Biofilm physiology and antimicrobial resistance Flashcards
(28 cards)
Intrinsic resistance
independent of antibiotic selective pressure and horizontal gene transfer; result of inherent structural or functional characteristics.
Acquired resistance
mutations in drug targets or transfer of resistance genes through phage-mediated transduction and mobile plasmids.
Tolerance
plays an important role in protecting bacteria during infections
Related to bacteria adaptation to environment, such as planktonic or sessile (biofilm) growth and presence of persister cells, and it can be due or not to mutations in target genes.
What does HGT stand for and features
Horizontal Gene Transfer
is promoted in biofilms; therefore possible that antibiotic resistance spreads more easily than in planktonic cultures
Intrinsic Transformation
Competent cells take up foreign DNA across their cell membrane and incorporate it into their own genome by genetic recombination
The rates of transformation were 10 to 600 times greater than those observed in cells in planktonic culture. Also shows that the biofilm matrix is no barrier to DNA penetration.
Transformation – new mechanism
Membrane vesicles: released from the cell surface by many Gram-negative, and some Gram-positive, bacteria and can contain proteins, polysaccharides and importantly for microbial adaptation, DNA.
DNA can be utilized by other competent bacteria for transformation. Virulence genes, plasmid located antibiotic resistance genes and gfp (encoding green fluorescent protein) have been shown to be exported from E. coli in vesicles and furthermore have been shown to successfully transform Salmonella
resistant determinants like _____, give survival advantage to bacteria due to ______
β-lactams,OMVs , and enzymes such as protease, endopeptidases, etc
antibiotic resistance traits in biofilms, thereby protecting from antibiotic carnage
gene transduction e.g.
Streptococcal phages have transferred resistance to tetracycline, chloramphenicol, macrolides, lincomycin, clindamycin and streptomycin, probably via generalized transduction of non-phage-encoded resistance genes. Similar for staphylococci.
Gram-negative Conjugation
Pili
made of protein, used for attachment to other bacteria for exchanging DNA (“sex”)
Gram-positive Conjugation
Conjugational transfer of a tetracycline bearing plasmid in Enterococcus faecalis. using plasmiud transfer - 100x higher than planktonic culture
Intergeneric conjugation: dual biofilm of Bacillus subtilus carrying a tetracycline resistant gene construct and a sensitive Staphylococcus species.Staphylococcus isolates resistant to tetracycline were recovered and were shown to be carrying the identical tetracycline resistant gene originally borne by the Bacillus
Antibiotic resistance. Prevention of access to target due to:
reduced permeability of the cell envelope
increased efflux activity
mutation in antibiotic target
enzymatic modification or inactivation of the drug (hydrolysis or transfer of a chemical group)
ability to form biofilms greatly enhance antibiotic resistance traits
Biofilms have a high-level resistance to:
Antibiotics and Biocides
Could be 1000x more resistant
How/why does biofilm resistance to anbtimicrobials occur? (3 ways to alter)
Genotypic e.g. tetracycline resistance
Phenotypic:
- marRAB locus (MarA transcription factor) up-regulates AcrAB-TolC efflux pump and down-regulates OmpF porin influx (intermediate clinical resistance)
- Quorum Sensing communities
—infection hot spots; intracellular infections of macrophages
- Global stress response factors (e.g. σ factors, chaperones)
- Surface attachment and biofilm formation (involve QS, σ)
- Slow growth rates in vivo (including biofilms)
—involve slow turnover of target e.g. penR and σ factor expression
Physical: Exopolysaccharide production (slime) by biofilms “shields” susceptible cells e.g. to aggressive oxidant biocides
Multidrug efflux transporters
Provide a means by which bacteria can confer intrinsic, low-level resistance to a diverse group of antibiotics
Provide a stepping stone to high-level biofilm resistance
Expression is regulated by the antibiotics they remove from the cell
Consequently, a high expression of multidrug efflux transporters confers a multidrug resistance (MDR) phenotype
Thus posing a serious therapeutic problem
MDR (miltidrug) divided into five major structural families
H+/drug antiporters
1. resistance-nodulation-cell division (RND; Gram-negative bacteria)
2. major facilitator superfamily (MF or MFS)
3. small multidrug resistance (SMR)
Na+/drug antiporters
4. multidrug and toxic compound extrusion (MATE, formerly DME)
ATP hydrolysis-linked drug transporters
5. ATP-binding cassette (ABC).
First four groups also known as secondary transporters, use the pre-stored energy of chemical gradients across the membrane
ABC transporters directly coupled with energy generation
Diffusion Summary
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.
But measurements of diffusion coefficients suggest that these solutes will typically diffuse at rates approximately 20 to 50% of their rate in water.
ADAPTIVE MECHANISMS OF BIOFILM PHYSIOLOGY
- Electrochemical - DLVO, PMF, sigma factors
- EPS lectin formation
- Adhesion to substratum
- Bioelectric effect - Co-aggrigation (lectin induction - stationary phase)
- Adhesion to pioneer species
- structured consortia - Maturation - homoserine lactones, AI-2,3; peptides
- cell density dependent quorum sensing - microenvironment colonisation
- by microaerophiles and anaerobes passive (convection) or chemotactic - Predator grazing and chemotaxis
- protozoa, nematodes, macrophages - Disaggregation
passive - sloughing
Active - daughter cells
- Nutritional status
- undocking: flagella induction - chemotaxis
Derjaguin, Landau, Verwey and Overbeek (DLVO) theory
Variation of free energy with particle separation. Net energy is given by sum of the double layer repulsion and van der Waals attractive forces that the particles experience as they approach
Variation of free energy with particle separation at higher salt concentrations showing the possibility of a secondary minimum.
Quorum Sensing
Bacteria in Biofilms are capable of cell to cell communication – Quorum Sensing (QS)
They can sense their state of population density
QS is characterised by an accumulation of autoinducing signal molecules in and around high density colonies
QS allows cells to co-ordinate their gene expression in a cell density dependant manner
QS controls the transcription of many virulence genes of pathogenic bacteria
Blocking QS signals would in theory render the Bacteria avirulent and/or susceptible to host attack and antimicrobial agents
Signals used in QS in Gram + and - bacteria
Gram negative =
N-acyl-l-homoserine lactones (AHL) AI-1 = furanone (E. coli, Salmonella, H. pylori)
AI-2 = adrenaline/noradrenaline AI-3
Gram positive = Auto inducing peptide (AIP)
MOSAIC OF MICROENVIRONMENTAL NICHES
Heterogeneity:
O2, redox - heterotrophic activity may lower m
facilitates: anaerobes,microaerophiles
EPS and products, pH, electrical
- corrosion
- nutrient attraction
- disinfectant repulsion (and charged antibiotics)
Altered Physiology and Resilience:
σ38, global stress response, hsp (chaperones), catalase
- attachment/detachment
- starvation temperature, disinfection, oxidative stress
quorum sensing - autoinduced acyl HSLs, AI-2, peptides
µ (growth rate) - slow, so fewer RNA/ribosomes
- slow cell wall turnover
- cell wall structure, porins, binding
proteins
Increase antibiotic resistance
Summary of antibiotic resistance mechanisms
Nutrient utilization
Stress response
Antimicrobial neutralization
persister formation
Toxin-antitoxin modules represent a major mechanism of persister formation e.g. in E. coli:
RelE, MazF toxins cause dormancy by cleaving mRNA
HipA toxin inhibits translation by phosphorylating elongation factor Ef-Tu (chronic infections have higher production)
TisB toxin forms a membrane pore, decrease in pmf and ATP
Nitric oxide in bacteria
Important signal molecule
Linked with iron acquisition, anaerobic growth, and quorum sensing which are important in biofilm growth
Produced by nitrite reductase, nirS during anaerobic respiration
Removed by nitric oxide reductase, norB
