BK - Biofilm physiology and antibiotic resistance I Flashcards
Differentiate between intrinsic and acquired antibiotic resistance, and antibiotic tolerance.
Intrinsic resistance: Independent of antibiotic selective pressure and horizontal gene transfer. Results from inherent structural or functional characteristics
Acquired resistance: Arises from mutations in drug targets or via transfer of resistance genes (e.g. through phage-mediated transduction or mobile plasmids)
Antibiotic Tolerance: Linked to adaptation (planktonic vs. biofilm growth, presence of persister cells). May or may not involve mutations in target genes. Helps bacteria survive during infections
What is Horizontal Gene Transfer (HGT) in bacteria?
- Process by which bacteria exchange genetic material
- Promoted in biofilms, enhancing the spread of antibiotic resistance
What are the three main mechanisms of HGT?
- Transformation – Uptake and integration of naked DNA
- Transduction – DNA transfer via bacteriophages
- Conjugation – Direct transfer through a physical connection (pilus)
How was Intrinsic Transformation demonstrated in Streptococcus pneumoniae? (2)
- Virulent but killed S. pneumoniae cells added to nonvirulent cultures induced virulence
- Competent cells incorporated foreign DNA via recombination, altering their genotype
What did Vitkovitch (2004) show about transformation in biofilms? (3)
- Biofilm-grown Streptococcus mutans transformed to erythromycin resistance by the addition of naked DNA or heat killed donor cells carrying the antibiotic resistance genotype.
- Transformation rates were 10–600 times higher than in planktonic cultures
- The biofilm matrix did not impede DNA penetration
How do Membrane Vesicles contribute to genetic transfer? (3)
- Released from Gram-negative (and some Gram-positive) bacteria
- Carry DNA (including resistance determinants) along with proteins and polysaccharides
- Fuse with recipient cells to transfer genetic material across species—and possibly kingdoms
What are some features of conjugation? (5)
- Predominantly involves gram negative bacteria
- Both fimbriae and pili appendages are made of protein
- Fimbriae: to surfaces (including host cells) and other bacteria
- Pili: to other bacteria for exchanging DNA (“sex”)
- Study demonstrated transfer of a tetracycline resistance gene between Bacillus subtilis and a sensitive Staphylococcus species within a dual-species biofilm (Intergeneric Conjugation)
What are some antibiotic resistance mechanisms? (5)
- Reduced permeability of the cell envelope
- Increased efflux activity
- Mutations in antibiotic targets
- Enzymatic modification or inactivation of the drug
- Biofilm formation, which creates a physical barrier
What are the Genotypic, Phenotypic and Physical mechanisms of resistance to antimicrobials?
Genotypic Resistance:
- Acquisition of resistance genes (e.g. tetracycline resistance)
Phenotypic Resistance:
e.g.
- Activation of the marRAB locus: MarA up-regulates the AcrAB-TolC efflux pump and down-regulates the OmpF porin, reducing drug entry while increasing export
- Formation of quorum sensing communities: Creates infection hotspots and supports intracellular infections (e.g. within macrophages)
- Global stress responses: Involves sigma factors and chaperones that help bacteria survive stress
- Biofilm formation
- Slow in vivo growth: Results in slower turnover of antibiotic targets (e.g. penR and sigma factor expression)
Physical Resistance:
- Exopolysaccharide production (slime) by biofilms “shields” susceptible cells e.g. to aggressive oxidant biocides
What roles do Multidrug Efflux Transporters play in resistance? (3)
- Actively pump antibiotics out of bacterial cells
- Confer intrinsic, low-level resistance and pave the way for higher-level biofilm resistance
- Their expression is regulated by the antibiotics they remove, leading to a multidrug resistance phenotype
What are the main families of bacterial multidrug efflux pumps?
H+/drug antiporters
- Resistance-nodulation-cell division (RND; Gram-negative bacteria)
- Major facilitator superfamily (MF or MFS)
- Small multidrug resistance (SMR)
Na+/drug antiporters
- Multidrug and toxic compound extrusion (MATE) family; which uses the sodium gradient
ATP hydrolysis-linked drug transporters
- ATP-binding cassette (ABC); which use ATP hydrolysis for energy
First four groups also known as secondary transporters, use the pre-stored energy of chemical gradients across the membrane
Are biofilms thick films?
- Indeed, oxidising biocides such as chlorine bind to the outer EPS layers and diffusion is limited.
- However, biofilm has water channels and antibiotic diffusion into the microcolonies is only partially reduced
Not a thick film
What role do P. aeruginosa rhamnolipid surfactants play in biofilm formation and maintenance? (4)
- Biosurfactants produced by P. aeruginosa
- Not essential for initial biofilm/channel formation but crucial for maintaining open channels around macrocolonies
- Likely function by modulating cell–cell interactions and bacterial attachment
- Production is quorum-sensing-dependent, induced at high cell densities to coordinate channel maintenance
What did studies using Rhodamine B reveal about biofilm diffusion? (3)
- Rhodamine B (fluorescent stain), similar in size to some antibiotics, reached the center of a biofilm microcolony within about 5 minutes
- Its effective diffusion rate was approximately 15% of that in pure water
Antibiotic Penetration: Even though the biofilm slowed down the diffusion of rhodamine B, it still penetrated the cell cluster relatively quickly.
How does the Biofilm Structure influence antibiotic penetration? (4)
- Biofilms are mostly water and solutes the size of most biocides and antibiotics can diffuse in the biofilm.
- They do not move as fast as they would in pure water because the cells, EPS, and other constituents of the biofilm hinder their mobility.
- Solutes typically diffuse at about 20–50% of their rate in water
- Water channels and rhamnolipid-maintained pathways (e.g. in Pseudomonas aeruginosa) help sustain some diffusion
What are the key Adaptive Mechanisms of Biofilm Physiology?
- Electrochemical interactions: EPS production, adhesion via DLVO forces, and lectin formation
- Co-aggregation: Clustering with pioneer species through lectin induction
- Maturation: Cell-density dependent quorum sensing using signaling molecules (e.g. homoserine lactones, AI-2, peptides)
How does the DLVO Theory relate to biofilm formation? (2)
- Describes the balance between electrostatic repulsion and van der Waals attraction among particles
- Bacteria overcome repulsive forces (using appendages) to attach to surfaces and form biofilms
What are some additional Adaptive Mechanisms of Biofilm Physiology?
Microenvironment colonization:
- Colonization by microaerophiles and anaerobes via passive convection or chemotactic movement
Predator Grazing and Chemotaxis:
- Interactions with protozoa, nematodes, and macrophages that influence biofilm dynamics
Disaggregation:
- Passive: Sloughing due to high shear forces or release of aged/dead cells
- Active: Release of daughter cells driven by nutritional status, undocking mechanisms, flagella induction, and chemotaxis
How does Biofilm Heterogeneity impact bacterial physiology? (3)
- Creates microenvironments with variable oxygen levels, pH, and nutrient availability
- Supports niches for anaerobes and microaerophiles
- Influences processes such as corrosion, nutrient attraction, and repulsion of disinfectants
In what ways do Altered Physiology and Resilience contribute to antibiotic resistance in biofilms? (3)
- Activation of global stress responses (e.g. σ38, chaperones, catalase); attachment/detachment, starvation, temperature, disinfection, oxidative stress
- Quorum sensing
- Reduced growth rates and slower cell wall turnover; Modifications in cell wall structure, porin expression, and binding proteins
These changes collectively enhance the survival of bacteria in the presence of antibiotics
