In Vitro Antibacterial Resistance Selection and Quantitation

Katherine Young1

1 Merck Research Laboratories, Rahway, New Jersey
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13A.6
DOI:  10.1002/0471141755.ph13a06s34
Online Posting Date:  October, 2006
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Abstract

The study of resistance is vital to the discovery and development of new antibacterial agents. Consider the case where the resistance frequency (the proportion of cells within a population that exhibit a resistance phenotype to the agent under study) of an agent is such that, in an average infection, a mutant resistant to the agent is likely to already exist. This compound will not be useful in the clinic as a single agent, since this mutant can survive its administration, leading to potential clinical failure. This unit provides protocols for determining the frequency and rate of resistance to antibacterial agents, as well as generating resistant mutants for mechanism of action determinations or for use in antibacterial discovery or development programs.

Keywords: growth curve; agar‐dilution MIC; resistance frequency; resistance rate; fluctuation test

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Determination of Growth Rate for use in Fluctuation Test
  • Basic Protocol 2: Agar Dilution to Determine Minimum Inhibitory Concentrations
  • Basic Protocol 3: Resistance Frequency Determination
  • Basic Protocol 4: Luria and Delbruck Fluctuation Test
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Growth Rate for use in Fluctuation Test

  Materials
  • Bacterial strain to be used in growth curve experiment
  • Appropriate agar medium (see recipe)
  • Appropriate liquid medium
  • Inoculating loops, disposable (plastic) or reusable
  • Shaking water bath or incubator
  • Dilution tubes: 16 × 100–mm glass tubes with metal closures or 17 × 100–mm, 14‐ml polypropylene round‐bottom tubes
  • Microtiter plate, optional
  • Microtiter plate reader with wavelength of 600 nm (Molecular Devices or equivalent), optional
  • Spin plater and reusable (glass) or disposable (plastic) spreaders, or 4‐mm‐diameter Pyrex beads (Fisher Scientific)

Basic Protocol 2: Agar Dilution to Determine Minimum Inhibitory Concentrations

  Materials
  • Bacterial strain to be used in agar‐dilution MIC
  • Appropriate agar medium, prepoured (see recipe) and in component form
  • Appropriate liquid medium
  • Antibacterial compound under study
  • Solvent (e.g., ethanol, methanol, DMSO)
  • Inoculating loops, disposable (plastic) or reusable
  • Shaking water bath or incubator
  • 45° to 50°C water bath
  • 50‐ml conical disposable (plastic) tubes or 50‐ to 100‐ml reusable (glass) bottles
  • 15 × 100–mm disposable (plastic) or reusable (glass) petri dishes

Basic Protocol 3: Resistance Frequency Determination

  Materials
  • Bacterial strain that will be used in resistance rate determination/resistance selection
  • Appropriate agar medium, prepoured (see recipe) and in component form
  • Appropriate liquid medium
  • Antibacterial compound under study
  • Appropriate solvent (e.g., DMSO, ethanol, methanol)
  • Inoculating loops, disposable (plastic) or reusable
  • Shaking water bath or incubator
  • Water bath set at 45° to 50°C
  • 500‐ml glass bottle
  • 15 × 100–mm disposable (plastic) or reusable (glass) petri dishes
  • Spin plater and disposable (plastic) or reusable (glass) spreaders, or 4‐mm‐diameter Pyrex beads (Fisher Scientific)

Basic Protocol 4: Luria and Delbruck Fluctuation Test

  Materials
  • Bacterial strain
  • Appropriate agar medium (see recipe)
  • Appropriate liquid medium
  • Spin plater and reusable (glass) or disposable (plastic) spreaders, or 4‐mm‐diameter Pyrex beads (Fisher Scientific)
  • Inoculating loops, disposable (plastic) or reusable
  • Shaking water bath or incubator
  • 50‐ml, conical, disposable (plastic) tubes or 50‐ to 100‐ml reusable (glass) bottles
  • 14‐ml glass or plastic tubes with caps
  • Spin plater and reusable (glass) or disposable (plastic) spreaders, or 4‐mm‐diameter Pyrex beads (Fisher Scientific)
  • Additional reagents and equipment for preparing counting plates and dilution tubes (see protocol 1), preparing agar plates infused with antibacterial compound (see protocol 2), and determining the resistance frequency (e.g., protocol 3)
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Figures

Videos

Literature Cited

   Difco Manual 1998. Difco Laboratories, Sparks, Maryland.
   Drlica, K. 2001. A strategy for fighting antibiotic resistance. ASM News 67:27‐33.
   Fujimoto‐Nakamura, M., Ito, H., Oyamada, Y., and Nishino, T. 2005. Accumulation of mutations in both gyrB and pare genes is associated with high‐level resistance to novobiocin in Staphylococcus aureus. Antimicrob. Agents Chemother. 49:3810‐3815.
   Griggs, D.J., Marona, H., and Piddock, L.J.V. 2003. Selection of moxifloxacin‐resistant Staphylococcus aureus compared with five other quinolones. J. Antimicrob. Chemother. 51:1403‐1407.
   Ince, D. and Hooper, D.C. 2000. Mechanisms and frequency of resistance to premafloxacin in Staphylococcus aureus: Novel mutations suggest novel drug‐target interactions. Antimicrob. Agents Chemother. 44:3344‐3350.
   Ince, D., Zhang, X., Silver, L.C., and Hooper, D.C. 2003. Topoisomerase targeting with and resistance to gemifloxacin in Staphylococcus aureus. Antimicrob. Agents Chemother. 47:274‐282.
   Kunkel, T.A. and Erie, D.A. 2005. DNA mismatch repair. Annu. Rev. Biochem. 74:681‐710.
   Kutterer, K.M., Davis, J.M., Singh, G., Yang, Y., Hu, W., Severin, A., Rasmussen, B.A., Krishnamurthy, G., Failli, A., and Katz, A.H. 2005. 4‐Alkyl and 4,4′‐dialkyl 1,2‐bis(4‐chlorophenyl)pyrazolidine‐3,5‐dione derivatives as new inhibitors of bacterial cell wall biosynthesis. Bioorg. Med. Chem. Lett. 15:2527‐2531.
   Luria, S. and Delbruck, M. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28:491‐511.
   NCCLS 1999. Performance Standards for Antimicrobial Susceptibility Testing, 9th informational supplement Vol. 19 (1). National Committee for Clinical Laboratory Standards Wayne, PA.
   NCCLS 2003. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard‐Sixth Edition M7‐A6. National Committee for Clinical Laboratory Standards. Wayne, PA.
   O'Neill, M.J., Smith, A., Heckelman, P.E., Obenchain, J.R. Jr., Gallipeau, J.A.R., and D'Arecca, M.A. 2001. The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 13th ed. Merck & Co., Inc., Whitehouse Stations, NJ.
   Pages, V., Janel‐Bintz, R., and Fuchs, R.P. 2005. Pol III proofreading activity prevents lesion bypass as evidenced by its molecular signature within E. coli cells. J. Mol. Biol. 352:501‐509.
   Perry, K.L., Elledge, S.J., Mitchell, B.B., Marsh, L., and Walker, G.C. 1985. umuDC and mutAB operons whose products are required for UV light‐ and chemical‐induced mutagenesis: UmuD, MucA, and LexA proteins share homology. Proc. Natl. Acad. Sci. U.S.A. 82:4331‐4335.
   Stent, G.S. 1971. The fluctuation test. In Molecular Genetics; An Introductory Narrative pp. 152‐157. W. H. Freeman and Co., San Francisco, California.
   Tippin, B., Pham, P., and Goodman, M.F. 2004. Error‐prone replication for better or worse. Trends Microbiol. 12:288‐295.
   Werngren, J. and Hoffner, S.E. 2003. Drug‐susceptible Mycobacterium tuberculosis Beijing genotype does not develop mutation‐conferred resistance to rifampin at an elevated rate. J. Clin. Microbiol. 41:1520‐1524.
   Yonezawa, Y., Kawamura, S., Yamato, M., and Nishioka, H. 2001. Mut‐Test to detect substances suppressing spontaneous mutation due to oxidative damage. Mutat. Res. 490:21‐26.
   Yun, H.‐J., Min, Y.‐H., Jo, Y.W., Shim, M.‐J., and Choi, E.‐C. 2005. Increased antibacterial activity of DW286, a novel fluoronaphthyridone antibiotic, against Staphylococcus aureus strains with defined mutations in DNA gyrase and topoisomerase IV. Int. J. Antimicrob. Agents 25:334‐337.
Key References
   Stent, G.S. 1971. See above.
  This book chapter gives a very thorough interpretation of the original fluctuation test carried out by Luria and Delbruck and explains the derivation of the key formula used to determine the rate of resistance.
Internet Resources
   http://www.sci.sdsu.edu/∼smaloy/MicrobialGenetics/problems/mutations/frequency/A
  Web site with problems and solutions in Molecular Genetics, a very useful page for beginners in this field.
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