Static Biofilm Cultures of Gram‐Positive Pathogens Grown in a Microtiter Format Used for Anti‐Biofilm Drug Discovery

Steven M. Kwasny1, Timothy J. Opperman1

1 Microbiotix, Anti‐Infectives R&D, Worcester, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13A.8
DOI:  10.1002/0471141755.ph13a08s50
Online Posting Date:  September, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

An in vitro assay is presented for culturing staphylococcal biofilms and biofilms of nonmotile Gram‐positive bacteria under static conditions in microtiter assay plates, and for the quantification of biofilm growth, using a simple staining procedure that measures amounts of bacterial cells and extracellular matrix. This basic assay can be adapted readily to study several aspects of biofilm formation, for high‐throughput screening to identify small molecule inhibitors of biofilm formation or biofilm‐defective mutants, and for quantifying the anti‐biofilm activity of biofilm inhibitors. Curr. Protoc. Pharmacol. 50:13A.8.1‐13A.8.23. © 2010 by John Wiley & Sons, Inc.

Keywords: biofilm; microtiter; Gram‐positive

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Basic Assay for Biofilm Formation of Nonmotile Gram‐Positive Bacteria
  • Alternate Protocol 1: Biofilm Formation on Alternative Surfaces, Such as Medical‐Grade Materials
  • Basic Protocol 2: Optimizing Biofilm Formation Assay Conditions
  • Basic Protocol 3: Screening Assay for Mutants or Compounds that Inhibit Biofilm Formation
  • Basic Protocol 4: A Quantitative Assay for Anti‐Biofilm Activity: The Minimal Biofilm Inhibitory Concentration (MBIC) Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Basic Assay for Biofilm Formation of Nonmotile Gram‐Positive Bacteria

  Materials
  • Gram‐positive bacterial strain(s) of interest
  • Sterile solid growth medium: tryptic soy agar (TSA; see recipe)
  • Sterile liquid growth medium: tryptic soy broth (TSB; see recipe)
  • Sterile 80% (v/v) glycerol (autoclaved; store at room temperature)
  • 40% (w/v) glucose (filter sterilize; store at room temperature)
  • Washing solution e.g., deionized H 2O, phosphate‐buffered saline (PBS; see recipe), or 0.9% saline (see recipe)
  • 95% ethanol (optional)
  • Bouin's fixative: dissolve 0.5 g picric acid in 37.5 ml deionized H 2O, then add 12.5 ml 37% formaldehyde and 2.5 ml glacial acetic acid
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.06% crystal violet (see recipe)
  • Sterile inoculating loops, toothpicks, and applicator sticks
  • 37°C bacteriological incubator containing rotary shaker or tube roller
  • Culture tubes (e.g., 16 × 150 mm)
  • 2‐ml cryogenic storage vials vial (e.g., Nalgene, cat. no. 5000‐0020)
  • 96‐well assay plates with lids (Costar 3595; flat bottomed, polystyrene, tissue culture treated, or equivalent)
  • Adhesive foil lids (Costar 6569, optional)
  • Plate washer: e.g., BioTek ELx405 or equivalent (optional)
  • Two 2‐liter beakers (optional)
  • Multichannel pipettor and pipetting reservoirs (optional)
  • Baking oven set at 60°C
  • Microtiter plate reader (Molecular Devices, optional)
  • Camera (optional)

Alternate Protocol 1: Biofilm Formation on Alternative Surfaces, Such as Medical‐Grade Materials

  • Silicone sealant (e.g., Silicone II, GE Sealants and Adhesives)
  • 30% (v/v) acetic acid
  • Nonreinforced, medical‐grade sheeting:
    • Silicone sheeting, 0.03‐in. thick (Cardiovascular Instrument Corp.; http://www.cinco7799.com/)
    • Polyurethane sheeting (Pellethane 55‐D, Specialty Silicone Fabricators)
  • No. 5 (∼1‐cm‐diameter) cork borer
  • 24‐well assay plate (Costar 3526)
  • Forceps

Basic Protocol 2: Optimizing Biofilm Formation Assay Conditions

  Materials
  • Library of small molecules stored in 96‐well plates (MicroSource Discovery Systems, http://www.msdiscovery.com/; ChemBridge Corporation, http://www.chembridge.com/; TimTec LLC, http://www.timtec.net/; ChemDiv Inc., http://us.chemdiv.com/)
  • Library of transposon‐insertion mutants stored in 96‐well plates (Tu Quoc et al., ; Boles et al., ; Xia et al., )
  • Dimethyl sulfoxide (DMSO)
  • 96‐pin replicator (Boekel Scientific, http://www.boekelsci.com/)
  • Additional reagents and equipment for preparing and assaying biofilms ( protocol 1) and optimizing biofilm formation assay conditions ( protocol 3)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 13.A0.1 Streaking bacteria on solid agar to obtain single, well‐isolated colonies. (A) Streaking plates. Plates can be streaked using a standard wire inoculating loop, a disposable plastic loop, a toothpick, or a long “picking stick.” The wire loop can be sterilized in a flame, but it must be cooled by touching the agar surface prior to making contact with bacteria. Single colonies can be isolated by repeated streaking as illustrated in the figure. (B) An example of the results obtained using this method. Single, well‐isolated colonies are indicated.
  •   FigureFigure 13.A0.2 Results of a biofilm assay using the strains listed in Table demonstrates the range of phenotypes of individual strains and the stimulatory effect of glucose. Biofilm cultures were grown in 0.5× TSB supplemented with 0.25% or 1% glucose (gluc) at 37°C for 18 hr. (A) Photographs of two representative rows from each tissue culture‐treated 96‐well assay that was stained with crystal violet. (B) The amount of crystal violet bound was quantified by measuring absorbance at 600 nm and plotting for each strain. Each bar represents the average of eight individual assay wells, and the error bars the standard deviation.
  •   FigureFigure 13.A0.3 Preparing assay plates for biofilm formation on alternative substrates.
  •   FigureFigure 13.A0.4 Optimization of assay conditions. (A) A biofilm growth curve of S. aureus ATCC 35556 grown in 0.5× TSB supplemented with 1% glucose. (B) Biofilms were grown in varying media conditions to identify optimal growth conditions and two methods of biofilm detection compared. Biofilms were grown at 37°C for 18 hr. Biofilms were stained and the amount of crystal violet bound to the bottom of each assay well measured directly as absorbance at 600 nm, or crystal violet was eluted from each well, diluted 1:10, and the absorbance measured at 600 nm. (C) Comparison of absorbance at 600 nm of neat versus a 1:10 dilution of eluted crystal violet demonstrates the linear range of the assay.
  •   FigureFigure 13.A0.5 An example of the screening results obtained using S. epidermidis 18972 and a mock screening plate. (A) A photograph of the crystal violet–stained mock screening plate containing nine compounds that are inhibitors of biofilm formation and two antibiotics. The remainder of the wells in columns 2 to 11 contained DMSO. Columns 1 and 12 contained the 0% INH (DMSO only) and 100% INH (100 µg chloramphenicol/ml; Cm100) controls, respectively. The wells containing anti‐biofilm and antibacterial compounds exhibited significant reductions in the intensity of crystal violet staining. (B) The OD600 values obtained for the planktonic cultures. Compounds that produced ≥40% inhibition of planktonic growth are highlighted in orange. The compound in well number C07, highlighted in blue, produced an OD600 of 2.64, which is significantly higher than those produced by the 0% INH (DMSO‐only) controls. This well contained gentian violet, an intensely colored compound that absorbs light at 600 nm. Beware of colored compounds that absorb at 600 nm, as they can mask antibacterial activity. (C) The OD600 values obtained for the crystal violet–stained biofilm cultures. Compounds that produced ≥80% inhibition of planktonic growth are highlighted in orange. (D) A table listing the compounds used in the mock screening plate, their location on the plate (row, column), and the screening call (HIT or antibacterial). Compounds that produced ≥80% biofilm inhibition and ≤40% planktonic growth inhibition were designated anti‐biofilm “HITS.” Compounds that produced ≥40% planktonic growth inhibition were designated as “Antibacterial” compounds. Seven of the anti‐biofilm compounds used in the mock screening plate were identified previously in a pilot screen using a compound library comprised of 2000 known bioactive chemicals (Spectrum Library; MicroSource Discovery Systems). MBX‐1240 and MBX‐1246 were identified in screen of compounds purchased from Chembridge Corp. (Opperman et al., ).
  •   FigureFigure 13.A0.6 An example of a Minimal Biofilm Inhibitory Concentration (MBIC) assay for eight anti‐biofilm compounds that were tested against S. epidermidis 18972. The assay plate in which biofilm cultures have been stained with crystal violet is shown in the photograph. The concentrations of each compound tested are indicated above the plate, and the compound tested in each row is indicated on the left side of the plate. The MBIC and MIC are defined as the lowest compound concentration that produces a ≥80% inhibition of biofilm growth or planktonic growth, respectively. The compounds used in this assay (Compounds 1 to 8), their chemical names and structures, and the MBIC and MIC values obtained as a result of the assay are also shown. Note that Gentian violet produced an MIC that was equal to the MBIC. Because this compound strongly absorbs light at 600 nm, the antibacterial activity of this compound became apparent only when it was diluted in the dose‐response assay. The compounds used in this assay are commercially available from MicroSource Discovery Systems. Alternatively, three of these compounds (Tomatidine, Actinonin, and Emodin) can be purchased from Sigma‐Aldrich. MBX‐1240 can be purchased from ChemBridge Corp.

Videos

Literature Cited

Literature Cited
   Baba, T., Takeuchi, F., Kuroda, M., Yuzawa, H., Aoki, K., Oguchi, A., Nagai, Y., Iwama, N., Asano, K., Naimi, T., Kuroda, H., Cui, L., Yamamoto, K., and Hiramatsu, K. 2002. Genome and virulence determinants of high virulence community‐acquired MRSA. Lancet 359:1819‐1827.
   Balaban, N., Giacometti, A., Cirioni, O., Gov, Y., Ghiselli, R., Mocchegiani, F., Viticchi, C., Del Prete, M.S., Saba, V., Scalise, G., and Dell'Acqua, G. 2003. Use of the quorum‐sensing inhibitor RNAIII‐inhibiting peptide to prevent biofilm formation in vivo by drug‐resistant Staphylococcus epidermidis. J. Infect. Dis. 187:625‐630.
   Boles, B.R., Thoendel, M., Roth, A.J., and Horswill, A.R. 2010. Identification of genes involved in polysaccharide‐independent Staphylococcus aureus biofilm formation. PLoS ONE 5:e10146.
   Branda, S.S., Vik, S., Friedman, L., and Kolter, R. 2005. Biofilms: The matrix revisited. Trends Microbiol. 13:20‐26.
   Chokr, A., Watier, D., Eleaume, H., Pangon, B., Ghnassia, J.C., Mack, D., and Jabbouri, S. 2006. Correlation between biofilm formation and production of polysaccharide intercellular adhesin in clinical isolates of coagulase‐negative staphylococci. Int. J. Med. Microbiol. 296:381‐388.
   Christensen, G.D., Bisno, A.L., Parisi, J.T., McLaughlin, B., Hester, M.G., and Luther, R.W. 1982. Nosocomial septicemia due to multiply antibiotic‐resistant Staphylococcus epidermidis. Ann. Intern. Med. 96:1‐10.
   Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton, D.M., and Beachey, E.H. 1985. Adherence of coagulase‐negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbiol. 22:996‐1006.
   Christensen, G.D., Baldassarri, L., and Simpson, W.A. 1995. Methods for studying microbial colonization of plastics. Methods Enzymol. 253:477‐500.
   CLSI. 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard, Seventh Ed., USA Vol. M7‐A7, vol. 26 (2). Clinical and Laboratory Standards Institute, Wayne, Pa
   Cramton, S.E., Gerke, C., Schnell, N.F., Nichols, W.W., and Gotz, F. 1999. The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect. Immun. 67:5427‐5433.
   Cramton, S.E., Gerke, C., and Gotz, F. 2001a. In vitro methods to study staphylococcal biofilm formation. Methods Enzymol. 336:239‐255.
   Cramton, S.E., Ulrich, M., Gotz, F., and Doring, G. 2001b. Anaerobic conditions induce expression of polysaccharide intercellular adhesin in Staphylococcus aureus and Staphylococcus epidermidis. Infect. Immun. 69:4079‐4085.
   Darouiche, R.O. 2001. Device‐associated infections: A macroproblem that starts with microadherence. Clin. Infect. Dis. 33:1567‐1572.
   Deighton, M.A., Capstick, J., Domalewski, E., and van Nguyen, T. 2001. Methods for studying biofilms produced by Staphylococcus epidermidis. Methods Enzymol. 336:177‐195.
   Gillaspy, A.F., Hickmon, S.G., Skinner, R.A., Thomas, J.R., Nelson, C.L., and Smeltzer, M.S. 1995. Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infect. Immun. 63:3373‐3380.
   Heilmann, C., Gerke, C., Perdreau‐Remington, F., and Gotz, F. 1996. Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect. Immun. 64:277‐282.
   Iordanescu, S. and Surdeanu, M. 1976. Two restriction and modification systems in Staphylococcus aureus NCTC8325. J. Gen. Microbiol. 96:277‐281.
   Jordal, P.B., Dueholm, M.S., Larsen, P., Petersen, S.V., Enghild, J.J., Christiansen, G., Hojrup, P., Nielsen, P.H., and Otzen, D.E. 2009. Widespread abundance of functional bacterial amyloid in mycolata and other gram‐positive bacteria. Appl. Environ. Microbiol. 75:4101‐110.
   Larsen, P., Nielsen, J.L., Dueholm, M.S., Wetzel, R., Otzen, D., and Nielsen, P.H. 2007. Amyloid adhesins are abundant in natural biofilms. Environ. Microbiol. 9:3077‐3090.
   Mack, D., Siemssen, N., and Laufs, R. 1992. Parallel induction by glucose of adherence and a polysaccharide antigen specific for plastic‐adherent Staphylococcus epidermidis: Evidence for functional relation to intercellular adhesion. Infect. Immun. 60:2048‐2057.
   Mack, D., Nedelmann, M., Krokotsch, A., Schwarzkopf, A., Heesemann, J., and Laufs, R. 1994. Characterization of transposon mutants of biofilm‐producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm production: Genetic identification of a hexosamine‐containing polysaccharide intercellular adhesin. Infect. Immun. 62:3244‐3253.
   Mack, D., Bartscht, K., Fischer, C., Rohde, H., de Grahl, C., Dobinsky, S., Horstkotte, M.A., Kiel, K., and Knobloch, J.K. 2001. Genetic and biochemical analysis of Staphylococcus epidermidis biofilm accumulation. Methods Enzymol. 336:215‐239.
   Merritt, J.H., Kadouri, D.E., and O'Toole, G.A. 2005. Growing and analyzing static biofilms. Curr. Protoc. Microbiol. 00:1B.1.1‐1B.1.17.
   Monzon, M., Oteiza, C., Leiva, J., and Amorena, B. 2001. Synergy of different antibiotic combinations in biofilms of Staphylococcus epidermidis. J. Antimicrob. Chemother. 48:793‐801.
   Novick, R. 1967. Properties of a cryptic high‐frequency transducing phage in Staphylococcus aureus. Virology 33:155‐166.
   O'Gara, J.P. and Humphreys, H. 2001. Staphylococcus epidermidis biofilms: Importance and implications. J. Med. Microbiol. 50:582‐587.
   O'Neill, E., Pozzi, C., Houston, P., Humphreys, H., Robinson, D.A., Loughman, A., Foster, T.J., and O'Gara, J.P. 2008. A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin‐binding proteins, FnBPA and FnBPB. J. Bacteriol. 190:3835‐3850.
   O'Toole, G.A. and Kolter, R. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: A genetic analysis. Mol. Microbiol. 28:449‐461.
   Opperman, T.J., Kwasny, S.M., Williams, J.D., Khan, A.R., Peet, N.P., Moir, D.T., and Bowlin, T.L. 2009. Aryl rhodanines specifically inhibit staphylococcal and enterococcal biofilm formation. Antimicrob. Agents Chemother. 53:4357‐4367.
   Pitts, B., Hamilton, M.A., Zelver, N., and Stewart, P.S. 2003. A microtiter‐plate screening method for biofilm disinfection and removal. J. Microbiol. Methods 54:269‐276.
   Rachid, S., Cho, S., Ohlsen, K., Hacker, J., and Ziebuhr, W. 2000a. Induction of Staphylococcus epidermidis biofilm formation by environmental factors: The possible involvement of the alternative transcription factor sigB. Adv. Exp. Med. Biol. 485:159‐166.
   Rachid, S., Ohlsen, K., Witte, W., Hacker, J., and Ziebuhr, W. 2000b. Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm‐forming Staphylococcus epidermidis. Antimicrob. Agents Chemother. 44:3357‐3363.
   Rice, K.C., Mann, E.E., Endres, J.L., Weiss, E.C., Cassat, J.E., Smeltzer, M.S., and Bayles, K.W. 2007. The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 104:8113‐8118.
   Rohde, H., Knobloch, J.K., Horstkotte, M.A., and Mack, D. 2001. Correlation of biofilm expression types of Staphylococcus epidermidis with polysaccharide intercellular adhesin synthesis: Evidence for involvement of icaADBC genotype‐independent factors. Med. Microbiol. Immunol. 190:105‐112.
   Rupp, M.E., Ulphani, J.S., Fey, P.D., Bartscht, K., and Mack, D. 1999a. Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial‐based infection in a mouse foreign body infection model. Infect. Immun. 67:2627‐2632.
   Rupp, M.E., Ulphani, J.S., Fey, P.D., and Mack, D. 1999b. Characterization of Staphylococcus epidermidis polysaccharide intercellular adhesin/hemagglutinin in the pathogenesis of intravascular catheter‐associated infection in a rat model. Infect. Immun. 67:2656‐2659.
   Rupp, M.E., Fey, P.D., Heilmann, C., and Gotz, F. 2001. Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter‐associated infection in a rat model. J. Infect. Dis. 183:1038‐1042.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainville, N.Y
   Seidl, K., Goerke, C., Wolz, C., Mack, D., Berger‐Bachi, B., and Bischoff, M. 2008. Staphylococcus aureus CcpA affects biofilm formation. Infect. Immun. 76:2044‐2050.
   Shanks, R.M., Donegan, N.P., Graber, M.L., Buckingham, S.E., Zegans, M.E, Cheung, A.L, and O'Toole, G.A. 2005. Heparin stimulates Staphylococcus aureus biofilm formation. Infect. Immun. 73:4596‐4606.
   Tu Quoc, P.H., Genevaux, P., Pajunen, M., Savilahti, H., Georgopoulos, C., Schrenzel, J., and Kelley, W.L. 2007. Isolation and characterization of biofilm formation‐defective mutants of Staphylococcus aureus. Infect. Immun. 75:1079‐1088.
   Wilson, M. 2001. Bacterial biofilms and human disease. Sci. Prog. 84:235‐254.
   Xia, G., Maier, L., Sanchez‐Carballo, P., Li, M., Otto, M., Holst, O., and Peschel, A. 2010. Glycosylation of wall teichoic acid in Staphylococcus aureus by TarM. J. Biol. Chem. 285:13405‐13415.
   Yarwood, J.M., Bartels, D.J., Volper, E.M., and Greenberg, E.P. 2004. Quorum sensing in Staphylococcus aureus biofilms. J. Bacteriol. 186:1838‐1850.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library