L14 I&I Biofilm formation and Cystic Fibrosis

Overview

This lecture covers what a biofilm is and how it forms, why biofilms matter medically (chronic infection, antibiotic tolerance), and then applies this to cystic fibrosis (CF): the CFTR defect that predisposes CF airways to infection, how the infecting organisms and their behaviour change over a patient’s life, and how CF lung infections are monitored, treated and controlled for transmission.

What Is a Biofilm?

• Planktonic bacteria are free-living, individual cells.
• A biofilm is a community of bacteria embedded in a self-produced matrix of extracellular polymeric substance (EPS): polysaccharides, proteins, lipids and extracellular DNA, adherent to each other and/or to a surface. If host-associated, the biofilm also incorporates host-produced fibrin and mucus.
• Biofilm formation sequence: (1) planktonic cells adhere to a surface, (2) they produce EPS, (3) the structure becomes increasingly complex, (4) it reaches maximum cell density, (5) it releases microcolonies that can migrate and seed new surfaces elsewhere.
• Two settings for this process: host-material embedded (e.g. CF airway mucus, wounds) and surface-attached (e.g. implants, catheters). In both, fast-growing/antibiotic-susceptible bacteria predominate early and slow-growing/tolerant bacteria predominate once the biofilm is mature.
• Mature biofilms have internal channels that carry nutrients and metabolites through the structure, producing a heterogeneous population with differential access to nutrients and different metabolic activity/division rates in different regions.
• Biofilms behave like a fortress for their residents: protection against desiccation, immune response and antimicrobials; capture of external resources; and an internal community with social cooperation, reuse of internal resources and an enhanced rate of gene exchange.

Why Biofilms Matter Medically

• Biofilms are a significant virulence mechanism for organisms including Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli; EPS protects them from antimicrobials, host defences and environmental stress.
• About 80% of all microbial infections in humans involve biofilms.
• Chronic infections: chronic wound infections (P. aeruginosa, S. aureus); osteomyelitis, endocarditis, prostatitis, otitis media, UTIs; CF lungs (P. aeruginosa).
• Medical device infections: IV catheters, in-dwelling urinary catheters, pacemakers, heart valves, stents, orthopaedic implants.
• Biofilm growth is the predominant mode of bacterial growth in chronic infection; planktonic growth is a transitional phase used for spread.
• Biofilms are often thousands of times more tolerant of antimicrobials than planktonic cells, via three mechanisms: restricted drug penetration into the matrix; reduced growth rate (oxygen/nutrient-deprived microenvironments slow growth, and most antimicrobials target rapidly replicating cells); and persister cells, a dormant subpopulation with extreme antimicrobial tolerance. This is a phenotypic, not genotypic, change, and persisters can repopulate the biofilm and cause relapse after treatment appears to have worked.
• Traditional antimicrobial susceptibility testing on planktonic bacteria cannot predict susceptibility of the same organism in a biofilm: achievable serum drug concentrations from standard dosing exceed the MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) thresholds that apply to planktonic bacteria, but fall well short of the MBIC (minimal biofilm inhibitory concentration) and MBEC (minimal biofilm eradication concentration) thresholds needed to treat the same organism as a biofilm.
• Quorum sensing coordinates this behaviour: bacteria constantly produce autoinducer molecules, and autoinducer level rises with cell density. At a critical threshold (“quorum”), autoinducers trigger coordinated repression or activation of gene expression, including biofilm production. This unified response maintains optimal biofilm size and coordinates virulence phenotypes, so the biofilm behaves more like a multicellular organism than a population of independent cells.
• Summary properties of an established medical biofilm: hard to culture; hard to treat (both host-defence-resistant and antibiotic-resistant); chronic and relapsing.

Treating Clinical Biofilms

• Remove any biofilm-infected object where possible (implants, catheters): for central line-associated bloodstream infections caused by multidrug-resistant organisms, central venous catheter (CVC) removal is recommended in most cases and is associated with markedly better survival than leaving the catheter in place; catheters can sometimes be preserved for coagulase-negative Staphylococcus spp. and Corynebacterium spp. infections if the patient is stable.
• Antimicrobial treatment strategy differs by stage: early infection is treated aggressively; chronic infection (e.g. endocarditis, osteomyelitis) is treated with prolonged or suppressive antimicrobial courses.

Cystic Fibrosis Pathophysiology

• CF is caused by mutation in the CFTR gene, which encodes a cyclic-AMP-regulated chloride ion channel.
• The defect causes defective ion transport across epithelial cell surfaces, including in the airways, leading to reduced airway surface liquid.
• This impairs mucociliary clearance (the mechanism that clears particles and microbes trapped in mucus), producing greatly increased susceptibility to bacterial infection.
• In normal airway epithelium, a normal airway surface liquid layer, normally beating cilia and functioning HCO3- transport keep mucociliary transport working; in CF, dehydrated airway surface liquid, flattened epithelial cells with low ciliary beat frequency, reduced HCO3- transport and an adherent mucus plug (which can block submucosal gland ducts, with neutrophils present) result in slow/impaired mucociliary transport.

  The source slide diagram comparing normal vs CF airway epithelium is cropped at its right edge in the lecture render; some panel content on that side was not captured in the transcript.

• CF-related lung disease combines chronic bacterial infection with inflammation. Exacerbations drive a repeating cycle of infection -> inflammation -> obstruction, which produces progressive structural damage -> bronchiectasis -> pulmonary insufficiency -> respiratory failure, i.e. progressive decline in lung function over time.

Microbiology of CF Lung Infection

• Major CF pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia complex. Other organisms implicated: Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Haemophilus influenzae, non-tuberculous mycobacteria, anaerobic bacteria, viruses and fungi (particularly Aspergillus spp.). CF airway infections are frequently polymicrobial.
• The dominant pathogen changes with age: S. aureus and H. influenzae dominate in younger children, while P. aeruginosa prevalence rises steadily with age and becomes the dominant pathogen in older patients.
• P. aeruginosa infection in CF typically progresses through phases: initial acquisition of an environmental isolate, which can be eradicated with treatment; subsequent intermittent infections, during which genetic adaptation occurs and bacterial clearance becomes impaired; and eventually chronic infection with emergence of a dominant clone that continues to adapt to its host. Once chronic, sputum P. aeruginosa density plateaus above the limit of detection despite antibiotic treatment, rather than clearing as it did during the intermittent phase.
• Adaptations that P. aeruginosa makes to the CF airway: transition to a biofilm mode of growth; production of a mucoid coating that helps it evade phagocytosis; altered expression of virulence factors; and enhanced resistance to antibiotics.
• Conversion to the mucoid phenotype (production of an alginate matrix) worsens prognosis: it forms part of a feedback loop in which lung infection drives inflammation (increased neutrophils/macrophages, ROS production), which promotes mutation to the mucoid type, which in turn drives chronic inflammation (the patient produces antibodies against alginate, and immune complexes contribute to ongoing lung damage).
• Outcomes: chronic P. aeruginosa infection is an independent risk factor for accelerated loss of pulmonary function and decreased survival. Burkholderia cepacia complex infection carries an even worse prognosis than chronic P. aeruginosa infection and is a contraindication to lung transplant. MRSA infection is also associated with worse survival. Lung function still declines with age in CF, but successive treatment-era cohorts (1990 vs 2000 vs 2010) show progressively better lung function at a given age, reflecting improving management over time.

Treatment and Surveillance in CF

• Prophylactic antibiotics to prevent acquisition of P. aeruginosa or S. aureus are not recommended.
• Regular surveillance cultures (e.g. three-monthly) are used to detect P. aeruginosa acquisition, guide treatment of acute exacerbations, and detect non-tuberculous mycobacteria (screened yearly).
• At first isolation of P. aeruginosa, eradication is attempted with inhaled tobramycin for 28 days.
• For chronic P. aeruginosa infection, chronic treatment uses inhaled antibiotics (tobramycin, aztreonam or colistin) given cyclically (28 days on, 28 days off), moving to continuous therapy if the patient is deteriorating.
• Acute exacerbations are treated (intravenously or orally) against the bacteria known to be present.
• Additional supportive measures promote airway clearance and reduce bronchial obstruction and inflammation.
• Non-tuberculous mycobacteria (NTM) in CF are mostly Mycobacterium avium complex and M. abscessus complex, detectable in 10-20% of patients, and can cause progressive inflammatory lung damage (increased cough, sputum production, shortness of breath, deteriorating lung function tests). Diagnosis is by microbiological testing (annual screening, or if symptoms are consistent) and high-resolution CT (nodular infiltrates, cavities). Treatment is an initial 8-12 weeks of combination antimicrobials, then maintenance until sputum culture is negative for 12 months.

Transmission and Infection Control

• A whole-genome sequencing study of 168 consecutive M. abscessus isolates from 31 infected patients over 42 months at a large UK adult CF centre was used to determine whether the multidrug-resistant organism (which is increasingly infecting CF patients) is being transmitted person-to-person. Three patterns of relatedness were found: (1) large genetic differences between isolates from different individuals, consistent with independent acquisition from an environmental source; (2) isolates from different individuals that group together but remain clearly segregated from one another, interpreted as a successful clone (seen in an M. abscessus subsp. abscessus cluster of 13 patients); (3) isolates from different individuals with near-identical genomic sequences, consistent with direct patient-to-patient transmission (seen in two M. abscessus subsp. massiliense clusters totalling 15 patients).
• Respiratory pathogens can be transmitted between CF patients within the health care system: highly transmissible strains of P. aeruginosa (associated with increased antibiotic use), and transmission of Burkholderia species, MRSA and M. abscessus have all been documented.
• Infection control measures to reduce transmission: clinicians wear gowns and surgical masks; patients wear surgical masks in the health care setting; patients with CF should not congregate with one another within or outside the health care setting, and are given single rooms in hospital.

Self-test

1. Define a biofilm and explain how it differs from planktonic bacterial growth.
2. Describe the five steps of biofilm formation, from planktonic cells to microcolony release.
3. Explain why the internal channel structure of a mature biofilm produces a heterogeneous bacterial population.
4. List three ways in which biofilms give bacteria increased tolerance to antimicrobials.
5. What are persister cells, and why does the tolerance they confer relapse after treatment rather than being permanent?
6. Distinguish MIC/MBC from MBIC/MBEC, and explain why standard antimicrobial susceptibility testing underestimates the dose needed to treat a biofilm infection.
7. Describe how quorum sensing controls biofilm-related gene expression as bacterial density rises.
8. A patient with a multidrug-resistant central line-associated bloodstream infection has their catheter removed. Based on the lecture’s evidence, what effect would this be expected to have on survival, and why?
9. Describe the mechanism by which the CFTR mutation leads to increased susceptibility to bacterial infection in the CF airway.
10. Describe the cyclical relationship between infection, inflammation and obstruction that drives CF lung disease, and its downstream structural consequences.
11. How does the dominant pathogen causing CF airway infection change as a patient ages, and name the major pathogens involved.
12. Describe the three phases of P. aeruginosa infection in CF (initial, intermittent, chronic) and what changes bacteriologically between the intermittent and chronic phases.
13. Explain how conversion of P. aeruginosa to the mucoid phenotype worsens prognosis via the inflammation-mutation-chronic inflammation cycle.
14. Outline the surveillance and treatment strategy for P. aeruginosa in CF, from first isolation through to chronic infection.
15. What did the whole-genome sequencing study of M. abscessus isolates find, and what three patterns of genetic relatedness did it distinguish?
16. List three infection control measures used to reduce pathogen transmission between CF patients in healthcare settings.

Answers

Reveal answers

1. A biofilm is a community of bacteria embedded in a self-produced matrix of extracellular polymeric substance (polysaccharides, proteins, lipids, extracellular DNA), adherent to each other and/or a surface (plus host fibrin/mucus if host-associated); planktonic bacteria are free-living individual cells, not community-embedded.
2. (1) Planktonic cells adhere to a surface; (2) they produce EPS; (3) the structure becomes increasingly complex; (4) it reaches maximum cell density; (5) it releases microcolonies that migrate to seed new surfaces elsewhere.
3. The channels give differential access to nutrients and metabolites across the structure, so bacteria in different regions have different metabolic activity and rates of cell division, producing a heterogeneous rather than uniform population.
4. Any three of: restricted penetration of antimicrobials into the matrix; reduced growth rate in oxygen/nutrient-deprived microenvironments (most antimicrobials target rapidly replicating cells); and the presence of persister cells with extreme antimicrobial tolerance.
5. Persister cells are a dormant subpopulation with extreme antimicrobial tolerance due to a phenotypic (not genotypic) change. Because the change is phenotypic and the cells are simply dormant rather than killed, they can repopulate the biofilm once treatment stops, causing relapse.
6. MIC/MBC (minimum inhibitory/bactericidal concentration) describe susceptibility of planktonic bacteria; MBIC/MBEC (minimal biofilm inhibitory/eradication concentration) describe the much higher concentrations needed against the same organism growing as a biofilm. Standard dosing achieves serum concentrations above MIC/MBC but well below MBIC/MBEC, so susceptibility testing based on planktonic thresholds underestimates the dose actually needed to treat a biofilm infection.
7. Bacteria constantly produce autoinducer molecules; as cell density rises, autoinducer concentration rises with it. At a critical threshold (“quorum”), autoinducers trigger coordinated repression or activation of gene expression across the population, including genes for biofilm production, producing a unified, multicellular-like response.
8. Survival would be expected to be markedly better with catheter removal: the lecture’s data show cumulative survival staying near 1.0 when the CVC is removed, versus declining to around 0.55-0.6 by day 30 when it is not removed, because removing the catheter eliminates the protected biofilm reservoir of multidrug-resistant organisms.
9. CFTR mutation impairs the cyclic-AMP-regulated chloride channel, causing defective ion transport across airway epithelium and reduced airway surface liquid; this impairs mucociliary clearance of trapped particles and microbes, resulting in greatly increased susceptibility to bacterial infection.
10. Infection drives inflammation, which drives airway obstruction, which in turn promotes further infection, forming a repeating cycle; this cycle drives progressive structural damage, then bronchiectasis, then pulmonary insufficiency, then respiratory failure.
11. In younger children, S. aureus and H. influenzae dominate; as patients age, P. aeruginosa prevalence rises steadily and it becomes the dominant pathogen in older patients. Major pathogens overall: S. aureus, P. aeruginosa, Burkholderia cepacia complex.
12. Initial: acquisition of an environmental isolate, which can be eradicated with treatment. Intermittent: subsequent infections with genetic adaptation and impaired clearance. Chronic: emergence of a dominant clone that keeps adapting; the key bacteriological change is that sputum P. aeruginosa density plateaus above the limit of detection despite antibiotics, rather than clearing as it does in the intermittent phase.
13. Lung infection drives inflammation (increased neutrophils/macrophages, ROS production); this promotes mutation of P. aeruginosa to the mucoid type, which produces an alginate matrix; this drives chronic inflammation, as the patient produces antibodies against alginate and the resulting immune complexes cause ongoing chronic inflammatory lung damage, worsening prognosis.
14. Regular (e.g. three-monthly) surveillance cultures detect acquisition; at first isolation, eradication is attempted with inhaled tobramycin for 28 days; once infection becomes chronic, cyclical inhaled antibiotics (tobramycin, aztreonam or colistin, 28 days on/off, or continuous if deteriorating) are used long-term, alongside treatment of acute exacerbations targeted at the organisms known to be present.
15. It examined 168 M. abscessus isolates from 31 CF patients over 42 months to look for person-to-person transmission. It distinguished: (1) large genetic differences between isolates from different patients, consistent with independent environmental acquisition; (2) isolates that cluster together but remain segregated from other patients’ isolates, consistent with a successful clone (13 patients); (3) near-identical isolates from different patients, consistent with direct patient-to-patient transmission (15 patients, two clusters).
16. Any three of: clinicians wearing gowns and surgical masks; patients wearing surgical masks in health care settings; patients with CF not congregating with one another in or outside health care settings; single hospital rooms for CF patients.