0.12</td>
			<td>DAL    0.25</td>
			<td>DAL      0.5</td>
			<td>DAL         1</td>
			<td>DAL         2</td>
			<td>Pos Ctrl.</td>
		</tr>
		<tr>
			<td>E</td>
			<td>DAL 0.002</td>
			<td>DAL 0.004</td>
			<td>DAL 0.008</td>
			<td>DAL 0.015</td>
			<td>Dal     0.03</td>
			<td>DAL    0.06</td>
			<td>DAL   0.12</td>
			<td>DAL    0.25</td>
			<td>DAL      0.5</td>
			<td>DAL         1</td>
			<td>DAL         2</td>
			<td>Pos Ctrl.</td>
		</tr>
		<tr>
			<td>F</td>
			<td>DAL 0.002</td>
			<td>DAL 0.004</td>
			<td>DAL 0.008</td>
			<td>DAL 0.015</td>
			<td>Dal     0.03</td>
			<td>DAL    0.06</td>
			<td>DAL   0.12</td>
			<td>DAL    0.25</td>
			<td>DAL      0.5</td>
			<td>DAL         1</td>
			<td>DAL         2</td>
			<td>Pos Ctrl.</td>
		</tr>
		<tr>
			<td>G</td>
			<td>DAL 0.002</td>
			<td>DAL 0.004</td>
			<td>DAL 0.008</td>
			<td>DAL 0.015</td>
			<td>Dal     0.03</td>
			<td>DAL    0.06</td>
			<td>DAL   0.12</td>
			<td>DAL    0.25</td>
			<td>DAL      0.5</td>
			<td>DAL         1</td>
			<td>DAL         2</td>
			<td>Pos Ctrl.</td>
		</tr>
		<tr>
			<td>H</td>
			<td>DAL 0.002</td>
			<td>DAL 0.004</td>
			<td>DAL 0.008</td>
			<td>DAL 0.015</td>
			<td>Dal     0.03</td>
			<td>DAL    0.06</td>
			<td>DAL   0.12</td>
			<td>DAL    0.25</td>
			<td>DAL      0.5</td>
			<td>DAL         1</td>
			<td>DAL         2</td>
			<td>Pos Ctrl.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>DAL</td>
			<td colspan="2">Dalbavancin</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="12">Concentrations are stated in µg/ml after 50 µl/well inoculation</td>
		</tr>
	</tbody>
</table>
Table 1. MIC Plate Format.  The 96 well plate format showing location of dalbavancin dilutions and positive control wells used as an example in this video publication
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>7</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Source Conc. (µg/ml)</td>
			<td>Volume Source (ml)</td>
			<td>Volume DMSO (ml)</td>
			<td>Intermediate Conc. in DMSO (µg/ml)</td>
			<td>Volume Intermediate Conc. (ml)</td>
			<td>Volume CAMHB + 0.004% P80* (ml)</td>
			<td>2X Conc. After 1:100 Dilution in CAMHB (µg/ml)</td>
			<td>Final Panel Conc. Post Inoculation (µg/ml)</td>
		</tr>
		<tr>
			<td>800</td>
			<td>1.0</td>
			<td>1.0</td>
			<td>400</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>4</td>
			<td>2</td>
		</tr>
		<tr>
			<td>400</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>200</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>2</td>
			<td>1</td>
		</tr>
		<tr>
			<td>400</td>
			<td>0.5</td>
			<td>1.5</td>
			<td>100</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>1</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>400</td>
			<td>0.5</td>
			<td>3.5</td>
			<td>50</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>0.5</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>50</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>25</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>0.25</td>
			<td>0.125</td>
		</tr>
		<tr>
			<td>50</td>
			<td>0.5</td>
			<td>1.5</td>
			<td>12.5</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>0.125</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>50</td>
			<td>0.5</td>
			<td>3.5</td>
			<td>6.25</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>6.25</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>3.13</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>0.03</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>6.25</td>
			<td>0.5</td>
			<td>1.5</td>
			<td>1.5</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>0.015</td>
			<td>0.008</td>
		</tr>
		<tr>
			<td>6.25</td>
			<td>0.5</td>
			<td>3.5</td>
			<td>0.8</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>0.008</td>
			<td>0.004</td>
		</tr>
		<tr>
			<td>0.8</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>0.4</td>
			<td>0.02</td>
			<td>1.98</td>
			<td>0.004</td>
			<td>0.002</td>
		</tr>
		<tr>
			<td colspan="5">This table has been modified from CLSI M100-S25, Table 8B3  </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2. Scheme for Preparing Dilutions of Water-Insoluble Antimicrobial Agents to be Used in Broth Dilutions Susceptibility Testing. Volumes of dalbavancin working solutions and diluents for preparation of final MIC dalbavancin solutions of 0.002-2 µg/ml.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Quality Control Strain</td>
			<td>Expected Dalbavancin MIC (µg/ml)</td>
		</tr>
		<tr>
			<td>S. aureus ATCC 29213</td>
			<td>0.03-0.12</td>
		</tr>
		<tr>
			<td>E. faecalis ATCC 29212</td>
			<td>0.03-0.12</td>
		</tr>
		<tr>
			<td>S. pneumoniae ATCC 49619</td>
			<td>0.008-0.03</td>
		</tr>
	</tbody>
</table>
Table 3. Expected Dalbavancin MIC Results for Quality Control Strains3, 11. CLSI quality control results for suggested strains to be tested along with clinical isolates in order to verify proper methodology

<img alt="Antimicrobial susceptibility results table for different bacteria; MIC values range from ≤0.03 to ≥4 μg/mL." src="/files/ftp_upload/53028/53028table4.jpg" data-altupdatedat="1787630408000" data-alt="Table 4"> 
Table 4. Distribution of Dalbavancin MIC Results (Number at each MIC) from USA and European 2011-2013 Surveillance Study15.  Dalbavancin MIC results from a published study of recent clinical isolates as an indicator of current dalbavancin in vitro activity.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
	</colgroup>
	<tbody>
		<tr>
			<td rowspan="2">Isolate #</td>
			<td colspan="3">Dalbavancin MIC (µg/ml)</td>
		</tr>
		<tr>
			<td>DMSO 1</td>
			<td>DMSO 2</td>
			<td>DMSO 3</td>
		</tr>
		<tr>
			<td rowspan="3">S. aureus ATCC 29213</td>
			<td>0.12</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>0.12</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td rowspan="3">E. faecalis ATCC 29212</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td rowspan="2">S. pneumoniae ATCC 49619</td>
			<td>0.015</td>
			<td>0.015</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>0.03</td>
			<td>0.015</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td colspan="4">DMSO 1 = Unopened flame sealed 10 ml ampules</td>
		</tr>
		<tr>
			<td colspan="2">DMSO 2 = Frequently opened 1L bottle</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">DMSO 3 = Frequently opened 1L bottle - sat on bench in uncovered beaker for 72 hr</td>
		</tr>
	</tbody>
</table>
Table 5. Dalbavancin MIC Results (µg/ml) using DMSO Stock and Intermediate Dilutions Preparation with DMSO from 3 Sources. Results of a dalbavancin MIC study that compared effect of using different DMSO solvents in preparation of the dalbavancin stock and intermediate dilutions.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td rowspan="2">Isolate Number</td>
			<td rowspan="2">Replicate Number</td>
			<td colspan="2">Dalbavancin MIC (µg/ml)</td>
		</tr>
		<tr>
			<td>DALB w/P80 in DMSO</td>
			<td>DALB w/o P80 in DMSO</td>
		</tr>
		<tr>
			<td rowspan="4">S. aureus ATCC 29213</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>4</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>S. aureus clinical DP80-002SA </td>
			<td>1</td>
			<td>0.03</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>S. aureus ATCC 700699</td>
			<td>1</td>
			<td>0.5</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td rowspan="4">S. pneumoniae ATCC 49619</td>
			<td>1</td>
			<td>0.03</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.03</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.03</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>4</td>
			<td>0.03</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>S. pyogenes clinical:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DP80-004PY</td>
			<td>1</td>
			<td>0.015</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>DP80-005PY</td>
			<td>1</td>
			<td>0.03</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>DP80-006PY</td>
			<td>1</td>
			<td>0.03</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>DP80-007PY</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>DP80-008PY</td>
			<td>1</td>
			<td>0.03</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>S. agalactiae clinical:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DP80-009AG</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.12</td>
		</tr>
		<tr>
			<td>DP80-010AG</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>DP80-011AG</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>DP80-012AG</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>S. dysgalactiae clinical DP80-013DY</td>
			<td>1</td>
			<td>0.03</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>S. pneumoniae clinical:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DP80-014SP</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>DP80-015SP</td>
			<td>1</td>
			<td>0.03</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>DP80-016SP</td>
			<td>1</td>
			<td>0.03</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>DP80-017SP</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.06</td>
		</tr>
	</tbody>
</table>
Table 6. Dalbavancin MIC Results using DMSO Stock and Intermediate Dilutions Preparation with and without 0.002% P-80. Results of a dalbavancin MIC study that compared effect of adding 0.002% P-80 to dalbavancin stock and intermediate dilutions.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td rowspan="3">Isolate </td>
			<td rowspan="3">Replicate Number</td>
			<td colspan="4" rowspan="2">Stock Dilution Tubes</td>
			<td colspan="4">Microtiter Plate (Plastic Type)</td>
		</tr>
		<tr>
			<td rowspan="2">Poly-propylene, Greiner</td>
			<td colspan="3">Polystyrene</td>
		</tr>
		<tr>
			<td>Glass</td>
			<td>Plastic</td>
			<td>Plastic 1hr</td>
			<td>Plastic 1hr / Intermediate 1hr</td>
			<td>Greiner</td>
			<td>Corning</td>
			<td>Nunc</td>
		</tr>
		<tr>
			<td rowspan="3">S. aureus ATCC 29213</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.06</td>
			<td>0.03</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td rowspan="3">E. faecalis ATCC 29212</td>
			<td>1</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.06</td>
			<td>0.03</td>
			<td>0.03</td>
			<td>0.06</td>
			<td>0.03</td>
		</tr>
		<tr>
			<td rowspan="2">S. pneumoniae ATCC 49619</td>
			<td>1</td>
			<td>0.015</td>
			<td>0.015</td>
			<td>0.015</td>
			<td>0.015</td>
			<td>0.008</td>
			<td>0.008</td>
			<td>0.008</td>
			<td>0.008</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.015</td>
			<td>0.015</td>
			<td>0.015</td>
			<td>0.015</td>
			<td>0.008</td>
			<td>0.015</td>
			<td>0.008</td>
			<td>0.008</td>
		</tr>
		<tr>
			<td colspan="10">Glass = Initial stock made in glass bottle, used immediately to make intermediate concentrations in plastic tubes, used immediately to make broth dilutions.</td>
		</tr>
		<tr>
			<td colspan="10">Plastic = Initial stock made in plastic centrifuge tube, used immediately to make intermediate concentrations in plastic tubes, used immediately to make broth dilutions.</td>
		</tr>
		<tr>
			<td colspan="10">Plastic 1 hr = Initial stock made in plastic centrifuge tube, let sit on bench for 1 hr then used to make intermediate concentrations in plastic tubes, used immediately to make concentrations in broth.</td>
		</tr>
		<tr>
			<td colspan="10">Plastic 1 hr / Intermediate 1 hr = Initial stock made in plastic centrifuge tube, let sit on bench for 1 hr then used to make intermediate concentrations in plastic tubes, let sit on bench for 1 hr then used to make concentrations in broth.</td>
		</tr>
	</tbody>
</table>
Table 7. Dalbavancin MIC Results using Stock Dilutions Preparation in Plastic and Glass Tubes and MIC Plate Plastic Types. Results of a dalbavancin MIC study that compared effect of using plastic or glass tubes when preparing the stock solutions and also effect of using different sources and types of plastic for microtiter plates.

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

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

The overall goal of the following procedure is to determine the minimum inhibitory concentration for dalbavancin. Using the broth micro dilution method with a focus on the key steps that are unique to Dalbavancin to do this. Stock and intermediate dilution of dalbavancin are made in DMSO and final dilution are made in cation on adjusted Mueller Hinton broth supplemented with polysorbate.The dilution are dispensed into a 96 well plate and inoculated with a standardized suspension of bacteria. The plate is then incubated for 16 to 24 hours and examined to determine the minimum amount of dalbavancin required to inhibit bacterial growth. Adherence to this standardized method will assure consistent results with both clinical isolates and as shown here, quality control isolates Dalbavancin is a semisynthetic lipo glycopeptide antimicrobial agent that was approved in May, 2014 by the FDA for the treatment of acute bacterial skin and skin structure infections caused by gram-positive organisms.This method describes the standardized broth, micro dilution susceptibility method for dassin, which includes the use of DMSO for preparing stock solutions in polysorbate 80 to alleviate adherence of the agent to plastic Visual demonstration of this method is helpful. As the stock and MIC plate solution preparation steps are not typical compared to most antimicrobial agents. These steps are unique to lipo Glycopeptide agents and are important to follow in order to achieve reproducible and accurate MIC results.Demonstrating the procedure will be Gina Fisher, a research scientist from my laboratory. Please note that appropriate safety precautions consistent with biosafety level two should be utilized when performing the following procedure. Begin by weighing out the appropriate amount of dalbavancin to prepare a stock solution of 800 micrograms per milliliter.Add the appropriate amount of NEAT DMSO to a sterile glass or plastic tube, then transfer the powder to the tube and vortex it.Next. Using the 800 microgram per milliliter DALBAVANCIN stock solution, prepare 100 x final MIC plate concentrations as shown in columns one through four. These are referred to as the intermediate concentrations.The total volume prepared will depend on the number of MIC panels. Finally, dilute the intermediate concentrations by one to 100 in cation unadjusted Mueller Hinten broth, also known as C-A-M-H-B, supplemented with 0.004%P 80. The resulting working concentration of DALBAVANCIN and P 80 is twice the final concentration.Since the addition of inoculum will result in a one to two dilution using a multi-channel pipette with sterile tips, dispense 50 microliters of each dilution of dalbavancin into the appropriate wells of the 96 well sterile micro dilution panel. The MIC plate format allows eight inocular to be tested per plate. If the panels will not be used immediately, apply plastic film to seal them, then place them in plastic bags and store them in a non defrosting freezer at at least minus 60 degrees Celsius until they are needed.In this demonstration, one freshly prepared plate will be inoculated. To prepare the inoculum, select several well isolated colonies from an 18 to 24 hour blood auger or other non-selective auger plate. Here, quality control s aria and EPHI callous strains are used.Touch the top of each colony with a sterile swab and transfer some of it to a tube containing one to five milliliters of C-A-M-H-B or saline. Keep transferring until the turbidity matches a 0.5 McFarland standard within 15 to 30 minutes of preparation, dilute the inoculum one to 100 in C-A-M-H-B to arrive at a final concentration of five times 10 to the fifth CFU per milliliter. The acceptable range is two to eight times 10 to the fifth CFU per milliliter.Immediately using a multi-channel pipette with sterile tips, transfer 50 microliters of the final inoculum to each appropriate well of the MIC panel plate here. Four rows are inoculated with each of the two isolates for a total of four replicates per isolate. Next, to perform a purity check, use a pipette to transfer one to 10 microliters of the solution in the positive growth control to a non-selective agar, such as try decay's soy agar with 5%sheep blood.Use a sterile loop to spread the solution over the surface of the plate. Turn the plate 90 degrees and streak again to assure that the inoculum is evenly distributed. Cover the plate and set it aside.In preparation for a colony count, use a single channel pipette to transfer 10 microliters of the solution in the growth control well to 10 milliliters of saline mix, and then transfer 100 microliters to a suitable non-selective agri medium, such as try toay soy agar with 5%sheep blood using a sterile loop, spread the solution over the entire agri surface. Repeat this process two times in different directions to assure even distribution of the inoculum. Next, stack up to four MIC panels in a plastic container ensuring that the top panel is tightly sealed with a lid.Then place a damp paper towel inside the plastic container and close it securely with the lid within 30 minutes of inoculation. Incubate the MIC panels, the colony count plate and the purity check plate in an ambient air incubator at 35 plus or minus two degrees Celsius for 16 to 20 hours. The next day, read the minimum inhibitory concentration as the lowest concentration that completely inhibits bacterial growth in the wells as detected by the unaided eye.Determine the number of colonies on the colony count plate. Multiply the number counted by the dilution factor. For example, 50 colonies times a dilution factor of 10, 000 equals five times 10 to the fifth CFU per milliliter.An acceptable range is 20 to 80 colonies, which equates to two to eight times 10 to the fifth colony forming units per ml. The concentration of the inoculum is an important variable to control the susceptibility testing in order to obtain accurate results. Finally, check the purity plate.If all colonies are similar to the colonies used as inoculum, then it 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. DAL bevans, MIC results were compared to those obtained using the agri dilution method as shown here.Agri MIC results for Dal bevans are typically two to eight fold higher than broth. Micro dilution, MIC results. This contrast vancomycin results, which are by broth and agri methods.Therefore, agri dilution is not recommended for testing of dalbavancin dalbavancin broth. Micro dilution MIC, results for quality control should be within expected ranges as shown here. The SS quality control organism, A TCC 2 9 2 1 3 with a very clear modal MIC of 0.06 micrograms per milliliter has been a good indicator of method variation.If the results obtained are outside of the expected ranges for the quality control organisms or trending to either the low or high end of the range, further validation of the method is warranted. In addition to validating the method using quality control organisms, dalbavancin, MIC data from regional antimicrobial surveillance programs can provide a basis for MIC results to expect in the clinical laboratory setting as shown here, Dal b Anson MIC, results for isolates collected in Europe and the USA in 2011 to 2013 were less than 0.25 micrograms per milliliter for 99.9%of SIUs and 100%of streptococci. Based on these surveillance data, it is evident that dalbavancin MIC results for staphylococci and streptococci greater than 0.12 micrograms per milliliter are rare.If higher numbers are seen, the test should be repeated and or sent to a reference laboratory for validation. The preparation of the stock dilution in DMSO and the addition of P 80 to the broth dilution at a concentration of 0.002%in the final MIC well are the critical steps to obtaining accurate and reproducible dalbavancin MIC results As always, but especially in this area of bacterial resistance development, accurate antimicrobial susceptibility results are critical to the clinician's choice of appropriate therapy.

a-reference-broth-microdilution-method-for-dalbavancin-vitro

Research

JoVE Journal

Immunology and Infection

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.

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

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.

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

Source: Koeth, L. M., et. al., A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically. J. Vis. Exp. (2015).This video demonstrates the broth microdilution method for determining the minimum inhibitory concentration (MIC) of a lipoglycopeptide antibiotic against Gram-positive bacteria. It covers bacterial colony isolation, dilution, inoculation of microplate wells with varying antibiotic concentrations, and verification of culture...

A Broth Microdilution Method for Antibiotic Susceptibility Testing of Gram-Positive Bacteria

This video demonstrates a microtiter plate-based method for determining the Minimum Inhibitory Concentration (MIC) of test antibiotics against bacteria. Upon adding a suspension of the bacterial pathogen Pseudomonas aeruginosa to a microtiter plate containing serial dilutions of a test antibiotic, and an inhibitor of the production of pyomelanin by the bacteria, the optical density of the wells is measured to compute the minimum inhibitory concentration of the antibiotic, and the impact of the...

A Microtiter Plate Method to Assess the Minimum Inhibitory Concentration of an Antibiotic

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).

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

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.

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

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

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

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.

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

Here, we present a protocol to identify potential anti-tuberculosis drug candidates through phenotypic screening, encompassing the chemical synthesis of a 4-aminoquinoline compound, determination of its minimum inhibitory concentration against Mycobacterium tuberculosis, and assessment of its cytotoxicity in mammalian cell lines.

Identification of Potential Anti-TB Candidates: A Step-by-Step Guide to Synthesis, MIC Determination, and Cytotoxicity Assessment in Mammalian Cells

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Learn the broth microdilution method for dalbavancin susceptibility testing in Gram-positive bacteria. This protocol ensures accurate MIC determination using DMSO stock solutions and polysorbate 80.

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

Explore More Videos