High Yield Overview

BIOFILM FORMATION

Structured Bacterial Communities | Extracellular Matrix Protection | 1000x Antibiotic Resistance | Implant-Associated Infections

1000xIncreased antibiotic MIC in biofilm

24-48hIrreversible attachment timeline

80%Of chronic infections involve biofilm

99.9%Of biofilm bacteria are dormant persisters

STAGES OF BIOFILM FORMATION

Stage 1: Attachment

PatternReversible adhesion (0-4 hours)

TreatmentAntibiotics still effective

Stage 2: Maturation

PatternIrreversible attachment, EPS production (4-48 hours)

TreatmentEarly debridement + antibiotics

Stage 3: Mature Biofilm

Pattern3D structure, persister cells (greater than 48 hours)

TreatmentRequires implant removal for cure

Critical Must-Knows

Biofilm = structured bacterial community embedded in self-produced extracellular polymeric substance (EPS)
1000-fold increase in MIC compared to planktonic bacteria - explains antibiotic failure
Persister cells (dormant, metabolically inactive) resist antibiotics that target dividing cells
Implant removal essential for cure of mature biofilm infections - antibiotics alone fail
DAIR (debridement, antibiotics, implant retention) only works for ACUTE infections (less than 3 weeks)

Examiner's Pearls

"
Biofilm bacteria communicate via quorum sensing (autoinducer molecules)
"
EPS matrix is polysaccharide (80% water, 10-15% eDNA, proteins, polysaccharides)
"
Rifampicin penetrates biofilm better than other antibiotics (used in PJI treatment)
"
Sonication of explanted implants releases biofilm bacteria - improves culture yield by 20-30%

Clinical Imaging

Imaging Gallery

(A,C) SEM and (B,D) fluorescent microscopy images of P. aeruginosa attached for 4 hours on the control (top) and black titanium (bottom) surfaces.Credit: Open-i / NIH via Open-i (NIH) (Open Access (CC BY))

Characteristic cell morphologies of MRSP biofilms and its surface coverage on titanium orthopaedic screws. The effect of fosfomycin against MRSP A12 strain on titanium orthopaedic screws was assessed — Characteristic cell morphologies of MRSP biofilms and its surface coverage on titanium orthopaedic screws. The effect of fosfomycin against MRSP A12 sCredit: DiCicco M et al. via BMC Microbiol. via Open-i (NIH) (Open Access (CC BY))

MRSP biofilm surface height profiles with corresponding AFM deflection mode images (Scale = 5 μm). (A), (B) MRSP A12 AFM image showing clusters of biofilms with extended chains exhibiting stable nanos — MRSP biofilm surface height profiles with corresponding AFM deflection mode images (Scale = 5 μm). (A), (B) MRSP A12 AFM image showing clusters of bioCredit: DiCicco M et al. via BMC Microbiol. via Open-i (NIH) (Open Access (CC BY))

Mouse surgical procedures.Anesthesia chamber with isoflurane inhalation (A); skin preparation (B); skin incision over the right knee (C); introduction of a 25-gauge needle retrograde into the femoral — Mouse surgical procedures.Anesthesia chamber with isoflurane inhalation (A); skin preparation (B); skin incision over the right knee (C); introductionCredit: Lovati AB et al. via PLoS ONE via Open-i (NIH) (Open Access (CC BY))

Critical Biofilm Exam Points

Biofilm Definition

Biofilm is a structured community of bacteria enclosed in self-produced extracellular polymeric substance (EPS) matrix, adherent to a surface. Fundamentally different from planktonic (free-floating) bacteria in physiology and antibiotic susceptibility.

1000x Antibiotic Resistance

Bacteria in biofilm are 1000-fold more resistant to antibiotics than planktonic bacteria. Not due to genetic resistance genes, but physical protection (EPS barrier) and metabolic dormancy (persister cells). This explains treatment failure despite in vitro susceptibility.

Implant Removal Necessary

Mature biofilm cannot be eradicated with antibiotics alone - physical removal of biofilm (implant exchange) essential for cure. DAIR only works if biofilm not yet established (less than 3 weeks, acute infections).

Persister Cells

Persister cells (0.1-1% of biofilm) are dormant, metabolically inactive bacteria resistant to all antibiotics. They survive treatment and cause relapse when conditions improve. Explains recurrence after stopping antibiotics.

Mnemonic

BIOFILMBIOFILM - Stages and Characteristics

Bacteria attach to surface

Initial reversible adhesion (0-4 hours)

Irreversible attachment

Bacterial adhesins bind tightly (4-24 hours)

Organize into microcolonies

3D structure begins to form

Form EPS matrix

Extracellular polymeric substance secreted (polysaccharide shield)

Impenetrable to antibiotics

1000x increased MIC, persister cells dormant

Long-term persistence

Chronic infection, requires implant removal for cure

Microenvironment created

pH gradients, nutrient channels, anaerobic zones

Memory Hook:BIOFILM forms in stages and creates impenetrable antibiotic resistance

Mnemonic

MATRIXMATRIX - Components of Biofilm EPS

Microbes embedded

Bacteria make up only 10-15% of biofilm volume

Adhesins for attachment

Surface proteins bind to implant (fibronectin-binding, collagen-binding)

Teichoic acids (Staph)

Cell wall components of gram-positive bacteria

Resistant to antibiotics

Physical barrier, altered pH, persister cells

Intercellular polysaccharide

PIA (polysaccharide intercellular adhesin) in S. epidermidis, encoded by ica genes

eXtracellular DNA (eDNA)

10-15% of matrix, provides structural support and antibiotic binding

Memory Hook:The MATRIX protects bacteria and makes them antibiotic-resistant

Mnemonic

PERSISTPERSIST - Why Biofilm Bacteria Survive Antibiotics

Physical barrier (EPS)

Matrix blocks antibiotic penetration

Enzymes degrade antibiotics

Beta-lactamases, aminoglycoside-modifying enzymes concentrated in biofilm

Reduced metabolic activity

Dormant bacteria not targeted by antibiotics (target dividing cells)

Slow growth rate

Nutrient limitation in biofilm depths slows division

Impaired antibiotic binding

eDNA and matrix components bind antibiotics

Small persister cell population

0.1-1% of bacteria are dormant persisters resistant to all antibiotics

Tolerance not resistance

Phenotypic tolerance (reversible) not genetic resistance (permanent)

Memory Hook:PERSIST explains why biofilm bacteria survive despite antibiotic susceptibility in vitro

Overview

Biofilm is a structured community of bacterial cells enclosed in a self-produced extracellular polymeric substance (EPS) matrix and adherent to an inert or living surface.

Historical context: Biofilms were first described by van Leeuwenhoek in 1684 observing "animalcules" on teeth. The modern concept of biofilm was established by Costerton in the 1970s-1980s, revolutionizing understanding of bacterial persistence and chronic infections.

Why biofilm matters clinically:

Prosthetic Joint Infections

Biofilm formation on implants explains why PJI cannot be cured with antibiotics alone. Mature biofilm requires implant removal for eradication. DAIR only effective if biofilm not yet established (acute infections less than 3 weeks).

Chronic Osteomyelitis

Biofilm on sequestrum (dead bone) and necrotic tissue protects bacteria from antibiotics and immune system. Explains need for surgical debridement of all necrotic material in addition to prolonged antibiotics.

Biofilm vs Planktonic Bacteria

Planktonic bacteria (free-floating) are what we test in microbiology lab (in vitro susceptibility). Biofilm bacteria are fundamentally different: 1000x higher MIC, metabolically dormant, physically protected. In vitro susceptibility does NOT predict in vivo efficacy for biofilm infections. This explains "antibiotic failure" despite sensitive organism.

Stages of Biofilm Formation

Biofilm Development Timeline

Stage 1: Reversible Attachment0-4 hours

Initial contact between planktonic bacteria and surface (implant, bone). Mediated by weak van der Waals forces, electrostatic interactions, and hydrophobic effects. Bacteria are still susceptible to antibiotics and mechanical forces (irrigation). This is a critical window for prevention - antibiotic prophylaxis effective at this stage.

Stage 2: Irreversible Attachment4-24 hours

Bacterial adhesins (surface proteins) bind tightly to host proteins (fibronectin, collagen, fibrinogen) adsorbed on implant. Bacteria begin producing extracellular polymeric substance (EPS). Attachment becomes permanent. Early debridement and antibiotics may still be effective.

Stage 3: Microcolony Formation24-48 hours

Bacteria proliferate and organize into microcolonies (clusters). EPS production increases, forming protective matrix. 3D architecture begins to develop with water channels for nutrient flow. Antibiotic penetration starts to diminish.

Stage 4: Mature Biofilm48 hours to days

Mature biofilm with complex 3D structure. EPS matrix fully developed (80% water, 10-15% polysaccharides/eDNA/proteins). Persister cells present (dormant, antibiotic-resistant). Quorum sensing coordinates bacterial behavior. Antibiotics largely ineffective - implant removal necessary.

Stage 5: Dispersal and SpreadDays to weeks

Bacteria detach from biofilm edges (planktonic dispersal) to colonize new sites. Triggers acute symptoms (bacteremia, sepsis). Dispersal can be triggered by nutrient depletion, quorum sensing signals, or external stress. Explains acute exacerbations of chronic infections.

Critical Time Window

First 24-48 hours post-implantation are critical. If bacteria attach and begin biofilm formation, chronic infection likely. Antibiotic prophylaxis most effective if given before incision (within 60 minutes) to prevent initial attachment. After 48 hours, biofilm maturation makes eradication difficult without implant removal.

DAIR Window

DAIR (debridement, antibiotics, implant retention) only successful if performed within 3 weeks of symptom onset for acute infections. After 3 weeks, mature biofilm established and implant removal required. Success rate of DAIR: 50-70% if acute (less than 3 weeks), less than 20% if chronic (greater than 3 weeks).

Biofilm Structure and Composition

Extracellular Polymeric Substance (EPS) Matrix

Composition (by weight):

Water: 80-90% of biofilm volume
Polysaccharides: 40-50% of dry weight (structural backbone)
eDNA (extracellular DNA): 10-20% of dry weight
Proteins: 20-30% of dry weight (enzymes, adhesins)
Lipids: 5-10% of dry weight
Bacterial cells: Only 10-15% of biofilm volume

Polysaccharides:

PIA (polysaccharide intercellular adhesin): S. epidermidis, encoded by ica genes
PNAG (poly-N-acetylglucosamine): Staphylococcus species
Alginate: Pseudomonas aeruginosa
Pel and Psl: Pseudomonas aeruginosa
Functions: Structural scaffold, adhesion, protection from desiccation and immune cells

Extracellular DNA (eDNA):

Released from lysed bacteria or actively secreted
Provides structural support (scaffolding)
Binds cationic antibiotics (aminoglycosides, polymyxins) - reduces penetration
Contains antibiotic resistance genes (horizontal gene transfer within biofilm)
DNase treatment can disrupt young biofilms (research application)

Proteins:

Adhesins: Bind to host proteins and implant surfaces
Enzymes: Beta-lactamases, proteases, nucleases
Amyloid fibrils: Structural support (Staphylococcus, E. coli)

ica Genes in Staphylococcus

ica operon (icaADBC) in S. epidermidis encodes enzymes for PIA synthesis. Bacteria lacking ica genes cannot form biofilm and are less virulent in prosthetic infections. icaA and icaD are essential genes. Biofilm-negative strains exist but are uncommon clinical isolates.

The EPS matrix is the key protective element of biofilm.

Mechanisms of Antibiotic Resistance in Biofilm

Biofilm bacteria are 1000-fold more resistant to antibiotics through multiple mechanisms:

Biofilm Antibiotic Resistance Mechanisms

Mechanism	Description	Effect	Clinical Implication
EPS barrier	Matrix blocks antibiotic diffusion	Reduced penetration to depths	Outer bacteria killed, inner survive
eDNA binding	eDNA binds cationic antibiotics	Aminoglycosides, polymyxins sequestered	Higher doses cannot overcome
pH gradients	Acidic microenvironments (pH 5-6)	Many antibiotics less active at low pH	Fluoroquinolones, aminoglycosides impaired
Enzyme degradation	Beta-lactamases concentrated in biofilm	Penicillins, cephalosporins destroyed	Even susceptible strains protected
Slow growth rate	Nutrient limitation slows division	Antibiotics target dividing cells	Dormant bacteria not killed
Persister cells	0.1-1% dormant, non-growing	Tolerant to ALL antibiotics	Cause relapse, require removal
Altered gene expression	Biofilm-specific genes upregulated	Efflux pumps, stress responses	Phenotypic resistance

Tolerance vs Resistance

Antibiotic tolerance (biofilm) is phenotypic and reversible - bacteria are genetically susceptible but protected by biofilm environment. Antibiotic resistance is genetic (genes like mecA, vanA) and permanent. Biofilm bacteria are TOLERANT not resistant - in vitro testing shows susceptibility, but in vivo treatment fails due to tolerance. This is why implant removal necessary.

Why antibiotics fail in biofilm infections:

Physical Barriers

EPS matrix, eDNA binding, and altered pH create physical and chemical barriers preventing antibiotics from reaching bacteria at MIC concentrations. Even high-dose IV antibiotics cannot penetrate to biofilm depths.

Metabolic Dormancy

Persister cells and slow-growing bacteria in nutrient-limited zones are metabolically inactive. Antibiotics target active processes (cell wall synthesis, protein synthesis, DNA replication) - dormant cells are untouched.

Clinical Implications in Orthopaedics

Biofilm in Prosthetic Joint Infections

Biofilm formation on implants:

Bacteria attach within hours of contamination (intraoperative or hematogenous)
Mature biofilm established by 48-72 hours
Coagulase-negative Staphylococci (S. epidermidis) are master biofilm formers
Biofilm provides sanctuary from antibiotics and immune cells

Treatment strategies based on biofilm maturity:

Acute infection (less than 3 weeks symptoms, less than 3 months post-op):

Biofilm not yet mature or well-established
DAIR (debridement, antibiotics, implant retention) possible
Success rate: 50-70% if treated early
Requires aggressive debridement, implant exchange of modular parts, biofilm-active antibiotics (rifampicin)

Chronic infection (greater than 3 weeks symptoms):

Mature biofilm established
Implant removal mandatory for cure
Two-stage exchange: Remove implant + antibiotic spacer, then reimplantation after 6-12 weeks
Suppressive antibiotics without removal: Temporary symptom control but eventual failure

Treatment Options by Infection Timing

Scenario	Biofilm Status	Treatment	Success Rate
Acute (less than 3 weeks)	Immature biofilm	DAIR + antibiotics + rifampicin	50-70%
Chronic (greater than 3 weeks)	Mature biofilm	Two-stage exchange	80-90%
Chronic (suppression)	Mature biofilm	Antibiotics alone (no surgery)	less than 20% (eventual failure)

Biofilm-active antibiotics:

Rifampicin: Best biofilm penetration, NEVER monotherapy (rapid resistance)
Fluoroquinolones: Moderate penetration, bactericidal
Linezolid: Good penetration, oral bioavailability
Daptomycin: Biofilm activity against Staphylococcus
Avoid: Vancomycin (poor biofilm penetration despite IV use)

Rifampicin in PJI

Rifampicin is the most biofilm-penetrating antibiotic for Staphylococcus. Used in PJI treatment (300-450mg PO twice daily) in combination (NEVER alone - resistance develops in 48 hours). Added after 2-5 days of primary antibiotic (if susceptible). Effective for both DAIR and suppression protocols.

Evidence Base

Biofilm Formation on Biomaterials

Costerton JW, et al. • Science (1999)

Key Findings:

Biofilms form on all implanted medical devices exposed to bacteria
Bacteria in biofilm 1000-fold more resistant to antibiotics than planktonic bacteria
Resistance due to EPS matrix barrier and persister cell dormancy, not genetic resistance
Biofilm bacteria survive despite in vitro antibiotic susceptibility

Clinical Implication: Established biofilm as major cause of implant-related infections. Explained why antibiotics fail despite susceptible organisms in culture. Led to understanding that implant removal necessary for cure.

Sonication Improves Culture Yield in PJI

Trampuz A, et al. • N Engl J Med (2007)

Key Findings:

Sonication of explanted implants increased culture sensitivity from 61% to 79%
Particularly helpful in patients who received antibiotics before cultures
Threshold greater than 50 CFU/mL in sonicate fluid = infection (94% specificity)
More sensitive than tissue cultures alone for detecting biofilm bacteria

Clinical Implication: Sonication should be standard for all explanted implants in revision surgery. Significantly improves detection of biofilm bacteria, especially in culture-negative cases.

Rifampicin for Staphylococcal PJI

Zimmerli W, et al. • JAMA (1998)

Key Findings:

Rifampicin combination therapy superior to non-rifampicin regimens for Staph PJI
Treatment success 100% (12/12) with rifampicin vs 58% (7/12) without rifampicin
Rifampicin penetrates biofilm and kills persister cells better than other antibiotics
Must combine with other antibiotic - rifampicin monotherapy leads to resistance in 48 hours

Clinical Implication: Rifampicin is essential component of Staphylococcal PJI treatment (DAIR or suppression). Best biofilm-penetrating antibiotic. Always use in combination, never alone.

DAIR Timing and Success

Osmon DR, et al. (IDSA Guidelines) • Clin Infect Dis (2013)

Key Findings:

DAIR success rate 50-70% if performed within 3 weeks of symptom onset
Success drops to less than 30% if delayed beyond 3 weeks (mature biofilm)
Requires aggressive debridement, modular component exchange, biofilm-active antibiotics
Patient selection critical: stable implant, susceptible organism, no sinus tract

Clinical Implication: DAIR only appropriate for acute infections (less than 3 weeks) before mature biofilm established. After 3 weeks, two-stage exchange necessary. Early diagnosis and treatment critical for implant retention.

Biofilm Viva Scenarios

Practice these scenarios to excel in your viva examination

VIVA SCENARIOStandard

Scenario 1: Define Biofilm and Antibiotic Resistance (~3 min)

EXAMINER

"What is a biofilm and why are bacteria in biofilm resistant to antibiotics?"

EXCEPTIONAL ANSWER

A biofilm is a structured community of bacterial cells enclosed in a self-produced extracellular polymeric substance, or EPS matrix, that is adherent to an inert or living surface. This concept was established by Costerton in the 1970s and revolutionized our understanding of chronic bacterial infections. Bacteria in biofilm are fundamentally different from planktonic, or free-floating, bacteria and are approximately 1000-fold more resistant to antibiotics through several mechanisms. First, the EPS matrix creates a physical barrier that impedes antibiotic penetration, preventing antibiotics from reaching bacteria in the deeper layers at sufficient concentrations. Second, extracellular DNA within the matrix binds to cationic antibiotics like aminoglycosides, sequestering them before they reach bacterial cells. Third, the biofilm creates pH gradients with acidic microenvironments where many antibiotics have reduced activity. Fourth, bacteria in the nutrient-limited deep layers of the biofilm have very slow growth rates or become dormant, and since most antibiotics target actively dividing cells, these dormant bacteria are not killed. Finally, there is a subpopulation called persister cells, which make up 0.1-1% of the biofilm, that are completely dormant and metabolically inactive, making them tolerant to all antibiotics regardless of mechanism of action. Importantly, this resistance is phenotypic tolerance, not genetic resistance - the bacteria are genetically susceptible, but protected by the biofilm environment. This explains why in vitro susceptibility testing does not predict clinical efficacy in biofilm infections.

KEY POINTS TO SCORE

Biofilm = structured bacterial community in EPS matrix adherent to surface

1000-fold increased antibiotic resistance compared to planktonic bacteria

Multiple resistance mechanisms (not single mechanism)

EPS matrix barrier: Blocks antibiotic penetration to deep layers

eDNA binding: Sequesters cationic antibiotics (aminoglycosides)

pH gradients: Acidic zones reduce antibiotic activity

Slow growth: Dormant bacteria not killed by antibiotics targeting division

Persister cells: 0.1-1% of biofilm, dormant, tolerant to ALL antibiotics

Phenotypic tolerance (reversible) not genetic resistance (permanent)

In vitro susceptibility does NOT predict in vivo efficacy for biofilm

COMMON TRAPS

✗Confusing tolerance with genetic resistance

✗Not mentioning multiple mechanisms (not just one)

✗Forgetting persister cells (critical concept)

✗Missing phenotypic vs genetic distinction

✗Not explaining why in vitro susceptibility misleading

LIKELY FOLLOW-UPS

"What are persister cells and why are they clinically important?"

"How does biofilm form on an implant over time?"

"Why is rifampicin particularly useful for biofilm infections?"

VIVA SCENARIOChallenging

Scenario 2: Biofilm in PJI Treatment (~4 min)

EXAMINER

"How does biofilm formation influence your treatment strategy for prosthetic joint infection, particularly the decision between DAIR and two-stage exchange?"

EXCEPTIONAL ANSWER

Biofilm formation is the central consideration when deciding between DAIR and two-stage exchange for prosthetic joint infections. The key determinant is whether a mature biofilm has already established, which is primarily a function of time. In acute infections - defined as symptoms for less than 3 weeks or infections occurring within 3 months of surgery - biofilm is either not yet formed or immature and not fully established. In these cases, DAIR is a viable option with success rates of 50-70%. The procedure involves aggressive debridement of all inflamed synovium and necrotic tissue, exchange of modular components like polyethylene liners and femoral heads, and thorough irrigation. This is combined with prolonged antibiotics including biofilm-active agents, particularly rifampicin for Staphylococcal infections. In contrast, chronic infections with symptoms for more than 3 weeks have mature, well-established biofilm that cannot be eradicated with antibiotics alone. The persister cells within the mature biofilm are dormant and completely tolerant to all antibiotics. In these cases, implant removal is mandatory for cure, typically via two-stage exchange with antibiotic spacer placement, 6-12 weeks of antibiotics, followed by reimplantation. Success rates for two-stage exchange are 80-90% compared to less than 20% for chronic suppressive antibiotics without implant removal. The decision tree is: acute infection less than 3 weeks with stable implant and susceptible organism - consider DAIR with biofilm-active antibiotics. Chronic infection more than 3 weeks, sinus tract present, or unstable implant - two-stage exchange mandatory. Patient factors also matter: DAIR requires good host immunity and compliance with prolonged antibiotics. The fundamental principle is that mature biofilm cannot be sterilized in situ - the physical structure must be removed.

KEY POINTS TO SCORE

Biofilm maturity determines treatment strategy (time-dependent)

Acute (less than 3 weeks): Immature biofilm, DAIR possible (50-70% success)

Chronic (greater than 3 weeks): Mature biofilm, implant removal required

DAIR requirements: Stable implant, susceptible organism, no sinus, acute timing

DAIR technique: Aggressive debridement, exchange modulars, biofilm-active antibiotics

Rifampicin essential for Staph (best biofilm penetration, always combine)

Two-stage exchange: Remove implant + spacer → 6-12 weeks antibiotics → reimplant

Two-stage success: 80-90% (gold standard for chronic PJI)

Suppression without removal: Less than 20% success (eventual failure)

Mature biofilm has persister cells - cannot be killed by any antibiotic

Physical removal of biofilm (implant exchange) is only definitive cure

COMMON TRAPS

✗Not defining acute vs chronic thresholds (3 weeks critical)

✗Missing DAIR success rate and appropriate patient selection

✗Forgetting rifampicin for Staphylococcal biofilm

✗Not mentioning persister cells as reason implant removal needed

✗Thinking antibiotics alone can ever cure chronic biofilm infection

LIKELY FOLLOW-UPS

"What makes rifampicin particularly effective against biofilm?"

"Why do we exchange modular components during DAIR?"

"What is sonication and how does it improve diagnosis?"

Management Algorithm

BIOFILM FORMATION

High-Yield Exam Summary

Core Definition

•Biofilm = structured bacterial community in EPS matrix adherent to surface
•Costerton 1970s-1980s established modern biofilm concept
•80% of chronic infections involve biofilm (implants, osteomyelitis)
•Fundamentally different from planktonic (free-floating) bacteria

Biofilm Formation Stages

•Stage 1 (0-4h): Reversible attachment, antibiotics still effective
•Stage 2 (4-24h): Irreversible attachment via adhesins, EPS begins
•Stage 3 (24-48h): Microcolony formation, 3D structure develops
•Stage 4 (48h+): Mature biofilm, persister cells, antibiotic resistance
•Critical window: First 24-48h before irreversible biofilm established

EPS Matrix Composition

•80-90% water, 10-15% bacterial cells (by volume)
•Dry weight: 40-50% polysaccharides, 10-20% eDNA, 20-30% proteins
•Polysaccharides: PIA in S. epidermidis (ica genes), alginate in Pseudomonas
•eDNA: Structural support, binds cationic antibiotics, gene transfer

Antibiotic Resistance Mechanisms

•1000-fold increased MIC compared to planktonic bacteria
•EPS barrier: Blocks antibiotic penetration to depths
•eDNA binding: Sequesters aminoglycosides, polymyxins
•pH gradients: Acidic zones (pH 5-6) reduce antibiotic activity
•Slow growth: Dormant bacteria not killed (antibiotics target division)
•Persister cells: 0.1-1% of biofilm, tolerant to ALL antibiotics
•Phenotypic tolerance (reversible) NOT genetic resistance

Persister Cells (Critical Concept)

•Dormant, non-growing bacteria (0.1-1% of biofilm)
•Tolerant to ALL antibiotics (not genetic resistance)
•Survive treatment, cause relapse when antibiotics stopped
•Located in nutrient-limited deep zones of biofilm
•Explain chronic relapsing infections despite susceptible organism
•Cannot be killed by antibiotics - require physical removal (implant exchange)

Clinical Implications - PJI

•Acute (less than 3 weeks): DAIR possible (50-70% success)
•Chronic (greater than 3 weeks): Implant removal required (mature biofilm)
•Two-stage exchange: 80-90% success (gold standard for chronic)
•Suppression without removal: Less than 20% success (eventual failure)
•Biofilm-active antibiotics: Rifampicin (best), fluoroquinolones, linezolid
•Rifampicin: NEVER monotherapy (resistance in 48h), always combine

Diagnostic Techniques

•5-7 tissue samples (MSIS criteria), ≥2 positive same organism = infected
•Sonication of explanted implant: Increases yield 20-30% (greater than 50 CFU/mL = infected)
•Extended incubation: 7-14 days for slow-growing biofilm bacteria
•16S rRNA PCR: Culture-independent, detects bacteria in culture-negative
•Culture-negative rate: 10-30% (prior antibiotics, dormant bacteria, biofilm)

Prevention Strategies

•Antibiotic prophylaxis: Within 60 minutes before incision (optimal 30 min)
•Prevents initial attachment (Stage 1), given BEFORE contamination
•Cefazolin 2g IV standard, redose if surgery greater than 4 hours
•Patient optimization: HbA1c less than 7.5%, smoking cessation, BMI less than 40
•Surgical technique: Minimize time, gentle handling, copious irrigation
•Antibiotic cement, silver coatings (research/emerging)

Key Numbers and Thresholds

•1000x: Increased MIC in biofilm vs planktonic
•24-48h: Irreversible biofilm attachment established
•3 weeks: Threshold for acute vs chronic PJI (DAIR vs exchange)
•0.1-1%: Persister cell frequency in biofilm
•50 CFU/mL: Sonication fluid threshold for infection (Trampuz)
•50-70%: DAIR success if acute (less than 3 weeks)
•80-90%: Two-stage exchange success for chronic PJI

Extracellular Polymeric Substance (EPS) Matrix

Composition (by weight):

Water: 80-90% of biofilm volume
Polysaccharides: 40-50% of dry weight (structural backbone)
eDNA (extracellular DNA): 10-20% of dry weight
Proteins: 20-30% of dry weight (enzymes, adhesins)
Lipids: 5-10% of dry weight
Bacterial cells: Only 10-15% of biofilm volume

Polysaccharides:

PIA (polysaccharide intercellular adhesin): S. epidermidis, encoded by ica genes
PNAG (poly-N-acetylglucosamine): Staphylococcus species
Alginate: Pseudomonas aeruginosa
Pel and Psl: Pseudomonas aeruginosa
Functions: Structural scaffold, adhesion, protection from desiccation and immune cells

Extracellular DNA (eDNA):

Released from lysed bacteria or actively secreted
Provides structural support (scaffolding)
Binds cationic antibiotics (aminoglycosides, polymyxins) - reduces penetration
Contains antibiotic resistance genes (horizontal gene transfer within biofilm)
DNase treatment can disrupt young biofilms (research application)

Proteins:

Adhesins: Bind to host proteins and implant surfaces
Enzymes: Beta-lactamases, proteases, nucleases
Amyloid fibrils: Structural support (Staphylococcus, E. coli)

ica Genes in Staphylococcus

The EPS matrix is the key protective element of biofilm.

Mechanism

Description

Effect

Clinical Implication

EPS barrier

Matrix blocks antibiotic diffusion

Reduced penetration to depths

Outer bacteria killed, inner survive

eDNA binding

eDNA binds cationic antibiotics

Aminoglycosides, polymyxins sequestered

Higher doses cannot overcome

pH gradients

Acidic microenvironments (pH 5-6)

Many antibiotics less active at low pH

Fluoroquinolones, aminoglycosides impaired

Enzyme degradation

Beta-lactamases concentrated in biofilm

Penicillins, cephalosporins destroyed

Even susceptible strains protected

Slow growth rate

Nutrient limitation slows division

Antibiotics target dividing cells

Dormant bacteria not killed

Persister cells

0.1-1% dormant, non-growing

Tolerant to ALL antibiotics

Cause relapse, require removal

Altered gene expression

Biofilm-specific genes upregulated

Efflux pumps, stress responses

Phenotypic resistance

Scenario

Biofilm Status

Treatment

Success Rate

Acute (less than 3 weeks)

Immature biofilm

DAIR + antibiotics + rifampicin

50-70%

Chronic (greater than 3 weeks)

Mature biofilm

Two-stage exchange

80-90%

Chronic (suppression)

Mature biofilm

Antibiotics alone (no surgery)

less than 20% (eventual failure)

Zone	Oxygen	Nutrients	Bacterial State	Antibiotic Susceptibility
Outer (aerobic)	High	Abundant	Actively dividing	Moderate susceptibility
Middle (microaerobic)	Moderate	Moderate	Slow growth	Low susceptibility
Deep (anaerobic)	Low/none	Depleted	Dormant (persisters)	No susceptibility

Technique	Sensitivity	Advantage	Limitation
Tissue cultures (5-7 samples)	70-80%	Standard, widely available	Misses dormant bacteria
Sonication	85-95%	Disrupts biofilm, higher yield	Requires explanted implant
Extended incubation (7-14 days)	75-85%	Detects slow-growers	Delay in diagnosis
16S rRNA PCR	85-95%	Culture-independent	Expensive, not widely available
Alpha-defensin	80-97%	Rapid (less than 1 hour)	False positives (metallosis, crystals)

Zone	Oxygen	Nutrients	Bacterial State	Antibiotic Susceptibility
Outer (aerobic)	High	Abundant	Actively dividing	Moderate susceptibility
Middle (microaerobic)	Moderate	Moderate	Slow growth	Low susceptibility
Deep (anaerobic)	Low/none	Depleted	Dormant (persisters)	No susceptibility

Technique	Sensitivity	Advantage	Limitation
Tissue cultures (5-7 samples)	70-80%	Standard, widely available	Misses dormant bacteria
Sonication	85-95%	Disrupts biofilm, higher yield	Requires explanted implant
Extended incubation (7-14 days)	75-85%	Detects slow-growers	Delay in diagnosis
16S rRNA PCR	85-95%	Culture-independent	Expensive, not widely available
Alpha-defensin	80-97%	Rapid (less than 1 hour)	False positives (metallosis, crystals)

Biofilm Formation in Orthopaedic Infections

Biofilm Formation in Orthopaedic Infections

BIOFILM FORMATION

STAGES OF BIOFILM FORMATION

Critical Must-Knows

Examiner's Pearls

Clinical Imaging

Imaging Gallery

Critical Biofilm Exam Points

Biofilm Definition

1000x Antibiotic Resistance

Implant Removal Necessary

Persister Cells

BIOFILMBIOFILM - Stages and Characteristics

MATRIXMATRIX - Components of Biofilm EPS

PERSISTPERSIST - Why Biofilm Bacteria Survive Antibiotics

Overview

Prosthetic Joint Infections

Chronic Osteomyelitis

Stages of Biofilm Formation

Biofilm Development Timeline

DAIR Window

Biofilm Structure and Composition

Extracellular Polymeric Substance (EPS) Matrix

Three-Dimensional Biofilm Architecture

Biofilm Zones and Bacterial State

Persister Cells - The Achilles' Heel of Treatment

Persister Problem

Quorum Sensing - Bacterial Communication

Mechanisms of Antibiotic Resistance in Biofilm

Biofilm Antibiotic Resistance Mechanisms

Physical Barriers

Metabolic Dormancy

Clinical Implications in Orthopaedics

Biofilm in Prosthetic Joint Infections

Treatment Options by Infection Timing

Biofilm in Chronic Osteomyelitis

Surgical Debridement Essential

Preventing Biofilm Formation

Diagnosing Biofilm Infections

Diagnostic Techniques for Biofilm Infections

Evidence Base

Biofilm Formation on Biomaterials

Sonication Improves Culture Yield in PJI

Rifampicin for Staphylococcal PJI

DAIR Timing and Success

Biofilm Viva Scenarios

Scenario 1: Define Biofilm and Antibiotic Resistance (~3 min)

Scenario 2: Biofilm in PJI Treatment (~4 min)

Management Algorithm

BIOFILM FORMATION

Core Definition

Biofilm Formation Stages

EPS Matrix Composition

Antibiotic Resistance Mechanisms

Persister Cells (Critical Concept)

Clinical Implications - PJI

Diagnostic Techniques

Prevention Strategies

Key Numbers and Thresholds

Biofilm Formation in Orthopaedic Infections

Biofilm Formation in Orthopaedic Infections

BIOFILM FORMATION

STAGES OF BIOFILM FORMATION

Critical Must-Knows

Examiner's Pearls

Clinical Imaging

Imaging Gallery

Critical Biofilm Exam Points

Biofilm Definition

1000x Antibiotic Resistance

Implant Removal Necessary

Persister Cells

BIOFILMBIOFILM - Stages and Characteristics

MATRIXMATRIX - Components of Biofilm EPS

PERSISTPERSIST - Why Biofilm Bacteria Survive Antibiotics

Overview

Prosthetic Joint Infections

Chronic Osteomyelitis

Stages of Biofilm Formation