Antimicrobial Resistance - S’s Version

• What are the two forms of antimicrobial resistance?

  1. Intrinsic resistance - inherited or natural resistance, e.g. chlamydia do not have peptidoglycan, so are not susceptible to penicillin.
  2. Acquired resistance - developed through the alteration of the microbial genome.
• Give three features of intrinsic resistance.

  1. Involves chromosomic genetic support.
  2. Affects almost all species strains.
  3. Existed before antibiotic use, e.g. Enterobacter sp. was already resistant to amoxicillin.
• Give three features of acquired resistance.

  1. Chromosomic, plasmidic or transposon genetic support.
  2. Affects a fraction of strains.
  3. Increased with antibiotic use (extended spectrum beta-lactamase producing E. coli).
• What are two ways alteration of the microbial genome occurs via?

  1. Vertical evolution - Darwinian, mutation and natural selection.
  2. Horizontal evolution - transfer of genes between microbes.
• What is acquired resistance and what are the two ways this occurs? Transfer of genes or genetic material between microbes.

  1. Transposons - small, mobile sequences of DNA that can move/be copied to other regions of the genome (either within the gene or other genes).
  2. Plasmids - circular, ‘mini chromosomes’ that replicate independently of chromosomal DNA.
• How can mutations arise to cause resistance in a strain?

  1. Spontaneous point mutations.
  2. Mistakes in DNA repair.
  3. Transposon insertion.
• How can genes duplicate to cause resistance in a strain?

  Homologous recombination - copying of a mutated gene.
• How can genes be transferred to cause resistance in a strain?

  1. Lysogenic Bacteriophage infection (transduction) - involves the transfer of DNA from one bacterium into another via bacteriophages.
  2. Pili mediated sex (conjugation) - involves transfer of DNA material via sexual pilus and requires cell-to-cell contact.
  3. Leaky bacterial uptake of nuclear material (transformation) - involves uptake of short fragments of naked DNA by naturally transformable bacteria.
• Name two sets of four bacteria that can genetically exchange their antimicrobial resistant genes.

  1st set:

  1. Pseudomonas
  2. Enterobacteriaceaa
  3. Vibrio cholerae
  4. Campylobacter

  2nd set:

  1. Staphylococci
  2. Enterococci
  3. Pneumococci
  4. Streptococci
• What does the minimum inhibitory concentration test (MIC) test for?

  The smallest concentration of antibiotic that inhibits the growth of an organism. It is an antimicrobial susceptibility test.
• What are the two means of the MIC test?

  1. Liquid media (dilution).
  2. Solid media (diffusion).
• What is the liquid media MIC test?

  Dilution in liquid broth:

  1. Tubes containing increasing antibiotic concentrations.
  2. Incubation for 18 hours at 37 degrees.
  3. Very tedious

  
• What is the solid media MIC test?

  Kirby-Bauer disc testing:

  1. Antibiotic-impregnated discs placed on agar plate at the interface between test organism and susceptible control organism.
  2. Resulting zones of inhibition compared.
  3. Susceptibility is inferred (standard tables).

  
• Give six mechanisms of resistance for microbes.

  1. Antimicrobial exclusion.
  2. Enzymatic degradation of the drug.
  3. Modification of the drug target.
  4. Target bypass.
  5. Enhanced production of the target.
  6. Efflux mechanisms.
• What is antimicrobial exclusion?

  1. Preventing the antimicrobial from entering the microbe, e.g. the outer membrane of gram-negative bacteria.
  2. The outer membrane acts as a barrier to extracellular compounds, i.e. gram-negative bacteria, hydrophobic molecules and high MW hydrophiles.

  
• What is enzymatic degradation?

  When the microbe prevents the drug from binding to the enzyme that aids its presence.
• Give an example of enzymatic degradation.

  1. Penicillins work on bacteria by binding to transpeptidase enzymes, stopping the process of peptidoglycan cross-linking.
  2. Binding occurs via the beta-lactam ring.
  3. Resistance of penicillins is mediated by beta-lactamases.
  4. Beta-lactamases hydrolyse the beta-lactam ring, preventing penicillin from binding the transpeptidases.
• What are the properties of beta-lactamases in gram-negative bacteria?

  1. Beta-lactamases are cell bound.
  2. They are found in the perisplasmic space, between the outer and inner membrane.
  3. Concentrated at a strategic location, so not a lot needs to be produced.
• What are the properties of beta-lactamases in gram-positive bacteria?

  1. Beta-lactamases are inducible - they are produced in response to the drug, which is economical for the bacteria.
  2. Beta-lactamases are extracellular as the pepide is exposed to the environment - they have long-range action; dilution of effectiveness.
• How do beta-lactams combat beta-lactamases?

  E.g. Methicillin - resistance to beta-lactamases is created through increased steric hindrance via increased methyl groups.

  
• How can beta-lactamases be inhibited by another drug?

  E.g. Clavulanic acid - is isolated from Streptomyces spp.. There is no antimicrobial activity when it binds to transpeptidases but causes irreversible acylation of beta-lactamases by binding so penicillin is able to be used.

  
• Give two other examples that use enzymatic degradation and how.

  1. Chloramphenicol - resistance is due to acylation by chloramphenicol acetyltransferases (CAT).
  2. Modification by, e.g. acetylation, phosphorylation or conjugation with a nucleotide.

  (acylation of chloramphenicol by CAT)

  
• How do glycopeptides act as antibacterials?

  1. Glycopeptides inhibit peptidoglycan synthesis.
  2. They bind to amino acids in the peptidoglycan, preventing extension.
  3. In particular they bind to acyl-D-alanyl-D-alanine.
• Give three features of glycopeptide intrinsic resistance.

  1. Gram-negative bacteria due to the outer membrane and drug exclusion.
  2. Some gram-positive bacteria.
  3. Those bacteria with intrinsic resistance to glycopeptides have precursors similar to acquired resistance.
• Give four features of glycopeptide acquired resistance.

  1. E.g. Enterococcus faecium.
  2. Resistance to vancomycin and teicoplannin is acquired from a plasmid.
  3. This results in the production of seven new polypeptides.
  4. Three of these polypeptides confer resistance through formation of a modified peptidoglycan precursor.
• What is the mechanism microbes use for resistance to glycopeptides?

  1. D-Ala - D-Ala is replaced by D-Ala - D-Lac. Lac = lactic acid, and the NH2 group is replaced by an OH group.
  2. This results in the loss of an H-bond critical for vancomycin and teicoplanin binding. This causes affinity for the antibiotics to reduce by 100x. Thus the peptidoglycan extension is no longer inhibited.
  3. Lac residue is lost during cross-linking of peptidoglycan, so therefore the bacteria is not deformed and is functional.
• How does trimethoprim resistance come about?


  When the target dihydrofolate reductase enzyme (DHFR) is modified.
• How does aminoglycoside resistance come about?

  Results from modifications to the structure of the bacterial ribosome.
• How does quinolone resistance come about?

  1. Can be a result of mutations in topoisomerase IV (DNA gyrase) reducing binding affinity.
  2. Can also be a result of production of novel proteins that bind to and mask the topoisomerase.
• What is meant when microbes do a drug target bypass?


  1. Antibiotic targets are mostly key steps in biological processes.
  2. Some microbes have intrinsic resistance to an antibiotic by not utilising the key step or by having an alternative.
  3. Microbes can therefore acquire resistance by acquiring mechanisms to side-step these steps.
• How can methicillin cause drug target bypass?

  Via the production of an additional transpeptidase not susceptible to penicillin.
• How can antifolates cause drug target bypass?

  Via sulfonamides - resistance has developed as bacteria have developed an alternative route for folic acid biosynthesis that doesn’t use dihydropteroate synthase enzyme (DHPS).

  
• What is meant when microbes use enhanced target production to resist drugs?

  Microbes develop resistance by increasing production of the drug target, which overwhelms the antibiotic.
• Give an example of a drug that can be a victim to enhanced target production.

  E.g. Trimethoprim - resistance to trimethoprim can result from an overproduction of the target, dihydrofolate reductase (DHFR).

  
• What are efflux pumps, where are they located, and what do they require?


  1. Transporter proteins that pump things out of the cell.
  2. Located in the cytoplasmic membrane.
  3. They are active transporters - so therefore require a source of energy to function (i.e. ATP or potential difference).

  
• What drugs have had resistance due to efflux mechanisms?


  1. Tetracylines
  2. Macrolides
  3. Quinolones
  4. Chloramphenical
• How does the efflux mechanism work in drug resistance?

  1. Over-expression of efflux pumps.
  2. Leads to antibiotic pressure.
  3. Mutator genes activate, causing more efflux pumps to form.
  4. Antibiotics are filtered out.
  5. Basal number of efflux pumps restored:

  
• Give an example of drug resistance in fungi.

  1. Azole anti-fungals, e.g. fluconazole.
  2. Inhibits the fungal cytochrome p450 enzyme, 14alpha-demethylase.
  3. Prevents the conversion of lanosterol to ergosterol - a major constituent of fungal cell membranes.
• Give three drug resistance mechanisms with azoles.

  1. Point mutations in 14alpha-demethylase enzyme gene, ERG11 —> leads to reduced affinity for azoles —> causes overexpression of the enzyme.
  2. Alterations in other enzymes of ergosterol biosynthetic pathway —> production of various sterols supporting growth —> cross-resistance to other azoles.
  3. Over-expression of the CDR and MDR efflux pump genes —> reduced drug accumulation in cell.
• What is the resistome?

  Collection of antibiotic resistance genes in a bacterial population.