Antibiotic Resistance and Susceptibility Testing

Antibiotic Resistance

Antibiotic resistance occurs when bacteria evolve mechanisms to withstand the effects of antibiotics, often due to misuse or overuse of these drugs.

• Bacteriostatic: Inhibits bacterial growth without killing the organisms.

• Bactericidal: Kills bacteria directly.

• Example: Prescribing antibiotics for viral infections can promote resistance.

Minimum Inhibitory and Bactericidal Concentrations

• MIC (Minimum Inhibitory Concentration): The lowest concentration of an antibiotic that inhibits visible growth of a microorganism.

• MBC (Minimum Bactericidal Concentration): The lowest concentration of an antibiotic that kills 99.9% of the original inoculum.

• If MBC is close to MIC, the agent is bactericidal; if much higher, the agent is bacteriostatic.

Antibiotic Dilution Example

Serial dilutions of antibiotics are used to determine MIC by observing the lowest concentration that prevents visible growth.

Disk Diffusion (Kirby-Bauer) Method

This method tests bacterial sensitivity to antibiotics by measuring the zone of inhibition around antibiotic disks on an agar plate.

• Requires standardization (Mueller-Hinton agar and McFarland standard).

• Cannot distinguish between bacteriostatic and bactericidal effects.

• Only determines efficacy for the tested bacteria-antibiotic combination.

Standardization Factors

• Sensitivity of the organism

• Antimicrobial molecule size and concentration

• Density of culture

• Diffusion rate of agent

• Incubation time and temperature

• Size of inoculum

• Interactions

Biofilm Formation

Biofilms

Biofilms are structured communities of bacteria encased in a self-produced matrix of polysaccharides, proteins, lipids, and DNA. They are common in environments with excess nutrients.

• Planktonic cells: Free-floating bacteria.

• Sessile cells: Bacteria attached to a surface.

Stages of Biofilm Development

1. Surface attachment: Planktonic cells attach to a surface.

2. EPS matrix production: Cells secrete extracellular polymeric substances (EPS) to anchor themselves.

3. Maturation: Biofilm grows and develops complex structures.

4. Dispersion: Cells leave the biofilm to colonize new surfaces.

5. Quorum sensing: Cell-to-cell communication regulates gene expression and biofilm behavior.

Disinfectants, Antiseptics, and DNA Damage

Disinfectants vs. Antiseptics

• Disinfectants: Used on inanimate objects to destroy bacterial cell membranes and interfere with metabolism.

• Antiseptics: Used on living tissue; must be less toxic and are often broad-spectrum.

Chemical-Induced DNA Damage

• Deaminating agents: Remove amino groups from nucleotides, causing mutations (e.g., cytosine to uracil).

• Cytosine deamination: Converts cytosine to uracil, leading to mutations in subsequent DNA replications.

Bacterial DNA Repair Mechanisms

Base Excision Repair (BER)

BER is a cellular mechanism that repairs damaged DNA throughout the cell cycle by removing small, non-helix-distorting base lesions from the genome.

UV Radiation and DNA Damage

UV Radiation

• UV light (especially UVC, 100–280 nm) is used to kill microbes by inducing DNA damage.

• Optimal germicidal wavelengths: 260–265 nm.

Mechanisms of UV-Induced DNA Damage

• Formation of photoproducts such as thymine dimers and thymine 6-4 photoproducts.

• These lesions block DNA replication and transcription.

Types of DNA Damage

• Thymine dimer: Covalent bond between adjacent thymines on the same DNA strand, blocking DNA polymerase.

• Thymine 6-4 photoproduct: Dimer between carbons 6 and 4 of adjacent thymines, distorting the DNA backbone.

Bacterial Protection Mechanisms

• Formation of endospores (highly resistant structures in Gram-positive bacteria such as Bacillus and Clostridium).

• DNA repair mechanisms (e.g., photoreactivation, excision repair).

Endospores

Formation and Structure

Endospores are dormant, tough, and non-reproductive structures produced by certain bacteria to survive extreme conditions.

Endospore Formation Cycle

• Initiated by nutrient deprivation or environmental stress.

• Involves asymmetric cell division, engulfment, cortex and coat formation, and maturation.

• Endospore is released when the mother cell lyses.

Types of Endospores

Endospore Structure

DNA Damage Repair Mechanisms

• Photoreactivation: Uses visible light to activate photolyase, which breaks thymine dimers.

• Nucleotide Excision Repair (NER): Removes damaged DNA segments and fills the gap using the undamaged strand as a template.

Bacterial Genetic Exchange

Conjugation

Conjugation is the direct transfer of DNA from one bacterium to another via cell-to-cell contact, typically mediated by a plasmid (e.g., F plasmid).

• Vertical gene transfer: Parent to offspring.

• Horizontal gene transfer: Between unrelated cells.

• Plasmids: Small, circular DNA molecules that replicate independently of the chromosome.

Example: F Plasmid Transfer

• F+ cell (donor) transfers F plasmid to F- cell (recipient), making it F+.

• Plasmids may carry genes for antibiotic resistance, virulence, or metabolism.

Summary Table: Growth on Selective Media

Transformation

Transformation is the uptake of free DNA from the environment by a bacterium. Cells must be competent (naturally or chemically induced) to take up DNA.

• Competence can be induced by CaCl2 treatment and heat shock.

• Plasmids must provide a selective advantage (e.g., antibiotic resistance) to be maintained.

Example: pGlo Transformation

• pGlo plasmid contains a gene for green fluorescent protein (GFP) under the control of the arabinose promoter.

• Only cells that receive the plasmid and are grown with arabinose will glow under UV light.

Results Interpretation

• Plates without plasmid: No growth in presence of ampicillin.

• Plates with plasmid: Growth in ampicillin; glowing colonies only with arabinose present.

Additional info: These topics correspond to Ch. 7 (Control of Microbial Growth), Ch. 8 (Microbial Genetics), Ch. 9 (Biotechnology & DNA Technology), and Ch. 20 (Antimicrobial Drugs) in standard Microbiology curricula.