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James N. Wilking
Assistant Professor, ChBE
Center for Biofilm Engineering
Montana State University
Center for Biofilm Engineering
CFP Biennial Meeting | 2016
Fundamentals of
Microbial Biofilms
Center for Biofilm Engineering at
Montana State University
2011, CLSM biofilm: K Hunt, MSU-CBE PhD student
wilkinglab.com
Center for Biofilm Engineering Bozeman, Montana
Free-swimming microbes vs. biofilms
Biofilms are all around us
Photos by CBE staff and students unless otherwise noted.
JW Costerton
Extracellular matrix: a hallmark of microbial biofilms
Extracellular matrix: a hallmark of microbial biofilms
Extracellular matrix: a hallmark of microbial biofilms
The extracellular matrix provides the biofilm with mechanical integrity
Biofilms undergo a “life-cycle”
Biofilms undergo a “life-cycle”
Biofilm growth and spreading
Biofilms form complex structures
Biofilms undergo a “life-cycle”
Biofilm detachment
Planktonic bacteria ready to be released!
Biofilms impact industry, environment & health
Center for Biofilm Engineering Industrial Associates
Consumer Products Church & Dwight
Colgate-Palmolive
Johnson & Johnson
Kimberly-Clark
Masco
Procter & Gamble
Reckitt Benckiser
Sherwin Williams Testing Laboratories WuXi AppTec*
Energy ExxonMobil
BP
Specialty Chemicals BASF
BCG Solutions *
Clorox
Dow Corning
Dow Microbial Control
Ecolab/Nalco
NCH Corp.
Novozymes A/S
Sani-Marc
Sample6 Technologies*
Sealed-Air
US Gov’t Programs/Labs NASA
Health Care/Biomedical 3M
Bard Access Systems
Bausch & Lomb
Baxter Healthcare
Bend Research*
Covidien
ICU Medical
Kane Biotech*
KCI
Next Science*
Semprus Biosciences*
Steris
W.L. Gore
Biofilm in oilfield pipeline system
Cooling tower biofilms
Health-related biofilms
Chronic wounds
Ellen Swogger, Garth James et al., MSU-CBE, 2005. Cover image on Wound Repair and Regeneration
16(1), January-February 2008, ISSN 1067-1927, Blackwell Publishing
Elbow prosthesis biofilm
Urinary catheter encrustation
Morris, N.S. and Stickler, D.J. (2001) BJU Int 88:192
Dental caries
Where ever moisture and nutrients are present, there will be biofilms!
Biofilm in a water distribution system
Biofilm in a water distribution system
Why not just eradicate them with antimicrobial agents?
Biofilms are more tolerant of antimicrobial agents than are free bugs
3
2
1
0
Time (min)
0 200 400 600 800
Biofilm
Planktonic Lo
g R
ed
uctio
n
Glutaraldehyde (50 mg/l) against P. aeruginosa
Biofilms are more tolerant of antimicrobial agents than are free bugs
-6-5-4-3-2-1012
0 25 50 75 100Time (h)
Log
(X/X
o)
MSU Center for Biofilm Engineering
control biofilm
planktonic
Tobramycin against P. aeruginosa
Biofilm Tolerance of Antimicrobial Agents Antimicrobial tolerance can be understood in light of three concepts
1. Slow penetration
Measure penetration with microelectrodes
hypochlorite
chloride
Time (sec)0 600 1200 1800 2400 3000 3600
Hydr
ogen
Pero
xide C
once
ntrati
on (m
M)
0
10
20
30
40
50
60
base of biofilm
bulk fluidBulk fluid
Base of biofilm
Time (seconds) Hyd
roge
n Pe
roxi
de C
once
ntra
tion
(mM
)
H2O2 catalase
H2O + 1/2O2
H2O2 fails to penetrate the biofilm
Chloride ions penetrate readily but hypochlorite ions do not
Time (sec) 0 200 400 600 800
Rel
ativ
e Pe
netr
atio
n
0.0
0.2
0.4
0.6
0.8
1.0
OCl-
Cl-
Hypochlorite penetrates biofilm slowly
Depth (microns) 0 200 400 600 800 1000 1200
0
200
400
600
800
1000
1200
5460 s
47 s
1511 s
2. Altered Microenvironment
Oxygen in P. aeruginosa Biofilm
Depth ( m m)
-50 0 50 100 150 200
Oxy
gen
Con
cent
ratio
n (m
g/l)
0
1
2
3
4
5
6
7
sterile control
untreated
GFP Expression in P. aeruginosa Biofilm
DNA Replication Pattern Biofilm
3. Stress Response
Catalase Induction in P. aeruginosa Biofilm
biofilm + H2O2
Time (min)
0 10 20 30 40 50 60
25
30
35
40
45
50
55
60
65
biofilm - untreated
planktonic + H2O2
katB-lacZ
S. epidermidis biofilm staining with Calcein AM green (CAM)
Time: 1 hr
Vel = 6 cm/sec
Imaging antimicrobial action
Imaging antimicrobial action
W. Davison
CAM-stained S. epidermidis biofilm treated with 10 mg/L sodium hypochlorite
Time: 1 hr
Vel = 6 cm/sec
Imaging antimicrobial action
W. Davison
CAM-stained S. epidermidis biofilm treated with 50 mg/L sodium hypochlorite
Time: 1 hr
Vel = 6 cm/sec
Options for Microbial Control
Biofilm control in industrial systems
Materials Compatibility
Antimicrobial access to the biofilm
Regular cleaning and antimicrobial dosing
Contact time
Measuring successful treatment
www.biofilm.montana.edu
Contents of these slides were provided by faculty, staff, and students in the Center for Biofilm Engineering at Montana State University
Thank you for your attention!
