Biofilms 101

Biofilms Made Easy: A Picture Tutorial

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

Biofilms Made Easy: A Picture Tutorial

Contents: I. Objectives & Goals II. Introduction/Background: Biofilms.. III. Micro 101 Universal Principles General Principles IV. Biofilms 201: We Live in a Microbial World V. Clinical Consequences. VI. Clinical Cases & Pictures (SEMs). VII. Biofilm Models (in vitro/in vivo) VIII. Rules: Dos and Donts.. IX. Glossary/Terms.. X. Key References/Web sites/ etc... XI. FAQs...

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18 30 38 46 50 51 52 54

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 (Dos and Donts) 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.

II. Introduction/Background: Biofilms Engineers were first to describe problems associated with microbial communities and filtration devices (water, sand, etc.) in mid-1980s Earliest literature (1980s) 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 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.

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

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 Bacteria An Interactive Continuum

Human Source Bioburden GIT Urogenital Mouth Skin 10 10 10 10 Ratio Diversity 1000:1 100:1 10:1 1:1 200 200 700 50
Bacteroides blackandwhites 11 correctus Campylobacter Dialister dualiste 8 euphemismium Eubacterium Fusobacterium frustratingia 6 Gemella gyratica Veillonella variabella 6 Xylanella fastidiosa Zymonoas mobilis

YOUR 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. 6

We Live in a Microbial World: Head to Toe Toe

The distinction between dental and medical microbiology is a manman-made fabrication via our simplistic attitude of a very complex total body ecosystem that is just now being uncovered uncovered There is no such thing as dental microbiology

West Virginia University

Biofilm Research Laboratory

The concept that stratification and separation occurs should be forever lost.

Microbiology and Disease

Transmission Planktonic Phenotype Sessile or Biofilm Phenotype

PP
Diseases & Symptoms

RATIO PBF:PP

PBF

Survival Antibiotic Resistance

ACUTE Organism Mediated

Center for Biofilm Research

CHRONIC Immunologic mediated

Microbes exist in 2 distinct life forms or phenotypes; 1 for survival, biofilms and the alternate life form, planktonic, for transmission.

The majority of prokaryotes are unculturable

Habitat
Seawater

Cultured (%)
0.001-0.1

Viable, but non-cultivable Freshwater 0.25 Mesotrophic lakes 0.1-1 VBNC Estuarine waters 0.1-3
Activated sludge Sediments Soil 1-15 0.25 0.3

Numbers based on direct cell counts.

Daims, H. University of Vienna. Department of Microbial Ecology.

West Virginia University Biofilm Research Laboratory

Out of the millions of microbes that inhabit the human body and the earth, most are uncultivable and represent viable but nonculturable classifications (VBNC).

Universal Microbial PRINCIPLE

Density or numbers = Infection
Infectious = Number(N)1 x Virulence2 Dentistry = Reduced 3 Disease Process Immunity

Medical = 0

1= mono/ poly/ eucaryotic/ procaryotic 2= planktonic/ sessile 3= local/ systemic

West Virginia University Biofilm Research Laboratory

In infectious diseases, the principle of infections can be simplified into a formula. Infections = numbers x virulence over the immunity.

Microorganism:
An Organism that Cannot be Seen Without the Use of a Microscope
Antonie van Leeuwenhoek (16321723) 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.

Types of Microorganisms
1. Viruses: Viruses smallest (0.02-0.3 m diameter); simplest: nucleic acid & protein coat (& lipoprotein envelope) 2. Bacteria: Bacteria 0.5-2.0 m diameter; prokaryotes; cellular; simple internal organization; binary fission. 3. Fungi: Fungi Yeasts and molds, non-photosynthetic, immotile; rigid cell wall 4. Protozoa: Protozoa most >2 m- 2 mm; eukaryotic; unicellular; non-photosynthetic; flexible cell membrane; no cell wall; wide range of sizes and shapes; hardy cysts (flagellates, amoebae, ciliates, sporozoans, microsporidia) 5. Algae: Algae Photosynthetic, Rigid cell wall, Wide range of sizes and shapes 2 micrometers and larger
The classifications and types of organisms observable under a microscope are usually listed as 5: Viruses, bacteria, fungi, protozoa and algae. 9

The Relative Size of Microbes

PROTOZOAN PARASITE
BACTERIUM VIRUS 0.1 um 1 X 2 UM

Cryptosopridium parvum 5 microns

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 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. 10

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, gramnegative red.

Microorganisms 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. 11

Normal Microflora
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 flora is a misused word and today better described as beneficial flora.

THE 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 antiinfectives should not reduce the benefits of normal flora.

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Terminology
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.

Host interaction with microbes

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

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. 13

Acquisition of the Oral Flora (Early)

Birth: Hours: 1 Year Sterile Streptococcus spp. Streptococci, staphylococci Neisseria, Veillonella Actinomyces, Lactobacilli Fusobacterium

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 (Later)

After tooth eruption: organisms favoring hard tissue e.g. Strep. sanguis and Strep. mutans, Actinomyces spp. Colonisation of crevicular tissues: anaerobic organisms e.g. Prevotella spp. Loss of teeth: a 2nd childhood microflora microflora Prosthetic appliance: similar to enamel plaque, 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. 14

Factors Affecting the Oral Ecosystem

Anatomical Salivary Crevicular fluid Microbial adherence bacteriocins metabolic products competition 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.

Bacteria in the Oral Cavity

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 plaque 4. 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
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. 15

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.

Distribution of Oral Bacteria

Species S. salivarius S. sanguis S. milleri S. mutans Lactobacilli Actinomyces Fusobacterium Capnocytophaga Treponema B. melaninogenicus P. gingivalis A.a Veillonella Saliva H M R R-L R-L L N N N N N N L Tongue H M R R L L N N N N N N L Plaque Supra- subgingival

H L-H L-H L-H M R R R R R R M

L N N L R-M R-M R-L R-H R-L N-L N-L M

H, high numbers; M, moderate; L, low; R, rare; N, usually absent

A list of the prokaryotic organisms often described as gram-positive or gramnegative. 16

Candida spp.
Genus comprised of 150 species with ~8 recognized as opportunistic pathogens of humans
C. albicans C. krusei C.guilliermondii C. kefyr C. lisitaniae C.tropicalis C.parapsilosis C.glabrata C.dubliniensis

Carriage rate ~65% of healthy individuals

A list of common yeast.

Caries Perio $50.6 Billion/yr

65.6% of WV Children have caries by age 8

Otitis Media $5 Billion/yr

Arteriosclerosis $5 Billion/yr

ORAL BIOFILMS

Infective Endocarditis $48,000/patient

REFERNCES: Low Birth VAP 1. NIH: Disease-specific estimates of direct and indirect costs of illness and NIH support. Weight http://ospp.od.nih.gov/ecostudies/COIreportweb.htm. $26 Billion/year 2. Shahbabies NP, et al. Direct medical costs associated with using vancomycin in methicillin-resistant staph. aurues $150,000/patient infections: an economic model. Thrushassociated pneumonia: results from a large US $2 Billion/yr 3. Kollef MH, et al. Epidemiology and outcomes of health-care database of culture-positive pneumonia. Chesi 2005. Throat Strep.

$600 million

West Virginia University

Biofilm Research Laboratory

Consequences of oral flora imbalance and systemic disease.

17

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

Biofilm Principle Being Attached Rather than Suspended, Makes a World of Difference
Given a choice, 99.9% of bacteria will form a BIOFILM

INTELLECTUAL DESIGN
West Virginia University Biofilm Research Laboratory

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.

3 COMPONENTS OF BIOFILMS
COMPONENTS & 3-D ARCHITECTURE PHYSIOLOGY GRADIENTS METABOLISM
+ 420
+

SURFACE O2 O2 Gradient

14 14

420

Eh Scale

pH Scale

BIOFILM
410
410

H2

Abiotic Enamel POCKET

Cells Biotic Tissue

PHYSICAL PROPERTIES VISCOELASTIC HYDRATED-POLYMER MATERIALS

There are 3 components of biofilms; its architecture, its physiologic gradients and its physical properties. 18

8-FACTORS THAT DEFINE BIOFILM DEVELOPMENT and ARCHITECTURE

2. Chronic Wound Model
Species colonization, community structure

(Anti-Infective) Hostile Forces

Genotypic Factors

The Physico-Chemical Environment THE BIOFILM COMMUNITY STRUCTURE AND EVOLUTION Mechanical Factors and Shear Forces

Cyclic Stage

BIOTIC Substratum (Sloughing)

Nutrient Energy, Resource

West Virginia University

Biofilm Research Laboratory

There are 8 features that define the architecture and physical features; 3 are critical - surface, organism and stress.
CROSS-TALK/CO-ADHESION Growth Stimulation
C. albicans and Pseudomonas C. albicans and CoNS

Strep. mutans and C. albicans

ANTI-FOOD CHAIN Growth Inhibition

Synergy

Staph. aureus and Pseudomonas

C. albicans and C. glabrata

Antagonism
West Virginia University Biofilm Research Laboratory

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.
West Virginia University Biofilm Research Laboratory

The biofilm is a developmental biology, primitive although focused on enzymes and resistance to steady states.

Component of Biofilms
Component
Water Microbial Cells Polysaccharide Proteins DNA & RNA Ions HOST

% of Matrix
- Up to 97% - 2-5% (many species) - 1-2% (neutral & Polyanionic) - <1-2% - <1-2% -? Fibrin, RBCs, WBCs

West Virginia University

Biofilm Research Laboratory

A major defense mechanism of the biofilm is that it is up to 97% a diluent.

20

SURFACE O2
+ + 420

O2 Gradient

14 14

420

Eh Scale

pH Scale

H2

410

410

Abiotic Enamel POCKET

Cells Biotic Tissue

A biofilms 3-dimensional architecture allows for gradients, often pH dependent, allowing pH regions to exist from pH 11 to pH 3.

Biofilms act as Hydrated Polymers

21

KEY PROPERTY OF A BIOFILM

Hydrated Polymer EXHIBTS Microbes VISCOELASTIC NOT material that has both elastic (solid)
and viscus (liquid-like) properties Seconds absorbs increased shear by behaving elastically Long periods shear is dissipated through viscus flow (no detachment) or streamlined to reduce drag
West Virginia University Biofilm Research Laboratory

Acts as Hydrogel (Extremely hydrated polymer gel)

CONSEQUENCES

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

3 2

Multi-Layered Dilutions of Antibiotics

Organization of microbes is intellectually established and represents multilayers effectively diluting out the activity of many antimicrobials. 22

West Virginia University

Biofilm Research Laboratory

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, antioxidants, lubricants, pigments, etc.
Center for Biofilm Research

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.

23

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

Center for Biofilm Research

The effect of force on the hydrated polymer can be significant in transmitting aggregates of the organism pool.

The Society of Rheology : 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?

West Virginia University

Biofilm Research Laboratory

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)

MICROBIAL STAGE CYCLE

STAGE
I-A I-B LAG II LOG III STATIONARY IV-A IV-B DEATH

STOP HERE

RATIO 1. ORGANISMS 2. PHENOTYPE

PROMOTE HERE

Complex Community

Intra Oral Sessile (PBF) and planktonic (PP) life forms are not mutually exclusive, but biofilms are the preferred growth vehicle West Virginia University Biofilm Research Laboratory

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 SUBSTRATUM Strep. mutans @ 24hrs in poloxamer Stage II: Early & Immature
Thickness 25 m

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. 25

A BIOTIC SUBSTRATUM P. gingivalis @ 24hrs in poloxamer Stage II: Synergy & Cooperation

Thickness 40 m Spiral Linked communities Substratum

Gram-Negative Organisms (or Late Stage Colonizers) often form a spiral, rising from the base to the environment.
A BIOTIC SUBSTRATUM P. gingivalis & S. mutans @ 72hrs in poloxamer Stage III: Complex and Diverse
Thickness 55 m

Domain 1

Domains Diverse Community Co-aggregation Quorum Sensing

Domain 2

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

26

A BIOTIC SUBSTRATUM In vitro analyses of gram positive organism B using poloxamer Stage I (microcolonies); Early & Immature

Quantification

1
Open & Loosly spaced

Microcolony Organization pattern Spatial Arrangement

Biofilm Thickness

Biovolume

3 4 Unstable

Complexity Heterogen Biomass

Substratum Coverage

Thickness 250 m

Images provided by Convatec

3D imaging of microcolonies forming early biofilm community.

A BIOTIC SUBSTRATUM In vitro analyses of gram positive organism A using poloxamer Stage I-II: Moderate
Microcolony Organization pattern

Quantification

2
Biofilm Thickness

Biovolume

1 pH 5

Community Function: Synergy, Coaggregation

3 pH 11

Spatial Arrangement Complexity Heterogen Biomass

Substratum Coverage

Thickness 250 m

Robust Association

Images provided by Convatec

3D image of intermediate coalescing biofilm.

27

A BIOTIC SUBSTRATUM In vitro analyses of gram positive organism A + B using poloxamer Stage III

Quantification

1 Crosstalk

Stable Integration of Function Microcolony & Increased BioBio-Diversity with Focused Marker Organisms Organization
pattern Spatial Arrangement Complexity Heterogen Biomass

Biofilm Thickness

2 Crosstalk

Biovolume

Substratum Coverage

Thickness 250 m

3
Images provided by Convatec

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

A BIOTIC SUBSTRATUM In vitro analyses of gram positive organism B using poloxamer under increased stress, Stage III-IV: Late/ Apoptosis or Necrosis Apoptosis Top Microcolony Organization pattern Spatial Arrangement Domain I
Biovolume

Quantification

Biofilm Thickness

Complexity Heterogen Biomass

Substratum Coverage

Domain II Channels Attachment

Images provided by Convatec

Thickness 250 m

Well-defined mature biofilm architecture.

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BIOTIC SUBSTRATUM 3D anaglyph Attachment Microcolony Organization pattern Vertical Orientation of Hyphae Spatial Arrangement Complexity Heterogen Biomass
Substratum Coverage

Quantification

Biofilm Thickness

Biovolume

Hyphae

Thickness 250 m

Malic et al (2006)

C. albicans (Yeast)

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

29

V. Clinical Consequences

Patho-Physiology of VAP and Significance of Lumenal Growth

1&2 Outside ET
Planktonic Life-Form; 2D only Zero 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

West Virginia University

Biofilm Research Laboratory

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 vitro To MACRO scopic in vivo AIR-WAY RESISTANCE

West Virginia University Biofilm Research Laboratory

30

Occlusions: A Significant Consequence of Biofilm Architecture

1. 2. 3. 1. OIL FLOW (Turbulent) 50 microns (5% diameter reduction) reduces oil flow by 33% in a 2 cm pipe. 33% curve of a pipe causes an additional 17%. Total = 50% reduction PICC LINES Occlusion is a significant problem. - 40% due to internal thrombus formation Resulting CRS is associated with increased morbidity/mortality, increased cost and increased LOS. ETT Occlusion is a significant problem WOB Feasibility Study

2.

1. 2.

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: biofilm prevention reduces attachment of secretions and thus prevents airway resistance buildup Metric: Metric pressure drop measurements i.e. smaller effective diameters produce higher pressures
Mucus Attached

Biofilm

ET Tube Inner Lumen Wall

West Virginia University

Biofilm Research Laboratory

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.

31

Extubated Endotrachs In Vivo Analysis 78 Patient tubes

Patient A: Half section

Patient A: Close-up

PHASE I

PTS-2000 Pressure Drop System

West Virginia University

Biofilm Research Laboratory

Extubated endotrach in accumulated accretion in spite of suctioning and nurse intervention at the time of extubation.

West Virginia University

Biofilm Research Laboratory

Data from the pressure-drop study showing reproducibility of the clean endotrach versus unrecognized consequences of biofilm and accretion buildup. 32

3-D Computational Fluid Dynamics (CFD)

Analysis of ET Tube Stage IV Dispersal Via Trachea
Lung End

ORAL FLORA

GI FLORA

LUNG FLORA

Endotracheal Tube 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

Center for Biofilm Research

In stage IV analysis of metastasizing fragments and recognition that 1-5 micron-size aggregates could integrate into the alveolar spaces of the lung.

33

Center for Biofilm Research

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

Center for Biofilm Research

Pathologic consequences in VAP, recognizing aggregates of 1-5 microns in the alveolar space.

34

PP

PB

ACUTE

CHRONIC

West Virginia University

Biofilm Research Laboratory

Shift in ratio from planktonic to biofilm phenotype is associated with more organisms of resistant biofilm.
Collateral Damage: Two Pathways to Patient Disease
30 Million Surgery
Device

KEY: Device
Infection (VAP) MICRO

(HOST) Airway Resistance MACRO PARTNERING

MICRO
colonies

MICRO colonies 3 Million ICU

Increases MACRO Airway colonies Resistance Occlusion: 100% Accretion Build-up

Endotrach Tube

M FI L Dispersal of Stage IV BIO INK Biofilm aggregates L / VIA NCE E N R HE TIO LAVA AD FEC CHS PRINCIPLES OF O N I LIKE K ATES RHEOLOGY TIAN STUL PO
3-6% INFECTION
(VAP)

1. 2.

Pressure Drop CT/VITAL

REORGANIZTION: MICRO VS. MACRO

Biofilm Research Laboratory

West Virginia University

Collateral damage, the significance of biofilm in 3 features: VAP, airway resistance and loss of function.

35

Paradigm Shift:
MEDICAL

DENTAL

DISEASE: MICROBE: THERAPY: MICROBIOLOGY:

Acute Infection One

(Kochs Postulates)

Chronic Infection Ecosystem

(Plaque Hypothesis)

Eradicate Pure Culture

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.

Antibiotic Resistance of Planktonic Organisms

Increasing Antibiotic Resistance
E. faecalis (resistant)
PCN Pneumonococci Multi GNR VRSA GNR MRD-Typhoid VRE MRD-Tb

Anxiety Threshold
MRSA -lactamase in Hemophilus PCN S. aureus Aminoglycoside Augmented PCN PCN Sulpha Aminoglycoside

Quirolones Cephalosporin sunge Vancomycin

Doxycycline (Stable)

1940

1950

1960

1970

1980

1990

2000

West Virginia University

Biofilm Research Laboratory

The increased antibiotic resistance paralleling the use of new antibiotics.

36

Colonization Resistance MIC:MBEC Using Calgary Device

1000 900 800 700 600 500 400 300 200 100 0

Planktonic vs. Sessile

Cefazolin Cloxacillin Imipenem Vancomycin Clindamycin Ceftazidime

MIC
West Virginia University

MBEC
Ratio May be Important Chemically: MIC MBEC
Biofilm Research Laboratory

Resistance associated with biofilms called colonization resistance where minimal inhibitory concentration planktonic is less than minimal biofilm elimination concentration (biofilm).

37

VI. Clinical Cases & Pictures (SEMs)

1.

2.

3.

Biofilms are everywhere, as they are the preferred means of survival.

1.

Oral/Dental Plaque Biofilm and Gingivitis

Plaque is the prominent biofilm example, linked to over $92 million in diseases per year. 38

Advanced 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 SEMs

Biofilms and the Nasal and Paranasal Sinus Epithelium Biofilm in Chronic Rhinosinusitis Patient

West Virginia University

Biofilm Research Laboratory

Oral Flora reservoir now include the sinus tracts which ping-pongs with oral microbes. 39

2.

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: (percutaneous coronary 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).

West Virginia University

Biofilm Research Laboratory

An aging population demands support.

The Growing Use of IMDs

Complex: - The frequency of use of the automatic implantable cardiodefibrillator (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
West Virginia University Biofilm Research Laboratory

IMDs are more and more difficult to treat once in place and require very expensive removal once infected by a biofilm.

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Culture Negative Biofilm Phenotype: Examples Viable, But NonNon-Cultable Cultable (VBNC)
Staph. aureus

West Virginia University

Biofilm Research Laboratory

Two cases, associated with biofilms, resulting in death at WVUH. Both were VBNC.

NICU Patient with Multiple Line Sepsis

West Virginia University

Biofilm Research Laboratory

The prefect We live in a Microbial World, and care givers providing a mixture of organisms. 41

West Virginia University

Biofilm Research Laboratory

Catheters and ETTs: the protected environment from host defenses

Line Sepsis Endoluminal Brush

West Virginia University

Biofilm Research Laboratory

A lumenal brushing of a line associated with Line Sepsis, organisms going from attached to free floating, and then attachedmetastasis.

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A Superficial Wound

Wounds are a growing problem with an aging population, but the importance of biofilms and pathogenicity are controversial.

Wounds: Soft Tissue

16 year old with Dystrophic Epidermolyses Bullosa (DEB). A group of heritable mechano-bullous skin diseases.

Pseudomonas and Staph. aureus

West Virginia University Biofilm Research Laboratory

Chronic wounds are NOT only seen in adults; this lad died at age 16 with sepsis related to skin biofilm.

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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. 44

3.

Environmental Biofilms SEMs

Co-Biofilm: Bacteria and Fungi in Ventilation Systems of Airplanes

Leaching Process in Mines Create Stress

www.mbec.ca
West Virginia University Biofilm Research Laboratory

The environment and abiotic surfaces are the perfect conditions for biofilms, constructed from the 99.9% of microbes we have never recovered or identified.

Agricultural Biofilms

Ref: www.mbec.ca
West Virginia University Biofilm Research Laboratory

Biofilms will continue to cause problems in the things we like. 45

VII. Biofilm Models (in vitro/in vivo)

Organisms Commonly forming Biofilms Arranged by IMD

DISEASE Blood Stream Infections (BSI)
POTENTIAL PATHOGENS
Staphylococcus aureus Coagulase Negative Staph Enterobacteriacae Pseudomonas aeruginosa Streptococcus pneumoniae* Streptococcus spp. Candida spp. Enterococcus spp. other bacteria Escherichia coli Enterococcus spp. Staphylococcus aureus Klebsiella spp. Proteus spp. Pseudomonas aeruginosa Candida spp. other bacteria Staphylococcus aureus Streptococcus pneumoniae Pseudomonas aeruginosa Klebsiella pneumoniae Enterobacteriacae Haemophilus influenzae Candida spp. Legionella spp. Bordetella pertussis

TRANSPORT DEVICES
Blood Culture Bottles

STORAGE
Room Temperature

REJECTION CRITERIA
Over or underinoculated No skin prep performed Line draws** Single draws

Urinary Tract Infections (UTI)

Sterile urine container or Urine Culture Tube

If delay to the laboratory of >2 hours, refrigerate

Unrefrigerated >2 hrs Received >24 hours after collection Fecal contamination No work up on cultures with >2 isolates

Lower Respiratory Tract Infections (LRI)

Sterile container (except B. pertussis) B. pertussisRegan Lowe transport or Coughplate using Regan Lowe agar

Gram stain showing contamination with saliva (>10 squamous pithelials/10X) Quality scores provide consistency of reporting

GI Infections

Salmonella spp. Shigella spp. Campylobacter spp. Escherichia coli (STEC) Clostridium difficile toxin Staphylococcus aureus Yersinia enterocolytica Center for Biofilm Research Vibrio spp.

Sterile Container Enteric Pathogen Vial Rectal swab (not for C. difficile)

Rrefrigerate (especially C. difficile)

Contaminated with urine Formalin-fixed

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

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Respiratory simulation The VEL Model

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

Zone X Zone Y Zone Z

Dental Cocktail

VAP Cocktail

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

West Virginia University Biofilm Research Laboratory

VEL model simulator, created as a result of a 50 patient ICU study, was based on replicating the stress of a mechanically ventilated patient.

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SHEAR FORCES
Microscopy

ZONE Y
Culture/ Non-culture

Ag Coating TYCO Healthcare

West Virginia University Biofilm Research Laboratory

Sheer forces were the most important and demanded analysis of the endotrach in 3 zones; A, B and C.

VENT PROFILES
ZONE X
A (NORMAL) REQUIRED PARAMETERS RANGE VALUE TIDAL VOLUME (ml) >750 800 RESPIRATORY RATE (breaths/min 10-15 12 FIO2 SET (%) 35-45 40 PEEP (cm of H2O) PIP (cm of H2O) FLOWRATE (litres per min or LPM)
West Virginia University

B (COPD) RANGE VALUE 750-650 700 16-25 20 46-65 50

C (ARDS) RANGE VALUE <650 600 >25 35 66-100 75

4.5-7 15-27 <55

5 20 50

8-15. 28-44 55-65

10 30 60

>=15 >35 >65

15 40 80

From 50 patient study

Biofilm Research Laboratory

Calculations by the School of Engineering in concert with respiratory therapy for establishing 3 ventilator profiles.

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Unsteady Pipe Flow

Unsteady shear stress prediction (Case B) (7 mm dia) (Note: Tau reverses direction during exhalation) 200 150 100
Shear stress, Pa

Tau smooth Tau rough

50 0 0 -50 -100 -150 -200 Time, sec 0.5 1 1.5 2 2.5 3

Center for Biofilm Research

Dynamics evaluated by School of Engineering for COPD in 7-mm endotrach.

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VII. Rules: Dos and Donts Dont select for overgrowth. Dont select for antibiotic resistance. Dont destroy normal flora. Be green. Be reusable. Be nontoxic. Save mixed flora.

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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.

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X. Key References and websites

WVU-Covidien Data Bank

West Virginia University John G. Thomas PhD and Staff

Data from 9 years of Collaborative Research, including: 1) Quantitative Imaging 2) Microbial Cultures 3) 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.

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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;284:131822. 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;50:2782. 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.

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XI. FAQs 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.

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