Antibiotic Mechanisms and the Evolution of Bacterial Resistance

Course Announcements and Introduction to Microbiomes

• Administrative Changes: Implementation of changes will occur at the end of the quarter. Detailed explanations will be sent via email; however, these shifts are not expected to impact students negatively.

• The Microbiome: Humans carry approximately $2\,\text{to}\,3\,\text{pounds}$ of bacteria within their bodies. These microorganisms are generally beneficial and perform essential functions for human health.

• Core Objective: The lecture shifts from the beneficial microbiome to the mechanisms of killing harmful bacteria, specifically focusing on the discovery of antibiotics and the escalating "arms race" of antibiotic resistance.

The Historical Impact of Bacterial Infections

• Long-term Human Threat: Bacteria have threatened human survival for the entirety of $\text{Homo sapiens'}$ existence and likely longer.

• Archaeological Evidence:

  • Otzi the Iceman: A frozen mummy discovered in the Alps, dated to $5,300\,\text{years}$ ago, was found to be carrying Lyme disease and $H.\,pylori$ (the bacterium responsible for stomach ulcers).

  • Brucella Infections: Evidence of these infections has been identified in the bones of human ancestors dating back $1.5 \times 10^6\,\text{years}$.

• The Bubonic Plague (Black Death): This bacterial infection killed between $30\%\,\text{and}\,60\%\,\text{of Europe's population}$ during the $14\text{th century}$.

• Pre-Antibiotic Reality:

  • Simple wounds or cuts could be fatal due to infection.

  • Childbirth was extraordinarily dangerous for both mothers and infants due to potential septic scenarios.

  • Tuberculosis (TB): Historically known for causing patients to cough up blood, it was a leading cause of death among young adults.

  • Common Ailments: Before treatments, conditions like strep throat, pneumonia, food poisoning, sexually transmitted infections (STIs), skin infections, and ear infections posed significantly higher mortality risks.

The Discovery and Rise of Antibiotics

• The "Happy Accident" (1928): Alexander Fleming, working in a London laboratory, left culture dishes unattended while on vacation. Upon return, he observed that a contaminating mold (a fungus) was killing the bacteria in the dish.

• Penicillin discovery: This observed antagonism led to the identification of penicillin, though it was not widely available until the $1940\text{s}$.

• World War II: The war created a massive demand for antibiotics to treat field injuries, which spurred the development of mass-production techniques.

• Revolutionary Status: Antibiotics were termed "miracle drugs" because they made surgeries survivable, childbirth safe, and previously fatal infections routinely treatable. This represents one of the most dramatic improvements in human welfare in history.

Antibiotic Design Principles and Mechanisms of Action

• Selective Toxicity: The fundamental design principle for any antimicrobial drug is to target a structure or process unique to the pathogen to avoid killing the host (the patient).

• Primary Bacterial Targets:

  • Cell Walls: Bacteria possess cell walls, whereas human cells do not. This is the most popular target for antibiotics.

  • Evolutionary Divergence: Targets can include shared structures that are evolutionarily distinct enough to be targeted selectively:

    • Ribosomes: Bacterial ribosomes differ significantly from human ribosomes.

    • DNA Synthesis Enzymes: Bacterial versions are distinct from human versions.

    • Metabolic Pathways: Bacteria synthesize their own coenzymes like folate (vitamin $B_9$), whereas humans must ingest it. Synthesis pathways for these compounds are prime targets.

• Mechanism of Penicillin:

  • Bacterial cell walls are dynamic, constantly breaking down and rebuilding during growth and division.

  • Penicillin blocks the rebuilding phase of the cell wall.

  • Lysis: Because bacteria are under positive internal pressure (swelling against the wall), a weakened wall causes the cell to burst, a process known as lysis.

Measuring Sensitivity: The Kirby Bauer Test

• Procedure: A "lawn" of bacteria is grown on an agar plate. Paper disks soaked in specific amounts/types of antibiotics are placed on the lawn. The antibiotic diffuses outward through the agar.

• Zone of Inhibition: If the bacteria are sensitive to the drug, a clear "halo" (area with no growth) appears around the disk. The size of this zone indicates the effectiveness of the concentration.

• Resistance Indicators: If the bacteria are resistant, they grow right up to the edge of the disk. This test is used clinically to determine which antibiotics will be effective for a specific patient's infection.

Patterns of Antibiotic Overuse and Resistance

• Agricultural Use: Approximately $70\%\,\text{of all antibiotic tonnage}$ in the United States is used in livestock. This is used to prevent disease in crowded conditions and to promote faster growth (though the mechanism for growth promotion is not fully understood). This is the largest contributor to the resistance problem.

• Medical Misuse:

  • Prescribing antibiotics for viral infections (colds, flus, sore throats), against which they have zero effect.

  • Patients failing to complete the full prescribed course of antibiotics.

  • Prophylactic overdose (taking drugs "just in case").

• Consumer Products: Antimicrobial ingredients in soaps and hand sanitizers often offer no benefit over plain soap. The FDA banned several of these ingredients from over-the-counter soaps in $2016$ to mitigate the evolution of resistance.

• The Evolutionary Cost: Overuse kills sensitive bacteria and leaves a "vacuum" for resistant strains to proliferate.

Mechanisms of Bacterial Resistance

1. Efflux Pumps: Membrane proteins physically pump the antibiotic out of the cell faster than it can diffuse in, keeping internal concentrations at sub-lethal levels (e.g., tetracycline resistance in $E.\,coli$).

2. Target Mutation: A random mutation changes the shape of the target protein (e.g., a ribosome or enzyme) so the drug cannot bind, but the protein remains functional (e.g., Tuberculosis resistance to rifampin/fluoroquinolones).

3. Drug Inactivation: Bacteria produce enzymes that chemically destroy the antibiotic. Beta-lactamases are enzymes that cleave the chemical bond in penicillin, rendering it useless.

4. Target Bypass/Replacement: The bacteria acquires a new gene that provides a backup version of the target protein. In MRSA, the $mecA$ gene encodes an alternative target protein that allows the bacteria to survive even when the original target is inhibited by methicillin.

The Case of MRSA and Superbugs

• Staphylococcus aureus (Staph a): A common resident of human skin and nostrils (found in $30\%\,\text{of the population}$). It becomes dangerous when it enters the bloodstream or soft tissues.

• Resistance Timeline:

  • $1940\text{s}$: Penicillin is the standard treatment.

  • $1950\text{s}$: $40\%$ of hospital staph isolates are penicillin-resistant.

  • $1960$: Methicillin (a modified penicillin) is introduced to defeat beta-lactamases.

  • $1990\text{s}$: Methicillin-resistant Staph aureus (MRSA) becomes widespread in hospitals and eventually the community.

• Multidrug Resistance: "Superbugs" accumulate multiple resistance mechanisms, often carried on plasmids (small, circular pieces of extra-genomic DNA).

• Clinical Mortality: The pharmaceutical pipeline for new antibiotics has slowed since the $1980\text{s}$, leading to rising mortality rates for infections with no remaining effective treatments.

• Contagiousness: Unlike cancer drug resistance, which is "private" (stays within one patient), bacterial resistance is contagious. One person's resistant infection can spread to the community.

Evolution by Natural Selection in Bacteria

• Fundamental Requirements:

  1. Variation: Individuals in a population must differ.

  2. Inheritance: Traits must be passible to offspring.

  3. Selection: Differential survival and reproduction based on traits.

  4. Time: Changes accumulate over generations.

• Selection vs. Creation: Natural selection does not create new traits. It acts on standing variation (genetic differences already present in the population before the antibiotic is introduced).

• Types of Selection:

  • Directional Selection: The environment favors one extreme of a trait. Antibiotic resistance is directional selection at maximum intensity, shifting the population toward high resistance.

  • Stabilizing Selection: The environment favors the average or median trait (e.g., human birth weight).

• Speed of Evolution: Bacteria reproduce much faster than humans, allowing us to witness the mechanical and inevitable process of evolution within a human timeframe.

Questions & Discussion

• Question: Why are people allergic to certain antibiotics (like penicillin) but not others?

• Response: Different antibiotics have different chemical compositions. Allergies, such as those to sulfa-based drugs, represent the body's negative immune reaction to a specific chemistry. Not all antibiotics share the same chemical base.

• Question: What happens if you do not take probiotics while on antibiotics? Do they kill all bacteria, and do probiotics ensure only good ones return?

• Response: Broad-spectrum antibiotics (like a "Z-Pak") act like a "nuclear bomb," killing bacteria indiscriminately. This creates a biological vacuum that can be filled by dangerous bacteria like Clostridium difficile (C. diff), which causes severe diarrhea. Probiotics are often recommended, but many are killed by stomach acid before they reach the gut. The balance is delicate.

• Question: How is penicillin derived from mold, given that mold often makes people sick?

• Response: Mold (fungi) and bacteria often compete for the same resources. Fungi like penicillin-producing species evolved to secrete antibiotics to kill bacterial competitors. Scientists identify these natural products and use chemistry to mass-produce the specific therapeutic compound for human use.