Microbial Metabolism, Growth, Control, and Antimicrobial Drugs: Study Guide

Microbial Metabolism

Metabolism: Anabolism and Catabolism

Metabolism refers to all the chemical reactions that occur within a living organism to maintain life. These reactions are divided into two main categories:

• Anabolism: The synthesis of complex molecules from simpler ones, requiring energy input. Example: Protein synthesis from amino acids.

• Catabolism: The breakdown of complex molecules into simpler ones, releasing energy. Example: Glycolysis, where glucose is broken down to pyruvate.

Enzymes: Composition, Mechanism, and Regulation

Enzymes are biological catalysts, usually proteins, that speed up chemical reactions without being consumed. Their activity is influenced by several factors:

• Composition: Most enzymes are proteins; some require non-protein cofactors (coenzymes or metal ions).

• Mechanism: Enzymes lower the activation energy of reactions by binding substrates at their active site, forming an enzyme-substrate complex.

• Factors Influencing Activity: Temperature, pH, substrate concentration, and presence of inhibitors.

Enzyme Inhibition

• Competitive Inhibition: Inhibitor resembles the substrate and competes for the active site.

• Noncompetitive Inhibition: Inhibitor binds to an allosteric site, changing the enzyme's shape and reducing activity.

Redox Reactions (Oxidation-Reduction)

Redox reactions involve the transfer of electrons between molecules:

• Oxidation: Loss of electrons.

• Reduction: Gain of electrons.

These reactions are essential in energy production pathways such as cellular respiration.

Phosphorylation Reactions Generating ATP

• Substrate-level phosphorylation: Direct transfer of a phosphate group to ADP from a phosphorylated intermediate.

• Oxidative phosphorylation: ATP generated by the transfer of electrons through the electron transport chain to oxygen, coupled with chemiosmosis.

• Photophosphorylation: ATP generated using light energy in photosynthetic organisms.

Cellular Respiration: Glycolysis, Krebs Cycle, Electron Transport Chain

• Glycolysis: Glucose is converted to pyruvate, producing 2 ATP and 2 NADH per glucose.

• Krebs Cycle (Citric Acid Cycle): Acetyl-CoA is oxidized, generating NADH, FADH2, and 2 ATP per glucose.

• Electron Transport Chain (ETC): Electrons from NADH and FADH2 are transferred through a series of carriers, driving ATP synthesis via chemiosmosis.

Overall equation for aerobic respiration:

$\mathrm{C_6H_{12}O_6} + 6\,\mathrm{O_2} \rightarrow 6\,\mathrm{CO_2} + 6\,\mathrm{H_2O} + 36-38\,\mathrm{ATP}$

ATP Yield from Glucose Oxidation

• Glycolysis: 2 ATP (net)

• Krebs Cycle: 2 ATP

• Oxidative Phosphorylation: 32-34 ATP

• Total: 36-38 ATP per glucose (in prokaryotes)

Aerobic vs. Anaerobic Respiration

• Aerobic Respiration: Uses oxygen as the final electron acceptor; produces more ATP.

• Anaerobic Respiration: Uses other inorganic molecules (e.g., nitrate, sulfate) as final electron acceptors; yields less ATP.

Fermentation

• Definition: Metabolic process that converts sugar to acids, gases, or alcohol in the absence of oxygen.

• Products: Lactic acid, ethanol, CO2, etc.

• ATP Yield: 2 ATP per glucose (from glycolysis only)

Biochemical Tests for Bacterial Identification

Biochemical tests detect metabolic capabilities (e.g., fermentation of sugars, enzyme production) to identify bacteria in the laboratory.

Phosphorylation Types: Oxidative vs. Substrate-level vs. Photophosphorylation

• Oxidative phosphorylation: Involves ETC and chemiosmosis.

• Substrate-level phosphorylation: Direct transfer of phosphate group.

• Photophosphorylation: Light-driven ATP synthesis in photosynthetic organisms.

Energy and Carbon Sources in Microorganisms

Group	Energy Source	Carbon Source
Chemoheterotroph	Chemicals	Organic compounds
Chemoautotroph	Chemicals	CO2
Photoautotroph	Light	CO2
Photoheterotroph	Light	Organic compounds

Microbial Growth

Temperature Classification of Microbes

• Psychrophiles: Grow best at 0–15°C

• Mesophiles: Grow best at 20–45°C

• Thermophiles: Grow best at 55–80°C

• Hyperthermophiles: Grow above 80°C

Osmotic Pressure and Microbial Growth

Osmotic pressure affects water availability. High salt or sugar concentrations can inhibit microbial growth by causing plasmolysis.

Nutritional Requirements for Microbial Growth

• Carbon

• Nitrogen

• Sulfur

• Phosphorus

Oxygen Requirements

• Obligate aerobes: Require oxygen

• Facultative anaerobes: Can grow with or without oxygen

• Obligate anaerobes: Cannot tolerate oxygen

• Aerotolerant anaerobes: Tolerate but do not use oxygen

• Microaerophiles: Require low oxygen levels

Protection from Toxic Oxygen

• Enzymes such as superoxide dismutase, catalase, and peroxidase neutralize toxic oxygen species.

Biofilms

Biofilms are communities of microorganisms attached to surfaces, encased in a self-produced matrix. They are significant in infections due to increased resistance to antibiotics and immune responses.

Culture Media

• Chemically defined media: Exact chemical composition is known.

• Complex media: Contains extracts and digests of yeasts, meat, or plants; composition varies.

Special Culture Techniques

• Anaerobic techniques: For growing obligate anaerobes (e.g., reducing media, anaerobic jars).

• Living host cells: For obligate intracellular organisms.

• Candle jars: For microaerophiles.

• Selective media: Suppress unwanted microbes, encourage desired ones.

• Differential media: Distinguish colonies based on metabolic reactions.

• Enrichment medium: Favors growth of a particular microbe.

Biosafety Levels

• BSL-1: No special precautions

• BSL-2: Moderate risk; lab coat, gloves, eye protection

• BSL-3: Biosafety cabinets, respiratory protection

• BSL-4: Sealed, negative pressure, full-body suits

Colony and Pure Culture Isolation

• Colony: A visible mass of microbial cells arising from one cell or spore.

• Streak plate method: Used to isolate pure cultures by spreading cells on an agar plate.

Bacterial Growth and Binary Fission

• Bacterial growth: Increase in number of cells, not cell size.

• Binary fission: Main method of reproduction in bacteria; one cell divides into two.

Phases of Microbial Growth

• Lag phase: Adaptation, no increase in cell number.

• Log (exponential) phase: Rapid cell division.

• Stationary phase: Growth rate equals death rate.

• Death phase: Cells die faster than they divide.

Generation time: Time required for a cell to divide.

Measuring Microbial Growth

• Direct methods: Plate counts, filtration, MPN (most probable number), direct microscopic count.

• Indirect methods: Turbidity, metabolic activity, dry weight.

The Control of Microbial Growth

Key Terms in Microbial Control

• Sterilization: Removal or destruction of all microbial life.

• Commercial sterilization: Sufficient heat treatment to kill Clostridium botulinum endospores in canned food.

• Disinfection: Destruction of vegetative pathogens on inanimate objects.

• Antisepsis: Destruction of vegetative pathogens on living tissue.

• Degerming: Removal of microbes from a limited area.

• Sanitization: Lowering microbial counts to safe public health levels.

• Bactericidal agents: Kill bacteria.

• Bacteriostatic agents: Inhibit bacterial growth.

• Asepsis: Absence of significant contamination.

Factors Influencing Antimicrobial Effectiveness

• Number of microbes

• Microbial characteristics

• Environment (organic matter, temperature, biofilms)

• Time of exposure

Physical Methods of Microbial Control

• Moist heat: Boiling, autoclaving, pasteurization

• Dry heat: Flaming, incineration, hot-air sterilization

• Filtration: Removes microbes from liquids or air

• Low temperature: Refrigeration, deep-freezing

• High pressure: Denatures proteins

• Desiccation: Removes water, inhibits growth

• Osmotic pressure: High salt/sugar inhibits growth

Autoclave Parameters and Limitations

• Standard conditions: 121°C, 15 psi, 15 minutes

• Limitations: Cannot sterilize heat-sensitive materials

Thermal Death Time and Point

• Thermal death time (TDT): Time to kill all microbes at a given temperature.

• Thermal death point (TDP): Lowest temperature at which all microbes are killed in 10 minutes.

Pasteurization

Reduces spoilage organisms and pathogens in food and beverages without sterilizing.

Radiation Types and Effects

Type	Example	DNA Damage Mechanism	Penetration
Ionizing	X-rays, gamma rays	Breaks DNA strands	High
Non-ionizing	UV light	Forms thymine dimers	Low

Chemical Methods of Control

• Phenols and derivatives: Disrupt membranes, denature proteins

• Halogens: Iodine (antiseptic), chlorine (disinfectant)

• Alcohols: Denature proteins, dissolve lipids

• Heavy metals: Denature proteins

• Quaternary ammonium compounds: Disrupt membranes

• Aldehydes: Inactivate proteins by cross-linking

• Ethylene oxide: Gaseous sterilizer

• Peroxygens: Oxidizing agents

Food Preservatives

• Sorbic acid, benzoic acid, nitrates: Inhibit microbial growth in foods

Microbial Resistance to Chemical Methods

Microbe Type	Relative Resistance
Prions	Most resistant
Endospores	Very resistant
Mycobacteria	Resistant
Gram-negative bacteria	Moderately resistant
Gram-positive bacteria	Less resistant
Enveloped viruses	Least resistant

Antimicrobial Drugs

History and Selective Toxicity

• Paul Ehrlich: Developed the concept of chemotherapy and the first synthetic antimicrobial (Salvarsan).

• Alexander Fleming: Discovered penicillin.

• Selective toxicity: Ability of a drug to target pathogens without harming the host.

Antibiotic-Producing Microbes

• Streptomyces, Bacillus (bacteria), Penicillium, Cephalosporium (fungi)

Antimicrobial Drug Terms

• Antibiotic: Substance produced by a microbe that inhibits another microbe.

• Narrow-spectrum antibiotic: Effective against a limited range of microbes.

• Broad-spectrum antibiotic: Effective against a wide range of microbes.

• Synthetic drugs: Chemically synthesized in the lab.

• Semi-synthetic drugs: Chemically modified natural antibiotics.

• Superinfection: Overgrowth of resistant microbes after antibiotic treatment.

Modes of Action of Antimicrobial Agents

• Rifampin: Inhibits RNA synthesis

• Penicillin: Inhibits cell wall synthesis

• Polymyxin B: Disrupts cell membrane

• Trimethoprim, Sulfanilamide: Inhibit folic acid synthesis

• Streptomycin, Chloramphenicol, Tetracycline: Inhibit protein synthesis

Advantages of Alternative Antibiotics

• Semi-synthetic penicillins: Broader spectrum, resistant to penicillinase

• Cephalosporins: Broader spectrum, less allergenic

• Vancomycin: Effective against penicillin-resistant Gram-positive bacteria

Antimycobacterial Agents

• Isoniazid, Ethambutol: Inhibit mycolic acid synthesis in Mycobacterium cell walls

Antifungal and Antiviral Drugs

• Antifungal: Target ergosterol synthesis, cell wall synthesis, nucleic acid synthesis

• Antiviral: Inhibit viral entry, nucleic acid synthesis, or assembly

Antibiotic Susceptibility Testing

• Kirby-Bauer test: Disk diffusion method to assess sensitivity

• E test: Determines minimum inhibitory concentration (MIC)

• Broth dilution test: Determines MIC and minimum bactericidal concentration (MBC)

MIC: Lowest concentration that inhibits visible growth. MBC: Lowest concentration that kills 99.9% of bacteria.

Mechanisms of Drug Resistance

• Enzymatic destruction of drug (e.g., penicillinase)

• Prevention of drug penetration

• Alteration of drug target site

• Rapid efflux (ejection) of the drug

Prevention of Drug Resistance

• Use antibiotics only when necessary

• Complete prescribed course

• Use combination therapy

• Infection control in healthcare settings

Synergism and Antagonism

• Synergism: Combined effect of drugs is greater than the sum of individual effects.

• Antagonism: One drug reduces the effect of another.

Alternative Therapies

• Antimicrobial peptides: Short proteins with broad-spectrum activity

• Phage therapy: Use of bacteriophages to treat bacterial infections