title: Spatial and temporal patterns of biocide action against Staphylococcus epidermidis biofilms.
abstract: The dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of Staphylococcus epidermidis was investigated using time-lapse confocal scanning laser microscopy. The technique allowed for the simultaneous imaging of changes in biofilm structure and disruption of cellular membrane integrity through the loss of an unbound fluorophore loaded into bacterial cells prior to antimicrobial challenge. Each of the four antimicrobial agents produced distinct spatial and temporal patterns of fluorescence loss. The antimicrobial action of chlorine was localized around the periphery of biofilm cell clusters. Chlorine was the only antimicrobial agent that caused any biofilm removal. Treatment with the quaternary ammonium compound caused membrane permeabilization that started at the periphery of cell clusters, then migrated steadily inward. A secondary pattern superimposed on the penetration dynamic suggested a subpopulation of less-susceptible cells. These bacteria lost fluorescence much more slowly than the majority of the population. Nisin caused a rapid and uniform loss of green fluorescence from all parts of the biofilm without any removal of biofilm. Glutaraldehyde caused no biofilm removal and also no loss of membrane integrity. Measurements of biocide penetration and action time at the center of cell clusters yielded 46 min for 10 mg liter(-1) chlorine, 21 min for 50 mg liter(-1) chlorine, 25 min for the quaternary ammonium compound, and 4 min for nisin. These results underscore the distinction between biofilm removal and killing and reinforce the critical role of biocide reactivity in determining the rate of biofilm penetration.
authors.affiliation: Center for Biofilm Engineering and Department of Chemical and Biological Engineering, Montana State University-Bozeman, Bozeman, MT 59717-3980, USA.
authors.forename: William M
authors.initials: WM
authors.lastname: Davison
authors.forename: Betsey
authors.initials: B
authors.lastname: Pitts
authors.forename: Philip S
authors.initials: PS
authors.lastname: Stewart
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects*; D002713:Chlorine / Q000494:pharmacology; D005434:Flow Cytometry; D005976:Glutaral / Q000494:pharmacology; D018613:Microscopy, Confocal; D009561:Nisin / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D013212:Staphylococcus epidermidis / Q000187:drug effects* / Q000254:growth & development*
chemical_list: D000890:Anti-Infective Agents; D000644:Quaternary Ammonium Compounds; D009561:Nisin; D002713:Chlorine; D005976:Glutaral
references.citation: J Microbiol Methods. 2003 Aug;54(2):269-76
references.pmid: 12782382
references.citation: Biotechnol Bioeng. 1996 Jan 5;49(1):93-100
references.pmid: 18623558
references.citation: Microbes Infect. 2002 Apr;4(4):481-9
references.pmid: 11932199
references.citation: J Appl Microbiol. 2004;97(2):262-70
references.pmid: 15239692
references.citation: Arch Ophthalmol. 1991 Aug;109(8):1155-7
references.pmid: 1867561
references.citation: Biotechnol Bioeng. 1998 Aug 5;59(3):261-72
references.pmid: 10099336
references.citation: J Appl Microbiol. 2001 Sep;91(3):525-32
references.pmid: 11556920
references.citation: Biocontrol Sci. 2007 Dec;12(4):149-53
references.pmid: 18198721
references.citation: Int Endod J. 2006 Nov;39(11):878-85
references.pmid: 17014526
references.citation: J Med Microbiol. 2008 Aug;57(Pt 8):966-973
references.pmid: 18628497
references.citation: Int J Artif Organs. 1993 Nov;16(11):777-88
references.pmid: 8150524
references.citation: J Antimicrob Chemother. 2008 Nov;62(5):1031-6
references.pmid: 18703525
references.citation: Antimicrob Agents Chemother. 2005 Feb;49(2):728-32
references.pmid: 15673757
references.citation: Microbios. 1996;85(345):223-30
references.pmid: 8699964
references.citation: J Appl Microbiol. 2001 Aug;91(2):248-54
references.pmid: 11473589
references.citation: Appl Environ Microbiol. 2008 Mar;74(6):1869-75
references.pmid: 18223108
references.citation: J Antimicrob Chemother. 2008 Mar;61(3):651-7
references.pmid: 18258612
references.citation: Appl Environ Microbiol. 2000 Feb;66(2):836-8
references.pmid: 10653761
references.citation: Appl Environ Microbiol. 1994 Dec;60(12):4339-44
references.pmid: 7811074
references.citation: Nat Rev Microbiol. 2009 Aug;7(8):555-67
references.pmid: 19609257
references.citation: J Med Microbiol. 2001 Jul;50(7):582-587
references.pmid: 11444767
references.citation: J Microbiol Methods. 2001 Mar 1;44(2):121-9
references.pmid: 11165341
references.citation: J Microbiol Methods. 2007 Aug;70(2):236-44
references.pmid: 17524505
references.citation: Appl Environ Microbiol. 2002 Sep;68(9):4194-200
references.pmid: 12200265
references.citation: Antimicrob Agents Chemother. 2002 May;46(5):1469-74
references.pmid: 11959584
references.citation: Lett Appl Microbiol. 2007 Dec;45(6):652-6
references.pmid: 17944843
references.citation: Appl Microbiol Biotechnol. 2006 Sep;72(2):368-76
references.pmid: 16397769
references.citation: Antimicrob Agents Chemother. 2009 Aug;53(8):3505-7
references.pmid: 19451285
references.citation: J Antimicrob Chemother. 2007 Aug;60(2):417-20
references.pmid: 17586808