21-22: Antimicrobial Chemotherapy and Clinical Microbiology

Antibiotics

• Minor infections that are easily treatable today used to be deadly just 60 years ago.
• The importance of antibiotics in treating diseases was recognized in the early 1940s.
• Antibiotics are compounds produced by one microbe species that inhibit growth or kill other microbes.

Antibiotic Discovery

• The usefulness of molds was known to ancient civilizations.
• Alexander Fleming discovered penicillin in 1929.
• Penicillin was initially forgotten due to its perceived instability.
• Howard Florey rediscovered penicillin in 1940, leading to its development as a therapeutic drug.
• Gerhard Domagk discovered sulfa drugs.
• Prontosil, a sulfa drug, doesn't directly affect bacteria on an agar plate.
• Prontosil was found successful in animal trials because it is metabolized by the body into sulfanilamide, which is the active antimicrobial agent.
• Some antimicrobial chemicals are inactive until converted by the body to an "active" form.
• Sulfanilamide is an analog of para-aminobenzoic acid (PABA).
• PABA is a precursor to folic acid.
• Sulfanilamide inhibits the conversion of PABA to folic acid, stopping bacterial growth.
• Bacteria use PABA to synthesize folic acid, which is essential for their growth.
• If bacteria cannot produce folic acid, they cannot grow because folic acid is needed for synthesis.

Selective Toxicity of Antibiotics

• Antibiotics must affect the infectious organism but not harm the patient.
• Many antibiotics have side effects at high concentrations.
• Chloramphenicol can interfere with our ribosomes and, at high levels, harm our bone marrow.
• Some antibiotics can cause allergic reactions because they are foreign substances to our bodies.
• Drugs should target microbial physiology, specifically something that humans don't have or do.
• Examples include peptidoglycan (the bacterial cell wall), differences in ribosome structure, and biochemical pathways missing in humans.

Spectrum of Activity

• Broad-spectrum antimicrobials are effective against many species.
• Narrow-spectrum antimicrobials are effective against few or a single species.
• Most antibiotics are discovered as natural products.
• Some antibiotics are modified artificially to increase efficacy or decrease toxicity to humans.
• Synthetic chemotherapeutic agents are clinically useful but chemically synthesized.

Types of Antibiotic Compounds

• Bactericidal antibiotics kill the target organism.
• Bacteriostatic antibiotics prevent the growth of the target organism, allowing the immune system to clear the infection.
• Many drugs only affect growing cells.
• Inhibitors of cell wall synthesis are only effective if the organism is building a new cell wall (e.g., Penicillin).
• Antibiotics must be taken until all cells leave the stationary phase.

Minimal Inhibitory Concentration (MIC)

• The minimal inhibitory concentration (MIC) is the lowest concentration of an antibiotic that prevents growth.

• MIC varies for different bacterial species.

• It's tested by diluting the antibiotic and observing the lowest concentration with no growth.

• At the MIC, organisms may still be alive but not growing.

• To test if organisms are still alive, the liquid is plated without the antibiotic.

  • If no colonies form, it indicates the Minimal Lethal Concentration(MLC).

Minimal Lethal Concentration (MLC)

• To find the MLC, plate the same volume from each dilution (each concentration is lower and lower).
• The MLC is the concentration at which no colonies grow.

Testing Antibiotic Efficacy

• The Kirby-Bauer disk diffusion test is used to test bacterial sensitivity to multiple antibiotics.
• Multiple disks soaked with different antibiotics are placed on an agar plate.
• The size of the cleared zones around the disks reflects the sensitivity of the bacteria to the antibiotic.

Disk Diffusion Test: Kirby-Bauer Method

• Disks soaked with specific drugs are placed on agar plates inoculated with a lawn of the test microbe.
• The drug diffuses from the disk into the agar, creating a concentration gradient.
• Cleared zones (no growth) are observed around the disks.
• The size of the clear zone is measured to determine the MIC.

Kirby-Bauer Assay and MIC

• The zone of clearing in the Kirby-Bauer test correlates with MIC values.
• The "zone of inhibition" is the edge of the no-growth zone.
• After incubating agar plates, measure the diameter of the zone of inhibition around disks.
• Compare this result with MIC values to determine if the antibiotic concentration is clinically useful.
• The MIC should be low enough to avoid causing side effects.

MIC and Drug Level in Tissue

• When using an antibiotic, drug concentration should be higher than the MIC to ensure effectiveness.
• As long as the drug concentration in tissue or blood remains higher than MIC, the drug will be effective.
• Drugs can be administered several times (multiple doses) and/or at higher doses.

Mechanisms of Antibiotic Action

• Antibiotics generally target microbe-specific physiology:
  • Cell wall synthesis/maintenance (peptidoglycan)
  • Cell membrane
  • DNA synthesis
  • RNA synthesis
  • Protein synthesis
  • Other metabolism

Mechanisms of Action of Antimicrobial Agents

• Cell wall synthesis: Penicillins, cephalosporins, bacitracin, vancomycin
• Protein synthesis: Chloramphenicol, tetracyclines, aminoglycosides, macrolides, lincosamides
• Cell membrane: Polymyxin, amphotericin, imidazoles (vs. fungi)
• Nucleic acid function: Nitroimidazoles, nitrofurans, quinolones, rifampin; some antiviral compounds, especially antimetabolites
• Intermediary metabolism: Sulfonamides, trimethoprim

Cell Wall Structure: Gram-Positive vs Gram-Negative

• Remember the Structures of Peptidoglycan?
  • Gram-negative: Direct cross-linking, thin layer
  • Gram-positive: Peptide interbridge, thick layer

Mechanisms of Antibiotic Action: Peptidoglycan (Cell Wall) Antibiotics

• Penicillins:
  • Competitive inhibitor of crosslink transpeptidation
  • The $\beta$-lactam is important in these compounds
  • Examples: Penicillin, Amoxicillin, Ampicillin, Carbenicillin
• Other cell wall synthesis inhibitors:
  • Vancomycin: binds ends of peptides; prevents crosslink formation (same step as penicillin, but different activity)
  • Cycloserine: blocks formation of peptide for crosslink
  • Bacitracin: blocks movement across membrane, disaccharide subunits don’t ever reach the periplasm

Beta-Lactam Mechanisms

• Cycloserine inhibits the formation of L-Ala.
• Amino acids sequentially add to N-acetylmuramic acid (NAM).
• D-Alanine peptide attaches.
• NAM pentapeptide transfers to bactoprenol.
• N-acetylglucosamine (NAG) links to NAM.
• Bactoprenol "flips," moving NAM-NAG to the outer side of the cytoplasmic membrane.
• Transglycosylase attaches a new disaccharide unit to the existing chain.
• A pentaglycine connects L-Lys on one side chain and the penultimate D-Ala on the other.
• One phosphate on liberated bactoprenol is removed, and the lipid moves back to the cytoplasmic side of the membrane.
• Bacitracin prevents bactoprenol from accepting new units of UDP-NAM.
• Vancomycin, penicillin, and cephalosporins inhibit peptide cross-linking.

Beta-Lactams

• Derived from Cys and Val.
• Different R groups change the antimicrobial spectrum and stability of the derivative penicillin.
• Penicillin resembles the “D-ala-D-ala” part of peptidoglycan.
• Allows the drug to bind to transpeptidase and transglycosylase, halting cell wall synthesis.
• Beta-lactamases:
  • Enzymes that cleave the $\beta$-lactam ring of $\beta$-lactams.
  • Enzyme is transported out of the cell into:
    • Surrounding media (Gram-positive bacteria)
    • Periplasm (Gram-negative bacteria)

Mechanisms of Antibiotic Action: DNA Synthesis and Metabolism

• Antibiotics interfering with DNA (synthesis or integrity):
  • Quinolones: i.e., nalidixic acid
    • Blocks bacterial DNA gyrase, preventing DNA replication
• Antibiotics that interfere with bacterial metabolism:
  • Sulfa drugs: Known as anti-metabolites; interfere with the synthesis of metabolic intermediates
    • Analogue of PABA, a vitamin B9 precursor, needed for DNA synthesis

Mechanisms of Antibiotic Action: RNA Synthesis Inhibitors

• Examples: Rifampicin and actinomycin D
• Inhibit transcription
• Bactericidal and most active against growing bacteria
• Rifampicin:
  • Blocks bacterial RNA Polymerase
• Actinomycin D:
  • Binds DNA and inhibits polymerase movement

Ribosomal Inhibitor Antibiotics

• Macrolides, chloramphenicols, lincosamides
  • Bind large subunit, block transfer of peptides
  • i.e., Erythromycin, azithromycin
• Tetracyclines
  • Bind small subunit, blocks binding of aminoacyl-tRNA
• Aminoglycosides
  • Prevent 30S and 50S subunits from binding each other
  • i.e., Streptomycin

Mechanisms of Antimicrobial Action: Disruption of Cytoplasmic Membranes

• Some drugs become incorporated into the cell membrane and damage it
  • Gramicidin
  • Polyenes (e.g., amphotericin B)
  • Azoles
  • Allyamines
  • Polymyxin

Mechanisms of Antibiotic Action: Disruption of Cytoplasmic Membranes (Gramicidin)

• Makes holes in the bacterial cell membrane, killing the cell
• Gramicidin:
  • Forms a cation channel, H+ leaks
  • Cell can’t maintain Proton Motive Force
  • Only use topically to treat or prevent infections because the drug can also damage human cell membranes

Antibiotic Resistance

• A growing problem due to antibiotic overuse.
• Overprescribed, taken too long, used in farm animal feed.
• Exerts strong selective pressures for drug-resistant strains.
• Many strains become multidrug-resistant.
• Over 80% of Streptococcus pneumoniae infections are now penicillin-resistant in some countries.

Methods of Antibiotic Resistance

• Destroy the antibiotic before it gets into the cell.
• Add modifying groups that inactivate the antibiotic.
• Modify the target so that the antibiotic no longer binds to it.
• Pump the antibiotic out: Use an efflux pump to move the antibiotic out of the cell.

Alternative Mechanisms of Antibiotic Resistance

• Alter the target to prevent antibiotic binding.
• Degrade the antibiotic to render it inactive.
• Modify the antibiotic to reduce its effectiveness.
• Pump the antibiotic out of the cell using efflux pumps.

Antibiotic Resistance Mechanisms

• Destroying the antibiotic:
  • Many bacteria make beta-lactamase, which destroys penicillin and its analogues.
  • This makes the bacteria resistant and lowers penicillin concentration, protecting nearby cells.
• Modifying the antibiotic:
  • Enzymes modify aminoglycoside antibiotics using acetylation, phosphorylation, and adenylation.
  • This changes the antibiotic so it no longer interferes with ribosomes.
• Altering the target:
  • Modify the target enzyme of penicillin or the ribosome.
  • This is the most common streptomycin-resistance mechanism.
• Drug efflux:
  • Uses a multidrug resistance transporter (MDR) to pump the drug out of the cell.
  • This often confers resistance to several different classes of antibiotics simultaneously.

How Antibiotic Resistance Happens

• Lots of germs, a few are drug resistant
• Antibiotics kill bacteria causing the illness, as well as good bacteria protecting the body from infection
• The drug-resistant bacteria are now allowed to grow and take over
• Some bacteria give their drug-resistance to other bacteria, causing more problems

What’s Causing the Rise in Antibiotic Resistance?

• Indiscriminate use of antibiotics:
  • Doctors prescribe antibiotics without knowing if an illness is bacterial.
  • Patients demand antibiotics for illnesses.
  • Doctors enable the patient instead of saying “no.”
• Feeding antibiotics to livestock to increase size (not for infection).
• Not finishing your prescription:
  • Not allowing enough time to completely remove the pathogen.
  • Allows time for drug resistance to be acquired.

Preventing the Emergence of Antimicrobial Resistance

• Give antibiotics in high concentrations.
• Give two or more drugs at the same time.
• Finish your prescriptions.
• Use antibiotics only when absolutely necessary.
• Possible future solutions:
  • Continued development of new drugs
  • Use bacteriophage to treat bacterial diseases (phage therapy)

Fighting Antibiotic Resistance

• Strategies to prevent or treat drug-resistant pathogens:
  • Chemically alter the antibiotic
  • Use more than one antibiotic
  • Use another drug to inactivate resistance enzymes (e.g., Clavulanic acid with penicillin).
    • Clavulanic acid is a $\beta$-lactam but not an antibiotic.
    • Competitively binds to $\beta$-lactamases secreted from penicillin-resistant bacteria.
    • Allows penicillin (or amoxicillin, etc.) to enter and kill the bacteria.

How Does Drug Resistance Develop?

• Bacterial transformation: Release of DNA containing antibiotic-resistance genes which are then acquired by a recipient cell.
• Bacterial transduction: Transfer of antibiotic-resistance genes via bacteriophages from a donor cell to a recipient cell.
• Bacterial conjugation: Transfer of antibiotic-resistance genes from a donor to a recipient cell via plasmids or transposons.

Sources of Resistance Genes

• Many bacteria have natural antibiotic resistance to protect themselves from their own antibiotics or against competitors in their environment.
• Acquired resistance:
  • Incomplete use and/or overuse of antibiotics leads to increased selective pressure on bacteria.
• Antimicrobial resistance mutations: very rare spontaneous mutations which confer resistance (usually changes the drug target).
• Horizontal gene transfer: getting resistance genes from another organism.
• R plasmid: Often carries multiple resistance genes.
• Mobile genetic elements: transposons, integrons.

Antiviral Agents

• Hard to find antivirals (they’re often toxic to us).
• Viruses use host enzymes, so it’s hard to target.
• There are few targets unique to viruses.
• Inhibition of viral uncoating and release (e.g., Influenza virus).
  • Amantadine inhibits viral uncoating, preventing entry of the virus into the host cell.
  • Zanamivir blocks neuraminidase, preventing the release of mature viruses.

Paxlovid (Nirmatrelvir + Ritonavir)

• Paxlovid uses Nirmatrelvir:
  • The drug blocks the catalytic site of SARS-CoV-2’s main protease, preventing protein processing.
• Paxlovid uses Ritonavir too:
  • Ritonavir slows your liver’s degradation of Nirmatrelvir; it is a CYP3A4 inhibitor.

Biosafety Containment Procedures

• BSL-1:
  • Low-risk microbes
  • Work can be performed on an open lab bench or table
  • Personal protective equipment are worn as needed
  • Example of risk group 1 organism: E. coli K-12, Saccharomyces spp.
• BSL-2:
  • Personal protective equipment is worn
  • Biological safety cabinet
  • Blood or human/animal tissues
  • Example of risk group 2 organism: Diarrheagenic E. coli, Samonella spp., Zika virus
• BSL-3:
  • Personal protective equipment is worn; respirators may be used
  • Biological safety cabinet
  • Entrance to the lab is through two sets of self-closing and locking doors
  • Example of risk group 3 organism: SARS, Mycobacterium tuberculosis
• BSL-4:
  • Full body, air-supplied, positive pressure suit
  • Treatments may not be available
  • There are a few BSL-4 facilities in the United States.
  • Example of risk group 4 organism: Ebola virus, Hantavirus

Principles of Clinical Microbiology

• Pathogen identification is critical for appropriate treatment.
• Antibiotics don’t work on all bacteria, and many bacteria are now drug-resistant.
• Antibacterials don’t work on viruses
• Anticipate likely downstream issues:
  • A sore throat could signal a mild infection or cause serious heart and kidney complications.
• Track spread of disease:
  • Allows faster treatment of others infected and identification of cause of infection.

Specimen Collection

• Taking samples from:
  • A. Urinary catheter
  • B. Throat swab
  • C. Sputum collection
  • D. Lumbar puncture

How to Identify the Pathogen?

• Use your knowledge of microbial physiology
• Different pathogens have different biochemistry, nutrient requirements, growth environments (pH, temperature, osmotic resistances, oxygen tolerance), and structures (cell wall, capsule, pilus, flagellar composition).

Determination by Structure

• Some specimens, such as spinal fluid, throat swabs, and sputum, can be directly stained with differential stains (i.e. Gram-stain).

How to Identify the Pathogen? Differential and Selective Media

• Grow on differential media
  • Strains grow, but differ in indicator color
  • Based on ability to utilize material on plate
  • Indicator dyes
• Grow on selective media
  • Removes non-pathogenic strains
  • Allows growth of pathogen
  • More cells makes testing easier
  • Ability to grow on medium helps identification

Determination by Metabolism

• Analytical profile index (API) strip technology:
  • Sample/culture with bacterium added to each well that performs biochemical reactions
  • Resulting reactions causes indicator color changes that indicates positive or negative reactions

API Strip Tests

• API strips include several biochemical tests in one strip with a separate well for each test.
• Each well contains chemicals needed for a different biochemical test.

Rapid Techniques for Pathogen ID

• Molecular detection methods for bacteria are typically more rapid than traditional culture-based methods (hours versus days or weeks).
• These detection methods are especially useful for viruses.
• They may also provide the only means of identifying newly recognized or emerging pathogens (e.g., SARS).

Molecular Identification Techniques

• PCR (Polymerase Chain Reaction):
  • Amplifies small fragment of DNA
  • Allows detection of tiny numbers of bacteria
  • Size of fragment can indicate species, strain
  • Samples from Clostridium botulinum toxin genes.

Nucleic-Acid Based Techniques

• Quantitative real time PCR
  • Used to quantify the amount of microbe-specific DNA using targeted primers
  • Can be used to quantify microbial RNAs using reverse transcriptase to generate complementary DNA from RNA templates

Identifications Based on Serology

• ELISA (enzyme-linked immunosorbent assay)
  • Can detect antigens or antibodies present in nanogram and picogram quantities.
  • Antigen from virus is attached to wells.
  • Patient serum is added
  • Antibody binds to viral antigen
  • Secondary antibodies have an enzyme attached.
  • Bind to Fc portion of serum antibodies
  • Enzyme reaction causes color change.
  • Indicates presence of antivirus antibodies in serum

Enzyme-Linked Immunosorbent Assay (ELISA)

• Ebola antigen is attached to an ELISA plate with albumin.
• Excess material is rinsed off, and patient serum is added.
• Human anti-Ebola antibodies from patient serum bind to Ebola antigen.
• Unbound serum is washed off, and conjugated antibody is added.
• Rabbit anti-human IgG antibody with an attached (conjugated) enzyme.
• Unbound conjugated antibody is washed off and substrate is added.
• The rate of conversion of substrate to colored product is proportional to the amount of anti-Ebola antibody present in the patient's serum.

Point-Of-Care Tests

• Point-of-care (POC) laboratory tests are designed to be used directly at the site of patient care, such as physicians’ offices and outpatient clinics, and can even be used at home.
• Commercial POC tests are widely available for the diagnosis of bacterial and viral infections and for parasitic diseases including malaria and COVID-19.
• Rely on immunochromatography to give results.