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Advantages and Disadvantages Advantages and Disadvantages of Different of Different BiofilmBiofilm ModelsModels
HenkHenk J. BusscherJ. BusscherUniversity Medical Center Groningen and University of GroningenUniversity Medical Center Groningen and University of Groningen
AntoniusAntonius DeusinglaanDeusinglaan 119713 AV Groningen, The Netherlands9713 AV Groningen, The Netherlands
A. Conditioning film formation(“pellicle”)
B. Mass transport
C. Initial adhesion
D. Co-adhesion
E. Anchoring through EPS excretion
F. Growth
Steps in biofilm formationSteps in Steps in biofilmbiofilm formationformation
Slime: The house of biofilm, basis for the protection offered by the biofilm mode of growthagainst predators, the host immune system and antimicrobials.
Photograph: Hans-Curt Flemming
The issues with oro-pharyngeal biofilm formation:
The issues with The issues with orooro--pharyngeal pharyngeal biofilmbiofilm formation:formation:
PREVENTION
REMOVAL
The model questions:The model questions:The model questions:
How far do you want to proceed: A, B, C, D, E, F?
Which strains and how many isolates?
Multiple strains and species at the same time?
Growth medium and substratum choices?
Antimicrobial burden?
Presence of shear and other detachment challenges?
Zig-Zag Talk with no answers:ZigZig--ZagZag Talk with no answers:Talk with no answers:
Chemical plaque control in vivo
Biodeterioraton of silicone rubber by singleand mixed-species oropharyngeal biofilms
Detergent-induced detachment ofinitially adhering single strains
and co-adhering pairs
Whole plaque prevention on Triclosan-coated sutures
SLS-induced detachment ofgrowing single strains
Antimicrobial effects on 2 h old versus 16 h old biofilms of single strains
Influence of co-adhesion sequence onmechanical removal of initially adhering bacteria
Busscher and Van der Mei, Clinical Microbiology Reviews 19(2006)127-141
Biofilm formation most often happens
under shear:
Phenomenon Shear rate (s-1) Reference
Flow of a film over a vertical plate 0.1 (5)
Blinking of the eye 0.35 (14)
Fluid flow in the oral cavity 0.1 – 50 (5)
On teeth, while biting an apple 200 (10)
Urinary cathether 15 (35)
Channels within a biofilm 60-300 (29)
Ship in harbor 50 (1)
Ship ‘navigating’ (turbulent flow) 125000 (1)
Tumbling or pouring 10-100 (5)
Bakker et al., Applied and Environmental Microbiology 69(2003)680-6287
Data analysesData analyses
flowtimeflowtime (seconds) (seconds)0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
0
1
2
3
4
5
6
7
101066 b
act
bact
per
cm
per c
m22
jj00 NNoo of of bactbact..at t at t 88
0
300
600
900
1200
1500
1800
j 0 (c
m-2
s-1
)
blank CR CTC CGC CoT CMC #27 CDAW
A. naeslundii T14V-J1
0
300
600
900
1200
1500
1800
j 0 (c
m-2
s-1
)
blank CR CTC CGC CoT CMC #27 CDAW
S. oralis J22
0
2
4
6
8
10
12
14
16
n 4h (
106 c
m-2
)
blank CR CTC CGC CoT CMC #27 CDAW0
2
4
6
8
10
12
14
16
n 4h (
106 c
m-2
)
blank CR CTC CGC CoT CMC #27 CDAW
A. naeslundii T14V-J1 S. oralis J22
0
20
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80
100
perc
enta
ge d
etac
hmen
t
CR CTC CGC CoT CMC CDAW0
20
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100
perc
enta
ge d
etac
hmen
t
CR CTC CGC CoT CMC CDAW
Dentifrice supernate induced detachment
Van der Mei et al., , J Clin Den 13(2002)44-4
0
10
20
30
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50
1 2 3 4 5 6 7 8 9 10
numbers of bacteria in aggregates
perc
enta
ge a
ggre
gate
dis
trib
utio
n S.oralis J22
S.sanguis PK1889
Initial co-adhesion involves 1 × 106 actinomyces/cm2 and 2.3 × 106 streptococci/cm2
Aggregate size distribution prior to detachment(actinomyces first, then streptococci)
Aggregate size distribution prior to detachmentAggregate size distribution prior to detachment((actinomycesactinomyces first, then streptococci)first, then streptococci)
0
10
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60
CR CTC CGC CoT CDAW
Perc
enta
ge la
rge
aggr
egat
es (%
)
Percentage large aggregates after dentifrice supernateinduced detachment and de novo deposition
Ca
Ca
CaF
CaF
CaF
CaF
Co-adhesion
Fluoride disrupt Ca-bonds,but not other interactions
CaF
CaF
CaF
CaF
SLS
Fluoride disrupt Ca-bonds,SLS breaks other interactions
CaF
CaF
Ca
Ca
Ca
Ca
PO3O PO3
PO3 O PO3
Fluoride prevents formatioof large aggregates inde novo deposition
Pyrophosphate enhancesformation of large aggregatesin de novo deposition
Fluoride and SLS in breaking of coFluoride and SLS in breaking of co--adhesive bondsadhesive bonds Fluoride and pyrophosphate in Fluoride and pyrophosphate in de novode novo depositiondeposition
Rose et al., The role of cation binding in microbial fluoride binding.Caries Research, 30 (1996) 458-464
Busscher et al., J Den 35(2007)294-301
Journal of Dental Research 86(2007)421-425
Sequence of Oral Bacterial Co-adhesion and Non-Contact Brushing
H.C. van der Mei,1 M. Rustema-Abbing,1 G.M. Bruinsma,1 B. Gottenbos,2 and H.J. Busscher.1
1 Department of Biomedical Engineering, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands;
2 Philips Research, Care & Health Applications, Professor Holstlaan 4, 5656 AA Eindhoven, The Netherlands.
Larger co-aggregates and approximately twofold more bacteria are found adhering prior to brushing when streptococci are deposited first.
The sequence streptococci first, followed by actinomes, leaves significantly larger aggregates after brushing than actinomyces first.
Removal of growing co-adhering pairs,is much more difficult than of initially adhering pairs.
Low Load Compression Testing of Biofilms Grown under Flow
Low Load Compression Testing of Biofilms Grown under Flow
A. naeslundi TV14-J1 followed by S. oralis J22
0
100
200
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9Strain
Stre
ss (P
a)
Stress-strain : S. oralis J22 followed by A. naeslundi TV14-J1
0
100
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400
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1000
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9Strain
Stre
ss (P
a)
S. oralis J22 followed by A. naeslundii TV14-J1
An S. oralis J22 base layer results in a stronger biofilm.
Constant Depth Film Fermentor???
An already compressed biofilm???An already compressed biofilm???
Remember Plax: the preRemember Plax: the pre--brushing rinse (0.45% SLS)brushing rinse (0.45% SLS)(But it did not work in vivo!)(But it did not work in vivo!)
Kozlovsky and ZuberyThe efficacy of Plax pre-brushing rinse: a review of the literature
Quintessence International 24(1993) 141-144
4 h adhering S. mutans HG985 65% detachment, 4 w% SLS
after 4 h growth in DMG 27% detachment
after 16 h growth in DMG 0% detachment
Landa et al., Recalcitrance of S. mutans biofilms toward detergent-stimulated detachment.European Journal of Oral Sciences 107 (1999) 263-243
Suci et al., Combined light microscopy and attenuated infra red spectroscopy for integration of biofilm structure,distribution and chemistry at the solid-liquid interface. Appl. Environ. Microbiol 63(1997)4600-4603
sulphate
Little penetration of SLS through a biofilm!
Effects of an 2 min AmF treatment on single species, oral biofilms grown for 2 and 16 h on AmF treated pellicles
S. mutans ATCC700610 S. mutans ATCC700610
S. oralis HM1 S. oralis HM1
16 h biofilm, prior 16 h biofilm, after
Confocal Scanning Laser Microscopy
As an advantage over plate-culturing identifying only viable organisms,CSLM allows distinguishing dead-life bacteria after appropriate staining
Problems of the application of CSLMto intact vs. dispersed biofilms:
- Complete penetration of the stain into the biofilm is difficult.
-A CSLM operator is always biased in the -search for “nice” pictures.
Therewith this microscopic technique becomes highlyobserver-dependent.
0.0E+00
1.0E+07
2.0E+07
3.0E+07
4.0E+07
5.0E+07
S. muta
ns N
SAmF
S. oral
is HM1AmF
S. muta
ns ATCC70
0610
AmFS. s
obrinu
s HG102
5AmF
A. nae
slundii
HM1
AmF
# ba
cter
ia (1
06 c
m-2
)
0
20
40
60
80
100
120
S. muta
ns N
SAmF
S. oral
is HM1AmF
S. muta
ns ATCC70
0610
AmF
S. sobri
nus H
G1025
AmFA. n
aeslu
ndii H
M1AmF
surfa
ce c
over
age
(100
%)
16 h biofilm2 h biofilm
Quantitative analysis of 2 min AmF treatmenton oral biofilms grown for 2 and 16 h on AmF treated pellicles
The artificial throat - an in vitro modelThe artificial throat - an in vitro model
Leunisse et al., The artificial throat: a new method for standardization of in vitro experimentswith tracheo-oesophageal voice prostheses. Acta Otolaryngol, 119 (1999) 604-608. Leunisse et al., The artificial throat: a new method for standardization of in vitro experimentswith tracheo-oesophageal voice prostheses. Acta Otolaryngol, 119 (1999) 604-608.
Experimental protocolExperimental protocol
• Inoculation (day 1-3)– single bacterial or yeast strain, combination
of stains: previously isolated from explantedvoice prostheses
• Perfusion scheme (day 4-7)– phosphate buffered saline: 3 times a day– growth medium: once a day– dietary components, salivary substitutes,
drugs, etc.
• Inoculation (day 1-3)– single bacterial or yeast strain, combination
of stains: previously isolated from explantedvoice prostheses
• Perfusion scheme (day 4-7)– phosphate buffered saline: 3 times a day– growth medium: once a day– dietary components, salivary substitutes,
drugs, etc.
Inoculation by a mixture of yeasts and bacteria:
C. tropicalis GB 9/9C. albicans GBJ 13/4AS. aureus GB 2/1S. epidermidis GB 9/6R. dentocariosa GBJ 52/2B
Inoculation by a mixture of yeasts and bacteria:
C. tropicalis GB 9/9C. albicans GBJ 13/4AS. aureus GB 2/1S. epidermidis GB 9/6R. dentocariosa GBJ 52/2B
Two lengths (5 cm each) of Triclosan-coated and uncoated suture materiawere lowered into the pooled, fresh human whole saliva
until the sutures were totally submerged.
Incubation was done for 4 h in an incubator shakerat 37°C and 60 rpm to simulate saliva flow around the sutures.
Half of all samples were subsequently immersed in 15 ml of CHX and hand shaken for 30 s.
Without Triclosan-coating With Triclosan-coating
– MR + MR – MR + MR
S. mutans 4.76 ± 0.51 0 ± 0a, b, c 5.17 ± 0.42 2.46 ± 0.54a, b
S. mitis / S. salivarius 4.74 ± 0.38 0 ± 0a, b, c 4.87 ± 0.49 1.12 ± 1.19a, b
Lactobacilli 5.08 ± 0.31 0 ± 0a, b, c 5.17 ± 0.64 1.93 ± 1.36a, b
Total number of micro-organisms 6.59 ± 0.13 2.27 ± 1.39a, b 6.77 ± 0.47 3.78 ± 0.82a, b
Bacterial Group
a p < 0.05, compared to the control group –MR, both in absence and presence of a Triclosan-coatingb p < 0.05, compared to the control group with Triclosan-coating and –MRc p < 0.05, compared to the control group with Triclosan-coating and +MR
Number of viable organisms (log-units) in biofilms on sutures
““From whence I concludeFrom whence I concludethat the Vinegar with which I that the Vinegar with which I washtwasht my Teeth,my Teeth,
killkill’’dd only those Animals which were on the outside of the scurf,only those Animals which were on the outside of the scurf,but did not pass thro the whole substance of itbut did not pass thro the whole substance of it””..
A. van Leeuwenhoek, 1684
ThreeThree--hundredhundred yearsyears later, we later, we addressaddress the the samesame questionsquestionswithwith a a millionmillion $ $ CLSMCLSM, , usingusing a a disperseddispersed ex ex vivovivo plaque model.plaque model.
9 h old biofilm 72 h old biofilm
control planktonicorganisms
biofilmorganisms
control planktonicorganisms
biofilmorganisms
CR 47 ± 17 2 ± 1 14 ± 6 49 ± 18 1 ± 0 28 ± 15
CTC 48 ± 14 4 ± 5 14 ± 11 29 ± 17 1 ± 0 14 ± 4
Total 66 ± 17 1 ±1 30 ± 19 48 ± 8 1 ± 0 30 ± 15
CDAW 66 ± 10 2 ± 2 30 ± 17 37 ± 10 1 ± 1 42 ± 12
CHX 55 ± 10 4 ± 3 18 ± 23 44 ± 12 1 ± 0 8 ± 2
treatment
PlanktonicPlanktonic versusversus BiofilmBiofilm effects of effects of dentifrice slurries (% live organisms)dentifrice slurries (% live organisms)
Triclosan
Van der Mei et al., J Clin Perio 33(2006)14-20
Model aspect Advantages Disadvantages
Initial adhesion Relatively simple; allows for fundamentalconclusions
Clinically less relevant
Use of flow chamber Highly relevant Time consuming
Use of selected strains Allows for fundamentalconclusions
One strain is more relevant thantwo;Co-adhesion more relevant thanadhesion;Multi-species biofilms mayshow unique features
Inclusion of growth (time) Yields more strongly adheringbiofilms;Mimicks better antimicrobialresistance;More relevant
Rapidly growing complexity
In terms of relevance, nothing is better than in vivo/ex vivo model.
In terms of fundamental questions,an in vivo model is too complicated.
Thank you for your attention!
(this presentation and references herein can be downloaded from www.bme-umcg.nl)
