Biofilm physiology and antimicrobial resistance

Question 1

Intrinsic resistance

Answer 1

independent of antibiotic selective pressure and horizontal gene transfer; result of inherent structural or functional characteristics.

Question 2

Acquired resistance

Answer 2

mutations in drug targets or transfer of resistance genes through phage-mediated transduction and mobile plasmids.

Question 3

Tolerance

Answer 3

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.

Question 4

What does HGT stand for and features

Answer 4

Horizontal Gene Transfer

is promoted in biofilms; therefore possible that antibiotic resistance spreads more easily than in planktonic cultures

Question 5

Intrinsic Transformation

Answer 5

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.

Question 6

Transformation – new mechanism

Answer 6

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

Question 7

resistant determinants like _____, give survival advantage to bacteria due to ______

Answer 7

β-lactams,OMVs , and enzymes such as protease, endopeptidases, etc

antibiotic resistance traits in biofilms, thereby protecting from antibiotic carnage

Question 8

gene transduction e.g.

Answer 8

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.

Question 9

Gram-negative Conjugation

Answer 9

Pili

made of protein, used for attachment to other bacteria for exchanging DNA (“sex”)

Question 10

Gram-positive Conjugation

Answer 10

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

Question 11

Antibiotic resistance. Prevention of access to target due to:

Answer 11

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

Question 12

Biofilms have a high-level resistance to:

Answer 12

Antibiotics and Biocides

Could be 1000x more resistant

Question 13

How/why does biofilm resistance to anbtimicrobials occur? (3 ways to alter)

Answer 13

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

Question 14

Multidrug efflux transporters

Answer 14

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

Question 15

MDR (miltidrug) divided into five major structural families

Answer 15

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

Question 16

Diffusion Summary

Answer 16

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.

Question 17

ADAPTIVE MECHANISMS OF BIOFILM PHYSIOLOGY

Answer 17

1. Electrochemical - DLVO, PMF, sigma factors
   - EPS lectin formation
   - Adhesion to substratum
   - Bioelectric effect
2. Co-aggrigation (lectin induction - stationary phase)
   - Adhesion to pioneer species
   - structured consortia
3. Maturation - homoserine lactones, AI-2,3; peptides
   - cell density dependent quorum sensing
4. microenvironment colonisation
   - by microaerophiles and anaerobes passive (convection) or chemotactic
5. Predator grazing and chemotaxis
   - protozoa, nematodes, macrophages
6. Disaggregation
   passive - sloughing
   Active - daughter cells
   - Nutritional status
   - undocking: flagella induction - chemotaxis

Question 18

Derjaguin, Landau, Verwey and Overbeek (DLVO) theory

Answer 18

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.

Question 19

Quorum Sensing

Answer 19

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

Question 20

Signals used in QS in Gram + and - bacteria

Answer 20

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)

Question 21

MOSAIC OF MICROENVIRONMENTAL NICHES

Answer 21

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

Question 22

Summary of antibiotic resistance mechanisms

Answer 22

Nutrient utilization

Stress response

Antimicrobial neutralization

persister formation

Question 23

Toxin-antitoxin modules represent a major mechanism of persister formation e.g. in E. coli:

Answer 23

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

Question 24

Nitric oxide in bacteria

Answer 24

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

Question 25

NO in biofilms

Answer 25

enhances biofilm removal using antimicrobials

increases P. aeruginosa biofilm susceptibility to antimicrobials

Question 26

MRSA

Answer 26

Gram-positive Staphylococcus aureus

5,000 out of 15,000 hospital acquired infections in England in 2002 were caused by MRSA:
Methicillin resistance Staphylococcus aureus

Reports of MRSA have rapidly increased over the last 6 years, from sporadic cases to being endemic in the UK

Last standby = vancomycin BUT intermediate and full resistance strains are now appearing – new therapy reqd

Question 27

SNP role in biofilmn/MRSA

Answer 27

SNP does not affect biofilm structure or apparent detachment

However, it does make MRSA much more susceptible to antimicrobials, particularly vancomycin