biofilms for everyone booklet

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Biofilms 101

BBiiooffiillmmss MMaaddee EEaassyy:: ““AA PPiiccttuurree TTuuttoorriiaall””

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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Biofilms Made Easy: “A Picture Tutorial”

Contents: Page:

I. Objectives & Goals……………………………………… 3

II. Introduction/Background: Biofilms…………………….. 4

III. Micro 101………………………………………………… 5

Universal Principles

General Principles

IV. Biofilms 201: “We Live in a Microbial World”………… 18

V. Clinical Consequences…………………………………. 30

VI. Clinical Cases & Pictures (SEM’s)……………………. 38

VII. Biofilm Models (in vitro/in vivo)………………………… 46

VIII. Rules: “Do’s and Don’ts”……………………………….. 50

IX. Glossary/Terms………………………………………….. 51

X. Key References/Web sites/ etc………………………... 52

XI. FAQ’s…………………………………………………...… 54

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I. Goals & Objectives:

Objectives:

• To provide an understanding of microbiology, generally, and biofilms,

specifically, upon design strategy.

In order to do this, we have selected approximately 60 PowerPoint slides

from over 1,500 used in 100 presentations by me, internationally, since

2001, which highlight significant principals and features of biofilms.

• Many of these originated from experimentation utilizing the VEL

(Ventilator-Endotrach-Lung) Model (now called Simulator), which helped

evaluate the efficacy of silver-coated endotracheal tubes, and subsequent

studies of sutures, ETT suction devices and chronic wounds.

• This educational tool also compliments our Micro Mini Educational Series,

and additional support are available on our WVU website:

http://www.hsc.wvu.edu/som/pathology/thomas/

Goals:

• To provide useful understanding of how biofilms are constructed, their

architecture and their “hydrated polymer-like” features.

• To unmask the consequences and collateral damage associated with

biofilms, specifically IMDs (Indwelling Medical Devices)

• To develop rules (Do’s and Don’ts) for design strategy in the evolving

“green” microbial ecology and probiotics era.

• The PowerPoints are arranged by five (V) Sections, and key points are

highlighted underneath each PowerPoint.

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II. Introduction/Background: Biofilms

• Engineers were first to describe problems associated with microbial

communities and filtration devices (water, sand, etc.) in mid-1980’s

• Earliest literature (1980’s) appeared in Engineering journals

• Engineers “coined” the name “biofilm” and “slime bacteria”

• Now, biofilm evolution, description and importance highlighted in medical/

scientific literature (1995 Present)

• Biofilms are the preferred means of microbial survival and growth and are

a global concerns associated with infinite number of problems:

engineering, medical, environmental, and Global Warming (below).

Center for Biofilm ResearchCenter for Biofilm Research

In the beginning, engineers were the first to recognize the importance of

biofilms in altering or reducing the effectiveness of selected devices. Although

biofilms are the preeminent form of microbes, their impact on medical and

dental applications did not occur until approximately the 1980s.

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III. Micro 101 UNIVERSAL MICROBIAL PRINCIPLES

Principle 1. Being Attached (Biofilm) rather than Suspended (Planktonic) makes

a world of difference, and given the opportunity, 99.9% of

microorganisms prefer attachment vs. free floating.

Principle 2. Sessile and planktonic life forms are not mutually exclusive,

existing simultaneously, and demonstrate the same growth cycle: I

(Lag), II (Log), III (Stationary), and IV (Death).

Principle 3. Shear Forces and Stress Patterns will affect the physical

morphology and dynamic behavior of the biofilm more than the

seven other influences.

Principle 4. Biofilm Structure = Function: multi-species biofilms are formed in

cascade fashion, Gram-Positive (Early) to Gram-Negative (Late),

bridged by Candida spp. (Universal Engineer or Co-Aggregate),

developing a more complex, resilient ecosystem with niches

(Evolution/Intellectual Design).

Principle 5. A Mixed Species Biofilm is more stable, robust, and cohesive than

Monospecies.

Principle 6. Infectious Diseases can be quantified where:

Infectious Disease = Number(N)1 x Virulence(V)2 Immunity(I)3

Principle 7. Ecological (Plaque) Hypothesis: Resident flora is distinct in 1)

health and 2) disease, where potential pathogens may be present

in low numbers; a major ecological shift is necessary for putative

pathogens to “out compete” resident flora, and achieve numerical

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dominance needed for disease. Critical Colonization or Synergistic

Threshold: Balanced flora between Biofilm and Planktonic

phenotypes is critical, recognizing the emerging role of targeted

synergistic isolates, particularly Candida albicans, Strep mutans,

and P. aeruginosa and the ratio of each towards a critical threshold

in oral or chronic wounds, respectively.

Principle 8. Biofilms share eight properties of another multi-cellular 3-D

community – Neoplasia – and resemble benign procaryotic, solid

tumors, which explain in part their resistance to standard antibiotic

interventions and limited success with anti-tumor drugs, particularly

those of anti-fungal legacy.

4 NF Reservoirs of Bacteria4 NF Reservoirs of BacteriaAn Interactive ContinuumAn Interactive Continuum

• GIT 1011 1000:1 200• Urogenital 108 100:1 200 • Mouth 106 10:1 700• Skin 106 1:1 50

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.

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““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.


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.

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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

(VBNC).

1= mono/ poly/ eucaryotic/ procaryotic2= planktonic/ sessile3= local/ systemic

Infectious = Number(N)1 x Virulence2

Disease Process Immunity3

Universal Microbial PRINCIPLEUniversal Microbial PRINCIPLEDensity or numbers = Infection

Medical = 0Medical = 0Dentistry = ReducedDentistry = Reduced

Biofilm Research LaboratoryWest Virginia University In infectious diseases, the principle of infections can be simplified into a

formula. Infections = numbers x virulence over the immunity.

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Microorganism: An Organism that Cannot be Seen Without the

Use of a Microscope

Antonie van Leeuwenhoek (1632–1723) was the first to see microorganisms from plaque samples

“animalcules”

The 1st microscope

Microbes were first identified by Antoin Van Leevenhook in 1632 when he

scraped plaque from a tooth surface and called the moving organisms

“animacules.” He was an early optometrist.

1. Viruses1. Viruses: smallest (0.02-0.3 µm diameter); simplest: nucleic acid & protein coat (& lipoprotein envelope)

2. Bacteria2. Bacteria: 0.5-2.0 µm diameter; prokaryotes; cellular; simple internal organization; binary fission.

3. Fungi3. Fungi: Yeasts and molds, non-photosynthetic, immotile; rigid cell wall

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.

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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.

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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.

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• 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.

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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.

1. Physical barriers (keratinized epithelium, mucous production, salivary flow)

2. Chemical compounds (salivary enzymes and antibacterials, gingival fluid secretions

3. Inflammatory reaction

Host interaction with microbes

There is a dynamic interface between the host human cells and those of

microbes, and we now understand that quorum sensing is a vehicle of

signaling shared between eukaryotic and prokaryotic cells.

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Acquisition of the Oral Flora Acquisition of the Oral Flora (Early)(Early)

Birth: SterileHours: Streptococcus spp.1 Year Streptococci, staphylococci

Neisseria, VeillonellaActinomyces, LactobacilliFusobacterium

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.

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Factors Affecting the Oral EcosystemFactors Affecting the Oral Ecosystem

AnatomicalSalivaryCrevicular fluidMicrobial

adherencebacteriocinsmetabolic productscompetition

Miscellaneous

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.

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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.

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IV. Micro 201: “We Live in a Microbial (Biofilm) World”

Biofilm PrincipleBiofilm Principle


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.

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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


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


Staph. aureus and PseudomonasStaph. aureus and Pseudomonas

Organisms that compose the biofilm in mixed species may be synergistic,

antagonistic or simply cooperative.

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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)

– Proteins - <1-2%– DNA & RNA - <1-2%– Ions - ?– HOST Fibrin, RBCs, WBCs

Biofilm Research LaboratoryWest Virginia University A major defense mechanism of the biofilm is that it is up to 97% a diluent.

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Abiotic Enamel

CellsBioticTissue

+

-

420

410

O2

H2

14

1

EhScale

pHScale

+420

-410

SURFACEO2 Gradient

POCKET

14

1

A biofilm’s 3-dimensional architecture allows for gradients, often pH

dependent, allowing pH regions to exist from pH 11 to pH 3.

Biofilms act asBiofilms act asHydrated Polymers Hydrated Polymers

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KEY PROPERTY OF A KEY PROPERTY OF A BIOFILMBIOFILM

Acts as Hydrogel(Extremely hydrated polymer gel)

EXHIBTSVISCOELASTIC – material that has both elastic (solid)

and viscus (liquid-like) propertiesCONSEQUENCES

Seconds – absorbs increased shear by behaving elasticallyLong periods – shear is dissipated through viscus flow

(no detachment) or streamlined to reduce drag

Hydrated PolymerHydrated PolymerNOT MicrobesNOT Microbes

Biofilm Research LaboratoryWest Virginia University The most unrecognized property is that the consequence of microbes existing

as a biofilm is that they respond as hydrated polymers, taking on physical

parameters of those materials.

MICROBIAL COMMUNITIES: Properties of which are greater than the sum of

component species

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33 MultiMulti--LayeredLayeredDilutions of AntibioticsDilutions of Antibiotics

Organization of microbes is intellectually established and represents

multilayers effectively diluting out the activity of many antimicrobials.

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Biofilm Research LaboratoryWest Virginia University The complex interaction of “Early and Late” shifting from a gram-positive

cocci to a gram-negative rod is extremely well choreographed.


Materials Concept Hypothesis• A biofilm is much like an uncured

rubber stock, or plastic in the melt in that it is structured as follows:

Polymer Mixture:

• Matrix: raw SBR or natural rubber

• Plasticizer: extenders, oils

• Fillers: carbon black, anti-oxidants, lubricants, pigments, etc.

Biofilm Mixture:

• Matrix: Glycocalyx from microbes

• Plasticizer: water

• Fillers: planktonic microbes, microbial colonies, hyphae

The structure of a biofilm is influenced by stress, measured in Reynolds units.

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Effects of Force on a Polymer Mixture

• Viscoelastic Properties– Viscous energy dissipation

• Deformation• Heat• Measurement: Viscosity

– Elastic energy dissipation• Oscillates until molecular friction

dissipates energy • Heat• Measurement: Elastic Modulus

• Most polymer mixtures have a compliment of both forms i.e. some viscous and some elastic response to force

• Viscoelasticity characterizes both components• Viscosity and Modulus of Elasticity are functions of both temperature and shear rate

The effect of force on the hydrated polymer can be significant in transmitting

aggregates of the organism pool.

The Society of Rheologyπαντα ρει : “Panta Rei”

Everything Flows

Greek Philosopher Heraclitus (540-480BC) of Ephesus:

“Everything flows and nothing abides; everything gives way and

nothing stays fixed.”

Will It Flow or Fracture?

Biofilm Research LaboratoryWest Virginia University Biofilms do flow.

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4 Stages of Plaque Biofilm Growth:I Attachment (Lag), II Growth (Log), III Maturity (Stationary), IV Dispersal (Death)

I-A I-BLAG

IILOG

IIISTATIONARY

IV-A IV-BDEATH

STOP HEREPROMOTE

HERE

STAGE

MICROBIAL STAGE CYCLE

RATIO1. ORGANISMS2. PHENOTYPE

Complex CommunityComplex Community


Intra Oral Sessile (PBF) and planktonic (PP) life forms are not mutually exclusive, but biofilms are the preferred growth vehicle

The life cycle of a free-floating planktonic or biofilm phenotype is

characterized by Lag, Log, Stationary and Death. It is cyclical and repetitive,

as rapid as 18 minutes and as slow as 48 hours.

A BIOTIC SUBSTRATUMStrep. mutans @ 24hrs in poloxamer

Stage II: Early & ImmatureThickness 25 µm

“Golfball” shape

Microcolonies

Loose association

Limited co-aggregation

Early biofilm configuration (or “pioneers”) is often spherical where the building

block is “microcolonies,” usually Gram-Positive Organisms.

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A BIOTIC SUBSTRATUMP. gingivalis @ 24hrs in poloxamer

Stage II: Synergy & Cooperation

Thickness 40 µm

Spiral

Linked communities

Substratum

Gram-Negative Organisms (or Late Stage Colonizers) often form a spiral,

rising from the base to the environment.

A BIOTIC SUBSTRATUMP. gingivalis & S. mutans @ 72hrs in poloxamer

Stage III: Complex and Diverse

Thickness 55 µm

Domains

Diverse Community

Co-aggregation

Quorum Sensing

Domain 1

Domain 2

A combination on an abiotic surface of Gram-positive and Gram-negative is

unique.

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Images provided by Convatec

A BIOTIC SUBSTRATUMA BIOTIC SUBSTRATUM

In vitro analyses of gram positive organism B using poloxamer

Stage I (microcolonies); Early & Immature

Microcolony

Organization pattern

Spatial Arrangement

Complexity Heterogen

Biomass

Quantification

Biofilm Thickness

Biovolume

Substratum Coverage

Thickness 250 µm

2211Open & Open & LooslyLoosly spacedspaced

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44

UnstableUnstable

3D imaging of microcolonies forming early biofilm community.


In vitro analyses of gram positive organism A using poloxamer

Stage I-II: Moderate

Thickness 250 µm


Microcolony


Spatial Arrangement


Biomass

Quantification

Biofilm Thickness

Biovolume

Substratum Coverage

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Community Community Function: Function: Synergy, Synergy,

CoaggregationCoaggregation11

pH 5pH 5

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pH 11pH 11

Robust AssociationRobust Association

3D image of intermediate coalescing biofilm.

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In vitro analyses of gram positive organism A + B using poloxamer

Stage III


Microcolony


Spatial Arrangement


Biomass

Thickness 250 µm

Quantification

Biofilm Thickness

Biovolume

Substratum Coverage

11

CrosstalkCrosstalk

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CrosstalkCrosstalk

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Stable Integration of Function Stable Integration of Function & Increased Bio& Increased Bio--Diversity with Diversity with

Focused Marker OrganismsFocused Marker Organisms

3D imaging of defined and well-developed biofilm multispecies.



In vitro analyses of gram positive organism B using poloxamer under increased stress, Stage III-IV: Late/ Apoptosis or Necrosis

Microcolony


Spatial Arrangement


Biomass

Quantification

Biofilm Thickness

Biovolume

Substratum Coverage Thickness

250 µm

TopTop

AttachmentAttachmentChannelsChannels

ApoptosisApoptosis

Domain IDomain I

Domain IIDomain II

Well-defined mature biofilm architecture.

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BIOTIC SUBSTRATUMBIOTIC SUBSTRATUM3D anaglyph3D anaglyph

Malic Malic et alet al(2006)(2006)

Microcolony


Spatial Arrangement


Biomass

Thickness 250 µm

Quantification

Biofilm Thickness

Biovolume

Substratum Coverage

C. albicansC. albicans (Yeast)(Yeast)

HyphaeHyphae

Vertical Vertical Orientation of Orientation of HyphaeHyphae

AttachmentAttachment

3D architecture of Candida albicans, a biphasic organism growing in biotic

substrata.

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V. Clinical Consequences

1&2 Outside ET• Planktonic Life-Form; 2D only•• Zero shear stress forcesZero shear stress forces• Significant immunological environment

• 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


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Occlusions: A Significant Consequence of Biofilm Architecture

OIL FLOW (Turbulent)OIL FLOW (Turbulent)1. 50 microns (5% diameter

reduction) reduces oil flow by 33% in a 2 cm pipe.

2. 33% curve of a pipe causes an additional 17%.

3. Total = 50% reduction

PICC LINESPICC LINES1. Occlusion is a significant problem.

- 40% due to internal thrombus formation

2. Resulting CRS is associated with increased morbidity/mortality, increased cost and increased LOS.

ETTETT1. Occlusion is a significant problem2. WOB Feasibility Study

Resistance attributable to biofilms in the form of reduced flow is universal.

Airway Resistance

• Increased airway resistance known to delay vent weaning and increase length of intubation–– Hypothesis:Hypothesis: biofilm prevention

reduces attachment of secretions and thus prevents airway resistance buildup

–– MetricMetric: pressure drop measurements i.e. smaller effective diameters produce higher pressures

Biofilm

Mucus Attached

ET Tube InnerLumen Wall

Biofilm Research LaboratoryWest Virginia University In the lumen of the endotrach, the hypothesis for increased airway resistance

is the adhesion of cellular components in pertinacious materials to the biofilm

which acts as a bridge to the surface of the endotrach.

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Extubated EndotrachsIn Vivo Analysis78 Patient tubes

Patient A: Half section Patient A: Close-up

PTS-2000Pressure DropSystem

PHASE I

Biofilm Research LaboratoryWest Virginia University Extubated endotrach in accumulated accretion in spite of suctioning and

nurse intervention at the time of extubation.

Biofilm Research LaboratoryWest Virginia University Data from the pressure-drop study showing reproducibility of the clean

endotrach versus unrecognized consequences of biofilm and accretion

buildup.

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3-D Computational Fluid Dynamics (CFD)Analysis of ET Tube Stage IV Dispersal Via Trachea

ORAL FLORA

GI FLORA

LUNG FLORA

Endotracheal Tube End

Lung End

Hypothetical drawing established by WVU Engineering in airflow dynamics in

carrying metastasizing fragments to the lung from the endotrach.


Stage IV. Degradation is probably the most important stage in pathogenesis of VAP

In stage IV analysis of metastasizing fragments and recognition that 1-5

micron-size aggregates could integrate into the alveolar spaces of the lung.

34


Overview of methods for biofilms to compete with single cells in survival and

dispersal.


Pathologic consequences in VAP, recognizing aggregates of 1-5 microns in

the alveolar space.

35

ACUTE CHRONIC

PP PB

Biofilm Research LaboratoryWest Virginia University Shift in ratio from planktonic to biofilm phenotype is associated with more

organisms of resistant biofilm.

Collateral Damage: Two Pathways to Patient DiseaseKEY: (HOST) Airway Resistance MACRO

PARTNERINGInfection(VAP)

MICRO

Device

MACROcolonies

Occlusion:Accretion Build-up

IncreasesAirway

Resistance

1. Pressure Drop2. CT/VITAL

Endotrach Tube

3 MillionICU 100%

3-6% REORGANIZTION: REORGANIZTION: MICRO VS. MACROMICRO VS. MACRO

INFECTION

“LAVALIKE”

MICROMICROcolonies

Dispersal of Stage IV Biofilm aggregates

Device

30 MillionSurgery

MICROcolonies

PRINCIPLES OFRHEOLOGY

BIOFILM

BIOFILM

LINKLINK

VIA VIA

ADHERENCE/

ADHERENCE/

INFECTION

INFECTION

ANTIANTI--KOCH

KOCH’’SS

POSTULATES

POSTULATES

(VAP)


Collateral damage, the significance of biofilm in 3 features: VAP, airway

resistance and loss of function.

36

DISEASE:DISEASE:

MICROBE:MICROBE:

THERAPY:THERAPY:

MICROBIOLOGY:MICROBIOLOGY:

ParPar··aa··digm Shift: digm Shift:

MEDICALMEDICAL

Acute Infection

One(Koch’s Postulates)

Eradicate

Pure Culture

DENTALDENTAL

ChronicInfection

Ecosystem(Plaque Hypothesis)

Maintain N.F.

Direct MIC

Marsh, PD. 2005. J. Clin. Perio. 33S:7.

Medical diseases with planktonic isolates need zero organism population in

marked contrast to biofilm diseases where normal flora may be of benefit.

1940 19901950 1960 1970 1980 2000

PCN ®S. aureus

PCNSulpha

AugmentedPCN

β-lactamasein Hemophilus

Aminoglycoside

Aminoglycoside®

Vancomycin

MRSA

Cephalosporinsunge

Quirolones

MRD-Tb

MRD-TyphoidMulti ® GNR

VRE

PCN ®Pneumonococci

GNRVRSA

‘Anxiety Threshold’

Incr

easi

ng A

ntib

iotic

Res

ista

nce

Antibiotic Resistance of Planktonic OrganismsAntibiotic Resistance of Planktonic Organisms

E. faecalis(resistant)

Doxycycline(Stable)

Biofilm Research LaboratoryWest Virginia University The increased antibiotic resistance paralleling the use of new antibiotics.

37

Colonization ResistanceColonization ResistanceMIC:MBEC Using Calgary DeviceMIC:MBEC Using Calgary Device

0100200300400500600700800900

1000

MIC MBEC

CefazolinCloxacillinImipenemVancomycinClindamycinCeftazidime

Planktonic vs. Sessile

Ratio May be Important Chemically: MICMBEC

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).


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


• 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


• 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.

45

www.mbec.ca

Leaching Processin Mines Create Stress

Co-Biofilm: Bacteria and Fungi in Ventilation

Systems of Airplanes

Environmental Biofilms Environmental Biofilms SEM’s


3.

• The environment and abiotic surfaces are the perfect conditions for

biofilms, constructed from the 99.9% of microbes we have never

recovered or identified.

Ref: www.mbec.ca

Agricultural BiofilmsAgricultural Biofilms


• Biofilms will continue to cause problems in the things we like.

46

VII. Biofilm Models (in vitro/in vivo)


Organisms Commonly forming Biofilms Arranged by IMD

DISEASEPOTENTIAL

PATHOGENSTRANSPORT

DEVICES STORAGEREJECTIONCRITERIA

Blood Stream Infections

(BSI)

Blood CultureBottles

RoomTemperature

Urinary TractInfections

(UTI)

If delay to thelaboratory of>2 hours,refrigerate

LowerRespiratory

Tract Infections

(LRI)

GI Infections Rrefrigerate(especially C. difficile)

Sterile ContainerEnteric Pathogen Vial

Rectal swab (not for C. difficile)

Salmonella spp.Shigella spp.Campylobacter spp.Escherichia coli (STEC)Clostridium difficile toxinStaphylococcus aureusYersinia enterocolyticaVibrio spp.

Staphylococcus aureusStreptococcus pneumoniaePseudomonas aeruginosaKlebsiella pneumoniaeEnterobacteriacaeHaemophilus influenzaeCandida spp.Legionella spp.Bordetella pertussis

Escherichia coliEnterococcus spp.Staphylococcus aureusKlebsiella spp.Proteus spp.Pseudomonas aeruginosaCandida spp.other bacteria

Staphylococcus aureusCoagulase Negative StaphEnterobacteriacaePseudomonas aeruginosaStreptococcus pneumoniae*Streptococcus spp.Candida spp.Enterococcus spp. other bacteria

Over or under–inoculatedNo skin prep performedLine draws**Single draws

Unrefrigerated >2 hrsReceived >24 hours after collectionFecal contaminationNo work up on cultures with >2 isolates

Gram stain showing contamination with saliva (>10 squamous pithelials/10X)

Quality scores provide consistency of reporting

Contaminated with urineFormalin-fixed

Sterile container (except B. pertussis)

B. pertussis-Regan Lowe

transportor “Cough”plate

using Regan Lowe agar

Sterile urine containerorUrine Culture Tube

Models used for studying biofilms are few and not very representative of

environment.

47

Respiratory simulation… The VEL Model


Dental Cocktail

• ••

•

•

•

•

•

•

••

•

Complex Biofilm Development in a Closed Ventilator-Endotrach-Lung (VEL) Model

Shown is 1 of 6 Stations Simulating a Multi-patient Intensive Care Unit

ZoneY

ZoneX

ZoneZ

VAP Cocktail

VEL model simulator, created as a result of a 50 patient ICU study, was

based on replicating the stress of a mechanically ventilated patient.

48

Ag CoatingTYCO Healthcare

Culture/Non-culture

MicroscopySHEAR FORCES

ZONE Y

Biofilm Research LaboratoryWest Virginia University Sheer forces were the most important and demanded analysis of the

endotrach in 3 zones; A, B and C.

VENT PROFILES

RANGE VALUE RANGE VALUE RANGE VALUETIDAL VOLUME (ml) >750 800 750-650 700 <650 600

RESPIRATORY RATE (breaths/min 10-15 12 16-25 20 >25 35FIO2 SET (%) 35-45 40 46-65 50 66-100 75

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


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

Costs. In: Pace JL, Rupp ME, Finch RG, eds. Biofilms, Infections, and Antimicrobial Therapy. Boca Raton, FL: CRC Press; 2006: 21-38.

http://www.hsc.wvu.edu/som/pathology/thomas/micromini.asp�

http://www.erc.montana.edu/�

53

Thomas JG, Posey SP. Biofilms. In: APIC Text of Infection Control and

Epidemiology - 3rd Edition. Washington, DC: APIC; 2009. Wilson A, Gray D, Thomas JG. Increases in Endotracheal Tube Resistance are

Independent of Duration of Intubation. CHEST. 2009. Accepted for Publication.

Costerton JW. Biofilm Theory Can Guide the Treatment of Device-Related

Orthopaedic Infections. Clin Ortho and Related Res. Aug 2005;437:7-11. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of

persistent infections. Science. 1999;284:1318–22. Hall-Stoodley L, Stoodley P. Biofilm formation and dispersal and the transmission

of human pathogens. Trends in Micro. Jan 2005;13(1):7-10. Kite P, Wilcox MH, Dobbins BM. Evaluation of a Novel Endoluminal Brush for the in

situ Diagnosis of Catheter Related Sepsis. J Clin Path. 1997;50:27–82. Kollef MH, Afessa B, Anzueto, et al. Silver-Coated Endotracheal Tubes and

Incidence of Ventilator-Associated Pneumonia; The NASCENT Randomized Trial. JAMA. August 2008;300(7):805-13.

Ramage G, Martinez JP, Lopez-Ribot JL. Candida biofilms on implanted

biomaterials: a clinically significant problems. FEMS Yeast Res. June 2006;6(7):979-86.

Walcott RD, Ehrlich GD. Biofilms and Chronic Infections. JAMA. June 2008;

299(22):2682-84.

54

XI. FAQ’s

1. Which came first, the planktonic free floating microorganism or attached

biofilms?

Biofilms appeared on the Earth 4.5 billion years ago, or 2 billion

years before planktonic isolates, primarily for transmission.

2. Are biofilms easy to culture and recover?

NO. Detection is best recognized by non-culture techniques and

recovery using standard laboratory methods is very poor, probably

less than 10%.

3. What is the most effective means of treatment?

Prevention. Biofilms to not like smooth, dry surfaces with zero

stress – a static environment.

4. What is the most overlooked feature of a biofilm?

pH. Biofilms are gradients of pH “niches” or communities, with

lower pH providing an unrecognized means of protection.

biofilms for everyone booklet

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