Biofilm Synergy: Architects of Survival

1
Biofilm Synergy Architects of Survival
The What
The UN WorkingProf. John G. ThomasWest
Virginia University, Cardiff University and
National University of SingaporeBarcelona
November 14, 2008
IMAGES http//www.tzaddik.us/mtstatic/images/un.g
if http//www.flagscollection.com/boutique/images/
OASCountries1.GIF
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The WHO John G.
Thomas, PhD

• RESEARCH
• Biofilms
• Dental
• Medical
• ICU-ET-VAP
• Vent Model
• Chronic Wound Model
• Antibiotic Resistance
• Electronic Surveillance
• Immunomodulation
• Outcomes
• Rapid Reporting
• Workflow via DRG

• ACADEMIC
• Professor/ SoM, SoD
• Director Micro/Virology, WVUH
• Director
• Oral/Facial Lab,
• WVUH
• Director
• Center for Biofilm Research
• Mentoring,
• Teaching

Hola!
2006 National Ski Patrol
National University of Singapore (NUS)
-Visiting Scholar Cardiff University
-Visiting Professor
Supported by and Consultant to Covidien
(formerly known as Tyco Healthcare)
3
The HOW
Can Oral Flora be Linked to Systemic Diseases,
Particularly HAP and VAP?
4
I. Biofilms We live in a Microbial World
5

PRINCIPLE 1
4 NF Reservoirs of BacteriaAn Interactive
Continuum
Human Source Bioburden Ratio Diversity
Bacteroides blackandwhites Campylobacter
correctus Dialister dualiste Eubacterium
euphemismium Fusobacterium frustratingia Gemella
gyratica Veillonella variabella Xylanella
fastidiosa Zymonoas mobilis

• GIT 1011 10001 200
• Urogenital 108 1001 200
• Mouth/Sinus 106 101 1000
• Skin 106 11 113

Community and Diversity
NIH Human Microbiome Project 2008
6
We live in a (oral) Microbial World Planktonic lt
Biofilm
7
Robert Koch

• The same organism must be present in every case
  of the disease.
• The organism must be isolated from the diseased
  host and grown in pure culture.
• The isolate must cause the disease, when
  inoculated into a healthy, susceptible animal.
• The organism must be re-isolated from the
  inoculated, diseased animal.

FREE FLOATING
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Imaging Techniques for Biofilms
BIOFILM ARCHITECTURE
ANALYTICAL IMAGING

FISH
FF
X
X
X
X
X
X
X
X
SEM Scanning Electron Microscopy FISH
Fluorescent in situ hybridization FF
Freeze Fraction SEM
X-RAY Zinc dust radiography BF Bright
Field
FC Flow Cytometry CLSM Confocal Laser
Scanning Microscopy
9
4 Stages of Plaque Biofilm Growth

I Attachment (Lag), II Growth (Log), III
Maturity (Stationary), IV Dispersal (Death)
MICROBIAL STAGE CYCLE
I-A I-B LAG
II LOG
III STATIONARY
IV-A IV-B DEATH
STAGE

• RATIO
• ORGANISMS
• PHENOTYPE

PROMOTE HERE
STOP HERE
Complex Community
Intra Oral Sessile (PBF) and planktonic (PP) life
forms are not mutually exclusive, but biofilms
are the preferred growth vehicle
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II. Biofilm Structure
11
Principle 2 Structure Equals Function
Optical sectioning
(X,Y,Z)

• Poloxamer F127
• co-polymer poloxyethylene/polyoxypropylene
• thermo-reversible gelation
• acts as a scaffold, supporting bacteria in 3
  dimensions
• biofilm formation due to
• close location of bacteria, co-aggregation, high
  densities

12
A BIOTIC SUBSTRATUMStrep. mutans _at_ 24hrs in
poloxamerStage II Early Immature
Thickness 25 µm Golfball
shape Microcolonies Loose association Limited
co-aggregation
13
A BIOTIC SUBSTRATUMStrep. mutans _at_ 24hrs in
poloxamerStage II Early Immature
Thickness 25 µm Golfball
shape Microcolonies Loose association Limited
co-aggregation
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A BIOTIC SUBSTRATUM P. gingivalis _at_ 24hrs in
poloxamerStage II Synergy Cooperation
Thickness 40 µm Spiral Linked
communities Substratum
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A BIOTIC SUBSTRATUM P. gingivalis _at_ 24hrs in
poloxamerStage II Synergy Cooperation
Thickness 40 µm Spiral Linked
communities Substratum
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A BIOTIC SUBSTRATUM P. gingivalis S. mutans _at_
72hrs in poloxamerStage III
Thickness 55 µm Domains Diverse
Community Co-aggregation Quorum Sensing
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A BIOTIC SUBSTRATUM P. gingivalis S. mutans _at_
72hrs in poloxamerStage III Complex and Diverse
Thickness 55 µm Domains Diverse
Community Co-aggregation Quorum Sensing
Domain 1
Domain 2
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Imaging The computer program COMSTATQuantificat
ion of Biofilm Structures

• Quantitative measurements instead of subjective
  observations
• The first true 3D biofilm quantification program
• Written as a script in MATLAB
• COMSTAT quantifies biofilm image stacks acquired
  by CLSM
• Biofilm thickness
• Biovolume
• Roughness
• Surface to volume ratio
• Substratum coverage
• Number of microcolonies
• Size of microcolonies
• Distribution of diffusion distances
• Fraktal dimensions
• Available from www.im.dtu.dk/comstat

19
A BIOTIC SUBSTRATUM In vitro analyses of Gram
positive organism B using poloxamer Stage I
(microcolonies) animation
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Quantification Biofilm Thickness
Biovolume Substratum Coverage

Thickness 250 µm
Images provided by Convatec
20
A BIOTIC SUBSTRATUM In vitro analyses of Gram
positive organism B using poloxamer Stage I
(microcolonies) Early Immature
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
2
1 Open
Loosly spaced
Quantification Biofilm Thickness Biovolume Subs
tratum Coverage
3
4 Unstable
Thickness 250 µm
Images provided by Convatec
21
A BIOTIC SUBSTRATUM In vitro analyses of Gram
positive organism A using poloxamer Stage I-II
Moderate
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Quantification Biofilm Thickness
Biovolume Substratum Coverage
Thickness 250 µm
Images provided by Convatec
22
A BIOTIC SUBSTRATUM In vitro analyses of Gram
positive organism A using poloxamer Stage I-II
Moderate
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Quantification Biofilm Thickness Biovolume Subs
tratum Coverage
2 Community Function Synergy, Coaggregation
3 pH 11
1 pH 5
Thickness 250 µm
Robust Association
Images provided by Convatec
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A BIOTIC SUBSTRATUM In vitro analyses of Gram
positive organism A B using poloxamer Stage
III Highly Complex
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Quantification Biofilm Thickness
Biovolume Substratum Coverage

Thickness 250 µm
Images provided by Convatec
24
A BIOTIC SUBSTRATUM In vitro analyses of Gram
positive organism A B using poloxamer Stage
III Highly Complex
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Stable Integration of Function Increased
Bio-Diversity with Focused Marker Organisms
1 Crosstalk
Quantification Biofilm Thickness Biovolume Subs
tratum Coverage
2 Crosstalk
Thickness 250 µm
3
Images provided by Convatec
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A BIOTIC SUBSTRATUM In vitro analyses of gram
positive organism B using poloxamer under
increased stress, Stage III-IV Late- Apaptosis
or Necrosis
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Quantification Biofilm Thickness
Biovolume Substratum Coverage
Thickness 250 µm
Images provided by Convatec
26
A BIOTIC SUBSTRATUM In vitro analyses of gram
positive organism B using poloxamer under
increased stress, Stage III-IV Late- Apaptosis
or Necrosis
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Apoptosis
Top
Quantification Biofilm Thickness Biovolume Subs
tratum Coverage
Domain I
Thickness 250 µm
Domain II
Channels
Attachment
Images provided by Convatec
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BIOTIC SUBSTRATUM 3D anaglyph
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Quantification Biofilm Thickness
Biovolume Substratum Coverage
Thickness 250 µm
Malic et al (2006)
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BIOTIC SUBSTRATUM 3D anaglyph
Attachment
Microcolony Organization pattern Spatial
Arrangement Complexity Heterogeneity Biomass
Quantification Biofilm Thickness Biovolume Subs
tratum Coverage
Vertical Orientation of Hyphae
Hyphae
Thickness 250 µm
Malic et al (2006)
C. albicans (Yeast)
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Mixed Species Biofilm is More Stable, Robust and
Cohesive than Monospecies
PRINCIPLE 3
Older, Less Response
Unstable Spatially and Temporally
Stable Spatially and Temporally
Some Stability
More Stability

• Homogeneous
• Very loose association
• Mutualism
• Symbiosis, function

• More robust
• associations

• Some stable
• associations, but
• most organisms
• capable
• of independence

Heterogeneous Development of Stable
associations, Ecto and endo- integration of
multi-species
Cross Talk
Synergy of Selected Isolates
Co-Aggregation
More stable, more heterogeneous
Bio-Diversity stability Survival
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III. VEL Model WVU and Cardiff University ICU
Study
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VEL Model
Biofilm Development in a Three Part Closed
Ventilation Endotrach Lung Model
X
Z
Y
Oral mixture
E. Test Lung Simulator with .1. Fixed
Compliance .2. Adjustable
Compliance . bi-lobed
C. Endotrach Tubing . Undisturbed
..Luminal Biofilm . 30
elevation (IHI)
D. Inoculation .Nebulizer with .Nosocomial
.Organisms .1. Staph. aureus .2.
C. albicans 3. MRSA.
.4. P. aeruginosa .5. S. maltophilia
A. Mechanical Ventilator with ..Adjustable
Parameters ..- Volume
..- Pressure
..- Flow FIO2
B. Inoculation Nebulizer .with
Pool of Oral Organisms .- Strep.
mutans .- P. gingivalis
- L. casei
.- F. nucleatum
Nosocomial mixture
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SYNERGYOral-VAP Co-Biofilm
Biphasic
Early Late
Are Oral and Nosocomial (VAP) Pathogens
Synergistic, demonstrating Co-Aggregation?

• Co-biofilm of
• Biofilm a)
• S. mutans
• Lactobacillus
• F. nucleatum
• Biofilm b)
• S. aureus
• C. albicans
• P. aeruginosa

1
Oral inoculums 48 hrs
2
Nosocomial (VAP) inoculums 48 hrs
33
Comparison of 7.5 Silver ETT to Hi-Lo
Mallinckrodt ETT on Lumenal Oral-VAP Complex
Co-Biofilms
Hi-Lo Mallinckrodt
7.5 Ag Guardian
Oral Mix 3-Species
VAP Mix 3-Species
Co-Biofilm of 6-Species
Co-Biofilm of 6-Species
gt50
gt 50
50
40
Approximate Number of cells/clusters or
micro-colonies/mm2
30
25
25
20
15
10
10
10
10
10
0
0
3
1
1
5
3
2
0
0
0
0
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
Number of Cells
Approximate number of cells adherent to ETTs,
colonized with 3-species Oral and/or VAP
Co-Biofilm after Gram Stains. The oral clinical
isolates included Strep. mutans, L. casei, and F.
nucleatum. The VAP clinical isolates included S.
aureus, C. albicans, and P. aeruginosa.
34
25 Patient Study Cardiff University
Michigan Lung COPD Resistance
7200 Ventilator COPD/ 7 Parameters
In Vitro Ventilator-Endotrach-Lung (VEL)
Model (3 thru 29 Days)
P. aeruginosa
Co-Biofilm Oral-Systemic
C. albicans
1
Strep. mutans
Silver vs. Hi-Lo ETT (Tyco Healthcare)
Molecular Methods FISH Microarray PCR
Concept Cross-talk Synergy
Biofilm 3-D Architecture
Lung Tissue VAP
Mouth/ Tooth
Extubated Endotrach Tube (ICU)
ETT Biofilm is Plaque Like (Oral)
4
3
2
Double Hit Concept Plaque Reservoir in
Stages Early Strep. mutans Late C. albicans
and P. aeruginosa
Co-Biofilm Organsims C. albicans S. mutans P.
aeruginosa
In Vivo (90 patients)
Design By John G. Thomas, PhD Lindsay Nakaishi,
BS WVU
35
25 Patient Cardiff study, extubated ETT
B
A
C

• Culture
• 2. PCR / DGGE
• 
  3. CLSM
  
  
  4. SEM / EA
  5.
  PNA-FISH
• 
  
  6. 18 Clinical Parameters

(A/ANA)
Spiral
Quantification
(16SrRNA)
36
Cardiff Study PCR / DGGE Community Diversity
Denaturant Urea
Formaldehyde
ETTs
DGGE Bands
P. gingivalis S. aureus P. gingivalis S. mutans
of Bands
20 18 14 12 20 35 40 ?
OTUs
Bacteria- 1 1 3 2 4 5
1 3 C. albicans- - - - -
- - -
Culture Results
37
Cardiff Study PCR / DGGE Community Diversity
Denaturant Urea
Formaldehyde
ETTs
DGGE Bands
of Bands
OTUs
20 41 20 38 20 31 40
Bacteria- 3 2 1
2 2 3 1 C.
albicans- - - - -
- -
Culture Results
38
Cardiff Study Sorted by Days Intubated
Late Colonization
Early Colonization
39
Endotrach SEMs from Cardiff Study
Diversity Among the Community Differences Among
the Patients
40
PNA-FISH
Staph. aureus Probe - Cardiff ICU Study
Undisturbed Biofilm Structure
1. Anaglyph composite
A B C
2. Thickness
A
B
C
3. Location Intensity
B
Top
A
C
Bottom
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Principle 4
Bi-Phasic Endotrach Colonization
HIGH
108
Oral / Dental
Microaerophelic Facultative Anaerobes
(-) Eh Anaerobic
Lunemal BIO- BURDEN
Hospital / Nosocomial
() Eh Aerobic
Gram Positive Cocci
Gram Negative Rods
3 Lactobacillus
Gram Positive Cocci
Gram Negative Rods
3 C. albicans
104
LOW
(5 Days)
1 S. mutans
4 S. aureus
2 P. gingivalis
5 P. aeruginosa
TIME
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The Bi-Phasic Origin
Animation Design by Lindsay Nakaishi
ORAL ETT LUNG
VAP
Oral

• ORAL Flora (Saliva) Initial or pioneering
  colonizers (Early)
• Nosocomial Flora (Systemic) Secondary colonizers
  (Late), attach to plaque on teeth, leech slowly

3 C. albicans
Change in Phenotype for VAP Organisms
2 P. gingivalis
Change in Phenotype for Oral Organisms
1 Strep. mutans
1
2
4 S. aureus 5 P. aeruginosa
Nosocomial Organisms
Oral Organisms
Planktonic Phenotype
Planktonic Phenotype
Biofilm Phenotype
Pooling of Oral Planktonic Phenotype
Biofilm Phenotype
Pooling of VAP Planktonic Phenotype
El-Solh AA, et al. Chest 20041261575-1582.
43
Using the latest digital imaging from
Vitrea--Automated Vessel Measurement, ETT tubes
are analyzed using their software for total
luminal volume isolating both the narrowest and
widest points within the lumen
44

Quantitative Analysis Capability of Software
45

Fly-Thru of ET Lumen
4 Hours Minimal Airway Resistance
24 Hours Maximum Airway Resistance
lt10 Decrease in Volume
44 Decrease in Volume
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IV. Ecological Equation
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Universal Principal 5ECOLOGICAL EQUATION

• ORAL ECOLOGICAL HYPOTHESIS EXPANDED
• Balanced oral flora between Biofilm and
  Planktonic phenotypes is critical, recognizing
  the emerging role of targeted synergistic
  isolates, particularly Candida albicans, Strep.
  mutans, Lactobacillus, and P. aeruginosa and the
  ratio of each towards a threshold.

• Critical (Synergistic) Colonization RATIO

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ECOLOGICAL EQUATION
RATIO
(CFUs / ml / cm2)
BALANCED
gt1
Clinical Synergistic Outcomes PHENOTYPE
Biofilm CFUs Planktonic
CFUs
UNBALANCED
Congo Red / Biofilm
lt1
Congo Red- / Planktonic
(CFUs / ml / cm2)
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The Structure of the UN
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Structure of the UN
Structure of a Biofilm
Vs.
Security Council
Strep. mutans Candida albicans Pseudomonas
aeruginosa
Committees
Fuso nucleatum P. gingivalis Lactobacillus casei
Commensals
General Assembly
RATIO
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V. Summary and Conclusion
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Summary/Conclusions

• Significant Systemic Disease originate in an
  unbalanced oral microbiota (Ecological Equation)
  when ratio of targeted synergistic, isolates are
  elevated in a Biofilm Phenotype.
• Consequences of oral biofilm imbalance should
  focus on global antibiotic resistance and the
  recalcitrant 3-D architecture of biofilms and the
  use of probiotics.
• Preventing Hospital-Acquired Pneumonia
  Developing Oral Health Guidelines for Patients on
  Ventilators is an absolute minimum for reducing
  Global VAP in hospitals.
  (Critical Care, 2008)
• Probiotics A Prescription Engineered by Nature
  Prof. John G. Thomas 10/2008

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THE LINK
Oral Care and Nosocomial Infections
aka. Hospital Associated
Oral Care via Probiotics
Nosocomial Infections
Pneumonia
VAP
Use of the probiotic Lactobacillus planatarum
299 to reduce pathogenic bacteria in the
oropharynx of intubated patients randomised
controlled open pilot study
Klarin et al. Critical Care.
200812R136
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We Live in a Microbial World Head to Toe

Gracias y adiós!
The distinction between dental and medical
microbiology is a man-made fabrication via our
simplistic attitude of a very complex total body
ecosystem that is just now being uncovered
There is no such thing as dental microbiology
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Micro Mini Series
A series of 30 minute lectures on Biofilms and
their importance in todays medicine. It is my
hope that these lectures will help shed light on
what a Biofilm is, and the complexity it presents
in todays medical and dental environments.
www.hsc.wvu.edu/som/ pathology/thomas

• Biofilms Architect of Survival.
• 2. Biofilms Architect of Chronic Diseases
  Head to Toe
• A Comprehensive Engineered
  Ventilator Endotrach Lung (VEL)
  Model.
• Diagnostic Microbiology, Pathology, and Biofilms
• Chronic Wound Care Changing Paradigms