Microbiology Exam Review: Antimicrobials, Drug Resistance & Subviral Agents

In Vivo vs. In Vitro

• In vivo

  • Definition: Experiments performed “within the living” organism (whole animal, human, or plant).

  • Environment: Natural, physiologic conditions (blood supply, immune responses, hormonal signaling, etc.).

  • Significance: Reveals systemic effects, pharmacokinetics, toxicity, immune interactions.

  • Limitations: Ethical constraints, high cost, more biological noise → harder to control variables.

• In vitro

  • Definition: Experiments performed “within glass”—i.e., in test tubes, petri dishes, microplates, cell-culture flasks.

  • Environment: Highly controlled, simplified, artificial.

  • Significance: Precise manipulation of variables, high-throughput screening, mechanistic clarity.

  • Limitations: May not faithfully replicate host physiology (no immune system, altered pH, no drug metabolism).

• Quick mnemonic: "Vivo = in the living; Vitro = in the vitro (glass)."

Prions

• Definition: Infectious misfolded proteins (PrP\textsuperscript{Sc}) lacking nucleic acids; propagate by inducing conformational change in native PrP\textsuperscript{C}.

• Properties: Highly resistant to heat, radiation, proteases, chemical disinfectants; extremely long incubation.

• Human / animal diseases (3 classic examples)

  1. Creutzfeldt–Jakob disease (CJD, vCJD)

  2. Kuru (ritualistic cannibalism among the Fore people)

  3. Bovine spongiform encephalopathy (BSE, “mad-cow”) → may jump to humans as vCJD

Satellite Viruses vs. Viroids

• Satellite viruses

  • Require a helper (usually unrelated) virus to coinfect the same host cell in order to replicate.

  • Example: Hepatitis D virus (HDV) needs hepatitis B virus (HBV) envelope proteins.

• Viroids

  • Infectious, naked circular ssRNA (≈ 250–400 nt) that lack capsid and do not encode proteins.

  • Replicate autonomously in plant nuclei/chloroplasts using host RNA polymerase.

  • Cause diseases in crops (e.g., potato spindle tuber viroid).

Chapter 9 (PowerPoint: first 5–6 slides)

⭑ Typical introductory slides review key control terms—know the precise differences.

• Sterilization: Complete removal/destruction of all microbial life incl. endospores and viruses (inanimate objects).

• Disinfection: Physical or chemical process destroying vegetative pathogens, not spores (inanimate surfaces).

• Antisepsis / Degerming: Chemicals applied to body surfaces to destroy/inhibit pathogens; mechanical removal of microbes from tissue.

• Sanitization: Cleansing technique that mechanically removes microbes and debris to safe public-health levels.

• Asepsis: Practices that prevent entry of infectious agents (surgical asepsis, handwashing, flame mouth of tubes).

• Sepsis: Microbial contamination or infection of blood/tissues.

• Bactericidal vs. Bacteriostatic: Killing vs. inhibiting growth.

Chapter 10 – Antimicrobials

Table 10.1 “Named Ideals” of Antimicrobial Drugs

• Selective toxicity: Harms microbe > host.

• Microbicidal, not microbistatic: Kills rather than merely inhibits.

• Soluble & active at low concentration: \le 1\;\text{µg mL}^{-1} preferred.

• Stability: Resists premature inactivation; active in tissues/fluids with variable pH & O\textsubscript{2}.

• No resistance development (or slow).

• Complements/assists host defense—doesn’t suppress immunity.

• Readily delivered: Oral, parenteral, topical.

• Reasonably priced & readily available.

Table 10.2 Essential Terms (with meaning)

• Broad-spectrum (extended-spectrum): Active against many Gram + and Gram – species.

• Narrow-spectrum: Target limited range (e.g., only Gram + cocci).

• Bactericidal vs. Bacteriostatic (see above).

• Minimum inhibitory concentration (MIC): Lowest concentration preventing visible growth after 18–24 h.

• Minimum bactericidal concentration (MBC): Lowest concentration killing \ge 99.9\% of inoculum.

• Synergism: Combined action > additive (\text{FIC}_\text{index} < 0.5) . Example: TMP + SMX.

• Antagonism: One drug blocks another (e.g., bacteriostatic + bactericidal targeting cell wall).

• Post-antibiotic effect (PAE): Persistent suppression after drug falls below MIC.

• Therapeutic index (TI) (see separate heading).

Zone of Inhibition (ZOI)

• Disk diffusion (Kirby–Bauer) assay on Mueller–Hinton agar.

• ZOI: Diameter (mm) of clear area where no colonies grow.

• Larger ZOI ⇒ microbe more susceptible at the test concentration.

• Compare to CLSI breakpoint chart (S/I/R categories).

• Which is better? Larger numerical ZOI → more susceptible → drug more likely effective.

Minimum Inhibitory Concentration (MIC)

• Serial two-fold dilutions (1, 0.5, 0.25, \dots \text{µg/mL}) inoculated with standard bacterial load (\approx 5\times10^5\;\text{CFU/mL}) .

• Lowest tube/well with zero turbidity = MIC.

• Clinical use: Guides dosage (serum level should exceed MIC for time-dependent drugs, or reach C\text{max}/MIC ≥ 10 for concentration-dependent drugs).

Therapeutic Index (TI)

• Quantifies drug safety.

• Classical formula: TI = \frac{\text{TD}{50}}{\text{ED}{50}} where TD\textsubscript{50} = toxic dose to 50 % of population; ED\textsubscript{50} = effective dose for 50 %.

• Newer microbiology texts: TI = \frac{\text{MIC for host}}{\text{MIC for pathogen}} (i.e., host toxicity ÷ pathogen inhibition).

• Bigger TI ⇒ safer because gap between effective and toxic dose is wide.

Selective Toxicity

• Concept introduced by Paul Ehrlich (“magic bullet”).

• Relies on structural / metabolic differences (peptidoglycan, 70S ribosome, ergosterol, folate synthesis).

• High selective toxicity → minimal host side effects.

Five Major Drug Categories by Mode of Action

1. Inhibition of cell-wall synthesis

   • β-lactams (penicillins, cephalosporins, carbapenems), glycopeptides (vancomycin), bacitracin.

2. Disruption of cell-membrane structure/function

   • Polymyxins, daptomycin; antifungal polyenes (amphotericin B, nystatin) and azoles alter ergosterol.

3. Inhibition of protein synthesis (70S ribosome)

   • Aminoglycosides, tetracyclines, macrolides (erythromycin, azithromycin), chloramphenicol, lincosamides.

4. Inhibition of nucleic-acid synthesis or integrity

   • Fluoroquinolones (DNA gyrase), rifamycins (RNA polymerase), metronidazole (DNA breaks under anaerobic conditions).

5. Antimetabolites / blockade of key metabolic pathways

   • Sulfonamides + trimethoprim (folate), isoniazid (mycolic acid), dapsone.

Common Drugs by Organism Type

• Bacterial infections

  • Gram + cocci: penicillin G/V, oxacillin, vancomycin.

  • Gram – rods: piperacillin-tazobactam, cefepime, gentamicin, ciprofloxacin.

  • Atypicals: doxycycline, azithromycin.

• Fungal infections

  • Azoles (fluconazole, itraconazole), echinocandins (caspofungin), polyenes (amphotericin B), allylamines (terbinafine).

• Protozoal infections

  • Metronidazole (Giardia, Trichomonas), chloroquine/mefloquine (Plasmodium), atovaquone-proguanil, artesunate, pentamidine (Trypanosoma).

• Helminth infections

  • Benzimidazoles (albendazole, mebendazole), ivermectin (Onchocerca, Strongyloides), praziquantel (schistosomes, tapeworms), pyrantel pamoate (pinworm).

Five Major Mechanisms of Drug Resistance in Microbes

1. Drug inactivation

   • β-lactamases, aminoglycoside-modifying enzymes, chloramphenicol acetyltransferase.

2. Decreased permeability / uptake

   • Porin mutations in Gram – outer membrane, thickened cell wall in VISA strains.

3. Efflux pumps

   • AcrAB-TolC in Enterobacteriaceae, MefA in Streptococcus.

4. Alteration / protection of target site

   • PBP2a (MRSA), methylation of 23S rRNA (macrolide resistance), DNA gyrase mutations (quinolones).

5. Bypass or substitution of metabolic pathway

   • Plasmid-encoded dihydropteroate synthase insensitive to sulfonamides; acquisition of folate from environment.

Probiotics vs. Prebiotics

• Probiotics: Live microorganisms (usually Lactobacillus, Bifidobacterium, Saccharomyces) administered to confer health benefit (restore gut flora after antibiotics, competitive exclusion of pathogens, produce bacteriocins).

• Prebiotics: Non-digestible food ingredients (inulin, fructooligosaccharides) selectively fermented by beneficial microbiota; “fertilizer” for probiotics.

• Synbiotics = combination product.

Superinfections

• Secondary infections arising when broad-spectrum therapy destroys normal flora, allowing resistant/opportunistic microbes to overgrow.

  • Common examples: Clostridioides difficile colitis after clindamycin/fluoroquinolones; vaginal candidiasis after broad-spectrum β-lactams.

• Prevention: Use narrow-spectrum agents when possible, consider probiotics, maintain infection-control practices.

Integrative & Ethical Points

• Balancing effective therapy vs. resistance evolution (antimicrobial stewardship).

• Selective toxicity framed within principle of non-maleficence in medicine.

• Prions highlight unique infection control challenges—require \ge 134^{\circ}\text{C} under pressurized steam w/ NaOH.

• Probiotic use intersects with regulations: classified as dietary supplements (USA) with less stringent oversight.

Key Equations & Numbers

• TI = \frac{TD{50}}{ED{50}}\;(>10=good)

• Example MIC determination: if growth in tubes up to 0.25\,\mu\text{g/mL} but not at 0.5\,\mu\text{g/mL} → MIC = 0.5\,\mu\text{g/mL}.

• ZOI interpretive standard (Staphylococcus vs. cefoxitin): \ge 22\,\text{mm}=S; \le 21\,\text{mm}=R.

Quick Review Checklist

• [ ] Can you distinguish in vivo from in vitro settings?

• [ ] List the 3 prion diseases and explain prion structure.

• [ ] Define satellite virus vs. viroid with an example.

• [ ] Accurately define sterilization, disinfection, antisepsis, sanitization, asepsis.

• [ ] Recall all 7 “ideal” drug characteristics (Table 10.1).

• [ ] Explain MIC, MBC, ZOI, TI and know which numerical value is preferable.

• [ ] Match 5 modes of action with representative drugs.

• [ ] Outline 5 resistance mechanisms.

• [ ] Differentiate probiotics, prebiotics, superinfections.

• [ ] Apply selective toxicity concept to antifungals vs. antibacterials.

Use this list to self-test before the exam!