Student Reader

Drug Resistance

How Can Resistance Occur?
  1. Underproduction of proteins required for drug activation.
  2. Underproduction of a membrane protein that is necessary for uptake of the drug (methotrexate resistance in Leishmania).
  3. Overproduction of the target enzyme resulting from DNA amplification: Dihydrofolate reductase-thymidylate synthetase (DHFR-TS) overproduction leading to methotrexate resistance.
  4. Mutation in a cellular protein that results in decreased affinity of the drug and its target.
  5. Overproduction of a membrane glycoprotein that pumps drugs out of the cell.

Introduction & History

Alexander Fleming discovered penicillin (the 1st antibiotic) in 1929. As more antibiotics have been developed, selective pressure for antibiotic resistance has increased. As genes encoding resistance are transferred between bacteria, antibiotics gradually become effective against fewer bacteria. As a result, new antibiotics must perpetually be discovered.

Approximately 2 million nosocomial infections/yr occur, resulting in ~80,000 patient deaths. Strains of Staph. aureus and Enterococcus resistant to conventional antibiotics are widespread, and the total cost of additional health care due to resistant strains is ~$5 billion.

Agriculture and aquaculture account for ~40-50% of all antibiotic use in the US. Tetracycline derivatives are used at subtherapeutic doses to augment the size of pigs and chickens, and to reduce farm animal infection rates. As a result, though, groundwater and manure from farms oftentimes carry Tetr (tetracycline resistant) bacteria which contaminate the ecosystem.

Currently, there are three precautions implemented to abate the development of antibiotic resistance:
  • Educating the medical community about infection prevention
  • Conservative use of antibiotics like vancomycin to slow evolution of antibioticr strains.

Spread of Resistance Genes

An example of a drug resistance plasmid is pB10. It was isolated in a waste water treatment plant and confers resistance to antimicrobials (amoxicillin, streptomycin, sulfonamides, and tetracycline) and mercury. It is a mosaic plasmid, meaning it has regions from several plasmids combined via recombination. It has at least 5 distinct mobile genetic elements (most conferring resistance).

Resistance genes can get around by gene transfer (plasmids, transposons, and conjugative transposons) or bacterial intermediaries (non-pathogenic strains are resistance gene reservoirs).

Example: Tetracyclines

Tetracycline antibiotics include doxycycline (clinical use) and tetracycline, chlortetracycline, and oxytetracycline (agricultural use). Tetracycline readily crosses the peptidoglycan layer of Gram-positive and outer membrane of Gram-negative bacteria and inhibits translation. Tetracycline is classified as a “broad spectrum”antibiotic because it is effective against a wide range of organisms. In Gram negative bacteria, tetracycline is transported though the outer membrane as a metal-chelatedcomplex (probably Mg2+) by the OmpFand OmpC porin channels. Accumulation of the cationic tetracycline-Mg complex in the periplasmis facilitated by the Donnanpotential of the membrane. Dissociation of the cationic complex allows the low molecular weight, lipophilictetracyclinesto penetrate the cytoplasmic membrane. In Gram-positive cells the peptidoglycan layer is porous to low molecular weight compounds allowing entry into the cell and bind the ribosome.

Mechanisms of resistance against tetracylines:
  • Effluxers: transporters move tetracyclines across cell membrane (often antiporters, with tetracycline out and H+ in). Effluxer proteins may have broad specificity, meaning they are mutlidrug transporters.
  • Ribosomal Protection: Proteins bind the ribosome to prevent tetracyclines from binding. tetM resembles EF-G and displaces tetracycline, but does not replace EF-G. Mutations to rRNA alter binding pockets where tetracyclines bind (16s rRNA).
  • Enzymatic: enzymes modify and inactivate tetracyclines
There are many Tetr genes. Those marked with asterisks are restricted to Gram-positive cells. Gene families are separated by semicolons.
  • Efflux: tetA, tetB, tetD, tetE, tetG, tetH, tetI, tetJ, tetZ*, tet30, tet31, tet33*, tet38, tet39; tetK*, tetL*; otrB*, tcr3*, otrC*; tetP(A); tetV; tetY; tet35
  • Ribosomal protection: tetM*, tetO*, tetS*, tetW*; tetQ*, tetT*, tet36*; otrA*, tetP(B), tet*; tet32*
  • Enzymatic: tetX, tet34, tet37
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