Characterization of the In Vitro Activity of Novel Lipoglycopeptide Antibiotics

Francis F. Arhin1, Adam Belley1, Geoffrey A. McKay1, Gregory Moeck1

1 The Medicines Company, Saint‐Laurent, Quebec, Canada
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 17.1
DOI:  10.1002/9780471729259.mc1701s16
Online Posting Date:  February, 2010
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Abstract

The increasing incidence of antibiotic resistance in human pathogens is of significant concern. Resistance to the widely‐used and highly effective glycopeptide antibacterial agent vancomycin, which has been in clinical use for over half a century, has emerged in staphylococci and enterococci. This has spurred the development of newer glycopeptide agents, some of which show activity against vancomycin‐resistant organisms. The newer agents currently being developed contain lipophilic side‐chains, which distinguish them from vancomycin; as such, they are categorized as lipoglycopeptides. Oritavancin, telavancin, and dalbavancin are lipoglycopeptides in late‐stage development to combat Gram‐positive bacteria including methicillin‐resistant Staphylococcus aureus (MRSA) and vancomycin‐resistant enterococci. This unit describes methods that may be used to assess the in vitro activities of lipoglycopeptides. The methods include susceptibility assays, time‐kill and time‐kill‐synergy assays, inhibition of synthesis of macromolecules, membrane perturbation assays, and measurement of activity against biofilms. Curr. Protoc. Microbiol. 16:17.1.1‐17.1.22. © 2010 by John Wiley & Sons, Inc.

Keywords: oritavancin; lipoglycopeptide; broth microdilution; time‐kill; synergy; biofilm; macromolecular synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Broth Microdilution Assay
  • Basic Protocol 2: Time‐Kill Assay
  • Basic Protocol 3: Macromolecular Synthesis Assay
  • Basic Protocol 4: Membrane Potential Assay
  • Basic Protocol 5: MBEC Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Broth Microdilution Assay

  Materials
  • Oritavancin diphosphate (The Medicines Company) or other lipoglycopeptide antibacterial agents, e.g., telavancin (Theravance), dalbavancin (Pfizer), teicoplanin (Sequoia Research Products)
  • 0.004% polysorbate‐80
  • Cation‐adjusted Mueller Hinton broth (CAMHB; see recipe)
  • CAMHB containing 5% lysed horse blood (for broth cultures of streptococci)
  • Test bacterial strain(s) in broth culture or as colonies on cation‐adjusted Mueller Hinton agar (CAMHA; see recipe)
  • CAMHA (see recipe) supplemented with 5% lysed sheep blood (for growth of streptococci on agar plates)
  • Brain‐heart infusion (BHI) medium (unit 9.4)
  • 0.9% sterile saline ( appendix 2A)
  • Quality control (QC) strains (Staphylococcus aureus, ATCC #29213; Enterococcus faecalis, ATCC #29212; Streptococcus pneumoniae, ATCC #49619)
  • BaSO 4 turbidity standard equivalent to 0.5 McFarland standard (preparation described in CLSI, ; see recipe)
  • DMSO‐resistant 96‐well plates (Becton Dickinson, cat. no. 352135; maximum 200 µl)
  • 96‐well deep‐well plates (Costar, cat. no. 3960; maximum 2 ml)
  • Multichannel pipettor
  • Untreated, 96‐well microtiter plates (Costar, cat. no. 3370) for broth microdilution assay (do not use tissue‐culture treated plates when testing oritavancin)
  • 35°C incubator (with 5% CO 2 when testing streptococci) or 35°C rotary incubator
  • Spectrophotometer

Basic Protocol 2: Time‐Kill Assay

  Materials
  • Appropriate medium
  • Test strain
  • Quality control (QC) strains (Staphylococcus aureus, ATCC #29213; Enterococcus faecalis, ATCC #29212; Streptococcus pneumoniae, ATCC #49619)
  • Cation‐adjusted Mueller Hinton broth (CAMHB; see recipe), 37°C
  • CAMHB containing drug (see protocol 1)
  • Drugs of interest
  • 25 mg/ml activated charcoal suspension in deionized water, sterile
  • 0.9% sterile saline ( appendix 2A)
  • Cation‐adjusted Mueller Hinton agar (CAMHA)
  • 14‐ml snap‐cap polypropylene tubes (Falcon, cat. no. 352059)
  • 37°C rotary incubator
  • Spectrophotometer
  • 125‐ml glass Erlenmeyer flasks
  • Cheesecloth plug
  • 96‐well deep‐well plates (Costar, cat. no. 3960; maximum 2 ml)
  • 125‐ml polymethylpentene Erlenmeyer flasks (Nalgene, cat. no. 4109‐0125)
  • AirPore tape sheet (Millipore, cat. no. 19571)
  • Multichannel pipettor
  • Omnitrays (rectangular polystyrene plates; Nunc, cat. no. 242811)

Basic Protocol 3: Macromolecular Synthesis Assay

  Materials
  • Oritavancin in water containing 0.004% polysorbate‐80 (see protocol 1)
  • Control antibiotics:
    • Ciprofloxacin (DNA synthesis inhibitor; Sequoia Research Products, cat. no. SRP03565c)
    • Rifampicin (RNA synthesis inhibitor; Sequoia Research Products, cat. no. SRP03565c SRP01277r)
    • Tetracycline (protein synthesis inhibitor; Sequoia Research Products, cat. no. SRP03565c SRP01575t)
    • Vancomycin (cell wall synthesis inhibitor; Sequoia Research Products, cat. no. SRP03565c SRP00975v)
  • Radioactively labeled precursors:
    • 3H‐thymidine (DNA; GE Healthcare, cat. no.TRK758‐1MCI)
    • 3H‐uridine (RNA; GE Healthcare, cat. no. TRK178‐1MCI)
    • 35S‐methionine (protein; GE Healthcare, cat. no. 25001825)
    • 3H‐N‐acetylyglucosamine (cell wall; GE Healthcare, cat. no. TRK376‐250UCI)
  • S. aureus RN4220 (restriction‐deficient mutant of S. aureus 8325‐4; Kreiswirth et al., )
  • Cation‐adjusted Mueller Hinton broth (CAMHB; see recipe)
  • M63 and supplemented M63 media, pre‐warmed (see reciperecipes)
  • Polysorbate‐80
  • Unlabeled thymidine, uridine, methionine, and N‐acetyl glucosamine
  • Sodium azide
  • 1%, 5%, and 10% (w/v) trichloroacetic acid (TCA) in water
  • 95% ethanol
  • Non‐aqueous scintillation fluid
  • Untreated 96‐well polystyrene microtiter plates
  • 1‐liter Erlenmeyer flasks
  • 35°C incubator with rotary shaker
  • Spectrophotometer
  • Centrifuge (e.g., Beckman GS‐6R)
  • 50‐ml conical polypropylene tubes
  • 96‐well deep‐well plates
  • Multichannel pipettor
  • 96‐well MultiScreen plate (Millipore, cat. no. MAFBNOB50)
  • 96‐well MultiScreen vacuum manifold (Millipore, cat. no. MSVMHTS00)
  • Liquid scintillation counter (Trilux 1450 Microbeta counter, PE Biosciences)

Basic Protocol 4: Membrane Potential Assay

  Materials
  • DiSC 3(5) dye (Invitrogen, cat. no. D‐306)
  • DMSO
  • S. aureus (ATCC #29213)
  • Cation‐adjusted Mueller Hinton broth (CAMHB; see recipe)
  • HEPES‐glucose buffer (see recipe)
  • 0.9% saline
  • Cation‐adjusted Mueller Hinton agar (CAMHA)
  • Control antibiotics: vancomycin (Sigma), daptomycin (Cubist)
  • Oritavancin diphosphate (The Medicines Company)
  • Polysorbate‐80
  • Spectrophotometer
  • 1.5‐ml polypropylene microcentrifuge tubes, sterile
  • Aluminum foil
  • Fluorescence microplate reader (e.g., Tecan Ultra or equivalent)
  • DMSO‐resistant 96‐well plate
  • 50‐ml sterile conical tubes (Falcon)
  • Multichannel pipettor and sterile trough
  • Black 96‐well microtiter plates (Corning, cat. no. 3686)

Basic Protocol 5: MBEC Assay

  Materials
  • Bacterial strains of interest (e.g., reference strains and clinical isolates)
  • Agar plates
  • 0.9% sterile saline
  • Tryptic soy broth (TSB) or TSB containing 1% glucose (units 3.1& 9.4)
  • Antibacterial agents
  • Cation‐adjusted Mueller Hinton broth (CAMHB; see recipe)
  • Multichannel pipettor
  • MBEC physiology and genetics assay plate (MBEC assay plate; Innovotech)
  • Humidified container
  • 37°C rotary incubator
  • 96‐well microtiter plates
  • Forceps
  • 2‐ml microcentrifuge tubes
  • Water bath sonicator (e.g., VWR Aquasonic model 550D)
  • 37°C incubator
  • Aluminum foil
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Figures

Videos

Literature Cited

Literature Cited
   Alborn, W.E. Jr., Allen, N.E., and Preston, D.A. 1991. Daptomycin disrupts membrane potential in growing Staphylococcus aureus. Antimicrob. Agents Chemother. 35:2282‐2287.
   Anderegg, T.R., Biedenbach, D.J., and Jones, R.N. 2003. Initial quality control evaluations for susceptibility testing of dalbavancin (BI397), an investigational glycopeptide with potent Gram‐positive activity. J. Clin. Microbiol. 41:2795‐2796.
   Arhin, F.F., Sarmiento, I., Parr, T.R. Jr., and Moeck, G. 2007. Mechanisms of action of oritavancin in Staphylococcus aureus. 47th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Chicago, Illinois. September 17‐20, 2007. Poster no. C1‐1471.
   Arhin, F.F., Sarmiento, I., Belley, A., McKay, G.A., Draghi, D.C., Grover, P., Sahm, D., Parr, T.R. Jr., and Moeck, G. 2008a. Effect of polysorbate‐80 on oritavancin binding to plastic surfaces—Implications for susceptibility testing. Antimicrob. Agents Chemother. 52:1597‐1603.
   Arhin, F.F., Tomfohrde, K., Draghi, D.D., Aranza, M., Parr, T.R. Jr., Sahm, D.F., and Moeck, G. 2008b. Newly‐defined in vitro quality control ranges for oritavancin broth microdilution testing and impact of variation in testing parameters. Diag. Microbiol. Infec. Dis. 62:92‐95.
   Arhin, F.F., Sarmiento, I., Parr, T.R. Jr., and Moeck, G. 2008c. Binding of oritavancin to surfaces impacts choice of vessels for oritavancin in vitro assays. 58th Annual meeting of the Canadian Society of Microbiologists; Calgary, Alberta, June 9‐12, 2007. Poster no. B1.
   Belley, A., Neesham‐Grenon, E., Arhin, F.F., McKay, G.A., Parr, T.R. Jr., and Moeck, G. 2008. Assessment by time‐kill methodology of the synergistic effects of oritavancin in combination with other antimicrobial agents against Staphylococcus aureus. Antimicrob. Agents Chemother. 52:3820‐3822.
   Belley, A., Neesham‐Grenon, E., McKay, G., Arhin, F.F., Harris, R., Beveridge, T., Parr, T.R. Jr., and Moeck, G. 2009. Oritavancin kills stationary‐phase and biofilm Staphylococcus aureus cells in vitro. Antimicrob. Agents Chemother. 53:918‐925.
   Blosser, R.S., Karlowsky, J.A., Loutit, J.S., Porter, S.B., Jones, M.E., Thornsberry, C., and Sahm, D.F. 2003. Evaluation of agar‐based susceptibility testing of oritavancin against Gram‐positive cocci. 103rd General Meeting of the American Society for Microbiology, Washington, D.C., Poster no. C‐70.
   Clinical and Laboratory Standards Institute. 2009a. Performance standards for antimicrobial susceptibility testing, 19th information supplement. M100‐S19. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania.
   Clinical and Laboratory Standards Institute. 2009b. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard, 8th edition. M7‐A8. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania.
   Higgins, D.L., Chang, R., Debabov, D.V., Leung, J., Wu, T., Krause, K.M., Sandvik, E., Hubbard, J.M., Kaniga, K., Schmidt, D.E., Gao, Q., Cass, R.T., Karr, D.E., Benton, B.M., and Humphrey, P.P. 2005. Telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity in methicillin‐resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 49:1127‐1134.
   Kaito, C., Kurokawa, K., Hossain, M.S., Akimitsu, N., and Sekimizu, K. 2002. Isolation and characterization of temperature‐sensitive mutants of the Staphylococcus aureus dnaC gene. FEMS Microbiol. Lett. 210:157‐164.
   Kreiswirth, B.N., Lofdahl, M.S., Betley, M.J., O'Reilly, M., Schlievert, P.M., Bergdoll, M.S., and Novick, R.P. 1983. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305:709‐712.
   Krogstad, D. and Moellering, R. Jr. 1986. Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 2nd ed. (V. Lorian, ed.) pp. 432‐492. Williams and Wilkins, Baltimore, Maryland.
   McKay, G.A., Beaulieu, S., Arhin, F.F., Belley, A., Sarmiento, I., Parr, T.R. Jr., and Moeck, G. 2009. Time‐kill kinetics of oritavancin and comparator agents against Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. J. Antimicrob. Chemother. 63:1191‐1199.
   McKay, G.A., Fadhil, I., Beaulieu, S., Ciblat, S., Rafai, Far A., Moeck, G., and Parr, T.R. Jr. 2006. Oritavancin disrupts transmembrane potential and membrane integrity concomitantly with cell killing in Staphylococcus aureus and vancomycin‐resistant Enterococci. 46th annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, CA. September 27‐30, 2006. Poster no. C1‐682.
   National Committee on Clinical Laboratory Standards. 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline. NCCLS document M26‐A [ISBN 1‐56238‐384‐1]. NCCLS, Wayne, Pennsylvania.
   Reynolds, P.E. 1989. Structure, biochemistry and mechanism of action of glycopeptide antibiotics. Eur. J. Clin. Microbiol. Infect Dis. 8:943‐950.
   Rybak, M.J. 2006. The pharmacokinetic and pharmacodynamic properties of vancomycin. Clin. Infect. Dis. 42:S35‐S39.
   Silverman, J.A., Perlmutter, N.G., and Shapiro, H.M. 2003. Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob. Agents Chemother. 47:2538‐2544.
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