Microbiology 2 - Antibiotics

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  1. What are antibiotics?
    Antibiotics are substances produced by microbes (or a similar substance produced by chemical synthesis) which in low concentration inhibits the growth of other microbes.
  2. What sources of antibiotics do we have?
    1. Microbes - e.g. Stretptomyces make tetracyline and streptomycin - Penicillium fungi makes penicillin.

    2. Synthesis - Chloramphenicol is produced by a synthetic process

    3. Semi-synthesis - Part of the molecule is produced by a microorganism and is then modified by a chemical process - many penicillins and cephalosporins are modified in this way.
  3. What is the main aim for tackling bacteria in antibiotic research?
    The aim is to try to identify targets that are only found in microbes but not in humans.
  4. What are β-Lactam antibiotics?

    What is the mode of action for β-Lactam antibiotics?

    How can cells be resistant to them?
    Beta-lactam antibiotics are a broad class of antibiotics, consisting of all antibiotic agents that contains a β-lactam ring in their molecular structures.

    A common example is penicillins.

    Penicillins have a core structure of 6-aminopenicillanic acid, but can differ in their R group, which determines the stability and antimicrobial spectrum of the penicillin.

    Naturally occurs as Benzylpenicillin (Penicillin G – is not effective orally) and Phenoxymethylpenicillin (Penicillin V – is effective orally) – both are sensitive to breakdown by enzymes called β-lactamases and both are poorly active against Gram negative bacteria.

    In 1959 chemists at Beechams isolated 6-aminopenicillanic acid (6-APA) i.e. the β-lactam structure minus the R sidechain

    (The sidechain can also be cleaved by an enzyme called penicillin amidase)

    6-APA is used as the building block for numerous ‘semisynthetic’ penicillin analogues that differ only in their sidechain – this imparts various properties:

    • e.g. Methicillin – is relatively resistant to β-lactamases
    • Ampicillin & Amoxycillin – are more active against Gram negatives (except Pseudomonas spp.)
    • Carbenicillin – is active against Gram negatives incl. Pseudomonas

    All β-lactam antibiotics inhibit the final stage of peptidoglycan synthesis in the bacterial cell wall, thereby causing them to burst. 

    • In a newly formed peptidoglycan sugar chain, the amino acid chain on the N-acetylmuramic acid has two D-Alanines at the end (dipeptide), one of which is cleaved allowing linking (transpeptidation). This is facilitated by transpeptidase.
    • β-lactam antibiotics are structurally similar to D-Ala-D-Ala dipeptides, and so react with traspeptidases to block activity (Penicillin binds transpeptidase and occupies the active site, preventing its function).

    By doing this, β-lactams therefore interfere with cell wall synthesis in growing cells.

    Cells which are not fast growing, or have already grown will therefore be resistant to β-lactams since they only prevent growth, and do not degrade already formed peptidoglycan. 

    Some bacteria become resistant because they produce β-lactamase.

    • Beta-lactamases are enzymes produced by some bacteria that provide resistance to β-Lactam antibiotics like penicillins, cephamycins, and carbapenems
    • Beta-lactamase provides antibiotic resistance by breaking the antibiotics' structure.
    • These antibiotics all have a common element in their molecular structure: a four-atom ring known as a β-Lactam.
    • Through hydrolysis, the lactamase enzyme breaks the β-Lactam ring open, deactivating the molecule's antibacterial properties.
  5. What is Vancomycin?

    How can bacteria become resistant?
    Vancomycin is a glycopeptide which, like the β-lactam antibiotics, interferes with cell wall synthesis.

    • The large hydrophilic molecule is able to form hydrogen bond interactions with the terminal D-alanyl-D-alanine amino acids of the NAM-peptides.
    • This binding of vancomycin to the D-Ala-D-Ala prevents cell wall synthesis of the long polymers of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) that form the backbone strands of the bacterial cell wall (fascilitated by transglycosylase), and it prevents the backbone polymers that do manage to form from cross-linking with each other.

    The resistant organism have  changed the D-Ala-D-Ala end of the peptidoglycan to either D-Ala-D-Lactate or D-Ala-D-Serine.  The result is the vancomycin cannot bind to this portion of the peptide and the transglycosylase can join the peptidoglycan sub-units together
  6. What is D-cycloserine?

    How do bacteria become resistant to it?
    D-cycloserine interferes in an early stage of peptidoglycan synthesis. It is used to treatMycobacterium tuberculosis infections

    It prevents the assemble of the D-Ala-D-Ala dipeptide.

    A racemase and a ligase (enzymes) are both needed to change L-alanine to D-alanine and then to join the two D-alanines together.

    D-cycloserine stops both enzyme from working by binding to their co-factor - pyridoxal phosphate.

    Mycobacteria have evolved a variety of resistance mechanisms, but one of the simplest is overproduction of the D-alanine racemase.

    • The overproduction of the enzyme is caused by a mutation in the promoter for the enzyme’s gene.
    • This leads to overexpression of the gene and thus the protein.
  7. What are sulfonamides and trimethoprim?

    How do bacteria develop resistance to them?
    They are both antibiotics which act synergistically to inhibit folate metabolism by blocking sequential steps in the process. The reduced form of folic acid is an essential coenzyme in nucleic acid synthesis, so this is prevented,

    Sulfonamides act as competitive inhibitors to the enzyme dhydropteroate synthetase.

    Trimethoprim acts as a competitive inhibitor to dihydrofolate reductase.

    Resistance to Trimethoprim: major cause of resistance is plasmid-encoded production of an altered dihydrofolate reductase (DfrB) enzyme. Enzyme can’t bind trimethoprim

    Other mechanisms can also exist - overproduction of normal chromosomally-encoded enzyme)

    Resistance to Sulfonamides: also caused by an altered enzyme. A plasmid encoded Dihydropteroate synthetase has a lower affinity for sulfonamides than the natural enzyme

    Increased production of para-aminobenzoic acid and increased synthesis of pteridine can also contribute to resistance
  8. What are nalidixic acid and ciprofloxacin?

    How can bacteria grow resistant to them?
    They are quinolones (The quinolones are a family of synthetic broad-spectrum antibacterial drugs) which target the DNA gyrase enzyme which is essential for DNA replication.

    DNA gyrase, often referred to simply as gyrase, is an enzyme that relieves strain while double-strand DNA is being unwound by helicase.

    The primary mechanism of resistance centres around alteration of the target to prevent the antibiotic from binding.

    DNA gyrase is a tetrameric enzyme composed of two A subunits and two B subunits, encoded by gyrA and gyrB.

    Spontaneous mutations can occur in either sub-unit of the enzyme, leading to resistance to the quinolones.

    In clinical isolates of resistant bacteria the mutations are nearly always found in the gyrA gene

    The mutations change the physical structure of the GyrA sub-unit in such a way to stop the quinolone from binding to it, when it is bound to the DNA.

    Other mechanisms that confer resistance to the quinolones include decreased uptake of the drug.
  9. How can changes in the cell membrane cause antibiotic resistance?
    All antibiotics need to get into the cell to cause disruption. There are two ways in which they can get across the cell membrane

    • Via porins – proteins which act as a pore through which molecules can diffuse
    • Via diffusion through the phospholipid bilayer

    Porins can mutate and become modified in such a way that antibiotics like quinolone cannot enter and therefore the cell becomes resistant.
  10. How can protein synthesis be disrupted by antibiotics?

    How can resistance occur?
    Protein synthesis in bacteria, while being similar to eukaryotes, is sufficiently different to provide viable targets for disruption.

    Two classes of antibiotics have been discovered and developed which target the 30S portion of the ribosome, but have different modes of action and different mechanisms of resistance.

    These are aminoglycosides and tetracylcines and they inhibit bacterial protein synthesis.

    Resistance to Aminoglycosides is through production of bacterial enzymes that modify the antibiotic, such as AAC, ANT, and APH (AAT modifies an amino group from one of the sugars).

    Aminoglycoside-modifying enzymes are often plasmid encoded, but are also associated with transposable elements.

    So acquisition of the resistance is again via HGT - Horizontal Gene Transmission.

    Other resistance mechanisms do exist, for example changes in the 30S ribosome complex stops streptomycin from binding, but does not stop types of other aminoglycosides from binding.
  11. How can microbial resistance arise?
    It arises through evolution due to the attack & counter-attack of m/o’s in complex niches (e.g. GI tract, soil etc.).

    Obviously rate of development of resistance is increased due to medical antibiotic use.

    Development of resistance may be linked to misguided prescribing of antimicrobials (e.g. for viral infections (esp. influenza)) – also linked to veterinary and agricultural use of antibiotics.
  12. Genetic resistence develops by two methods, what are these?
    Vertical evolution – spontaneous mutation in the chromosome

    Horizontal evolution – transfer of resistance genes e.g. by transformation, conjugation or transduction – resistance genes may be on a plasmid.

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  13. What role does natural selection play in the process of antibiotic resistance?
    Evolution of antibiotic resistance is not in response to antibiotic exposure.  Evolution is occurring all the time and it’s the exposure of the population to lethal and sub-lethal levels of antibiotics which apply the selective pressure.

    This pressure allows organism within the population, which are fitter due to mutations or gene acquisition to thrive.
  14. What are multidrug efflux pumps
    Efflux pumps are proteinaceous transporters localized in the cytoplasmic membrane of all kinds of cells.

    They are active transporters, meaning that they require a source of chemical energy to perform their function.

    Some are primary active transporters utilizing Adenosine triphosphate hydrolysis as a source of energy, whereas others are secondary active transporters (uniporters, symporters, or antiporters) in which transport is coupled to an electrochemical potential difference created by pumping hydrogen or sodium ions from or to the outside of the cell.

    They can pump the antibiotic out of the cell faster than it gets in.
Card Set:
Microbiology 2 - Antibiotics
2014-10-07 17:09:03
Microbiology Antibiotics
Microbiology 2 - Antibiotics
Microbiology 2 - Antibiotics
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