T13

Lecture Topic: Controlling Microbial Growth and Antimicrobial Resistance

Methods to Control Microbial Growth

  • Physical Sterilization

    • Heat - autoclaving, pasteurization

    • Radiation - UV, gamma, x-ray

    • Filter - HEPA, membrane filters

  • Chemical Agents

    • Antimicrobials for internal use - antibiotics, antivirals, antifungals

    • Sterilants - kill everything including spores (bleach, hydrogen peroxide vapor)

    • Disinfectants - kill microbes on surfaces

    • Antiseptics - safe for skin

Not all methods work equally well on all microbe types (bacteria vs viruses vs fungi)

1. Bacteria (Vegetative Cells)

What kills them easily

  • Autoclaving

  • Boiling (depending on organism)

  • Disinfectants (bleach, alcohol, Lysol)

  • UV radiation

  • Antibiotics

  • Pasteurization (reduces load, not sterilization)

Why they're killable

  • They rely on membranes, proteins, and DNA that are easily damaged by heat or chemicals.

Exceptions

Some bacteria are unusually resistant (Mycobacterium, Pseudomonas)

  1. Bacterial Endospores (Hardest to Kill)

Examples: Bacillus anthracis, Clostridium botulinum

What can kill spores

  • Autoclaving (121°C, 15 psi, 15–20 min)

  • Dry heat (>200°C)

  • Chemically: ethylene oxide, high-concentration bleach

  • Gamma radiation

What CAN'T kill spores

  • Alcohol

  • UV (penetrates poorly)

  • Most disinfectants

  • Pasteurization

Why they’re so hard to kill

Spores have:

  • Thick protein coats

  • Dehydrated cytoplasm

  • Calcium-DPA stabilizing DNA

  • Almost no metabolism

3. Viruses (Enveloped vs. Non-enveloped)

Viruses are NOT all the same. Their structure determines what kills them.

Enveloped viruses

(e.g., influenza, HIV, coronaviruses)

Easiest to kill.

What kills them:

  • Alcohol

  • Detergents/soap

  • Disinfectants

  • UV

  • Heat

Why?
Their weak spot is the lipid envelope — once destroyed, virus cannot infect.

Non-enveloped viruses

(e.g., norovirus, adenovirus, poliovirus)

Much harder to kill.

What kills them:

  • Bleach

  • Autoclaving

  • High heat

  • Gamma radiation

What does NOT kill them well:

  • Hand sanitizers

  • Low-level disinfectants

  • Soap alone

Why?
They lack a lipid envelope. Their protein capsid is extremely stable.

4. Fungi

Vegetative fungi (hyphae, yeast)

Moderately easy to kill.

What kills them:

  • Alcohol

  • Bleach

  • Heat

  • Antifungals

Fungal spores (conidia)

More resistant.

What kills them:

  • Autoclaving

  • Strong disinfectants

  • UV (higher exposure)

5. Parasites (protozoa, helminths)

Varies widely.

Protozoan cysts are very resistant (like Giardia).
They require:

  • Filtration

  • Strong disinfectants

  • Boiling

Helminths (worms) are easier to kill but harder to detect.

Effectiveness Comparison

  • Determination through quantitative measure called Decimal Reduction Time (D)

  • D: Time required to reduce microbial population by 90% (10-fold reduction / 1log)

  • Formula:
    D = -(tf - t0) / (log Nf - log N0)

Variables:

  • Nf = final population or concentration

  • N0 = initial population or starting concentration

  • tf - t0 = exposure time or time treated (minutes)

Factors affecting D-value:

  • Microbe type

  • agent concentration

  • pH

  • temperature

  • various abiotic factors

A low D-value = more effective agent (kills faster)

Decimal Reduction Time (D) vs Generation Time (g)

  • Definitions:

    • D-value: Time for the population to decrease by 90%

    • g-value: Time for 1 cell to divide into 2 cells or population to double

    • Formulas:

    • Decimal Reduction Time:
      D = -(tf-t0) / (log Nf - log N0)

    • Generation Time:
      g = (tf - t0) / 3.3 (log Nf - log N0)

Calculating D-Values

  • Example Calculations:

    • Calculation of D for Agent A for E. coli:

    • Steps:

      1. Given Summary: D=−(7−3)(log⁡104−log⁡106)D = -\frac{(7-3)}{(\log 10^4 - \log 10^6)}

      2. Compute:
        D=−−4(4−6)D = -\frac{-4}{(4-6)}
        D=−−4−2=2 minutesD = -\frac{-4}{-2} = 2 \text{ minutes}

Interpreting D-value graphs

  • Steeper slope = larger slope = kills faster = lower D-value

  • Flatter slope = smaller slope = kills slower = higher D-value

  • Agent with lowest D = most effective = kills 90% very quickly

  • Agent with highest D = least effective = takes longer to kill 90%

  • D-value = 1 / slope

Types of Physical Sterilization Techniques

  • Heat Sterilization:

    • Widely used method for controlling microbial growth

    • Mechanism: High temperatures cause denaturation of proteins

    • Autoclave:

    • Uses steam under pressure, raising water temperature to 121°C without boiling (121C at 15psi)

    • Effectiveness due to high temperature, not just pressure

    • Sterilizes liquids, metal tools, glassware

  • Pasteurization:

    • reduces microbial load in heat-sensitive liquids/fluids

    • Example: Milk treated at 63-72°C for 10-30 minutes

    • Important note: Does not achieve sterilization (not all organisms are killed)

    • Does NOT kill spores

  • Radiation:

    • Different types: UV, X-ray, gamma rays

    • Mechanism: Modifications and breaks in DNA

    • Applications: Medical field, food industry for sterilization

    • UV radiation - causes DNA breaks and thymine dimers; used for surfaces; cannot penetrate solids or opaque materials

    • Ionizing radiation (gamma, x-ray) - creates radical that damage DNA; penetrates food and medical supplies; used for sterilizing spices, meats

  • Filtration:

    • Utilized for heat-sensitive liquids and gases

    • How it works: Pores are small enough to block organisms but permit liquid/gas passage

    • Types:

      • Depth filters

      • HEPA filters

      • Membrane filters

  • Dry heat

    • For killing endospores you need >200C, such as baking glassware

Application of Physical Sterilization Techniques

  • Scenario-Based Applications:

    • To sterilize heat-labile thin plastic: Filtration

    • Sterilizing a 1M glucose solution: Filtration

    • Best method for sterilizing tap water for experiments: Filtration

    • Thick metal rods used for stirring: Autoclaving

    • Glassware used for studying Clostridium botulinum: Autoclaving

    • To disperse endospores: Filtration

    • Air conditioning system: Filtration

    • New fruit juice production: Pasteurization

Chemical Growth Control: Antibiotics Terminology

  • Definitions:

    • Antibiotics : natural or semi-synthetic antibacterials

    • Antimicrobials: Compounds that inhibit or kill microbes; umbrella term for antibacterials, antivirals, antifungals, antiparasitics 

    • Antibacterials: Type of antimicrobials specifically targeting bacteria

    • Antifungals: Target ergosterol or fungal cell wall

    • Antivirals: Target specific viral enzymes or entry

    • Antiparasitics: kills protozoa/helminths

    • Chemotherapy agents: drugs targeting cell growth (can be antimicrobial or anticancer)

BACTERIOSTATIC VS BACTERICIDAL VS BACTERIOLYTIC

Bacteriostatic

  • Stops growth

  • Cells remain alive if drug removed

Bactericidal

  • Kills cells

  • Total cell count unchanged; viable count falls

Bacteriolytic

  • Cells burst

  • Both viable count and total cell count drop

CHEMICAL AGENTS FOR EXTERNAL USE
Sterilants

  • Kill all microbes + spores

  • Hydrogen peroxide vapor, ethylene oxide, bleach

Disinfectants

  • For surfaces

  • Kill pathogens but not spores

  • Lysol, chlorine gas

Sanitizers

  • Reduce the microbial load

  • Lower strength alcohol gels

Antiseptics

  • Safe for living tissue

  • Alcohol wipes, iodine

ANTIBIOTICS (INTERNAL USE)

Broad-spectrum examples:

  • Tetracyclines

  • Aminoglycosides

  • Quinolones

  • Carbapenems

Narrow-spectrum examples:

  • Isoniazid (Mycobacterium only)

  • Polymyxins (Gram-negative)

  • Vancomycin (Gram-positive)

Why microbes produce antibiotics?

→ Kill competitors in the environment.

SYNTHETIC ANTIMICROBIALS

Growth factor analogs

  • Mimic vitamins or amino acids

  • Block pathways

Examples:

  • Sulfa drugs – block folate synthesis

  • Isoniazid – blocks mycolic acid in Mycobacterium

NATURAL ANTIBIOTICS

Made by:

  • Streptomyces (majority)

  • Penicillium (β-lactams)

  • Bacillus

CELL WALL INHIBITORS

β-lactams (penicillin, cephalosporins):

  • Inhibit transpeptidation

  • Cause cell lysis

  • Mainly effective on Gram-positives

Cell wall is a good target because humans lack peptidoglycan.

Tuskegee experiment included as historical context for withholding penicillin treatment.

MEMBRANE INHIBITORS

Examples:

Daptomycin

  • Forms membrane pores

  • Works on Gram-positive

Polymyxins

  • Bind LPS

  • Work on Gram-negative

  • Toxic to kidneys and nerves

Platensimycin

  • Blocks fatty acid synthesis

Membrane is a good target because disrupting it kills cells fast

QUANTIFYING ANTIMICROBIAL ACTIVITY

1. Viable cell counts

  • Measures growth over time

  • Shows static vs cidal vs lytic

  • Allows D-value calculation

2. Minimum inhibitory concentration (MIC)

  • Lowest concentration that visually inhibits growth

  • Depends on temp, pH, inoculum size

3. Disc diffusion (Kirby-Bauer)

  • Zones of inhibition measured

  • Larger zone = more sensitive

ANTIVIRAL DRUG TARGETS

Which is best universal target?
→ RNA-dependent RNA polymerase (RdRp) because many viruses use it.

Zanamivir/Tamiflu → block viral release
AZT/Tenofovir → block reverse transcriptase
Fusion inhibitors → stop viral entry
Protease inhibitors → stop maturation of viral proteins

ANTIFUNGAL DRUGS

Challenge: fungi are eukaryotic → fewer unique targets.

Major targets:

  • Ergosterol (instead of cholesterol)

  • Chitin in cell wall

  • Beta-glucans

Examples:

  • Miconazole

  • Tinactin

ANTIMICROBIAL RESISTANCE

Microbes resist drugs by:

5 major mechanisms

  1. Lack target

  2. Impermeable to drug

  3. Inactivate drug (β-lactamases)

  4. Modify target (mutated PBP)

  5. Efflux pumps

Resistance can be chromosomal mutations or horizontal gene transfer (plasmids).

MDR / XDR / PDR PATHOGENS

  • MDR: resistant to ≥3 drug classes

  • XDR: resistant to nearly all

  • PDR: resistant to all

ESKAPE pathogens = major hospital threats:

  • Enterococcus faecium

  • Staphylococcus aureus

  • Klebsiella pneumoniae

  • Acinetobacter baumannii

  • Pseudomonas aeruginosa

  • Enterobacter spp.

PREVENTING RESISTANCE

Key strategies:

  • Correct antibiotic selection

  • Narrow-spectrum when possible

  • Avoid overuse in agriculture

  • Develop new antibiotics

  • Increase vaccination

Important note from slides:
→ Resistance does NOT disappear immediately after stopping drug use
(because genes are often stable on plasmids).

Mechanisms of Action and Resistance

  • Resistance Mechanisms:

    • Intrinsic Resistance: Does not require external genetic changes

    • Acquired Resistance: Developed through genetic alterations (e.g., plasmids)

  • Patient Safety Concerns:

    • Example: The Tuskegee Experiment highlighted serious ethical violations in healthcare, affecting trust in medical practices

Conclusion: Addressing Antimicrobial Resistance

  • Prevention Strategies:

    • Use antibiotics judiciously to limit the development of resistance

    • Target appropriate organisms and avoid unnecessary broad-spectrum antibiotics

    • Encourage the design and identification of new antibiotics

    • Improve vaccination efforts to decrease disease incidence and antibiotic use

  • Global Health Implications:

    • “A post-antibiotic era, where common infections can be deadly, is a real possibility,” (Keiji Fukuda, WHO).

1. Compare and contrast physical sterilization vs chemical means of controlling microbial growth

Physical sterilization

  • What it is: Uses physical agents like heat, radiation, or filtration to remove or kill microbes.

  • Main mechanisms:

    • Heat (moist/dry): denatures proteins, disrupts membranes, damages DNA.

    • Radiation: UV causes DNA damage (thymine dimers); ionizing radiation (gamma/X-ray) creates radicals that break DNA and other molecules.

    • Filtration: physically removes microbes by size; doesn’t kill them.

  • Typical uses:

    • Autoclaving media, glassware, metal tools.

    • Pasteurization of milk/juices (reduction, not full sterilization).

    • Dry heat for glassware / things contaminated with spore-formers.

    • Radiation for medical supplies, some foods.

    • Filtration for heat-labile liquids and air (HEPA).

  • Pros: Often reliable, no toxic residues, can achieve full sterilization (autoclave, ionizing radiation, dry heat).

  • Cons: Heat can damage materials/foods; radiation requires special equipment; filtration doesn’t kill, only removes.

Chemical methods

  • What they are: Use chemicals to kill or inhibit microbes.

  • Major categories:

    • Sterilants: kill all microbes + spores (e.g., ethylene oxide gas, strong bleach, H₂O₂ vapor).

    • Disinfectants: kill most microbes on inanimate surfaces (e.g., chlorine, Lysol).

    • Sanitizers: reduce microbial load but don’t sterilize (low-level alcohols, some sprays).

    • Antiseptics: safe for living tissue (alcohol, iodine, chlorhexidine).

    • Internal drugs (“antibiotics” broadly): antibacterials, antivirals, antifungals, antiparasitics.

  • Mechanisms:

    • Disrupt membranes, denature proteins, modify or damage DNA, inhibit key enzymes or pathways.

  • Pros: Can be used on surfaces, skin, and inside the body; targeted drugs (e.g., β-lactams) can hit specific microbial structures.

  • Cons: Toxicity to humans/animals at high doses, residues, resistance development, may not kill spores (except true sterilants).

Key contrast:

  • Physical methods = heat/radiation/filtration; generally no chemical residues, many achieve true sterilization.

  • Chemical methods = molecules applied to surfaces/skin or used internally; may or may not sterilize; resistance can evolve.

2. Write the decimal reduction time equation

Decimal reduction time (D) is the time required to reduce a microbial population by 90% (1 log)

Where:

  • D = decimal reduction time

  • t0, tf​ = initial and final times (minutes)

  • N0, Nf​ = initial and final cell concentrations (CFU/mL)

3. Apply the D-value equation to a problem set of data

Use the formula step-by-step:

Example (like the worked slide):
A culture starts at N0 =10^6 CFU/mL. After 4 minutes of treatment (from 3 to 7 min), Nf=10^4 CFU/mL. What is D?

General steps for any data set:

  1. Pick two time points and their populations.

  2. Subtract times to get tf−t0t_f - t_0tf​−t0​.

  3. Take log10 of each population.

  4. Plug into D equation.

  5. Repeat for other agents/conditions if needed.

4. Interpret the results of multiple decimal reduction time calculations

When you calculate D for several agents:

  • Lower D = more effective / more “potent” killing

    • Kills 90% of cells in less time.

  • Higher D = less effective

    • Takes longer to achieve the same log reduction.

Example using Agents A, B, C against E. coli on your slide:

  • If A: D = 2 min, B: D = 6 min, C: D = 10 min →

    • Agent A is most efficacious, Agent C least.

  • If two agents have very similar D values, their killing efficacy is roughly the same under those conditions.

You can also:

  • Compare same agent, different microbes to see which microbe is more resistant.

  • Compare same agent, different conditions (pH, temp) to see when it works best.

5. Define how heat affects microbes at the molecular level

Heat damages microbes mainly by:

  1. Protein denaturation and coagulation

    • Heat disrupts hydrogen bonds, ionic bonds, hydrophobic interactions.

    • Enzymes lose their 3-D shape → metabolism stops → cell death.

  2. Membrane disruption

    • Increased fluidity → membranes become leaky.

    • Loss of proton gradients and ion balance → energy failure.

  3. Nucleic acid damage

    • High temperatures cause DNA strand breaks and base modifications.

    • Interferes with replication and transcription.

  4. Dehydration/oxidation (especially in dry heat)

    • Removes water necessary for biological reactions.

    • Promotes oxidative damage to macromolecules.

Moist heat (autoclave) primarily kills via rapid protein denaturation and membrane disruption, while dry heat adds oxidation and dehydration to the mix.

6. List the ways heat is used to control microbial growth

From the slides (plus detail):

  1. Moist heat sterilization (Autoclaving)

    • 121°C, 15 psi, ~15–20 min

    • Kills all microbes, including endospores.

  2. Dry heat sterilization

    • Hot air ovens, >160–200°C for 1–2 hours.

    • Used for materials that tolerate high dry temperatures; good for spores.

  3. Pasteurization

    • Moderate heat (e.g., 63–72°C for 10–30 min).

    • Reduces microbial load and kills pathogens in heat-sensitive liquids, but does not sterilize.

  4. (Sometimes mentioned) Boiling or low-level moist heat

    • 100°C for short periods kills many vegetative cells but not spores; more “disinfection” than sterilization.

7. List the types of things that can be sterilized with each method of heat

Autoclaving (moist heat)

  • Culture media, agar, broth

  • Glassware (flasks, pipettes)

  • Surgical instruments and metal tools

  • Some plastics that tolerate 121°C

  • Biological waste (autoclave bags)

Dry heat (hot air oven, >200°C)

  • Glassware (petri dishes, pipettes, flasks)

  • Metal instruments (forceps, needles)

  • Materials contaminated with endospores (e.g., Clostridium botulinum glassware)

  • Powders, oils, waxes that cannot be autoclaved

Pasteurization

  • Heat-sensitive liquids, especially foods:

    • Milk, cream

    • Dairy products (some cheeses, ice cream mix)

    • Fruit juices (e.g., dragon fruit juice from the example)

    • Beer, wine, liquid eggs

Remember: pasteurization = not sterile, just safer.

8. Define how radiation affects microbes at the molecular level

Radiation in your slides is divided into UV and ionizing radiation.

Ultraviolet (UV) radiation

  • Non-ionizing; low penetration.

  • Absorbed by DNA → forms pyrimidine dimers (especially thymine dimers).

  • Distorts DNA helix; blocks replication and transcription.

  • Leads to lethal mutations if unrepaired.

  • Mainly used for surface decontamination (benches, air in hoods).

Ionizing radiation (gamma rays, X-rays, high-energy electrons)

  • High energy; deep penetration.

  • Ejects electrons from molecules → generates reactive ions and free radicals (e.g., hydroxyl radicals).

  • Radicals and direct hits cause:

    • DNA single- and double-strand breaks

    • Protein and membrane damage

    • Oxidative damage to many cellular components

  • At sufficient doses, this leads to irreversible cell death.

9. List products that can be subjected to radiation to reduce microbial contamination

From the slide:

  • Foods particularly susceptible to contamination, including:

    • Hamburger (ground beef)

    • Chicken

    • Spices

Also commonly irradiated (even if not listed explicitly on your slide):

  • Certain fruits and vegetables (delay ripening, kill insects)

  • Dried foods (herbs, teas)

  • Ready-to-eat meals for immunocompromised patients

  • Medical products: syringes, catheters, surgical gloves, implants

The goal is to:

  • Reduce pathogens (e.g., Salmonella, E. coli O157:H7)

  • Extend shelf life

  • Do so without heating the product.

10. Describe harmful effects of irradiating food, if they are known

According to your slide, food irradiation is approved by the WHO and used in the USA for certain foods.

What the evidence shows

  • At doses used for food, irradiation does not make food radioactive.

  • Major health organizations (WHO, FAO, CDC, FDA) consider it safe when properly controlled.

  • The main molecular changes are similar to those produced by other preservation methods (cooking, canning): small changes in fats, proteins, and vitamins.

Possible / theoretical “harmful” effects or drawbacks

  • Nutrient loss: Some sensitive vitamins (A, E, some B vitamins) can be partially degraded; this is usually small and similar to heat treatment.

  • Quality changes: At high doses, texture or flavor can be affected (off-flavors, changes in color).

  • Chemical byproducts: Small amounts of radiolytic products form, but studies show they’re present at very low levels and similar to compounds formed during normal cooking.

  • Public perception: People may avoid irradiated foods due to fear of “radiation,” which can reduce its use even when it would improve safety.

Ways to be better stewards of antimicrobials

Q: What are key strategies for antimicrobial stewardship?

  1. Use antibiotics only when necessary

  2. Choose narrow-spectrum drugs when possible

  3. Complete full treatment courses

  4. Avoid using antimicrobials in household products unnecessarily

  5. Reduce agricultural overuse

  6. Improve vaccination rates

  7. Rotate antibiotics (population-level strategy)

  8. Develop new antibiotics and alternatives