A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.
The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.
Pierce
Christopher G
CG
Department of Biology and South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, Texas 78249, USA.
Uppuluri
Priya
P
Tristan
Amanda R
AR
Wormley
Floyd L
FL
Mowat
Eilidh
E
Ramage
Gordon
G
Lopez-Ribot
Jose L
JL
D018441:Biofilms / Q000254:growth & development*; D018929:Cell Culture Techniques / Q000379:methods*; D025141:Drug Resistance, Fungal; D005562:Formazans; D005658:Fungi / Q000187:drug effects / Q000254:growth & development*
D005562:Formazans
Arch Oral Biol. 2004 Oct;49(10):789-98
15308423
J Med Microbiol. 2007 Sep;56(Pt 9):1205-1212
17761484
Antimicrob Agents Chemother. 2002 Nov;46(11):3591-6
12384370
Antimicrob Agents Chemother. 1992 Aug;36(8):1619-25
1416843
Antimicrob Agents Chemother. 2006 Mar;50(3):1021-33
16495265
FEMS Microbiol Lett. 2007 Jul;272(2):172-81
17490429
APMIS. 2006 Apr;114(4):298-306
16689830
J Antimicrob Chemother. 2002 Jun;49(6):973-80
12039889
Antimicrob Agents Chemother. 1998 Aug;42(8):2146-9
9687427
Med Mycol. 2007 Jun;45(4):321-46
17510856
J Med Microbiol. 2006 Aug;55(Pt 8):999-1008
16849719
Antimicrob Agents Chemother. 1998 Aug;42(8):1900-5
9687381
Mycopathologia. 2005 Apr;159(3):353-60
15883718
Science. 1999 May 21;284(5418):1318-22
10334980
Antimicrob Agents Chemother. 2002 Nov;46(11):3634-6
12384379
Rev Iberoam Micol. 2008 Mar;25(1):37-40
18338926
Eukaryot Cell. 2004 Apr;3(2):536-45
15075282
J Clin Microbiol. 1998 May;36(5):1450-2
9574729
Antimicrob Agents Chemother. 2001 Sep;45(9):2475-9
11502517
Curr Opin Infect Dis. 2002 Dec;15(6):569-74
12821832
J Clin Microbiol. 2003 Jan;41(1):506-8
12517908
Antimicrob Agents Chemother. 2003 Nov;47(11):3657-9
14576141
J Microbiol Methods. 2001 Sep;46(3):193-208
11438184
Eukaryot Cell. 2005 Apr;4(4):633-8
15821123
Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004 Jul;98(1):53-9
15243471
J Microbiol Methods. 2005 Dec;63(3):287-95
15936097
Mycopathologia. 2006 Jan;161(1):3-10
16389478
Emerg Infect Dis. 2002 Sep;8(9):881-90
12194761
Public Health Rep. 1996 May-Jun;111(3):226-35
8643813
Curr Biol. 2005 Jun 21;15(12):1150-5
15964282
Antimicrob Agents Chemother. 2002 Jun;46(6):1773-80
12019089