Week 14

Methods in Microbial Ecology (II)

1 sub-topics · Pages 572–585

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1. Introduction

📖 Lecturer's Note

Biofilms are the dominant mode of microbial life in natural and engineered environments. Their formation is coordinated by quorum sensing — a cell-density-dependent communication system in which bacteria release and detect small signalling molecules (AHLs in Gram-negatives, oligopeptides in Gram-positives) to collectively regulate gene expression. Understanding biofilm development and dispersal, diversity metrics (Shannon H', Simpson D), and the ecological principles governing structured communities is essential for managing biofilm-related problems in water systems, medical devices, and industrial processes.

✏️ Fill in the Blank

1. A structured community of microorganisms attached to a surface and encased in a self-produced extracellular matrix is called a _______.

Show Answer Biofilm

2. The mechanism by which bacteria sense population density and coordinate group behaviour through secreted signal molecules is called _______.

Show Answer Quorum sensing

3. The primary signal molecules used in quorum sensing of Gram-negative bacteria are N-acyl _______ lactones (AHLs).

Show Answer Homoserine

4. Subpopulations of non-growing bacteria within a biofilm that survive bactericidal antibiotic treatment without being genetically resistant are called _______ cells.

Show Answer Persister

5. The Shannon diversity index formula is H' = −Σ(pᵢ ln pᵢ), where pᵢ is the _______ abundance of species i.

Show Answer Relative (proportional)

6. The technique that measures lipid biomarkers extracted from environmental samples to estimate microbial biomass and community structure is called _______ analysis.

Show Answer PLFA (phospholipid fatty acid)

7. A microbial community that attaches to surfaces and is embedded in an extracellular polymeric substance (EPS) matrix is called a _______.

Show Answer Biofilm

8. The process of introducing microorganisms with known degradation capabilities into a contaminated site is called _______.

Show Answer Bioaugmentation

🔘 Multiple Choice

1. Biofilms are significantly more resistant to disinfectants than planktonic (free-floating) cells because:

  • A) Biofilm cells grow faster and produce more protective proteins
  • B) The extracellular matrix limits diffusion of disinfectants; slow-growing persister cells tolerate chemical stress; altered gene expression in biofilm mode
  • C) Biofilm cells are genetically resistant to all antibiotics
  • D) Disinfectants are quickly neutralised by the solid surface
Show Answer Correct: B) The extracellular matrix limits diffusion of disinfectants; slow-growing persister cells tolerate chemical stress; altered gene expression in biofilm mode

2. The Shannon diversity index (H') takes into account:

  • A) Only the number of species (richness)
  • B) Only the relative abundance of the most dominant species
  • C) Both the number of species (richness) and the evenness of their distribution
  • D) The phylogenetic distance between species
Show Answer Correct: C) Both the number of species (richness) and the evenness of their distribution

3. In quorum sensing, N-acyl homoserine lactones (AHLs) are used as signalling molecules by:

  • A) Gram-positive bacteria only
  • B) Gram-negative bacteria (as their primary QS signal class)
  • C) All bacteria and archaea equally
  • D) Only biofilm-forming eukaryotic microorganisms
Show Answer Correct: B) Gram-negative bacteria (as their primary QS signal class)

4. The extracellular polymeric substance (EPS) matrix of biofilms serves all of the following functions EXCEPT:

  • A) Structural support and cohesion of the biofilm community
  • B) Diffusion barrier limiting access of antimicrobials
  • C) Nutrient storage and enzyme concentration near cells
  • D) Direct energy generation via EPS fermentation for all biofilm cells
Show Answer Correct: D) Direct energy generation via EPS fermentation for all biofilm cells

5. Quorum sensing inhibition (QSI) is considered a promising anti-virulence strategy. The main advantage over conventional antibiotics is:

  • A) QSI compounds kill bacteria more rapidly than antibiotics
  • B) By targeting virulence rather than growth, QSI exerts weaker selection pressure for resistance and may be tolerated by the host microbiome
  • C) QSI compounds are effective against all bacterial species without dose limitation
  • D) QSI blocks all bacterial communication, preventing any evolution of resistance mechanisms
Show Answer Correct: B) By targeting virulence rather than growth, QSI exerts weaker selection pressure for resistance and may be tolerated by the host microbiome

6. Biofilm formation in Pseudomonas aeruginosa in CF patient lungs is clinically significant because:

  • A) Biofilm P. aeruginosa can be eradicated by standard doses of tobramycin given orally
  • B) Chronic biofilm infection triggers persistent inflammation, lung damage, and the biofilm is highly tolerant to antibiotics and host immune clearance
  • C) P. aeruginosa biofilm produces exotoxins that directly damage the bronchial epithelium without causing inflammation
  • D) Biofilm formation occurs only in immunocompromised patients and resolves with restoration of immunity
Show Answer Correct: B) Chronic biofilm infection triggers persistent inflammation, lung damage, and the biofilm is highly tolerant to antibiotics and host immune clearance

7. Evenness (Pielou's J) approaches 1.0 when:

  • A) Species richness is maximised regardless of abundance distribution
  • B) All species in the community are present in approximately equal abundances
  • C) Only a single species dominates the community
  • D) The Simpson index equals the Shannon index
Show Answer Correct: B) All species in the community are present in approximately equal abundances

8. The four stages of biofilm formation in correct order are:

  • A) Maturation → Attachment → EPS production → Dispersal
  • B) Reversible attachment → Irreversible attachment → Maturation → Dispersal
  • C) Dispersal → Attachment → EPS production → Maturation
  • D) Attachment → Dispersal → EPS production → Maturation
Show Answer Correct: B) Reversible attachment → Irreversible attachment → Maturation → Dispersal

9. In Vibrio fischeri, quorum sensing controls bioluminescence. At high cell density:

  • A) AHL concentration decreases, repressing luciferase gene expression
  • B) AHL accumulates → binds LuxR transcription factor → LuxR-AHL complex activates lux operon → luciferase produced → bioluminescence
  • C) AHL is degraded by host enzymes, signalling V. fischeri to reduce virulence
  • D) Bioluminescence is constitutively expressed regardless of cell density
Show Answer Correct: B) AHL accumulates → binds LuxR transcription factor → LuxR-AHL complex activates lux operon → luciferase produced → bioluminescence

10. Which method would be most appropriate for in situ visualisation of the spatial arrangement of different microbial species within a biofilm?

  • A) Plate count on selective media
  • B) CLSM (confocal laser scanning microscopy) combined with FISH using species-specific fluorescent probes
  • C) 16S amplicon sequencing of biofilm homogenate
  • D) Transmission electron microscopy of unstained biofilm sections
Show Answer Correct: B) CLSM (confocal laser scanning microscopy) combined with FISH using species-specific fluorescent probes

11. NanoSIMS (nanoscale secondary ion mass spectrometry) is used in microbial ecology to:

  • A) Amplify rare DNA sequences from single cells
  • B) Map the distribution of stable isotopes at nanometre resolution within individual cells
  • C) Measure optical density of microbial suspensions
  • D) Determine 16S rRNA gene sequences at single-cell resolution
Show Answer Correct: B) Map the distribution of stable isotopes at nanometre resolution within individual cells

12. Which statistical ordination method is most commonly used to visualise differences in microbial community composition based on Bray-Curtis dissimilarity?

  • A) Principal Component Analysis (PCA)
  • B) Non-metric Multidimensional Scaling (NMDS)
  • C) Linear discriminant analysis (LDA)
  • D) ANOVA
Show Answer Correct: B) Non-metric Multidimensional Scaling (NMDS)

13. Raman microspectroscopy of single bacterial cells can reveal:

  • A) The complete genome sequence of the cell
  • B) Chemical composition (e.g., lipids, nucleic acids, proteins) and metabolic activity indicators without labelling
  • C) The expression level of all genes simultaneously
  • D) The phylogenetic lineage based on 16S rRNA fingerprint
Show Answer Correct: B) Chemical composition (e.g., lipids, nucleic acids, proteins) and metabolic activity indicators without labelling

14. In amplicon sequencing, alpha diversity refers to:

  • A) Differences in community composition between samples
  • B) Diversity within a single sample (richness and evenness)
  • C) The total diversity across all samples in a study
  • D) Phylogenetic distance between dominant taxa
Show Answer Correct: B) Diversity within a single sample (richness and evenness)

15. Catalysed reporter deposition FISH (CARD-FISH) is an improvement on standard FISH because it:

  • A) Does not require fluorescent dyes
  • B) Amplifies the fluorescent signal, allowing detection of low-ribosome-content (slow-growing) cells
  • C) Sequences the 16S gene within intact cells
  • D) Only works on Gram-positive bacteria
Show Answer Correct: B) Amplifies the fluorescent signal, allowing detection of low-ribosome-content (slow-growing) cells

16. The UniFrac metric used in beta diversity analysis is unique because it:

  • A) Counts only the number of shared OTUs between samples
  • B) Incorporates phylogenetic distances between taxa into the diversity measure
  • C) Normalises for differences in sequencing depth without rarefaction
  • D) Only measures evenness, not richness
Show Answer Correct: B) Incorporates phylogenetic distances between taxa into the diversity measure

💬 Open-Ended Questions

1. Describe the four stages of biofilm formation: initial attachment, irreversible attachment, maturation, and dispersal. At which stage is biofilm removal most feasible, and what strategies can be used?

Hint / Guidance Stage 1 (reversible attachment): surface conditioning film (proteins, polysaccharides); motile cells approach surface; initial contact by van der Waals/electrostatic forces + appendages (flagella, pili); removable by washing. Stage 2 (irreversible attachment): type IV pili retraction; EPS production (alginate in P. aeruginosa, Pel, Psl); quorum sensing activation; flagella downregulated. Stage 3 (maturation): 3D mushroom/tower structures with water channels; metabolic gradients (O₂ limitation deeper); subpopulation differentiation; maximum biomass. Stage 4 (dispersal): environmental cues (nutrient depletion, NO, cis-2-decenoic acid); cells revert to planktonic mode; EPS degradation (alginate lyase). Prevention most feasible: Stage 1 (anti-fouling coatings — quaternary ammonium, silver nanoparticles; surface hydrophobicity modification). Removal strategies: mechanical scrubbing; enzymatic (DNase, dispersin B, alginate lyase); QS inhibitors; D-amino acids (disrupt biofilm matrix).

2. How do pathogenic bacteria use quorum sensing to regulate virulence factor expression? Give two specific examples and explain the therapeutic potential of quorum sensing inhibition.

Hint / Guidance P. aeruginosa: las system (3-oxo-C12-HSL + LasR) activates elastase (lasB), alkaline protease (apr), exotoxin A, biofilm EPS; rhl system (C4-HSL + RhlR) activates rhamnolipid (destroys PMNs), pyocyanin (oxidative stress). QS is hierarchical: las activates rhl. Staphylococcus aureus: agr system (peptide-based QS — AIP activates RNAIII); at high density, RNAIII represses surface proteins (surface colonisation phase) and activates secreted toxins (α-toxin, TSST-1, Panton-Valentine leukocidin) and proteases (tissue invasion phase). QSI strategies: (1) AHL structural analogues (brominated furanones — competitive inhibitors of LuxR-type receptors); (2) Acylase/lactonase enzymes degrade AHLs; (3) Plant-derived compounds (garlic, quercetin); (4) AI-2 pathway inhibition (broad-spectrum). Advantage: anti-virulence approach — bacteria remain viable but non-virulent → less selection for resistance; host microbiome unharmed.

3. A community of microorganisms in a lake sediment has a Shannon index H' = 3.2, while an adjacent contaminated site has H' = 1.1. Interpret these values ecologically and explain what factors might account for the difference.

Hint / Guidance H' = 3.2: high richness + high evenness → mature, diverse community; resistant to perturbations (functional redundancy); representative of undisturbed sediment with many trophic guilds (methanogens, SRBs, fermenters, heterotrophs, chemolithotrophs). H' = 1.1: low diversity → highly uneven community dominated by 1–2 tolerant/specialist taxa; likely stress-selected. Contamination drivers: organic loading → dominance by fermenters; heavy metals → selection for metal-tolerant specialists (Desulfovibrio, Geobacter with metal reduction ability); petroleum hydrocarbons → Alcanivorax/Marinobacter bloom. Ecological consequences: loss of functional redundancy; ecosystem services impaired (nutrient cycling); recovery slow because diversity acts as insurance. Monitoring: H' as indicator of ecosystem health; legal thresholds in EU Water Framework Directive biological quality elements.

4. Describe the structural components of the EPS matrix in biofilms. How does each component contribute to biofilm stability and antimicrobial tolerance?

Hint / Guidance Exopolysaccharides: provide bulk structure and cohesion; can be charged (alginate, negative → binds cationic antibiotics); neutral (cellulose, Psl); act as carbon/energy reserve; P. aeruginosa overproducing alginate (mucoid phenotype) in CF lungs. Extracellular DNA (eDNA): released by cell lysis; backbone of biofilm structure; negatively charged → chelates cations disrupting AMPs; DNASE I disperses biofilms. Proteins: adhesins (BapA); enzymes (amylases, lipases) for nutrient acquisition; structural proteins (CdrA cross-links Psl). Lipids: outer membrane vesicles contribute to matrix; surfactants (rhamnolipids of P. aeruginosa). Ions: Ca²⁺, Mg²⁺ cross-link EPS chains providing mechanical stability. Together: diffusion limitation (Cl₂, tobramycin concentration drops >10-fold into biofilm); micro-niches (pH, O₂ gradients → persister formation in deep zones); cooperative/competitive metabolic interactions.

5. Explain how confocal laser scanning microscopy (CLSM) combined with fluorescent dyes and genetic reporters is used to study biofilm structure and physiology.

Hint / Guidance CLSM: collects optical sections at different z-planes → 3D reconstruction without physical sectioning; avoids artifacts. Fluorescent tools: (1) FISH probes — identify specific taxa in situ; (2) GFP/RFP reporters — gene promoter::GFP fusion reveals when/where genes expressed (e.g., algD::GFP reports alginate production zones); (3) Live/dead staining (SYBR Green/PI) — membrane integrity indicator; (4) Redox-sensitive dyes (resazurin → resorufin) — metabolic activity mapping; (5) Lectin staining — specific polysaccharide components of EPS. Quantitative analysis: COMSTAT software measures biofilm thickness, surface area, roughness, volume-to-biomass ratio. Limitations: fluorescent probes may not penetrate deep into mature biofilms; fixation may alter structure; limited depth penetration (~100 µm). Super-resolution (STED, STORM) now enabling nanoscale biofilm architecture.

6. What is meant by 'community succession' in microbial ecology? Describe primary succession in a newly cooled volcanic lava field as an example.

Hint / Guidance Succession: directional change in community composition over time as organisms modify their environment, facilitating arrival of new colonists. Primary succession: colonisation of bare, sterile substrate. Lava field example: (1) Pioneer colonisers: cyanobacteria (Chroococcidiopsis, Gloeocapsa) — photosynthesis + N₂ fixation; tolerate UV, desiccation; produce EPS → begin soil formation. (2) Heterotrophs: use cyanobacterial EPS/cells; Gram-positive spore-formers (Bacillus), Actinobacteria. (3) Lichens (fungal-cyanobacterial/algal symbiosis): physical weathering of rock; mineral release. (4) Mosses/ferns: with Rhizobium-like associates; further soil organic matter accumulation. (5) Higher plants → complex rhizosphere microbiome. Facilitation mechanism: each stage modifies pH, nutrient content, moisture retention, making environment suitable for next successional stage. Final climax community determined by local climate.

7. Explain why biofilm infections on medical devices (catheters, prosthetic valves, implants) are so difficult to treat. What prevention strategies are used in clinical practice?

Hint / Guidance Why difficult: (1) 100–1000× MIC required for biofilm killing vs. planktonic MIC — achievable only with toxic doses systemically; (2) Persister cells survive any antibiotic exposure; (3) Regrowth from small numbers of survivors; (4) Immune evasion: biofilm protects from phagocytosis, complement; (5) Genetic diversity within biofilm → antibiotic-resistant mutants selected; (6) Biofilm acts as septic focus shedding planktonic cells. Device-associated infections: catheter-associated UTI (Staphylococcus epidermidis, E. coli), CLABSI (central line-associated bloodstream infection), prosthetic valve endocarditis (S. aureus). Prevention: (1) Antimicrobial-impregnated catheters (silver, chlorhexidine); (2) Antibiotic lock therapy for CLABSI; (3) Surface modification (anti-adhesion coatings, hydrophilic polymers); (4) Strict aseptic technique during insertion; (5) Minimise duration of device use (bundle approach); (6) Anti-biofilm enzymes in device coatings (DNase, dispersin B). Treatment often requires device removal.

8. Compare the Simpson diversity index (D) and Shannon index (H') in terms of their calculation, sensitivity, and ecological interpretation.

Hint / Guidance Simpson D: probability that two randomly chosen individuals belong to different species. D = 1 − Σ(nᵢ(nᵢ−1)/N(N−1)). Range 0–1; 1 = maximum diversity. Sensitive to abundant species (dominant species have large effect). Shannon H': H' = −Σ(pᵢ ln pᵢ). Range 0–ln(S); higher = more diverse. Based on information theory; more sensitive to rare species than Simpson. Comparison: H' gives more weight to rare species presence; Simpson gives more weight to dominant species. For highly uneven communities: Simpson better reflects dominance; Shannon better reflects total diversity including rare taxa. Evenness: Pielou's J = H'/ln(S); normalises Shannon for different richness; comparable between sites. When to use each: H' preferred in ecology for general diversity; Simpson preferred when concerned with ecological dominance. Modern preference: often report both; also report Faith's Phylogenetic Diversity for phylogeny-aware comparison.

9. What is the 'Anna Karenina principle' in host microbiome research and how does it relate to dysbiosis?

Hint / Guidance Anna Karenina principle (AKP) in microbiome science (Zaneveld et al. 2017): 'All healthy microbiomes are alike; each dysbiotic microbiome is dysbiotic in its own way' (paraphrasing Tolstoy's novel). Concept: healthy microbiomes across individuals are more similar in composition than diseased microbiomes; disease/stress increases stochastic community assembly (unpredictable, dispersal-limited outcomes) vs. deterministic assembly in healthy state. Evidence: gut microbiome of healthy individuals clusters tightly in ordination space; IBD, obesity, antibiotic-treated patients show highly variable, dispersed microbiomes. Mechanism: perturbation disrupts stable attractors → community falls into different alternative stable states depending on chance colonisation events. Relevance: explains why FMT works for C. difficile (restores deterministic healthy state) but inconsistent for complex diseases (IBD, obesity) — different patients may need different communities. Beta diversity dispersion (betadisper) tests AKP in real datasets.

10. What is single-cell genomics (SCG) and how does it complement metagenomics for studying uncultured microorganisms?

Hint / Guidance SCG: (1) individual cells isolated by flow cytometry or micromanipulation; (2) whole-genome amplification (MDA using phi29 polymerase or MALBAC); (3) library preparation + sequencing. Produces single-amplified genomes (SAGs). Complements metagenomics: SCG gives complete (or near-complete) genome of single organism without binning ambiguity; confirms gene associations within one cell (metagenomics cannot); can resolve strain-level variation. Limitations: MDA amplification bias (uneven coverage, chimeras); low cell input; contamination risk; typically <70% complete. Used to characterise Candidate Phyla Radiation (CPR), DPANN Archaea, SAR11 clade organisms.

11. How is flow cytometry used in microbial ecology? What parameters can be measured and how does it help overcome limitations of culture-based methods?

Hint / Guidance Principle: cells pass single-file through laser beam; scatter (size, granularity) and fluorescence detected per cell at thousands/second. Parameters: cell size (forward scatter); complexity (side scatter); nucleic acid content (SYBR Green/DAPI staining); viability (propidium iodide — only penetrates dead cells); specific taxa (fluorescent antibodies); actively respiring cells (CTC dye); GFP-reporter strains. Advantages over culture: counts all cells in minutes; distinguishes viable/non-viable; works on non-culturable organisms; can sort populations for downstream analysis (FACS); detects phytoplankton by autofluorescence. Limitations: no species ID without specific probes; cannot detect intracellular metabolism directly.

12. Describe how isothermal microcalorimetry can be used to study microbial activity in complex environmental samples. What advantages does it offer over conventional methods?

Hint / Guidance Principle: measures heat flow (µW to mW) from metabolic reactions in real time; all metabolic reactions produce or consume heat; no sample pre-treatment required; can use intact soil, sediment, gut content, biofilms. Parameters: total heat production (J/g organic matter); growth rate (from thermogram shape); inhibition/stimulation by compounds. Advantages: label-free, non-invasive, works on opaque/turbid samples; real-time continuous monitoring; detects dormant cells reactivation; applicable to mixed communities. Applications: soil organic matter decomposition rates; biofilm antimicrobial testing; microbial activity in extreme environments; food spoilage monitoring. Limitation: cannot identify which organisms are active; low sensitivity for very low biomass samples.

13. What is environmental transcriptomics (metatranscriptomics) and what does it reveal that metagenomics cannot?

Hint / Guidance Metatranscriptomics: extract total RNA from environment → deplete rRNA → reverse transcribe → sequence → map to reference genomes or metagenome. Reveals: actively transcribed genes at time of sampling (not just what's there but what's doing); identifies expressed metabolic pathways, stress responses, growth phases; detects active viruses (RNA viruses); reveals niche partitioning (different community members express different pathways in same habitat). What metagenomics misses: dormant organisms have genomes but low transcription; regulation at RNA level (riboswitches, sRNAs) not captured by DNA; metatranscriptomics shows gene expression dynamics across diel cycles (ocean), seasons (soil), microhabitats. Challenges: RNA degrades rapidly; rRNA dominates (>95%) requiring depletion; snapshot in time; requires large sample volumes.

14. Explain how microfluidics and lab-on-chip technologies are transforming single-cell microbiology. Give two specific applications.

Hint / Guidance Microfluidics: manipulate nanolitre–picolitre volumes in microfabricated channels (PDMS chips); enable high-throughput single-cell isolation, culture, and analysis. Applications: (1) Droplet microfluidics for cultivation — cells encapsulated in water-in-oil droplets (~10 pL); each droplet = independent micro-fermentation vessel; screen millions of conditions; isolate previously unculturable organisms by providing diffusible co-culture factors (ichip concept related); (2) Single-cell whole-genome sequencing — microfluidic chips (Fluidigm C1) capture individual cells, lyse, amplify WGA in isolated chambers, prepare sequencing libraries; removes contamination risk of bulk WGA; produces thousands of SCGs per run. Future: integrated chips combining cell lysis, amplification, sequencing, and metabolite detection on single device.
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