1 Biofilms 101 B B i i o o f f i i l l m m s s M M a a d d e e E E a a s s y y : : “ “ A A P P i i c c t t u u r r e e T T u u t t o o r r i i a a l l ” ” Understanding the Impact of Microbiology, “Focusing” on Biofilms. John G. Thomas, PhD, Sara B. Posey, MPH, and Staff. WVU School of Medicine Department of Pathology Biofilm Research Laboratory for Translational Studies Morgantown, WV 26506-9203
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Human Source Bioburden Ratio DiversityBacteroides blackandwhitesCampylobacter correctusDialister dualisteEubacterium euphemismiumFusobacterium frustratingiaGemella gyraticaVeillonella variabellaXylanella fastidiosaZymonoas mobilis
YOUR FRIENDSYOUR FRIENDS
The human body is a continuum of microbes, although there are 4 recognized
reservoirs that contain the highest concentrations and continually provide
resident microbiota.
7
““We Live in a Microbial World: Head to ToeWe Live in a Microbial World: Head to Toe””
““The distinction between dental and medical The distinction between dental and medical microbiology is a manmicrobiology is a man--made fabrication via our made fabrication via our simplistic attitude of a very complex total body simplistic attitude of a very complex total body ecosystem that is just now being uncoveredecosystem that is just now being uncovered””
There is no such thing as dental microbiologyThere is no such thing as dental microbiology
Biofilm Research LaboratoryWest Virginia University The concept that stratification and separation occurs should be forever lost.
Center for Biofilm ResearchCenter for Biofilm Research
Planktonic Phenotype
Diseases&
Symptoms
Sessile or BiofilmPhenotype
SurvivalAntibiotic Resistance
ACUTEOrganism Mediated
CHRONICImmunologic mediated
PPBFBFPPPP
Microbiology and DiseaseMicrobiology and Disease
Transmission
RATIO PBF:PP
Microbes exist in 2 distinct life forms or phenotypes; 1 for survival, biofilms
and the alternate life form, planktonic, for transmission.
8
The majority of prokaryotes are The majority of prokaryotes are unculturableunculturable
0.3Soil
0.25Sediments
1-15Activated sludge
0.1-3Estuarine waters
0.1-1Mesotrophic lakes
0.25Freshwater
0.001-0.1Seawater
Cultured (%)Habitat
Numbers based on direct cell counts.
Daims, H. University of Vienna. Department of Microbial Ecology.
“Viable, but non-cultivable”VBNC
Biofilm Research LaboratoryWest Virginia University
Out of the millions of microbes that inhabit the human body and the earth,
most are uncultivable and represent viable but nonculturable classifications
4. Protozoa4. Protozoa: most >2 µm- 2 mm; eukaryotic; uni-cellular; non-photosynthetic; flexible cell membrane; no cell wall; wide range of sizes and shapes; hardy cysts (flagellates, amoebae, ciliates, sporozoans, microsporidia)
5. Algae5. Algae: Photosynthetic, Rigid cell wall, Wide range of sizes and shapes 2 micrometers and larger
Types of Microorganisms
The classifications and types of organisms observable under a microscope
are usually listed as 5: Viruses, bacteria, fungi, protozoa and algae.
10
VIRUS 0.1 um
BACTERIUM
1 X 2 UM
PROTOZOAN PARASITE
Cryptosopridium parvum
5 microns
The Relative Size of MicrobesThe Relative Size of Microbes
Relative comparison of viruses, bacteria and parasites: The largest of the
microorganisms is a mold estimated at over 30 miles long in upper Michigan.
Prokaryotic (bacteria) vs. Eukaryotic CellsProkaryotic (bacteria) vs. Eukaryotic Cells
Bacteria versus human cells or prokaryotic versus eukaryotic cells. They
share many features but bacteria have a cell wall. Note: Although fungi and
yeast are described as microorganisms, they are a eukaryote possessing no
cell wall.
11
Bacterial Cell Wall
The bacterial cell wall can be complex or simple, and the constituents help
describe 2 primary shapes within the microbial world; gram-positive cocci and
gram-negative rods. Positive and negative refers to the retention of dyes
used to stain the organisms to make them visual. Gram-positive blue, gram-
negative red.
Microorganisms of the Oral CavityMicroorganisms of the Oral Cavity
• Bacteria (>700 different groups)
• Fungi (primarily Candida spp)
• Mycoplasma (wall-less bacteria)
• Protozoa
• Viruses (acellular, replicate within living cells)
The diversity of microbes in the oral cavity with 1 additional class of isolates
called mycoplasma, often referred to as CWD or cell wall deficient bacteria.
12
• The "normal microflora flora" colonize the oral cavity, skin, gastrointestinal tract, upper respiratory tract and parts of the genitourinary tract (urethra and vagina)
• Colonization by normal flora can be neutral or beneficial. Harmful outcomes are also possible; can lead to disease and invasion of other parts of the body (opportunistic pathogens)
‘‘Normal MicrofloraNormal Microflora’’
“Normal flora” is a misused word and today better described as beneficial
flora.
THE BENEFITS OF THE NORMAL FLORATHE BENEFITS OF THE NORMAL FLORA
•The normal flora synthesize and excrete vitamins (B-vitamins, Lactobacilli and Streptococci)•The normal flora prevents colonization by pathogens •The normal flora may antagonize other bacteria •The normal flora can stimulate the development of certain tissues•The normal flora can stimulate the production of cross-reactive antibodies
The benefits of “normal flora” or “beneficial flora” are significant. New anti-
infectives should not reduce the benefits of normal flora.
13
TerminologyTerminology
‘Commensal’ microorganisms: frequently isolated from the human body, not associated with disease
‘Pathogen’: frequently cause human infection when present in the body
‘Opportunistic pathogen’: commensal that cause infection under certain conditions
The terms Commensal, Pathogen and Opportunistic need to be clearly
highlighted.
Oral health is an equilibrium between endogenous bacteria and the oral defense system.
Acquisition of oral flora depends upon exposure at the time of birth, cesarean
or vaginal. Shortly thereafter, caregivers provide the next insult of
microorganisms.
Acquisition of the Oral Flora Acquisition of the Oral Flora (Later)(Later)
After tooth eruption: organismsAfter tooth eruption: organismsfavoring hard tissue favoring hard tissue e.g.e.g. Strep. sanguis Strep. sanguis and and Strep. mutansStrep. mutans, , ActinomycesActinomyces spp.spp.
Colonisation of crevicular tissues: anaerobic Colonisation of crevicular tissues: anaerobic organisms organisms e.g.e.g. PrevotellaPrevotella spp.spp.
Loss of teeth: Loss of teeth: ““ a 2nd childhood microfloraa 2nd childhood microflora””
Prosthetic appliance: similar to enamel Prosthetic appliance: similar to enamel plaque, may harbor large numbers of yeastplaque, may harbor large numbers of yeast
Organisms acquired reflect the surfaces for which organisms can adhere, and
there is a dramatic shift with the acquisition of enamel or teeth abiotic
surfaces.
15
Factors Affecting the Oral EcosystemFactors Affecting the Oral Ecosystem
There are a number of factors affecting the oral ecosystem. Please note:
Approximately 70% of patients are on medications over-the-counter (OTC)
that influence the oral ecosystem dramatically.
1. 108 bacteria/day are shed in saliva 2. Plaque bacteria comprise 5% of the salivary
flora 3. >300 species can be isolated from dental
plaque4. 1 mg of dental plaque contains about 106
bacteria 5. The flora of clinically healthy gingiva is
composed mainly of aerobic and facultative anaerobic bacteria
Bacteria in the Oral CavityBacteria in the Oral Cavity
The number of organisms and their interface with each other is significant,
although the oral cavity has the least number of organisms in the 4 primary
reservoirs.
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‘Unculturable’ Bacteria in the Mouth
It has been estimated only about 0.4% of all bacteria have been identified. It is also known that >40% of the bacteria present in the oral cavity and 99% in the environment are unculturable.
VBNC (viable but not culturable) describes the fact that most scientists
believe less than 0.5 of the microbial world has been cultured or is culturable.
Part of this may be due to biofilm.
PlaqueSpecies Saliva Tongue Supra- subgingival
S. salivarius H HS. sanguis M M H LS. milleri R R L-H NS. mutans R-L R L-H NLactobacilli R-L L L-H LActinomyces L L M R-MFusobacterium N N R R-MCapnocytophaga N N R R-LTreponema N N R R-HB. melaninogenicus N N R R-LP. gingivalis N N R N-LA.a N N R N-LVeillonella L L M M
H, high numbers; M, moderate; L, low; R, rare; N, usually absent
Distribution of Oral Bacteria
A list of the prokaryotic organisms often described as gram-positive or gram-
negative.
17
Candida spp.Genus comprised of 150 species with ~8 recognized as opportunistic pathogens of humans
C. albicans C.tropicalisC. krusei C.parapsilosisC.guilliermondii C.glabrataC. kefyr C.dubliniensisC. lisitaniae
Carriage rate ~65% of healthy individuals
A list of common yeast.
Caries65.6% of WV Children have
caries by age 8Perio$50.6 Billion/yr
Arteriosclerosis$5 Billion/yr
Low Birth Weight babies
$2 Billion/yr ThrushStrep. Throat$600 million
VAP$26 Billion/year$150,000/patient
Infective Endocarditis
$48,000/patient
Otitis Media$5 Billion/yr
ORAL BIOFILMSREFERNCES:1. NIH: Disease-specific estimates of direct and indirect costs of illness and NIH support.
http://ospp.od.nih.gov/ecostudies/COIreportweb.htm.2. Shah NP, et al. Direct medical costs associated with using vancomycin in methicillin-resistant staph. aurues
infections: an economic model.3. Kollef MH, et al. Epidemiology and outcomes of health-care associated pneumonia: results from a large US
database of culture-positive pneumonia. Chesi 2005.
Biofilm Research LaboratoryWest Virginia University Consequences of oral flora imbalance and systemic disease.
18
IV. Micro 201: “We Live in a Microbial (Biofilm) World”
Biofilm PrincipleBiofilm Principle
Biofilm Research LaboratoryWest Virginia University
Being Attached Rather than Suspended,Makes a World of Difference
““INTELLECTUAL DESIGNINTELLECTUAL DESIGN””
Given a choice,99.9% of bacteria
will form a BIOFILM
The fundamental of biofilms is that being attached rather than being
suspended is a mechanism of survival. The unique 3-dimensional
organization has recently been referred to as “intellectual design.”
Abiotic Enamel
CellsBioticTissue
+
-
420
410
O2
H2
14
1
EhScale
pHScale
+420
-410
SURFACEO2 Gradient
POCKET
14
1
BIOFILM
COMPONENTS& 3-D
ARCHITECTURE
PHYSIOLOGYGRADIENTS
METABOLISM
PHYSICAL PROPERTIESVISCOELASTIC
HYDRATED-POLYMER MATERIALS
3 COMPONENTS OF BIOFILMS
There are 3 components of biofilms; its architecture, its physiologic gradients
and its physical properties.
19
Nutrient Energy, Resource
BIOTIC Substratum (Sloughing)Mechanical Factors and Shear Forces
The Physico-Chemical Environment
THE BIOFILM COMMUNITYSTRUCTURE AND EVOLUTION
Species colonization, community structure
Cyclic Stage
(Anti-Infective) Hostile Forces Genotypic Factors
Biofilm Research LaboratoryWest Virginia University
8-FACTORS THAT DEFINE BIOFILM DEVELOPMENT and ARCHITECTURE
2. Chronic Wound Model
There are 8 features that define the architecture and physical features; 3 are
critical - surface, organism and stress.
Syne
rgy
Antagonism
C. albicans and PseudomonasC. albicans and Pseudomonas
C. albicans and C. albicans and CoNSCoNS
Strep. mutans and C. albicansStrep. mutans and C. albicans
C. albicans and C. C. albicans and C. glabrataglabrata
CROSS-TALK/CO-ADHESIONGrowth Stimulation
AN
TI-FOO
D C
HA
ING
rowth Inhibition
Biofilm Research LaboratoryWest Virginia University
Staph. aureus and PseudomonasStaph. aureus and Pseudomonas
Organisms that compose the biofilm in mixed species may be synergistic,
antagonistic or simply cooperative.
20
Definition: Biofilm
• A primitive type of developmental biology in which spatial organization of the cells within the matrix optimizes the utilization of the nutritional resources available
• An immobilized enzyme system in which the milieu and the enzyme activities are constantly changing and evolving to appropriate steady state.
• The steady state can be radically altered by applying physical factors such as high sheer.
Biofilm Research LaboratoryWest Virginia University The biofilm is a developmental biology, primitive although focused on
enzymes and resistance to steady states.
Component of Biofilms
• Component % of Matrix– Water - Up to 97%– Microbial Cells - 2-5% (many species)– Polysaccharide - 1-2% (neutral & Polyanionic)
• Low bioburden• Low virulence• High antibiotic concentration
3&4 Inside ET Lumen• Sessile Life-Form; 3D (Co-Habitation)• Significant Shear Force• Cyclical community• Limited immunological environment• High bioburden• High virulence; new phenotype• Low antibiotic concentration
Patho-Physiology of VAP and Significance of Lumenal Growth
Biofilm Research LaboratoryWest Virginia University The environment faced by microbes on the outside of the endotrach versus
the inside is one reason the biofilm survived so readily in the lumen.
Micro scopic in vitroTo
MACRO scopic in vivo
AIR-WAY RESISTANCE
Biofilm Research LaboratoryWest Virginia University
31
Occlusions: A Significant Consequence of Biofilm Architecture
Biofilm Research LaboratoryWest Virginia University Resistance associated with biofilms called colonization resistance where
minimal inhibitory concentration planktonic is less than minimal biofilm
elimination concentration (biofilm).
38
VI. Clinical Cases & Pictures (SEM’s)
1. 2.
3.
• Biofilms are everywhere, as they are the preferred means of survival.
Oral/Dental Plaque Biofilm and Gingivitis
1.
• Plaque is the prominent biofilm example, linked to over $92 million in
diseases per year.
39
Advanced PeriodontitisAdvanced Periodontitis
• Not that uncommon a presentation in dental clinics in Appalachia and the
reason for the Surgeon Generals 2000 Report on Oral Care Accessibility
and Inequality.
The Cradle of Rhinosinusitis SEM’s
Biofilms and the Nasal and Paranasal Sinus Epithelium
Biofilm in Chronic Rhinosinusitis Patient
Biofilm Research LaboratoryWest Virginia University • Oral Flora “reservoir” now include the sinus tracts which “ping-pong’s” with
oral microbes.
40
The Growing Use of IMDs• The last few years have witnessed
an explosive growth in the use of IMDs including simple and complexes devices.
• Simple (catheters and stents): - Catheters: In the United
States 200 million catheters of all types are used annually.
- Coronary stent: (percutaneouscoronary intervention procedures) increased from 9,933 (2.7%) in 1991 to 28,133 (79%) in 1999.
- Hundreds of thousands of implantations are performed each year in dental practice (a fraction of the number of synthetic material implanted into humans in all fields of medicine).
Biofilm Research LaboratoryWest Virginia University
2.
• An aging population demands support.
The Growing Use of IMDsComplex:
- The frequency of use of the automatic implantable cardio-defibrillator (ICD) has increased more than 100-fold since it was first approved about 15 years ago.
- Today over 100,000 hip and 150,000 knee replacements are performed in the United States annually.
- Gold M (2000) Cardiology Clinic, 18:375-389- Utah Hip and Knee Center (2002) History of total joint
replacement
Biofilm Research LaboratoryWest Virginia University • IMDs are more and more difficult to treat once in place and require very
expensive removal once infected by a biofilm.
41
Staph. aureus
Culture Negative Biofilm Phenotype: ExamplesCulture Negative Biofilm Phenotype: Examples““Viable, But NonViable, But Non--CultableCultable”” (VBNC)(VBNC)
Biofilm Research LaboratoryWest Virginia University • Two cases, associated with biofilms, resulting in death at WVUH. Both
were VBNC.
NICU Patient with Multiple Line Sepsis
Biofilm Research LaboratoryWest Virginia University
• The prefect “We live in a Microbial World,” and care givers providing a
mixture of organisms.
42
Biofilm Research LaboratoryWest Virginia University • Catheters and ETT’s: the protected environment from host defenses
Line Sepsis Endoluminal Brush
Biofilm Research LaboratoryWest Virginia University
• A lumenal brushing of a line associated with “Line Sepsis,” organisms
going from attached to free floating, and then attached…metastasis.
43
A Superficial Wound
• Wounds are a growing problem with an aging population, but the
importance of biofilms and pathogenicity are controversial.
16 year old with Dystrophic EpidermolysesBullosa (DEB). A group of heritable
mechano-bullous skin diseases.
Wounds: Soft TissueWounds: Soft Tissue
Pseudomonas and Staph. aureusBiofilm Research LaboratoryWest Virginia University
• Chronic wounds are NOT only seen in adults; this lad died at age 16 with
sepsis related to skin biofilm.
44
Acute Wound SEM
James Wound Repair and Regeneration Vol 16, 2008 • Wounds are colonized with biofilms, usually multiple organisms (>8)
Chronic Wound SEM
James Wound Repair and Regeneration Vol 16, 2008
• Wound biofilms reflect with organism community and the stress/nutrient
(pH) availability, which determine the 3-D structure.
PIP (cm of H2O) 15-27 20 28-44 30 >35 40FLOWRATE (litres per min or
LPM) <55 50 55-65 60 >65 80
REQUIRED PARAMETERS A (NORMAL) B (COPD) C (ARDS)
10 >=15 15PEEP (cm of H2O) 4.5-7 5 8-15.
From 50 patient study
ZONE X
Biofilm Research LaboratoryWest Virginia University Calculations by the School of Engineering in concert with respiratory therapy
for establishing 3 ventilator profiles.
49
Center for Biofilm ResearchCenter for Biofilm Research
Unsteady Pipe FlowUnsteady shear stress prediction (Case B)
(7 mm dia) (Note: Tau reverses direction during exhalation)
-200
-150
-100
-50
0
50
100
150
200
0 0.5 1 1.5 2 2.5 3
Time, sec
She
ar s
tres
s, P
aTau smooth
Tau rough
Dynamics evaluated by School of Engineering for COPD in 7-mm endotrach.
50
VII. Rules: Do’s and Don’ts
• Don’t select for overgrowth.
• Don’t select for antibiotic resistance.
• Don’t destroy normal flora.
• Be green.
• Be reusable.
• Be nontoxic.
• Save mixed flora.
51
IX. Glossary/Terms Commensal microorganisms: frequently isolated from the human body, not associated with disease Pathogen: frequently cause human infection when present in the body Opportunistic pathogen: commensal microorganisms that cause infection under certain conditions Normal microflora flora (beneficial flora): colonize the oral cavity, skin, gastrointestinal tract, upper respiratory tract and parts of the genitourinary tract (urethra and vagina) Colonization: by normal flora can be neutral or beneficial. Harmful outcomes are also possible; can lead to disease and invasion of other parts of the body (opportunistic pathogens) VBNC (Viable But Not Culturable): the 99.9% of bacteria in the environment and >40% in oral cavity which cannot be cultured by traditional methods; may be partially due to biofilm. Reynolds units: a unit of Dynamic Viscosity used to measure stress, in this case, in a biofilm. Greater than 5,000 is considered significant in biofilm structure.
52
X. Key References and websites…
WVU-CovidienData Bank
West Virginia University John G. Thomas PhD and Staff
Data from 9 years of Collaborative Research, including:1) Quantitative Imaging
2) Microbial Cultures3) Anti-infectives
4) Others-UNDER CONSTRUCTION-
• Micro Mini Educational Series:
http://www.hsc.wvu.edu/som/pathology/thomas/micromini.asp • Coming Soon! The 19th and 20th Annual John G. Thomas Microbiology
Symposium Lecture Series. Available at: SOLE.wvu.edu. For information about access: 304-239-1584.
• Center for Biofilm Engineering: http://www.erc.montana.edu/
Thomas JG, Posey SP. Emergence of Oral/Dental Microbiology. ADVANCE for
Administrators of the Laboratory. June 2009;18(6):35-38. Thomas JG, Posey SP, Namsupak A. Probiotics: The Link Between Beneficial
Oral Bacteria and Total Health. Sherman Oaks, CA. Health Pointe Press; 2009.
Thomas JG, Nakaishi L, Corum L. Ch 14. Consequences of Biofilms on
Indwelling Medical Devices: Cost and Prevention. In: Manivannan G, ed. Disinfection and Decontamination: Principles, Applications, and Related Issues. Boca Raton, FL: CRC Press; 2008:289-338.
Thomas JG, Litton I, Rinde H. Ch 2. Economic Impact of Biofilms on Treatment