Antimicrobial Resistance - M’s Version

• Where does antimicrobial resistance come from?

  Healthcare

  Community

  Food/Farms

  The world

  
• What are the problems of antimicrobial resistance?

  Some bacteria are resistant to nearly all current antibiotics

  
  

  Unchecked, by 2050, the global death toll will rise to ~10 million
• What are the forms of antimicrobial resistance?

  Intrinsic resistance–inherited or natural resistance (e.g. Chlamydia do not have peptidoglycan, so are not susceptible to penicillins)

  Acquired resistance–developed through alteration of the microbial genome
• Compare & contrast intrinsic & acquired resistance.

  Intrinsic resistance:- Chromosomic genetic support- Affect almost all species strains- Existed before antibiotic use

  Acquired resistance:- Chromosomic, plasmidic or transposon genetic support.- Affects a fraction of strains- Increases with antibiotic use

  
• How can you get alteration of the microbial genome in acquired resistance?

  2 routes:

  ‘Vertical evolution’(Darwinian, mutation and natural selection)

  ‘Horizontal evolution’(transfer of genes between microbes)
• How does transfer of genes between microbes happen?

  Usually will include either:

  Transposons– small, mobile sequences of DNA that can move/be copied to other regions of the genome, either within the gene or to other genes

  Plasmids– circular, ‘mini chromosomes’ that replicate independently of chromosomal DNA
• How does resistance evolve in a strain?

  Mutations can arise through:spontaneous point mutations; mistakes in DNA repair; transposon insertionGenes can be duplicated/amplified by:homologous recombination; other forms of recombination eventGenes can be transferred by:lysogenic bacteriophage infection (transduction); pili mediated sex (conjugation); transformation (‘leaky’ bacterial uptake of nuclear material)
• Is genetic exchange of antimicrobial resistance genes limited to their own species?

  No, it can happen between members of different species as well

  
• How do you test for antimicrobial susceptibility?

  Measure theMIC(Minimum inhibitory concentration)- this can be assessed through 2 methods:

  1. Liquid media (dilution)2. Solid media (diffusion) e.g. disc diffusion (Kirby-Bauer) or E-tests

  
• What would the setup be if you were measuring MIC in a liquid medium?

  
• Describe Kirby-Bauer disc testing.

  Antibiotic-impregnated discs are placed on an agar plate at the interface between test organism and susceptible control organism

  
  

  Resulting zones of inhibition are compared, use of controls Susceptibility is inferred (from standard tables)

  
  

  The more resistant, the smaller the zone of inhibition

  
• What are the 6 main mechanisms of antibiotic resistance?

  Antimicrobial exclusion

  Enzymatic degradation of the drug

  Modification of the drug target

  Target bypass

  Enhanced production of the target

  Efflux mechanisms
• What is antimicrobial exclusion?

  Preventing the antimicrobial from entering the microbe, so no destruction is caused

  e.g.

  the outer membrane of Gram-negative bacteria

  The outer membrane acts as a barrier to extracellular compounds and only allows small hydrophilic solutes to pass through the OM through channels/pores.

  This means antibiotics for these bacterias need to be small
• What is the consequence of the outer membrane of Gram-negative bacteria acting as a barrier?

  Large(e.g. glycopeptides) and hydrophobic agents (e.g. macrolides, rifamycins) cannot readily penetrate and diffuse across the outer membrane

  Small, hydrophilic solutes can pass through the outer membrane through aqueous channels/pores formed by transmembrane proteins (porins)
• What is an example of enzymatic degradation being a mechanism of antibiotic resistance?

  Resistance in penicillin

  
  

  Penicillins work by irreversibly binding to the transpeptidase enzymes, stopping the process of peptidoglycan cross linking. This means we lose the structural integrity.

  
  

  Binding occurs via the β-lactam ring

  
• What is resistance to penicillin mediated by and how?

  Mediated byβ-lactamases

  β-lactamaseshydrolyse the β-lactam ring, preventing penicillin from binding the transpeptidases

  Gram-negative bacteria produce one type of β-lactamase, whilst Gram-positive bacteria produce a wide variety
• How do the β-lactamases differ in Gram-negative & Gram-positive bacteria?

  
• How can you overcome β-lactamases?

  Develop resistantβ-lactams:E.g. methicillin- resistance to β-lactamases is created through increased steric hindrance

  Inhibit theβ-lactamases with another drug:E.g.clavulanic acid- isolated fromStreptomyces spp.& there is no antimicrobial activity when it binds to transpeptidasesBUTcauses irreversible acylation of β-lactamases, causing isolation of B-lactamases = inactivating it.
• What are some other examples of enzymatic degradation?

  Chloramphenicol- resistance is usually due to acetylation by chloramphenical acetyl transferases (CAT), this causes it to become inactive

  Aminoglycosides- modification by e.g. acetylation, phosphorylation or conjugation with a nucleotide
• How does modification of drug target induce resistance?

  Glycopeptides inhibit peptidoglycan synthesis

  
  

  They bind to amino acids in the peptidoglycan, preventing extension (in particular, they bind acyl-D-alanyl-D-alanine)

  
  

  BUT

  
  

  you can have resistance to glycopeptides which will result in antibiotic resistance
• What are the 2 main methods of glycopeptide resistance?

  
• What is the modified peptidoglycan precursor and how does it result in antibiotic resistance?

  D-Ala - D-Ala is replaced by D-Ala – D-Lac (Lac= lactic acid, with the NH2 group replaced by an OH group)

  This results in the loss of an H-bond critical for vancomycin & teicoplanin binding

  Affinity for the antibiotics reduces by ~100xThus peptidoglycan extension is no longer inhibitedLac residue is lost during cross-linking of peptidoglycan
• Give more examples of when modification of drug target has led to antibiotic resistance.

  1.Trimethoprimresistance occurs when the target dihydrofolate reductase enyzme (DHFR) is modified

  2.Aminoglycosideresistance can result from modifications to the structure of the bacterial ribosome

  3.Quinoloneresistance can be a result of mutations in topoisomerase IV (DNA gyrase) reducing binding affinity or can also be a result of production of novel proteins that bind to & mask the topoisomerase
• How does drug target bypass work in order to achieve antibiotic resistance?

  Antibiotic targets are mostly key steps in biological processes

  
  

  Some microbes have intrinsic resistance to an antibiotic by not utilising the key step or by having an alternative.

  
  

  Microbes can acquire resistance by acquiring mechanisms to side-step these steps
• Give some examples of drug target bypass.

  Methicillin- production of an additional transpeptidase which is not susceptible to penicillins, meaning that you can then carry on cross-linking the peptide chains because you're no longer dependent on the penicillin-sensitive transpeptidases

  Sulfonamides- Resistance has developed as bacteria have developed an alternative route for folic acid biosynthesis that doesn’t use the dihydropteroate synthase enzyme (DHPS)
• How does enhanced target production lead to antibiotic resistance?

  Some microbes develop resistance by increasing production of the drug target to overwhelm the antibiotic.

  e.g. trimethoprim-resistance to trimethoprim can result from an overproduction of the target, dihydrofolate reductase (DHFR)
• Efflux mechanisms involve efflux pumps. What are they?


  Efflux pumps are transporter proteins that pump things out of the cell & are located in the cytoplasmic membrane (they are "active" transporters so require a source of energy to function i.e. ATP or p.d.).

  
  

  There are many different types of efflux pumps that fall into 5 different groups, based on protein sequence & energy source utilised
• What has drug resistance via efflux mechanisms arisen to?

  Tetracyclines, macrolides, quinolones, & chloramphenical
• How do efflux mechanisms work?

  It's usually a stress-mediated response so first there will be antibiotic stress which will activate the efflux pumps & pump the antibiotic out

  
  

  Because there's a high conc. of antibiotic causing the stress, that causes a stress response meaning you get over expression of the efflux pumps

  
  

  So more of them are activated, antibiotic is all boosted out of the cell

  
  

  The more efflux pumps there are, the more antibiotic can be removed
• How do azoles work?

  Inhibits the fungal cytochrome p450 enzyme, 14α- demethylase

  This prevents the conversion of lanosterol to ergosterol– a major constituent of fungal cell membranes
• How can resistance to azoles develop?

  1. Point mutations in the 14a-demethylase enzyme gene, ERG11

  
  

  2. Alterations in other enzymes of ergosterol bio synthetic pathway

  
  

  3.Overexpression of the CDR and MDR efflux pump genes.

  
• What is a resistome?

  Collection of antimicrobial resistance genes.

  
  

  There are different resistome profiles which helps track resistance within a large population

  
• How can we combat antibiotic resistance?

  Use the correct antibiotic

  Use the correct dose of antibiotic

  Use multiple antibiotics

  Use a secondary, non-antibiotic drug to reduce resistance

  Use of bacteriophages to disrupt resistance genes
• What are bacteriophages?

  Bacteriophages are natural viruses of bacteria which are specific to individual bacterial species

  AND:

  there isnochance of developing resistance

  
• By what mechanisms can we use bacteriophages as 'antibiotics'?

  1. Used as a direct antibiotic : phage enters and replicates within the cell before lysing the cell(LYTIC CYCLE)

  2. Remove/antagonise resistance genes :Lysogenic cycle