Untitled Quiz

Microbial Growth and Control

• Lecture 2 covered disinfection and sterilization methods in clinical microbiology labs
• It included antimicrobial agents, their modes of action, and efficacy in clinical settings
• Discussed antimicrobial sensitivity testing, its importance in clinical microbiology, and specific methods, including agar dilution and broth dilution
• Also covered modifications, common supplementary testing like D-test and B-lactamase test, and screening tests for specific bacteria like MRSA, VRE, ESBL, and CPE.

Disinfection vs. Sterilization

• Sterilization destroys all forms of life (all or nothing).
• Disinfection eliminates specific microorganisms from inanimate objects.
• Antiseptics eliminate or reduce bacteria on living tissue.

Types of Microorganisms and Resistance to Killing

• Prions are the most resistant to killing.
• Bacterial spores are highly resistant.
• Mycobacteria are also highly resistant.
• Nonlipid viruses are more resistant than lipid viruses.
• Fungi are more resistant than bacteria.
• Bacteria are more resistant than lipid viruses.
• Lipid viruses are the least resistant.

Methods of Disinfection/Sterilization

• Physical Methods
  • Heat (dry or moist, including boiling and pasteurization)
  • Filtration
  • Radiation
  • Lyophilization
  • UV
• Chemical Methods
  • Alcohols (50-70%)
  • Aldehydes
  • Halogens
  • Heavy Metals
  • Detergents
  • Phenolics
  • Gases

The Autoclave

• Typical specifications include a pressure of 15 psi at 121°C for 15-20 minutes.
• This process destroys vegetative microorganisms, bacterial endospores, and viruses.

Chemical Agents

• Table 4.3 details various chemical agents commonly used as disinfectants and antiseptics.
  • Provides agent, action, application, and precautions for each type, including alcohols, aldehydes, halogens, heavy metals, detergents, phenolics, etc.

Antimicrobial Agents

• Organisms are eradicated by agents that inhibit or kill them.
• Antimicrobial agents are a group of substances, either natural or synthesized, targeting organisms.
• Antibiotics are derived from other microbial organisms.
• Bacteriostatic agents halt bacterial growth.
• Bactericidal agents typically kill the organisms.

Antimicrobial Mode of Action

• Targets include cell walls, membranes, protein synthesis, DNA/RNA synthesis, and metabolic pathways.

Antimicrobial Mode of Action - Structural Integrity

• Includes cell walls , cell membrane, and other components
• Inhibiting the molecules that make up these structures.

Antibacterial agents - Organization by Mode of Action

• Organizes various antimicrobial agents by their action on cell walls, protein synthesis, genetic material, cell membrane, and metabolic processes.

Inhibition of Bacterial Cell Wall Biosynthesis

• Specific integral enzymes are needed to build and shape the cell walls.
• Transpeptidases (also known as penicillin-binding proteins or PBPs) cross-link the bacterial cell wall.
• Drugs are designed to inhibit these enzymes.

The Cell Envelope Structure

• Gram-positive and gram-negative bacteria have different cell envelope structures, which may lead to variation in response to different antibiotics.
  • Explains lipoteichoic acid, murein, Gram-positive cell wall, proteins, phospholipids, O Antigen, outer membrane, and periplasmic space in Gram-negative structures.

B-Lactam Antibiotics

• B-Lactam antimicrobial agents contain a four-membered, nitrogen-containing ring.
• They bind to and inhibit transpeptidases (PBPs), inhibiting cell wall synthesis.
• Gram-negative cells require porins to pass through their cell walls for β-lactams to reach PBPs.
• Various classes of β-lactams exist with different activity spectra.
  • Provides tables including Penicillins, Cephalosporins, Carbapenems, and Monobactams including details of categories, substrates, inhibitors, representative enzymes and various other characteristics.

Glycopeptides

• Bactericidal agents bind to the end of peptidoglycans, interfering with transpeptidation, inhibiting cell wall synthesis and growth.
• Large molecules and do not easily penetrate the cell walls of Gram-negative organisms or parts of the human body (examples include the blood-brain barrier).
• Has toxic side effects.
• Vancomycin is an example.
• Spectrum of activity is limited to Gram-positive organisms.

Cell Membrane Inhibitors - Polymyxin

• Usually act like detergents, increasing cell membrane permeability.
• Macro and micro-molecules leak from the membrane, causing the death of the bacteria cell.
• Example is Polymyxin B, and Colistin.
• More effective against Gram-negative bacteria.

Protein Synthesis Inhibitors

• Target protein synthesis and disrupt cellular processes.
• Bind to 30S or 50S ribosomal subunits.
• Includes aminoglycosides, MLS group (macrolides-lincosamides-streptogramins), ketolides, oxazolidinones, streptogramins, chloramphenicol, tetracyclines, and glycylglycines.

Aminoglycosides

• Bactericidal, binds to 30S ribosomal subunit.
• Prevents docking of aminoacyl-tRNA and contributes to misreading genetic code.
• Large, cannot cross blood-brain barrier.
• Side effects include: nephrotoxicity, ototoxicity, and potentially nerve damage.
• Examples are gentamicin, tobramycin, streptomycin, and kanamycin.
• Primarily effective against Gram-negative bacteria.

Chloramphenicol

• Bactericidal, binds to 50S ribosomal subunit.
• Prevents aminoacyl-tRNA docking.
• Serious side effects: bone marrow toxicity (aplastic anemia).
• Limited clinical use.

Glycylcyclines (Tigecycline)

• Higher affinity for 30S ribosomal subunit than tetracyclines.
• Increased effectiveness against tetracycline-resistant organisms.
• Side effects associated with increased mortality.
• Wide spectrum activity against various pathogens.
• Used to treat atypical infections and complicated intra-abdominal infections, skin infections, and community-associated pneumonias.

Macrolide-Lincosamide-Streptogramin (MLS) Group

• Protein synthesis inhibitors effective against gram-positive bacteria and some gram-negative.
• Bacteriostatic or bactericidal based on concentration, includes Erythromycin, Azithromycin, Clarithromycin and Clindamycin.

Oxazolidinones (Linezolid)

• Binds to 50S subunit and blocks initiation and translocation.
• Long-term therapy can lead to significant hematologic and neurologic toxicity.
• Narrow spectrum, mostly used against MRSA, VRE, and Mycobacterium tuberculosis.

Tetracyclines

• Bacteriostatic, binds to 30S subunit.
• Reversible binding; inhibits tRNA rotation.
• Causes premature release of peptides from ribosomes.
• Wide spectrum including atypical bacteria like Mycoplasma, some intracellular organisms(Treponema pallidum, Chlamydia, Rickettsia), and spirochetes bacterium(Borrelia, Vibrio cholerae).

Metronidazole

• Nitroimidazole antibiotic, bactericidal.
• Nitro group is reduced in bacterial cytoplasm to cytotoxic compounds that disrupt DNA.
• Effective against anaerobic bacteria.
• Can be used to treat some protozoan infections.

Quinolones

• DNA/RNA synthesis inhibitors
• Inhibit DNA gyrase and topoisomerase IV enzymes.
• Associated with many toxic effects, including aortic dissections and tendinitis.
• Broad spectrum, used in treatment of several bacterial infections, including Enterobacteriaceae, Pseudomonas, Staphylococci, and Enterococci.

Rifamycin (Rifampin)

• DNA/RNA synthesis inhibitors, binds to DNA-dependent RNA polymerase and inhibits RNA synthesis.
• Synthetic derivative of rifamycin B.
• Targets DNA transcription.
• Narrow spectrum effective against Gram-positive organisms.
• Used in combination therapy for tuberculosis.

Folic Acid synthesis Inhibitors (Sulfonamides, Trimethoprim)

• Inhibits bacterial folic acid synthesis
• Sulfonamides block dihydropteroate synthase.
• Trimethoprim blocks dihydrofolate reductase.
• These combined agents are synergistic, have broad-spectrum activity.

Microbial Resistance Mechanisms

• Intrinsic Resistance:
  • Naturally present in bacteria.
  • Inherited vertically.
  • Predictable after organism identification.
  • Example is Gram-negative bacteria's resistance to vancomycin.
• Acquired Resistance:
  • Derived from exogenous DNA, including plasmids, transposons, and bacteriophages.
  • Major concern in healthcare environments.
  • Example is ESBL resistance.
  • Explain possible causes that might lead to resistance
  • Include Loss of drug affinity for the target site.
  • Impermeable bacterial cell wall
  • Enzymatic inactivation of the antibiotic
  • Increased antibiotic efflux

Antimicrobial Susceptibility Testing (AST)

• Employs to determine which antimicrobial agents will effectively treat an infection.
• Common methods are disk diffusion and dilution (broth macrodilution and microdilution).
• Includes important considerations such as testing the correct organism, presence of other organisms, testing medium, quality of samples, and patient characteristics to yield accurate results.
• Clarifies important factors, the choice of antimicrobials, reporting considerations, and potential contraindications depending on the characteristics of the patient and test conditions.

Antimicrobial Susceptibility Testing (AST) Methods

• Broth Methods:

  • A dilution method.
  • Determines MIC (minimum inhibitory concentration).
  • Compares MIC to standards to determine susceptibility, intermediate, and resistance categories. Includes tables of MIC interpretive standards for different organisms.

• Agar Methods (Macrodilution, Microdilution, Disk Diffusion, Agar Dilution): Detailed steps, guidelines, interpretations, and considerations for each mentioned method.

  • Explains how to determine the zone of inhibition by measuring with a ruler, comparing it to established zone size, and classifying it as susceptible, intermediate, or resistant based on the growth of the bacteria. Provides instructions on interpreting the test for certain organisms and what to look for when microbes like Proteus swarm. Explains factors to consider during the test like purity of sample and how the testing conditions should be set up.

AST Standard Elements

• Includes the need for pure culture, log-phase organisms, standard inoculum, specific media, antibiotic selection, and quality control procedures for accurate and reliable results.

VRE, D-test, and ESBL Screen

• Individual methods used to detect different resistance mechanisms of microbes.

CPE Screen

• Specific test to screen for resistance to carbapenems, especially frequently encountered in organisms like Klebsiella pneumoniae.

Gradient Diffusion Test

• Also known as Epsilometer test or Etest; uses antibiotic impregnated strips, gradients of antimicrobial along the strip. Explains how to interpret results by the elliptic inhibitory zones the agent forms.

Beta-Lactamase Tests

• Used to detect β-lactamases, important as it's the most common method of antibiotic resistance.
• Commonly used chromogenic methods (using discs treated with nitrocefin) to identify the enzymatic inactivation of the antibiotic.