Antibiotics and Bacterial Cell Structures

BIO 418 Antibiotics

• Course taught by Seyhun Yurdugül
• Lecture I: Introduction
• Lecture II: Mode of action of antibiotics and their uptake into the bacteria
• Lecture III: Inhibitors of Protein Synthesis
• Lecture IV: Peptidoglycan Synthesis Inhibitors
• Lecture V: Penicillin-Binding Proteins (PBPs)

Content Outline (Lecture I)

• Definition
• Historical background
• Classification of antibiotics
• Examples

Abbreviations (Lecture I, III)

• m/o: microorganisms
• AB: Antibiotics
• DNA: Deoxyribonucleic acid
• APUA: Alliance for the Prudent Use of Antibiotics
• S: Svedberg unit
• mRNA: messenger RNA
• tRNA: transfer RNA

Definition (Lecture I)

• Any chemical (synthetic or biological) agent designed to eliminate microorganisms.

What are Antibiotics? (Lecture I, II)

• Chemical compounds typically derived from molds or bacteria.
• Capable of killing or inhibiting bacterial growth.
• Many now synthetically made; others naturally produced.
• Effective only against bacterial infections, not viral.

Historical Background (Lecture I)

• Since 1940, considered the most efficient and desirable means of fighting syndromes.
• Once described as "magic bullets" for eliminating bacteria without major side effects on treated individuals.
• Overuse has led to consequences, not "harmless".
• Antibiotics are quite costly.

Different Situations of Use (Lecture I)

• Usage not limited to fighting human illnesses.
• Widely used throughout the ecosystem (veterinary practices, food-animal production, and selected plants).
• In animals, used to prevent or cure infections.
• Long-term exposure to low doses for selecting resistant bacteria in treated animals and indirectly in humans consuming undercooked meal.

Classification of Antibiotics (Lecture I, II, III)

• Based on several factors.
• Bacterial spectrum (broad vs. narrow).
• Route of administration (injectable, oral, topical).
• Type of activity (bactericidal vs. bacteriostatic).
• Chemical structure (most useful).

Antibacterial Agents (Lecture I)

• Disinfectants
• Antiseptics
• Preservation agents
• Antibiotics

Disinfectants (Lecture I)

• Usually too toxic, irritant, or corrosive for body surfaces or tissues.
• Suitable for removing microorganisms from equipment or inanimate environments.

Antiseptics (Lecture I)

• Include bacterial inhibitors that are relatively free from toxic effects.
• Can be applied to body surfaces or exposed tissues.
• Agents that assist and don't impair natural body defense systems.

Preservation Agents (Lecture I)

• Frequently added to pharmaceuticals, cosmetics, and food products.
• Used to inhibit microbial contamination and proliferation.

Examples of Antibiotics (Lecture I,II)

• Penicillins: Oldest class, common chemical structure with cephalosporins, beta-lactam antibiotics; generally bacteriocidal (kill bacteria).
• Cephalosporins: Usually preferred for surgical prophylaxis, cross the blood-brain barrier, used for meningitis and encephalitis.
• Fluoroquinolones: Synthetic antibacterial agents, readily interchangeable with traditional antibiotics.
• Tetracyclines: Share a chemical structure with four rings, effective against various microorganisms (rickettsia, amoebic parasites).
• Macrolides: Prototype is erythromycin, spectrum and use similar to penicillin, newer members (azithromycin, clarithromycin) useful for lung penetration, used to treat Helicobacter pylori infections.
• Others: Aminoglycosides, Lincosamides (e.g., clindamycin, lincomycin) are effective against anaerobic pathogens.

Structure of Bacteria (Lecture I)

• Gram (+): retain iodine-crystal violet stain when treated with organic solvents.
• Staining response depends on bacterial cell wall morphology and composition.

Cell Wall (Lecture I)

• Surrounds the inner cytoplasmic membrane.
• Maintains cell shape and protects the membrane from rupture.
• Gram (+): peptidoglycan and teichoic acid.
• Gram (-): peptidoglycan and lipopolysaccharide.

Capsules (Lecture I)

• Discrete, tightly bound polysaccharide layers.
• Distinct from extracellular mucoid substance (glycocalyx, muco-exopolysaccharide).
• Example: Pseudomonas aeruginosa.
• Glycocalyx structure provides a biofilm structure.

Peptidoglycan (Lecture I)

• Mechanical stability of cell walls.
• Polymer of disaccharide repeating units of two different N-acetylated amino sugars.
• One amino sugar has a short peptide chain attached.
• Sugars: N-acetyl glucosamine and N-acetyl muramic acid.
• Selective target for antibiotic action (e.g., β-lactam).

Cytoplasmic Membrane (Lecture I)

• Lies directly outside the cytoplasm.
• Acts as a selective permeability barrier between the cytoplasm and the cell environment.
• Contains phospholipids and proteins.
• Provides the matrix by which metabolism, solute transport, flagellar movement, and ATP generation are linked.

Deoxyribonucleic Acid (DNA) (Lecture I)

• Bacterial chromosome: single, circular DNA.
• No surrounding membrane.
• 18 different gene products participate in E. coli chromosome replication (e.g., DNA gyrase, target of quinolone antibiotics).
• Example: Acridines.

Spore Structure (Lecture I)

• Under extreme conditions.
• Spore formers: Bacillus, Clostridium, Sporolactobacillus, Sporosarcina, Desulfomaculum spp.
• Germ cell (protoplast or core) surrounded by cortex; cortex surrounded by spore coats.
• Bacterial spores are most resistant to inactivation by chemicals or physical agents (heat, radiation).
• Under normal conditions: Germination

Main Target of Antibiotics (Lecture I)

• Preventing step 4 of bacterial infection (damage to host tissue).
• Either by killing pathogens or slowing their growth to a point where host defense mechanisms can clear the infection.
• Attempts to develop antibiotics that interfere with bacterial attachment (step 1).

Inhibitors of Nucleic Acid Synthesis (Lecture II)

• Growth depends on DNA and RNA synthesis.
• Three major categories:
• Interruption of nucleotide metabolism (interference with synthesis or interconversion).
• Interaction with DNA to form a complex or cause strand breakage.
• Antibiotics that directly inhibit enzymatic processes in nucleic acid synthesis.

Sulfonamides (Lecture II)

• Structural analog of p-(4)-aminobenzoic acid (PABA).
• Binds more tightly to dihydropterate synthase (DHPS) enzyme than PABA itself.
• Examples: Sulphamethoxazole and trimethoprim completely inhibit bacterial dihydrofolate reductase (DHFR).

Inhibitors of RNA Polymerase (Lecture II)

• Example: Rifampicin (semi-synthetic).
• Binds and specifically inhibits bacterial DNA-dependent RNA polymerase, inhibiting initiation processes.
• No effect on nuclear or mitochondrial DNA-dependent RNA polymerase.

Inhibitors of DNA Gyrase (Lecture II)

• Enzyme involved in bacterial DNA replication (also known as DNA topoisomerase II).
• Quinolone group antibiotics have particular interest in inhibiting DNA gyrase.
• Examples: Nalidixic acid, norfloxacin, enoxacin, ofloxacin, ciprofloxacin.

Uptake of Antibiotics (Lecture V)

• Antibiotic molecules must cross one or two bacterial membranes.
• Methods of uptake:
• Self-promoted uptake–Destabilization and disorganization of outer membrane as a result of displacement of divalent cations. Polycationic antibiotics often use this method.
• Passive diffusion–Occurs through water-filled pores (porins), influenced by molecular size, charge, and lipophilic properties of the permeating molecule.
• Active transport–Involves specific carrier proteins that uses energy (ATP or proton motive force) to take antibiotics across the concentration gradient

Types of Penicillin-Binding Proteins (PBPs) (Lecture V)

• Several PBPs in bacterial species (usually at least four).
• Usually numerically designated (e.g., PBPs 1-7 in E. coli K12).
• Minor components of the bacterial cell membrane.
• Catalyze transpeptidase, carboxypeptidase, and endopeptidase reactions.

E.coli PBPs (Lecture V)

• Most studied set of PBPs.
• Primarily divided into High molecular weight and Low molecular weight groups
• High molecular weight: PBPs 1-3
• Catalyzes the polymerization of disaccharide units into glycan chains (transglycosylation) and cross-linking of their pentapeptide side chains (transpeptidation).
• Inhibition prevents the formation of a strong peptidoglycan layer, causing cell death
• Low molecular weight: PBPs 4-7

Other Mechanisms of Inhibition (Lecture V)

• Lytic death – Peptidoglycan degradative enzymes (autolysins) of some bacteria hydrolyzing bonds in the glycan or peptide side chains of peptidoglycans. Together with antibiotics like penicillin, cause cell lysis.
• Non-lytic death – Observed in bacteria lacking autolytic activity. Peptidoglycan is 'nicked', resulting in a limited amount of cell wall damage.

Other Main Targets (Lecture V)

• Peptidoglycan carboxypeptidases and transpeptidases.
• Main targets for β-lactams.
• Normal substrate is D-alanyl-D-alanine moiety of the pentapeptide.
• β-lactams bind covalently to the same group on the PBPs that the normal substrate binds.

General Information About Vancomycin (Lecture IV)

• Class: Glycopeptide antibiotic
• Type: Stage III inhibitor
• Synthetic antibiotic.
• Indications: Effective against methicillin-resistant Staphylococcus, penicillin allergy, and resistant microorganisms.
• Use with other antibiotics to treat serious infections.

History and Background (Lecture IV)

• Introduced in hospitals ~40 years ago.
• Strains exhibiting penicillin resistance.
• Vancomycin-resistant enterococci (VRE) emerged in 1987.

Mechanism of Action (Lecture IV)

• Inhibits bacterial cell wall synthesis.
• Binds to the D-alanyl-D-alanine residues of peptidoglycan monomers.
• Prevents cross-linking, resulting in a weak cell wall.
• Intense osmotic pressure ruptures the cell.

Spectrum of Action (Lecture IV)

• Effective against Gram-positive organisms (Listeria, Rhodococcus, Peptostreptococcus).
• Bacteriostatic against Enterococcus.

Mechanism of Resistance (Lecture IV)

• Enterococcus: Vancomycin precursor has decreased affinity (D-ala-D-ala replaced by D-ala-D-lac).
• Staphylococcus aureus (VISA/hVISA): Increased amount of precursor with decreased affinity, thicker cell wall.

Pharmacokinetics (Lecture IV)

• Not absorbed from the GIT (except in antibiotic-associated colitis).
• Given intravenously.
• Widely distributed (including CSF).
• Eliminated entirely by glomerular filtration; accumulates in renal impairment.
• Half-life approximately 8 hours

Clinical Uses (Lecture IV)

• Serious Staphylococcus infections (methicillin resistant, penicillin allergy) and other resistant microorganisms.
• Prophylaxis for major surgical procedures in hospitals with high rates of infections due to MRSA or MRSE.
• Treatment of antibiotic-associated colitis with metronidazole preferred.
• Vancomycin resistant Staphylococcus use Daptomycin.
• Dose based on total body weight and renal function (15-20 mg/kg).
• Normal renal function = q12 dosing.
• Goal through concentrations (10-15 mcg/mL for bacteremia/skin and soft tissue infections; 15-20 mcg/mL for osteomyelitis, meningitis, pneumonia).

Side Effects (Lecture IV)

• Fever, rashes
• Thrombophlebitis at injection site.
• Ototoxicity and nephrotoxicity (high concentration).
• Hypotension and red-man syndrome (rapid infusion).

Alternatives to Vancomycin (Lecture IV)

• Skin and Soft Tissue – Trimethoprim-sulfamethoxazole (TMP/SMX), Doxycycline/minocycline, Clindamycin (if no inducible resistance; NOT levofloxacin)
• Urinary Tract Infections –TMP/SMX, linezolid (or not adequate urinary concentration); Tetracycline (if susceptible).
• Other options may include fluoroquinolones (if susceptible in vitro)
• Daptomycin, Linezolid, Tigecycline, Ceftobiprole.

This quiz covers key concepts regarding antibiotics and their mechanisms of action on bacterial cells. It includes questions about cytoplasmic membranes, cell wall structures, and specific antibiotic agents targeting DNA and other bacterial processes. Test your knowledge on the resilience of spore-forming bacteria and the differences between Gram-positive and Gram-negative bacteria.