Antimicrobial Drugs, Resistance, and Recombinant DNA Technology

Antimicrobial Drugs & Resistance

Definitions and Key Concepts

Understanding the terminology and foundational concepts is essential for studying antimicrobial drugs and resistance mechanisms.

• Chemotherapeutic agent: A chemical used to treat disease.

• Antimicrobial: An agent that kills or inhibits the growth of microorganisms.

• Antibiotic: An antimicrobial produced naturally by microorganisms.

• Semi-synthetic antibiotic: A natural antibiotic that has been chemically modified to improve effectiveness or overcome resistance.

Historical Figures in Antimicrobial Discovery

• Paul Ehrlich: Proposed the "magic bullet" concept and developed Salvarsan for syphilis.

• Alexander Fleming: Discovered penicillin in 1929.

• Selman Waksman: Discovered many antibiotics (e.g., streptomycin), especially from soil microbes like Streptomyces.

Sources of Antibiotics

• Soil bacteria (notably Streptomyces)

• Fungi

• Plants

• Marine organisms

Spectrum of Activity

• Broad spectrum: Effective against many types of bacteria.

• Narrow spectrum: Effective against specific bacteria.

Bacteriostatic vs. Bactericidal

• Bacteriostatic: Inhibits bacterial growth; relies on the host immune system to eliminate pathogens.

• Bactericidal: Kills bacteria directly.

Selective Toxicity

Selective toxicity refers to a drug's ability to harm the microbe without damaging the host. This is easier to achieve with bacteria due to:

• Presence of peptidoglycan in bacterial cell walls

• 70S ribosomes (distinct from eukaryotic 80S ribosomes)

• Unique metabolic pathways

• Fungi and protozoa are eukaryotic and more similar to human cells, making selective toxicity harder

Therapeutic Index

• Therapeutic Index (TI): The ratio of the toxic dose to the effective dose of a drug.

$\text{Therapeutic\ Index} = \frac{\text{Toxic\ Dose}}{\text{Effective\ Dose}}$

• A higher TI indicates a safer drug.

Major Drug Targets

Antimicrobial drugs target essential structures or functions in microbes:

1. Cell wall synthesis

2. Plasma membrane integrity

3. Protein synthesis (ribosomes)

4. Metabolic pathways

5. Nucleic acid synthesis

Best selective toxicity: Cell wall, 70S ribosomes, and bacterial-specific metabolism.

Synergism

• When two drugs are used together and are more effective than either alone.

Examples:

• Augmentin: Combination of amoxicillin (inhibits cell wall synthesis) and clavulanic acid (inhibits beta-lactamase enzymes).

• TMP-SMZ (Bactrim): Combination of trimethoprim and sulfamethoxazole; both block folic acid synthesis at different steps, preventing DNA synthesis.

Mechanisms of Action of Major Antimicrobials

• Beta-lactams: Block cell wall cross-linking (e.g., penicillins, cephalosporins).

• Tetracyclines, Aminoglycosides, Macrolides: Inhibit protein synthesis (translation) at the ribosome.

• Isoniazid: Blocks mycolic acid synthesis (used for tuberculosis).

• Polymyxin B: Disrupts cell membrane integrity.

• Sulfonamides: Block folic acid synthesis pathway.

• Rifampin: Inhibits transcription (RNA synthesis).

• Quinolones: Inhibit DNA gyrase (DNA replication).

Antimicrobial Susceptibility Testing

• Disk Diffusion (Kirby-Bauer) Test: Antibiotic disks are placed on an agar plate inoculated with bacteria. A large zone of inhibition indicates sensitivity; no zone indicates resistance.

Development and Mechanisms of Resistance

• How resistance develops:

  1. Mutation

  2. Gene transfer (e.g., R plasmids)

• Resistance mechanisms:

  • Efflux pumps (pump drug out of cell)

  • Enzymatic drug destruction (e.g., beta-lactamases)

  • Target modification (alteration of drug target)

  • Decreased drug uptake

Factors Contributing to Rising Resistance

• Overuse and misuse of antibiotics

• Incomplete courses of treatment

• Agricultural use (e.g., antibiotics in animal feed)

Antibiotics in animal feed: Promotes resistant bacteria, which can transfer to humans. The industry claims use is safe when regulated, but concerns remain.

Antifungal, Antiprotozoal, and Antiviral Drugs

Fungi and protozoa are harder to treat due to their eukaryotic nature, making them more similar to human cells.

• Antifungals:

  • Polyenes (e.g., Amphotericin): Damage cell membranes; can cause kidney toxicity.

  • Azoles: Block membrane synthesis; used for yeast infections.

• Antiprotozoals:

  • Chloroquine, Mefloquine (antimalarials)

  • Flagyl (metronidazole): Targets anaerobic metabolism.

• Antivirals:

  • Viruses are hard to treat due to their intracellular lifestyle, use of host machinery, latency, and rapid mutation.

  • Acyclovir: Nucleoside analog that stops viral DNA replication.

  • AZT: Blocks reverse transcriptase; resistance is common.

  • HAART (Highly Active Antiretroviral Therapy): Combination of 3–4 HIV drugs to prevent resistance.

Biotechnology & DNA Technology

Gene Cloning: Cloning a Eukaryotic Gene in Bacteria

Gene cloning allows for the production and study of specific genes and proteins. The process involves several key steps:

1. Extract mRNA from cells containing the gene of interest.

2. Use reverse transcriptase to convert mRNA into complementary DNA (cDNA).

3. Cut both the plasmid and cDNA with restriction enzymes.

4. Join the cut cDNA and plasmid by complementary base-pairing (sticky ends).

5. Use ligase to seal the DNA, forming covalent bonds.

6. Transform the recombinant plasmid into host bacteria.

7. Add antibiotic to select for host cells containing the plasmid.

8. Use a DNA probe to identify the correct clone.

Eukaryotic Gene Structure and cDNA Production

• Exons: Coding regions of a gene.

• Introns: Noncoding regions; absent in mature mRNA.

• Translation: Process occurring at the ribosome, converting mRNA to protein.

• cDNA: Produced from mRNA template using reverse transcriptase; lacks introns.

• cDNA library: Contains only actively transcribed genes (no introns).

Plasmid Features

• Origin of replication: Required for plasmid replication.

• Restriction enzyme cut site: Area accepting foreign DNA.

• Antibiotic resistance gene: Used as a selection marker.

Restriction Enzymes and DNA Joining

• Restriction enzymes: Cut DNA at specific sequences, often leaving sticky ends.

• Sticky ends: Single-stranded overhangs that facilitate complementary base pairing.

• Ligase: Seals DNA by forming covalent bonds between sugar-phosphate backbones.

• Recombinant DNA: DNA formed by joining DNA from different sources.

Transformation and Selection

• Transformation: Introduction of plasmid DNA into bacteria (e.g., using calcium chloride and heat shock).

• Selection: Antibiotic is added; only bacteria with the plasmid (and resistance gene) survive.

Screening Colonies

• DNA probe: Used to detect colonies with the gene of interest by complementary base pairing.

• Confirmation: Determined by nucleotide sequencing.

Cloning Vectors and Libraries

• Cloning vector: A DNA molecule (e.g., plasmid) used to carry foreign DNA into a host cell.

• Genomic library: Contains fragments of all DNA in an organism’s genome.

• cDNA library: Contains only coding sequences (exons) of eukaryotic genes.

Polymerase Chain Reaction (PCR)

• PCR: Technique to amplify (make multiple copies of) a DNA molecule.

• Does not require large amounts of starting DNA.

• After n cycles, the number of double-stranded DNA molecules is $2^n$.

• After 3 cycles: $2^3 = 8$ double-stranded DNA molecules.

Southern Blotting

• DNA fragments are transferred from a gel to a nitrocellulose filter to attach them to a permanent surface for probing.

• Does not involve addition of heat-stable DNA polymerase.

Blue-White Screening

• Bacteria transformed with recombinant plasmid and cultured on media with ampicillin and X-gal produce white colonies (indicating successful insertion of foreign DNA).

Applications and Ethical Considerations

• Recombinant DNA technology is used for gene cloning, protein expression, and gene identification, but not for culturing unknown organisms.

• Genetic technology enables screening for genetic conditions, raising privacy and ethical issues.

• Legislation may be needed to protect individuals’ genetic information privacy.

Selected Q&A and Key Points

Concept	Key Point
Cloning vector example	Plasmid
Restriction enzyme function	Makes staggered DNA cuts (sticky ends)
DNA ligase absence	Base-pairing occurs, but sugar-phosphate backbone is not connected
PCR	Amplifies DNA; does not require large starting amounts
Transformation method	Calcium chloride and heat shock for E. coli
Genomic vs. cDNA library	Genomic: all DNA; cDNA: only coding sequences (no introns)
Southern blotting	Transfers DNA to filter for probing
Blue-white screening	White colonies = recombinant plasmid
Antibiotic resistance	Occurs with indiscriminate use; not limited to gram-negative bacteria
Antibiotics in animal feed	Promotes resistant bacteria; survivors repopulate host
Actinomycin D	Binds G-C pairs, interferes with transcription
Transposase	Enzyme that moves genes between chromosome and plasmid

Summary Table: Mechanisms of Antimicrobial Action

Drug Class	Target	Example(s)	Mechanism
Beta-lactams	Cell wall	Penicillins, Cephalosporins	Block cross-linking of peptidoglycan
Tetracyclines	Protein synthesis	Tetracycline	Block translation at ribosome
Aminoglycosides	Protein synthesis	Streptomycin	Block translation at ribosome
Macrolides	Protein synthesis	Erythromycin	Block translation at ribosome
Isoniazid	Mycolic acid synthesis	Isoniazid	Used for TB
Polymyxin B	Cell membrane	Polymyxin B	Disrupts membrane
Sulfonamides	Metabolic pathway	Sulfa drugs	Block folic acid synthesis
Rifampin	RNA synthesis	Rifampin	Blocks transcription
Quinolones	DNA replication	Ciprofloxacin	Blocks DNA gyrase

Additional info:

• Some explanations and context have been expanded for clarity and completeness.

• Tables have been reconstructed to summarize key comparisons and mechanisms.