Microbiology Exam #3

Which protocol completely eliminates microorganisms, including endospores and viruses?

Sterilization

Which protocol is for living tissues?

Antisepsis + Degerming

Which protocol is for non-living surface uses?

Disinfection + Sanitization + Sterilization

Which protocol uses only mild chemicals, like handwashing?

Degerming

Which protocol is to ensure safe public health levels?

Sanitization

Cleaning laboratory benches

• Chlorine bleach, phenols (e.g., Lysol), glutaraldehyde

Disinfection

Commercial dishwashing of eating utensils

• Industrial-strength cleaners containing

  quaternary ammonium compounds

Sanitization

Preparation of surgical equipment and needles used for injection

• Pressurized steam (autoclave), chemicals,

  radiation

Sterilization

Cleaning skin before surgery

• Boric acid, isopropyl alcohol, hydrogen

  peroxide, iodine (Betadine)

Antisepsis

Handwashing

• Soap, alcohol swab

Degerming

Cidal Agents

• Bactericide ~ bacteria

• Fungicide ~ fungi

• Viricide ~ virus

Kill microorganisms

Static Agents

• Bacteriostat

• Fungistat

Prevent growth

D-value

Decimal reduction time; time it takes to kill 90% of the population when exposed to a specific microbial control protocol

• Used to evaluate microbial control effectiveness

Factors impacting D-value

• Time of Exposure

• Microbial Population Size

• Concentration of Agent

• Temperature

• Presence of Organic matter (biofilm/blood)

• Microbial Resistance

BSL-1 (Biosafety Level)

Microbes are not known to cause disease in healthy hosts and pose minimal risk to workers and the environment.

BSL-2 (Biosafety Level)

Microbes are typically indigenous and are associated with diseases of varying severity. They pose moderate risk to workers and the environment.

BSL-3 (Biosafety Level)

Microbes are indigenous or exotic and cause serious or potentially lethal diseases through respiratory transmission.

BSL-4 (Biosafety Level)

Microbes are dangerous and exotic, posing a high risk of aerosol-transmitted infections, which are frequently fatal without treatment or vaccines. Few labs are at this level.

Standard aseptic technique

BSL-1

Requires lab coats, gloves, and autoclaves

BSL-2

Requires respirators and biological safety cabinets (BSCs)

BSL-3

Requires full-body pressure suits, HEPA- filtered air, and isolated buildings

BSL-4

Incineration

Dry-Heat Sterilization

Autoclave

Moist-Heat Sterilization

Pasteurization

Reduce pathogens while maintaining food quality

HTST Pasteurization

Milk heated at 72 ℃ for 15 seconds, then bottled and refrigerated

UHT Pasteurization

Milk heated at 138 ℃ for 2 or more seconds, then sealed in airtight containers for up to 90 days without refrigeration

Refrigeration

• Slows metabolism

• Slows growth

• Does not sterilize food

Freezing

• Stops growth

• May kill some microbes

Pascalization

High-pressure processing (100–800 MPa) to kill microbes in food

Desiccation (Drying)

Removes water needed for metabolism

Lyophilization (Freeze-drying)

Rapid freezing and vacuum-dried, long- term preservation

Osmotic Pressure

High concentrations of salt or sugar draw water out

of cells

UV light

Nonionizing Radiation

• Surface only

• Poor penetration

X-rays and Gamma rays

Ionizing Radiation

• Penetrate surfaces; cause double-strand DNA breaks

• Used for heat-sensitive clinical items

HEPA Filters (Filtration)

Removes microbes, endospores, and viruses as air flows through them

Membrane Filters (Filtration)

Liquid filtration for heat-sensitive solutions (0.2 μm typical)

Phenolics

Denature proteins and disrupt membranes

Iodine

Oxidizes macromolecules

• Betadine is a solution of the iodophor. It is commonly used as a topical antiseptic on a patient’s skin before incision during surgery.

Chlorine

Used for disinfection, strong oxidant

Fluorine

Bacteriostatic (stop bacterial growth without killing), strengthen tooth enamel

Alcohols

Denature proteins and disrupt membranes

• Work best in aqueous solutions

• Longer contact time (evaporate more slowly than 100% ethanol) improve microbial killing

Soap/Detergent (Surfactant)

Mechanical removal (rather than killing) via emulsification (~ not antiseptic)

Quats (Surfactant)

Cationic detergents that disrupt membranes via plasma membrane infiltration

• Nontoxic and common in household cleaners

Bisbiguanides

Cationic (positively charged), bacteriostatic at low concentration, bactericidal at high concentration. Can also act against enveloped virus

• Topical only (skin/mucous membranes)

Alkylating Agents

Inactivate enzymes and nucleic acids

• Highly explosive, need careful use and proper aeration after treatment

Peroxygens

Strong oxidizing agents, create free radicals that damage cellular macromolecules

• Highly effective, commonly used, no environmental hazard

Disk-Diffusion Method

A disk-diffusion assay is used to determine the effectiveness of chemical agents against a particular microbe

Use-Dilution Test

To determine a chemical’s disinfection effectiveness on an inanimate surface

In-Use Test

Determines contamination of disinfectant currently in use

Chemotherapy

Refer to the use of chemicals or drugs to treat any disease

Antimicrobial Drugs

Target infectious microorganisms by destroying structures or interfering with enzymes

Bacteriostatic Drugs

Reversible inhibition of growth (requires patient strong immune system)

Bactericidal Drugs

Kills target bacteria (essential for immunocompromised patients or life-threatening infections)

Narrow-spectrum

Targets specific subsets (prevents "collateral damage" to microbiota)

Broad-spectrum

Targets a wide variety; used for unknown pathogens or polymicrobic infections. Can lead to a Superinfection (e.g., C. diff or Yeast infections)

Slow Diagnosis

Empiric broad-spectrum treatment

Rapid Diagnosis

Targeted narrow-spectrum treatment

Synergistic

Two drugs work better together (e.g., trimethoprim and sulfamethoxazole) → each drug only provides bacteriostatic inhibition but combined are bactericidal

Antagonistic

Drugs interfere with each other (e.g., Antacids increase pH → affecting antimicrobial absorption)

Mechanisms of Antibacterial Drugs

• Inhibit cell wall biosynthesis

• Disrupt Membranes

• Inhibit protein synthesis

• Inhibit nucleic acid synthesis

• Antimetabolites

Inhibitors of Cell Wall Biosynthesis

Target: Peptidoglycan( structural component of bacterial cell walls)

• Selective Toxicity: High. Human cells do not have cell walls or peptidoglycan, so these drugs specifically target bacteria

Peptidoglycan structure

Peptidoglycan is composed of cross-linked chains of peptidoglycan monomers

• A peptidoglycan monomer consists of two joined amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a pentapeptide coming off of the NAM

β-lactam group (Inhibitors of Cell Wall Biosynthesis)

β-lactam inhibits transpeptidases (aka PBP ~ penicillin binding proteins)

• Blocks the crosslinking of peptide chains during the biosynthesis of new peptidoglycan in bacterial cell wall

• Weaken bacterial cell wall

• Cell lysis

Glycopeptide (Vancomycin)

Effective mainly against Gram-positive bacteria; too large to cross the Gram-negative outer membrane

• Binds the D-Ala-D-Ala terminus of peptidoglycan precursors

• Blocks both Transglycosylation and Transpeptidation

• Unlike β-lactams, does not bind PBPs

Bacitracin

Blocks bactoprenol ~ membrane carrier that transports peptidoglycan precursors across the cytoplasmic membrane → prevents cell wall building blocks from reaching the growing cell wall

• Commonly used topically (e.g., Neosporin) due to potential nephrotoxicity (kidney damage)

Inhibitors of Protein Biosynthesis

Target: Bacterial ribosome (70S)

• Moderate to High. Human cytoplasmic ribosomes are 80S, allowing for differentiation. However, human mitochondria have 70S ribosomes, which can lead to side effects

Inhibitors of Membrane Function

Lipids of the cytoplasmic or outer membrane

• Low. Human cells also have membranes, making these drugs more likely to have toxic side effects (like kidney or nerve damage)

Inhibitors of Nucleic Acid Synthesis

Enzymes involved in DNA replication or RNA transcription

• High. Bacterial enzymes for these processes are structurally different from human versions

Inhibitors of Metabolic Pathways (Antimetabolites)

Essential metabolic enzymes

• High. Humans get their folic acid from food; bacteria must synthesize it

Types of Metabolic Pathway Target

• Folic acid synthesis

• Mycolic acid synthesis

Folic acid synthesis (Broad Spectrum)

Inhibits the enzyme involved in production of dihydrofolic acid. Inhibits the enzyme involved in the production of tetrahydrofolic acid

Mycolic acid synthesis (Narrow Spectrum)

Interferes with the synthesis of mycolic acid

Fungi

Targets: Ergosterol, fungal cell wall

Protozoa

Targets: Folic acid synthesis, DNA synthesis, metabolism

Helminths

Targets: Microtubules, neuromuscular function, ATP production

Viruses

Targets: Viral enzymes, genome replication, entry/release

Antifungal Drugs

Ergosterol a good target

• Inhibit ergosterol synthesis

• Bind ergosterol in the cell membrane and create pores that disrupt the membrane

Antiprotozoan Drugs

act as antimetabolites, disrupting pathways required for growth and replication

• Inhibit electron transport in mitochondria

• Inhibit folic acid synthesis

• Produces damaging reactive oxygen species

• Inhibit DNA synthesis

• Inhibit heme detoxification

Antihelminthic Drugs

kill worms by causing starvation, paralysis, or impaired cell division

• Inhibit microtubule formation, reducing glucose uptake

• Block neuronal transmission, causing paralysis and starvation

• Inhibit ATP production

• Induce calcium influx

• Inhibit RNA synthesis

Antiviral Drugs

target specific stages of the viral life cycle rather than general cellular processes

Why is HIV difficult to treat

• Infects human immune cells

• Integrates into host genome

• Mutates rapidly due to error-prone reverse transcriptase

Evolution of Drug Resistance

• Natural Evolution

• Selective Pressure

• Genetic Acquisition

Natural Evolution

Microbes have evolved for millennia to overcome antimicrobial compounds produced by competitors in nature

Selective Pressure

Human use (and misuse) of drugs provides a selective pressure that accelerates evolution

• Overuse and misuse of antimicrobials

• Subtherapeutic dosing (not enough to kill, just enough to "train" the bacteria)

• Patient non-compliance (stopping a 10-day course after 5 days)

Genetic Acquisition

Antibiotics do not create resistance; they select for resistant microbes already present or newly mutated.

Vertical Transfer

Spontaneous chromosomal mutations passed to subsequent generations

Horizontal Gene Transfer (HGT)

Resistance genes found on plasmids or transposons are shared between bacteria (even different species)

Porin Channels

Gram-negative bacteria change the structure or number of channels to keep drugs out

Drug modification/ inactivation

Bacteria produce enzymes that chemically alter or destroy the drug

MIC

Minimal Inhibitory Concentration (lowest dose that stops growth)

MBC

Minimal Bactericidal Concentration (lowest dose that kills 99.9%)

Dilution Tests

Labs often use 96-well microdilution trays. This allows them to test multiple different drugs and different concentrations all at once using automated dispensing and "plate readers" (spectrophotometers)

Etest

Combines diffusion zone of inhibition and dilution using a plastic strip with a gradient

• It can indicate MIC but not MBC

Antibiogram

is a compilation of local susceptibility data from a specific hospital or region

Signs

Objective and measurable

• Can be directly observed by a clinician

• Examples: Fever (temp), heart rate, blood pressure, presence of antibodies

Symptoms

Subjective

• Felt or experienced by the patient; cannot be objectively measured

• Examples: Nausea, loss of appetite, pain level

Syndrome

A specific group of signs and symptoms characteristic of a particular disease.

Classification of Diseases: Infectious

Caused by the direct effect of a pathogen

Classification of Diseases: Communicable

Capable of being spread person-to-person