6/10-Computer: Advanced Microbiology: Pathogenesis, Immunology, and Research Dynamics

University Classifications and Research Funding Dynamics

• Carnegie Classifications (R1 vs. R2):

  • R1 Universities: Distinguished by their ability to secure extensive external grant funding. Examples include Harvard, Yale, University of Colorado, University of Virginia, Vanderbilt, UT Memphis, and Texas A&M. These institutions often bring in approximately $1 \times 10^9$ (one billion dollars) in research grants annually. In these environments, job security is frequently tied directly to the acquisition of external funding.

  • R2 Universities: Designated as institutions striving to elevate their research profiles. These schools typically receive less than $7,000,000$ per year in grant funding. Smaller state schools often fall into this category.

  • East Tennessee State University (ETSU) Context: ETSU is classified as an R2 institution because its total grant funding is below the $7,000,000$ threshold. However, it is positioned in the top tier of R2 schools due to the presence of professional schools in Medicine, Public Health, and Pharmacy.

• Administrative and Funding Challenges in Research:

  • Disparity in research intensity: A lab-based research professor may work $60$ or more hours per week in the laboratory, whereas administrators may equate this effort to humanities research (e.g., English or History) which may require fewer hours of active lab-based investigation.

  • Funding Scale: A small grant for a laboratory researcher (e.g., $10,000$) may be exhausted within an hour due to high overhead and material costs, whereas the same amount may be considered significant in other disciplines.

  • Infrastructure and Beautification: Controversy often exists regarding the allocation of university funds toward "beautification projects" (e.g., a $63,000,000$ new building) while existing lab facilities (like the Brown building) suffer from structural issues, aging equipment, or a lack of maintenance.

Practical Laboratory Realities and Technical Skills

• Maintenance and Equipment Issues:

  • Labs often operate with equipment dating back to the 1990s, such as older thermocyclers used for Polymerase Chain Reaction (PCR). Modern thermocyclers complete processes in $1.5$ to $2$ hours, whereas manual heat-block methods used in previous decades took $10$ hours.

  • Universities may neglect service contracts for essential equipment like autoclaves (which are necessary to run a microbiology lab effectively), forcing researchers to perform their own repairs (e.g., replacing o-seals and bearings).

• Career Preparation for Technicians:

  • Mastery of lab equipment is vital for securing employment as a technician (e.g., at Crown Labs) for students who do not immediately enter medical school.

  • Employers prioritize hands-on experience (knowledge of specific equipment) over a high Grade Point Average (GPA) if the student cannot perform practical tasks.

Introduction to the Domains of Life and Microbiota

• Three Domains of Life:

  • Archaea: A domain often overlooked in introductory courses but critical to human health. They are part of the normal human microbiota and can protect against conditions such as colitis, irritable bowel syndrome (IBS), Crohn’s disease, and potentially colorectal cancer.

  • Prokaryotes: Organisms lacking a true nucleus.

  • Eukaryotes: Organisms characterized by a true nucleus and complex structural organization.

• Eukaryotic Etymology:

  • Eu-: Greek/Latin for "true."

  • -kary: Greek root for "nucleus."

Pathogenic Protozoa and Parasites

• Eukaryotic Pathogens:

  • These include Protozoa like malaria (Plasmodium) and trypanosomes.

  • Coccidia: Parasites affecting dogs, cats, chickens, and mammals. Common examples include Cryptosporidium and Cyclospora.

  • Environmental Resistance: Cysts from these parasites are highly resistant to chlorine. Outbreaks in water supplies (even chlorinated pools) can lead to illness upon ingestion of contaminated water.

The Lifecycle and Ecology of Malaria

• Vector and Transmission:

  • Malaria is a vector-borne disease transmitted by the Anopheles mosquito.

  • The mosquito injects sporozoites into the host.

• Intra-host Cycle:

  • Liver Stage: Sporozoites travel to the liver, infect liver cells, and transform into merozoites, which replicate asexually (cloning).

  • Blood Stage: Merozoites infect Red Blood Cells (RBCs). This stage causes clinical symptoms: fever, chills, and general malaise.

  • Red blood cells lack a nucleus; therefore, the visual presence of genetic material in an RBC smear indicates infection (or other skip-borne pathogens like Ehrlichia or Anaplasmosis).

• Host Definitions:

  • Indeterminate (Intermediate) Host: The host where asexual reproduction occurs (Humans).

  • Determinate (Definitive) Host: The host where sexual reproduction occurs (Mosquitoes).

• Ecological and Historical Factors:

  • The U.S. reduced malaria by draining swamps and using pesticides (e.g., DDT). Historically, half of John Smith’s group in Jamestown died from malaria.

  • Factors like climate change and mosquito adaptation (e.g., Tiger mosquitoes living in flower boxes) are leading to a resurgence in the Mediterranean.

Mechanisms of Chloroquine and Treatment Challenges

• Mechanism of Action:

  • Chloroquine is used to treat malaria. It works by preventing the acidification of vesicles inside the infected host cell.

  • Acidification (increased $H^+$/lowered pH) normally signals the parasite to transition to the next lifecycle stage. By blocking this, chloroquine inhibits replication.

• Side Effects and Risk Assessment:

  • Since human immune cells (macrophages, neutrophils) also rely on vesicle acidification to kill pathogens, taking chloroquine can make a patient more susceptible to other infections.

  • Side effects can include vivid dreams, hallucinations, or a "jumpy" sensation.

• FDA Requirements: Drugs require a "rational mechanism of action" (knowing exactly what it affects in a cell) for approval.

Prokaryotic Classification and Structure

• The Gram Stain:

  • Developed by Hans Christian Gram in the 1880s.

  • Gram-Positive: Characterized by a thick peptidoglycan cell wall. Stains purple.

  • Gram-Negative: Characterized by a thin peptidoglycan layer and an outer membrane containing Lipopolysaccharide (LPS). Traditionally does not contain LPS in all species (e.g., Borrelia).

Pathogenesis of Borrelia burgdorferi (Lyme Disease)

• Biological Characteristics:

  • Morphology: A spirochete (corkscrew shape).

  • Genome: Highly unusual and segmented; consists of one small chromosome and up to $21$ linear and circular plasmids. These "plasmids" are often essential for survival, contradicting the traditional definition of a plasmid as "dispensable."

• Transmission and Infection Cycle:

  • Transmitted by the Ixodes tick genera.

  • Use of Outer Surface Proteins (OSPs):

    • OspA: Used by the bacteria to stick to the tick gut receptor (Trospa).

    • OspC: Required for mammalian infection; the bacteria switches from OspA to OspC during migration from the tick gut to the salivary glands.

• Immune Evasion and Lymphatic Disruption:

  • Borrelia disrupts the architecture of lymph nodes, specifically targeting subcapsular sinus macrophages (the "fly paper" of the immune system).

  • It interferes with the communication between B cells and T cells in the germinal centers. This leads to immunocompromise; infected individuals may not respond well to vaccines and are more susceptible to secondary infections (e.g., Pseudomonas pneumonia).

  • Antigenic Variation: The bacteria can change its surface proteins to avoid detection by antibodies.

Chronic Lyme Disease Controversy

• Clinical Debate: Many physicians claim a 10-14 day course of doxycycline cures the infection. However, research in non-human primates and mice shows that Borrelia can persist in lymph nodes and tissues (e.g., the brain) for the host's entire life if early treatment is missed.

• Diagnostic Signs: The Erythema Migrans (bullseye rash) is an inflammatory response to the bacteria moving through the skin. The rash expands because the bacteria move faster than immune cells.

Pseudomonas aeruginosa: The Opportunistic Pathogen

• General Characteristics:

  • Habitat: Ubiquitous in human environments; can even be found in bottled water.

  • Heat Tolerance: Grows at $42^\circ C$.

  • Identification: Produces distinct smells (tortilla chips or grapes) and pigments. It fluoresces green under UV light.

• Clinical Manifestations:

  • Burn Wounds: A leading cause of death in burn patients. ERs use UV light to detect colonizing Pseudomonas.

  • Eye Infections: The leading cause of infections in contact lens wearers; it can grow in lens solution.

  • Cystic Fibrosis (CF) and COPD: The primary cause of death in CF patients due to bronchiectasis.

• Biofilm Transformation in CF:

  • In the lungs of CF patients, Pseudomonas can mutate (specifically the MUC A or MuK gene) to become mucoid.

  • Mucoid strains overproduce alginate (a polysaccharide capsule), creating a thick, snot-like biofilm that is highly antibiotic-resistant and prevents mucociliary clearance.

Cystic Fibrosis (CF) Mechanisms

• Genetic Basis: A recessive mutation in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene.

• Physiological Impact:

  • In healthy cells, CFTR transports Chloride ions ($Cl^-$) out of the cell; water follows by passive diffusion to thin the mucus.

  • In CF, the absence of this transport results in thick, sticky mucus that traps bacteria and prevents cilia from functioning.

Mycobacterium tuberculosis (TB)

• Global Epidemiology:

  • Currently the number one infectious disease killer globally. In 2023, approximately $1.8 \times 10^6$ people died from TB.

  • The COVID-19 pandemic set TB progress back by decades due to disrupted surveillance and treatment.

  • Synergy with HIV: HIV-infected individuals have a $10\%$ chance of developing active TB every year, compared to a $10\%$ lifetime risk for immunocompetent individuals.

• Treatment and Prevention:

  • Requires a grueling 3-6 month regimen of Isoniazid, Pyrazinamide, and Ethambutol.

  • BCG Vaccine: An attenuated version of Mycobacterium bovis. It is not used in the U.S. because it interferes with PPD skin tests and does not protect adults from pulmonary TB; it only protects children from miliary TB (meningitis).

Biosafety Levels (BSL)

• BSL-2: Standard lab safety.

• BSL-3: Required for pathogens like Mycobacterium tuberculosis and Burkholderia pseudomallei. Involves high maintenance costs ($1M+$/year), specialized air filtration, and protective gear (e.g., PAPRs).

• BSL-4: Required for lethal viruses like Ebola or Marburg. Researchers wear full Tyvek suits with external air supplies. There are only about four or five such facilities in the U.S. (e.g., Galveston, UT; Walter Reed).

Questions & Discussion

• Queston: Can Coccidia be found in saltwater?

• Response: Yes, they are highly resistant to harsh environmental conditions.

• Question: How long does it take for blood to filter through the spleen?

• Response: In mice, bacteria can be tracked to the spleen within three hours. For humans, it is relatively quick, but repeat infections from the liver act like a "time-release" parasite, making treatment take longer.

• Question: Can you take Chloroquine as a preventative?

• Response: Yes, people traveling to endemic areas take it to prevent the malaria cycle from establishing.

• Question: Can you get TB from raw milk?

• Response: Yes, Mycobacterium bovis (bovine TB) can be transmitted via unpasteurized milk, which is a significant risk for those consuming raw dairy products.