Lecture 5: Antibiotic Resitance Flashcards
What are antibiotics
Compounds produced by bacteria, plants, chemist that kill or inhibit bacteria growth
They originiated as anitmicrobial agents in the environment
antibiotics dervied from bacteria
bacterias create sometimes own antibiotics (to extablish a niche) and have resistance mechanisms to protect themselves
other bacteria will respond to these compounds (move away, expresss resistance mechanisms, get eliminated from the environment)
What are antibiotic targets and what is resistance
Target: specific to a bacterial cell (cell wall, bacterial ribosome)
Resistance: occurs when interaction no longer occurs
What does that antibiotic binding do
Prevents proper function causing either:
- the death
- growth inhibition
of the bacteria
Bacterial strcutures/processes targeted by antibiotics
Cell wall integrity
Cell wall synthesis
Protein synthesis
DNA synthesis
DNA Gyrase
RNA poymerase
phospholipid membranes
General antibiotic resistance mechanisms
1) Alteration of outer membrane (prevent antibiotic from getting INTO cell)
2) Up-regulation of drug efflux pumps
3) Alteration of the target
4) Inactivation of the antibiotic (cleavage on outside of the cell or Acyl group added once inside the cell)
How does antibiotic resistance arise (2 general ways) and 2 other ways
1) Mutation and selection
- Random mutations naturally occur at low frequency (DNA polymerase make mistaje ever 10-8)
- Antibiotic resistant mutants can be SELECTED for by the ENVIRONMENT
2) Bacterial cells producing antibiotics have intrinsic resistance mechanisms
3) Horizontal transfer of genes that have resistance
- Transformation (DNA from environment)
- Transduction (Bacteriophage)
- Conjugaison (pilus)
4) the more ofter bacteria is exposed to antibiotic the more likely they will develop resistance (not always the case, some have intrinsic like soil dwelling to protect against their own antibiotics)
Soil bacteria resistance antibiotics
Expermiment showed that soil dwelling bacteria were resistant to many antibioics
Potential that these mechanisms could be transfered to pathogenic bacteria (and has happened in the past)
Need to study the soil resistome and understand the resistance mechanisms
What is the antibiotic resistome
Group of all existing antibiotic resistance genes (known or unknown) in the world
Factors that cause the development of resistance related to exposure to antibiotics
Overuse of antibiotics (in humans + in agriculture)
Non-compliance: Patients dont finish antibiotic presciption
Natural exposure to antimicrobial agents
What are reasons of overuse of antibiotics in humans
used to treat viruses
Long term antibiotics of patient has acne, UTI, recurrent ear infections
Resistance spread going up rapidly for what antibiotic and by what %
Went up 60% since discovered MRSA
Antibiotics in the environment where do they come from
Soil organisms produce antimicrobiol agents naturally
Humans and animals excrete antibiotics
Acquired vs intrinsic resistance
A: Resistance gene not present in genome. Acquired either through:
- Mutation and selection
OR
- Transfered from other bacteria
I: Opposit for I
4 classes of antibiotics and what they target
Cell wall: B-lactams
Macrolides and Aminoglycosides: Ribosomes
Quinolones: DNA replication
B-lactam
- Target and desiption of structure
- What it does
- Types of B-lactams
Gram + cell wall (giant peptidoglycan layer) is the type of bacteria it acts on
Peptidoglycan is made of:
- NAG
- NAM
- Tetrapide chains are fixed to NAM
- Transpeptidation are cross linking between tetrapeptides
Bactericidal (kills bacteria)
Penicillin. Cephalosporins and carbapenems are all B-lactams
Mechanism of action of B-lactams
Inhibitis peptidoglycan synthesis by binding penicillin-binding proteins (transpeptidases)
B-lactam resistance
Loss or diminished expression of outer membrane proteins
Alteration in penicllin binding proteins so that they have reduced affinity for B-lactams
Production of B-lactamases (hydrolyze the B-lactam rings)
Antibiotics that inhibite protein synthesis
Macrolides and aminoglycosides
Macrolides
Binds to large ribosomeal sununit
Mostly bacteriostatic (inhibit bacterial growth) but can be bacteriacidal in HIGH concentrations
Resistance to Macrolides
Active efflux pumps
Alteration of antibiotic binding site by methylation (doesnnt change the ribosomal function)
Aminoglycosides
Bactericidal
binds to Small subunit
Includes ribosomal dissociation
Resistance to aminoglycosides
Dcreased permeability of outer membrane
Active efflux
Inactivation of aminoglycosides by the addition of acyl or phosphate group
Inhibitor of nucleic acid synthesis
Quinolones
Revise before exam
Quinolones function
Revise before exam
Targer DNA gyrase or topoisomerase IV or both and tertiary negative supercoiling (those 2 enxymes restore proper conformation of DNA after replication fork)
Bactericidal
3 generations of quinolones (1st generation is Nalidoxic acid)
Resistance to the quinolones
Revise before exam
Alteration of targets (topo IV and DNA gyrase) - doesnt change their function
Efflux/perneability changes of the outer membrane
Plasmid based qnr genes: encodes protein that probably binds DNA gyrase and topoisomerase to protect them from quinolones
Some antibiotic resistant pathogens of concern
MRSA (Methicillin resistant) and P.aeruginosa (multidruf resistant)
MRSA
Revise before exam
Methicillin is a B-lactam
MRSA is a major hospital aqured infection
Its resistance is due to the presence of mecA gene that encodes a variant of penicillin binding protein that does not bind to B-lactams so the transpeptidation step can occur
Where is mecA located
Revise before exam
on the SCCmec and there are 11 major tupes of these that all have that gene but differe in additional drug resistance genes
p.aeruginosa resistance
Revise before exam
Can be resistant to many drugs because of aquired and intrinsic resistance
Has limited outer membrane permeability (intrinsic)
Efflx pumps (intinsic and aquired)
- 4 maj RND efflux pumps: MexAB, MexXY, MexCDm, NexEF
- Intrisic: MexAB, MexXY
- Aquired: OVEREXPRESSION of MexAB (because mecR gene is mutated so no repression of MexAB) , MexXY (in CF patients, MexZ is a negative regulator and is mutated) and MexCD
RND efflux pumps
Revise before exam
Made of 3 different compounants:
OMP is no OM
MPF: intermembrane
Transporter in IN
Major p.aeruginosa efflux pumps and their substrate (what they pump out)
MexAB: fluoroquinolones, aminoglycosides, B-lactames
MexCD: Fluoroquinolones, macrolides
MexEF: fluoroquinolones
MexXY: fluoroquinolones, aminoglycosides
eDNA (found in biofilm matrix) and antibiotic resistance
Found in biofilm matrix due to active release od DNA from cell and cell death
it has an anionic nature which results in low Mg 2+ in matrix which causes alteration of LPS which resukts in make outer membrane more impermeable to certain antimicrobial agents
There are genes that are expressed only in biofilms whose gene products protect biofilm cells from antibiotics
Hypothesis
How to identify genes taht are important for biofilm-specific antibiotic resistance
- screen 10 000 random transposon-insertion mutants
- Let biofilm grow and add antibiotics
- Identify the ones that survived
- Use that cadidates in several other secondary screens (growth, biofilm formation, plaktonic antibodic resistance)
After screening what is found
isolated 6 mutants
Properties are:
- Grow as well as the WT
- Have same planktonic resistance level as the WT
- Make a biofilm
- More susceptible to Tb (antibiotic) compared to WT in biofilm
THis is an example if intrinsic antibiotic resistance in biofilms
How to confirm biofilm specific antibiotic phenotypes
Revise before exam
Delete gene that is thought to be important, biofilm will be smaller in mutant 1
mutant 1 has a Tn5 insertion in a predicted glucosyltransferase (similar to the ndvB gene that synthesises a cyclic sugar molecule called cyclodextrin)
ndvB gene is expressed in WT biofilm but not in WT planktonic
*never dive below
Possible roles of ndvB-derived glucans
Revise before exam
Biofilm achritecture (when doing a 3D reconstruction there is no change between WT and ndvB absent mutant)
Sequestration (the cyclodextrin molecule has a barrel like structure where hydrophobic molecules can hide from water so that might be where antibiotic is attaching. tests indicated that ndvB mutant is defective of glucacan synthesis. a in virto interaction assay is done to see if p.aeruginosa glucans and Tb interact. Tb flows through tube when glucagon is not present. Glucacans interact with Tb and prevent Tb from reaching target in cytoplasm)
Signaling: glucagon can affect the expression of genes important for protecting cells agains the cytotoxic effects of ROS
What are biofilm sepcific antibiotic resistance due to
MUltiple intrinsic resistance and not due to mutattion
Targeting virulence factors
preventing virulence:
- Inhibition of toxin functions
- Inhibition of secretion (ex: toxin)
- Controlling regulation of virulence gene expression)
- Inhibition of adhesion
Bacteriophages
promising therapy
Bacteria attacking gram - bacteira
B.bacteriovorus
