Antibiotics and Bacterial Genetics

Antibiotics, Antibiotic Resistance and Bacterial Genetics

Learning Outcomes

  • Differentiate between broad and narrow spectrum antibiotics.
  • Identify the targets of the various classes of antibiotics and understand how each antibiotic class affects cell growth.
  • Recognize the structure of antibiotics from each class.
  • Discuss the different mechanisms of resistance to each class of antibiotics.
  • Define the term “horizontal gene transfer” and how it applies to bacteria.
  • Discuss the 4 forms of horizontal gene transfer and how each occurs.
  • Understand how horizontal gene transfer leads to the formation of genetic diversity and understand the role this plays in the formation of novel pathogenic bacteria.

Characteristics of an Ideal Antimicrobial Compound

  • Toxic to microbes without toxicity to host cells/organism.
  • Microbicidal (kills microbes) vs. microbistatic (halts microbe growth).
  • Soluble and potent enough to function at low concentrations in tissues.
  • Long-acting enough to be functional (not excreted or broken down too quickly).
  • Avoids antimicrobial resistance development.
  • Works together with host activities/host defenses.
  • Active in tissues or blood.
  • Readily transported to infected areas.
  • Affordable.
  • Does not disrupt the host’s health (i.e., no allergic reactions)

Targets for Antimicrobial Drugs

  1. Inhibition of cell wall synthesis:
    • Examples: penicillins, cephalosporins, bacitracin, vancomycin.
  2. Inhibition of protein synthesis:
    • Examples: chloramphenicol, erythromycin, tetracyclines, streptomycin.
    • Process: Transcription (DNA to mRNA) to Translation (mRNA to Protein).
  3. Inhibition of nucleic acid replication and transcription:
    • Examples: quinolones, rifampin.
  4. Injury to plasma membrane:
    • Examples: polymyxin B, daptomycin.
  5. Inhibition of essential metabolite synthesis:
    • Examples: sulfanimide, trimethoprim.
  • Antimicrobials target any microorganism, while antibiotics specifically target bacteria.

Antibiotic Spectrum

  • Antibiotics are NOT a one-size-fits-all solution.
  • Different types of bacteria have varying susceptibilities to different classes of antibiotics.
  • Gram-negative bacteria are generally less susceptible to many antibiotics than gram-positive bacteria.
  • Pseudomonas has an unusual outer membrane that resists penetration by many drugs.
  • Bacteria with substantial polysaccharide coatings are often more resistant to killing by antibiotics.
  • Antibiotics that target a large variety of microorganisms are called broad spectrum.

Antibiotics that Target Bacterial Cell Walls

  • Most bacterial cell walls contain a rigid girdle of peptidoglycan.
  • Penicillin and cephalosporin block the synthesis of peptidoglycan cross-links by inhibiting DD-transpeptidase. This causes the cell wall to lyse if the cells are actively growing/remodeling. Act as lysins
  • Original penicillins do not penetrate the Gram-negative outer membrane well and are less effective against those bacteria.
  • Broad-spectrum penicillins and cephalosporins can cross the cell membranes of Gram-negative bacteria.

Beta-Lactam Antibiotics

  • Large group of diverse molecules.
  • Share a common beta-lactam ring.
  • R-group characteristics change the specificity of the drug.
  • Important members of this class:
    • Penicillins
    • Cephalosporins
    • Carbapenems
    • Semisynthetic penicillins (e.g., ampicillin, amoxicillin, methicillin, oxacillin, carbenicillin)
  • Examples:
    • Meropenem (carbapenem)
    • Cefotaxime (3rd gen cephalosporin)
    • Methicillin
    • Penicillin G (Benzylpenicillin)

Penicillins

  • Penicillin G and Penicillin V are the most important natural forms.
  • Penicillins were the primary treatment choices for Gram-positive cocci infections (e.g., Staphylococci and Streptococci).
  • Penicillins are the primary antibiotics administered for some Gram-negative infections (e.g., Meningococci and Syphilis).

Cephalosporins

  • First identified in 1945, produced by aerobic fungus Acremonium (previously called Cephalosporium).
  • Most commonly prescribed antibiotics.
  • Up to 5 generations (number varies between countries and agencies).
  • Effective against Gram-positive and Gram-negative infections.
  • Resistant to penicillinases and less allergenic than penicillin.

Vancomycin

  • Glycopeptide produced by bacterium Amycolatopsis orientalis.
  • Used in the treatment of Gram-positive infections ONLY!
  • Narrow-spectrum, effective against penicillin & methicillin-resistant staphylococcal infections, & C. diff infections; relatively toxic, requires intravenous administration.
  • Binds to the D-alanine D-alanine of the pentapeptide precursor, blocking the transpeptidation reaction.

Antibiotics That Inhibit Protein Synthesis

  • Inhibit 70S ribosomes of prokaryotes.
  • Most are bacteriostatic (except aminoglycosides).
  • Classes:
    • Aminoglycosides: streptomycin, gentamicin, amikacin, neomycin, tobramycin, kanamycin
      • Changes shape of 30S portion, causing code on mRNA to be read incorrectly resulting in truncated proteins
    • Tetracyclines: tetracycline, doxycycline, minocycline
      • Interfere with attachment of tRNA to mRNA–ribosome complex
    • Chloramphenicol
      • Binds to 50S portion and inhibits formation of peptide bond
    • Macrolides: erythromycin, azithromycin, clarithromycin
      • Binds to 50S portion and inhibits formation of peptide bond
  • Process: Translation

Aminoglycosides

  • Composed of 2 or more amino sugars and an aminocyclitol (6C) ring.
  • Products of various species of soil actinomycetes in genera Streptomyces & Micromonospora.
  • Broad-spectrum, inhibit protein synthesis, especially useful against aerobic Gram-negative rods & certain Gram-positive bacteria.
  • Streptomycin – bubonic plague, tularemia, TB.
  • Gentamicin – less toxic, used against Gram-negative rods.
  • Tobramycin & amikacin – newer drugs used against Gram-negative bacteria.

Tetracyclines and Chloramphenicol

  • Broad-spectrum, block protein synthesis.
  • Produced by Streptomyces.
  • Doxycycline & minocycline are taken for STDs, Rocky Mountain spotted fever, Lyme disease, typhus, acne & protozoa.
  • Chloramphenicol is highly lipid soluble, thus able to penetrate the blood-brain barrier.
  • Used in the treatment of meningitis, VRE infection, and ocular infections.

Macrolides

  • Produced by Streptomyces.
  • Broad-spectrum, fairly low toxicity.
  • Protein synthesis inhibitors (attach to ribosomes).
  • Taken orally for Mycoplasma pneumoniae, legionellosis, Chlamydia, pertussis, diphtheria and as a prophylactic prior to intestinal surgery.
  • For penicillin-resistant gonococci, syphilis, acne.
  • Erythromycin – macrolide (macrocyclic) large lactone ring with sugars.
  • Newer semi-synthetic macrolides: clarithromycin, azithromycin.

Antibiotics That Interfere with Nucleotide Synthesis

  • Quinolones and fluoroquinolones target DNA gyrase.
    • Examples: ciprofloxacin, norfloxacin, nalidixic acid.
    • Used to treat UTIs, respiratory and soft tissue infections.
  • Metronidazole:
    • Induces dsDNA breaks.
    • Used to treat Giardia and anaerobic infections.
  • Rifamycins block RNAP elongation.
    • Rifampin (a.k.a. rifampicin).
    • Used to treat mycobacterial infections.
  • Trimethoprim/sulfamethoxazole:
    • Target folate biosynthesis (precursors for nucleic acid synthesis).

Antibiotics That Cause Injury to the Plasma Membrane

  • Lipopeptides:
    • Cause structural changes in the membrane, followed by arrest of the synthesis of DNA, RNA, and protein.
    • Examples:
      • Daptomycin (lipoglycopeptide).
      • Polymyxin B (topical, combined with bacitracin and neomycin in over-the-counter preparations).
      • Colistin (Polymyxin E) - Last resort antibiotic for Gram-neg.
  • Isoniazid – inhibits an enzyme important for cell wall and lipid precursor synthesis; affects mycolic acids.
  • Triclosan (a.k.a. Irgasan) – interferes with fatty acid synthesis.

Antibiotic Resistance Mechanisms in Bacteria

  • Four ways to circumvent the action of antibiotics:
    1. Alteration of antibiotic target:
      • Mutation in ribosomes, gyrase, penicillin-binding proteins, etc.
    2. Enzymatic destruction or inactivation of drug:
      • Beta-lactamases.
    3. Reduction of penetration of drug:
      • Mutation or downregulation of expression of outer membrane porins.
    4. Efflux of antibiotic:
      • Broad substrate efflux pumps like AcrAB-TolC.
      • Specific substrate pumps like TetA.

Enzymatic Destruction – Beta-Lactamases

  • Lactam ring in beta-lactam antibiotics is targeted for cleavage by beta-lactamases.
  • Enzymes that destroy carbapenems are called carbapenemases.
  • Many classes exist:
    • Ambler classes A, C, and D are serine-type; Ambler class B are metalloproteases.
  • Highly variable specificity.
  • ESBLs have activity against most 3rd generation cephalosporins and nearly all first and second-generation drugs.
  • Types include: SHV, CTX, TEM, and OXA.

Clavulanic Acid Mechanism of Action

  • Clavulanic acid is an inhibitor of beta-lactamases and is often administered with beta-lactam antibiotics to improve their efficacy.

Inactivation of Aminoglycosides

  • Aminoglycoside N-acetyltransferases (AACs): N acetylation
  • Aminoglycoside O-phosphotransferases (APHs): O phosphorylation
  • Aminoglycoside O-nucleotidyltransferases (ANTs): O adenylation

Inactivation of Chloramphenicol

  • CAT (chloramphenicol acetyltransferase) covalently attaches an acetyl group to chloramphenicol.
  • This blocks chloramphenicol binding to ribosomes.

Mechanism of Vancomycin Resistance

  • Susceptible bacteria:
    • Vancomycin binds to D-Ala-D-Ala, preventing cross-linking.
  • Resistant bacteria:
    • One enzyme (VanH) catalyzes the conversion of pyruvate to D-lactate.
    • A second enzyme (VanA or VanB) leads to the formation of D-Ala-D-lactate; vancomycin cannot bind D-Ala-D-lactate.
    • A third enzyme (VanX) cleaves any D-Ala-D-Ala formed by the usual pathway back to D-Ala, thus preventing incorporation of D-Ala-D-Ala into cell wall peptides.

Penetration and Efflux

  • Gram-negative outer membranes block large molecules like antibiotics.
  • Antibiotics enter cells through outer membrane porins.
  • OmpF is a major entry porin for antibiotics in Enterobacteriaceae.
  • A reduction in the number of porin molecules in the membrane reduces the susceptibility of Gram-negatives to antibiotics.
  • Efflux pumps can pump antibiotics out of cells before they reach their targets.
  • AcrAB-TolC effluxes multiple classes of antibiotics.
  • TetA effluxes tetracyclines.

Gene Transfer in Bacteria

Two types of gene transfer:

  • Vertical gene transfer – genes passed down through generations (your parents, grandparents, etc, mutations in bacteria).
  • Horizontal gene transfer – exchange of genetic material between members of the same generation.
    • Genes acquired horizontally come in many forms and some require recombination.

The Mosaic Nature of Genomes

  • A surprise arising from bioinformatic studies is the mosaic nature of all microbial genomes.
  • Example: Escherichia coli’s genome is rife with genomic islands, inversions, deletions, and paralogs and orthologs.
  • This is the result of heavy horizontal gene transfer, recombinations, and a variety of mutagenic and DNA repair strategies.

Recombination

  • Two different DNA molecules in a cell can recombine by one of two main mechanisms:
    • Generalized recombination requires that the two recombining molecules have a considerable stretch of homologous DNA sequences.
    • Site-specific recombination requires very little sequence homology between the recombining DNA molecules (e.g., lambda phage).
      • But it does require a short sequence recognized by the recombination enzyme.

Horizontal Gene Transfer Mechanisms

  • Transformation – Transfer of “naked” DNA.
  • Transduction – Transfer of genes via bacteriophage.
    • Generalized.
    • Specialized.
  • Conjugation – Transfer of plasmids between bacteria.
  • Hfr formation – Acquisition of chromosomal genes onto plasmids.

Gene Transfer - Transformation

  • Transformation is the process of importing free DNA into bacterial cells.
  • Initially discovered by Frederick Griffith in 1928 – called transforming factor.
  • DNA was identified as the “transforming factor” by Avery, MacLeod, and McCarty in 1941.
  • Transformation provided the first clue that gene exchange can occur in microorganisms.

Gene Transfer by Conjugation

  • Conjugation is the transfer of DNA from one bacterium to another, following cell-to-cell contact.
  • Often called “bacterial sex.”
  • It is typically initiated by a special pilus protruding from the donor cell.

Conjugation

  • Conjugation requires the presence of special transferable plasmids.
  • These usually contain all the genes needed for pilus formation and DNA export.
  • A well-studied example in Escherichia coli is the fertility factor (F factor).
    • Contains two replication origins:
      • oriV: used in nonconjugating cells.
      • oriT: used during DNA transfer.
  • Conjugation begins with contact between the donor cell, called the F+ cell, and a recipient F– cell.

Integration of F-Plasmid into the Chromosome

  • Plasmids are extra chromosomal DNA molecules that carry nonessential (for life) genes.
  • They can be transferred between bacterial species.
  • The F-factor plasmid can integrate into the chromosome.
  • The cell is now designated Hfr, or high-frequency recombination strain.
  • An Hfr cell is capable of transferring chromosome parts into a recipient cell.
  • Genes are transferred in order. The entire chromosome takes about 100 minutes to transfer.
  • The process can be used to map genes.

Mis-Excision of F-Plasmid Creates F'-Plasmid

  • An integrated F factor can excise from the chromosome.
  • Aberrant excision results in an F′ factor or F′ plasmid, which carries chromosomal genes.
  • This mechanism can create plasmids carrying antibiotic resistance determinants or virulence factors (e.g., toxins, protein secretion systems, siderophores, etc.).

Gene Transfer by Phage Transduction

  • Transduction is the process in which bacteriophages carry host DNA from one cell to another.
  • This occurs accidentally as an offshoot of the phage life cycle.
  • There are two basic types:
    • Generalized transduction: can transfer any gene from a donor to a recipient cell.
    • Specialized transduction: can transfer only a few closely linked genes between cells.

Generalized Transduction

  1. A phage infects the donor bacterial cell.
  2. Phage DNA and proteins are made, and the bacterial chromosome is broken into pieces.
  3. Occasionally during phage assembly, pieces of bacterial DNA are packaged in a phage capsid. Then the donor cell lyses and releases phage particles containing bacterial DNA.
  4. A phage carrying bacterial DNA infects a new host cell, the recipient cell.
  5. Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells.

Bacteriophage Life Cycles - Lysogeny

  • Phage DNA circularizes and enters the lytic cycle or lysogenic cycle.
  • Phage DNA integrates within the bacterial chromosome by recombination, becoming a prophage.
  • Lysogenic bacterium reproduces normally.
  • Occasionally, the prophage may excise from the bacterial chromosome by another recombination event, initiating a lytic cycle.

Specialized Transduction

  • Specialized transduction is restricted to moving host genes flanking 1.
  • Integration of phages into the chromosome is typically stable and can last indefinitely.
  • Excision of phage from the genome precedes the lytic cycle.
  • The “decision” to excise from the genome depends on DNA repair status (i.e., DNA damage occurs).
  • Chemical agents that cause DNA damage can induce prophages into a lytic cycle.

Review question

A kidney-damaging toxin produced by Enterohemorrhagic E. coli (EHEC) O157:H7 is encoded by a lysogen found in the chromosome. Predict what would happen to a person with an EHEC O157:H7 infection who was treated with ciprofloxacin (a fluoroquinolone) antibiotic. (Hint: think about the amount of toxin that would be produced and the target of the toxin)Hint: Ciprofloxacin will induce a lytic cycle because it damages DNA. The cell will then produce the toxin, which damages the kidneys. This would not be helpful to the patient.