Control of Microorganisms

Control of Microorganisms

• Microorganisms are controlled for various reasons, including preventing corrosion, controlling plant pathogens, treating human pathogens and preventing food spoilage.

Definitions of Frequently Used Terms

• Antisepsis: Chemicals applied to body surfaces to destroy or inhibit pathogens.
• Sanitation: Reduction of microbial population to safe levels based on public health standards.
• Disinfection: Destruction or removal of pathogens, but not bacterial endospores. Usually used on inanimate objects.
• Sterilization: Complete removal or destruction of all viable microorganisms. Used on inanimate objects, including endospores.
• Chemotherapy: Chemical used to kill or inhibit growth of microorganisms within host tissues.

Reduction of Microbial Numbers

• The decline in microbial numbers follows exponential patterns for different types of control. Antisepsis, sanitation and disinfection and sterilization all have different curves showing log reduction over time.

What Does Log Kill Mean?

• 1 log reduction = 90% kill
• 3 log reduction = 99.9% kill
• The approximate number of bacteria in the mouth is similar to the number of people on Earth (approximately 7.5 billion).
• 3 log reduction reduces bacterial population from 6.1 x 10⁹ to 6 x 10⁶.
• Note: log reduction is a transient reduction

The Pattern of Microbial Death

• Microbial death does not occur instantly.
• Population decline occurs exponentially.
• Decimal reduction time is the time to kill 90% of a population.
• Persister cells are viable but nonculturable (VBNC), meaning they are intact, have low metabolic rate, do not divide, and cannot form colony-forming units.
• VBNC cells can recover and regain the ability to reproduce and cause infections.

Conditions Influencing Antimicrobial Agent Activity

• Larger populations take longer to kill compared to smaller populations.
• Different species have different sensitivities to antimicrobials.
• Higher concentrations of antimicrobials kill microorganisms more rapidly.
• Longer exposure times lead to more microbes killed.
• Higher temperatures kill more microbes.
• Local environment factors such as pH, viscosity and organic matter affect the effectiveness of antimicrobials. Organisms in biofilms are less susceptible to many antimicrobials.

Physical Control of Microbes

• Heat and Radiation are methods for controlling microbes.

Moist Heat

• Moist heat is an effective method for killing viruses, fungi, protists, and bacteria.
• It degrades nucleic acids, denatures proteins, and disrupts cell membranes.
• Spores are more resistant to heat and moisture than vegetative cells.

Autoclaving

• Uses pressurized steam at temperatures above 100°C for sterilization. This method effectively sterilizes closed containers and microorganisms (including spores).
• Quality control procedures use Geobacillus stearothermophilus strips to ensure proper operation during autoclaving.
• Important to understand the sterilization time required for different container sizes during autoclaving.

Why is an autoclave essential for microbiologists?

• Microbiologists need sterility to maintain and conduct experiments, and aseptic technique and sterilization are important parts of a microbiologist's work, especially when applying for an industry job.

Pasteurization

• Invented by Louis Pasteur in 1864.
• This process uses controlled heating to reduce bacterial numbers without sterilizing a product.
• It's widely used in the food industry (milk, dairy, beer).
• Choice of pasteurization method depends on product. High-temperature-short-time and low-temperature-long-time treatments are used depending on the food.

Dry Heat Sterilization

• Dry heat is less effective than moist heat sterilization.
• It requires higher temperatures and longer exposure times (e.g. 160-170°C for 2 to 3 hours).
• Dry heat is not usually applicable to food. It oxidizes cell constituents and denatures proteins.
• It's suitable for sterilizing solids that are unaffected by moist heat.

Ionizing Radiation

• Gamma radiation penetrates deep into objects, destroying bacterial endospores.
• It's not always effective against viruses.
• It is used for sterilizing antibiotics, hormones, sutures, plastic and food products (such as meat, eggs).

Ultraviolet (UV) Radiation

• UV radiation with a wavelength of 260nm is effective against bacteria (DNA absorbs).
• It causes thymine dimers preventing replication and transcription.
• UV radiation does not penetrate glass, dirt films and water. It's limited to surface decontamination.

Chemical Control of Microbes

Chemical Agents

• Phenols: Used as laboratory and hospital disinfectants; tuberculocidal, effective in presence of organic materials, and long-lasting. Act by denaturing proteins and disrupting cell membranes. Disagreeable odor. Triclosan is used in hand sanitizers and toothpastes.
• Alcohols: Among the most widely used disinfectants and antiseptics; ethanol and isopropanol are the most common. Bactericidal, fungicidal, but not sporicidal. Inactivate some viruses and denature proteins. Most effective at 70% solution with water.
• Halogens: Iodine is a skin antiseptic, used in surgical preparations. Oxidizes cell constituents and iodinates proteins. At high concentrations may kill spores. Issues with skin damage, staining and allergies. Chlorine is important in water disinfection, swimming pools, and other food applications; oxidizes cells and is sporicidal. Can react with organic matter to form harmful substances.
• Heavy metals: (e.g., mercury, silver, arsenic, zinc, copper) combine with and inactivate proteins; may precipitate proteins. Silver coins were once used to preserve foods. Used as skin infection treatment (ointment). Use is often limited due to toxicity.
• Quaternary Ammonium Compounds: Detergents with antimicrobial activity. Amphipathic organic cleansing agents. Safe and easy to use, but are inactivated by hard water and soap. Target cell integrity by killing most bacteria, but not endospores.
• Aldehydes: Formaldehyde and glutaraldehyde. Highly reactive molecules, toxic; combine with and inactivate nucleic acids and proteins. Sporicidal. Can be used for chemical sterilants.
• Sterilizing Gases: Sterilize heat-sensitive materials; microbicidal and sporicidal. Ethylene oxide sterilization is done in autoclave-like equipment.

Mechanical Control of Microbes

• Filtration: Removes microorganisms, applicable to sensitive materials like air (HEPA filters), food (milk, juices) and research labs.

Filtering Liquids

• Uses porous membranes with defined pore sizes to remove microorganisms. Pore size of 0.2 microns adapted to bacteria.

Filtering Air

• HEPA filters are used in surgical masks, cotton plugs on culture vessels and lab safety equipment, such as laminar flow biological safety cabinets.

Biological Control of Microbes

• Emerging field showing great promise. Natural control mechanisms include predation by Bdellovibrio, toxin-mediated killing using bacteriocins, bacteriophages (bacteria viruses), and viral-mediated lysis using pathogen-specific bacteriophage lysins.

Bacteriophage products:

• Bacteriophages express lysins that target the peptidoglycan.
• Virion-associated peptidoglycan hydrolases create holes.
• Endolysins degrade peptidoglycan at the end of the cycle.
• Enzymes classes are being explored as antibacterial agents.

The Development of Chemotherapy

• Paul Ehrlich (1904): Developed the concept of selective toxicity. Identified dyes that treated African sleeping sickness.
• Sahachiro Hato (1910): identified arsenic compounds to treat syphilis.
• Gerhard Domagk, Jacques and Therese Trefouel (1935): discovered sulfonamides and sulfa drugs.

Penicillin

• First discovered by Ernest Duchesne (1896) but lost; rediscovered by Alexander Fleming (1928).
• Fleming observed penicillin activity on contaminated plates.
• Florey, Chain, and Heatley demonstrated its effectiveness.
• Fleming, Florey, and Chain were awarded the Nobel Prize in 1945.

Chemotherapeutics

• Antibiotics: Used for treating bacterial or viral infections.
• Bactericidal agents kill bacteria.
• Bacteriostatic agents inhibit bacterial growth.

General Characteristics of Antimicrobial Drugs

• Selective toxicity: ability of drug to kill the pathogen without harming the host.

• Narrow spectrum: targets a few pathogens.

• Broad spectrum: targets many pathogens.

• Therapeutic dose: the ideal level of a drug to have therapeutic effect.

• Side effects: may be toxic to humans.

• Therapeutic index: ratio between toxic and therapeutic doses, a higher index is better.

• Effectiveness: MIC (minimal inhibitory concentration): lowest concentration of a drug that inhibits pathogen growth. Minimal bactericidal/lethal concentration (MBC): lowest concentration of drug that kills a pathogen.

Terminology of Chemical Control

• "-static": The agent will prevent growth of the type of organism (e.g., bacteriostatic, fungistatic)
• "-cide": The agent will kill the type of organism in question (e.g., fungicide, bacteriocide)

Ability of Drug to Reach Site of Infection

• The mode of administration (e.g., oral, topical, intravenous, intramuscular) determines how easily a drug reaches the site of infection.

Determining Antimicrobial Potency

• Dilution susceptibility tests: used for determining the lowest concentration of antibiotic needed to prevent bacterial growth or kill bacteria.
• Disc diffusion tests/Kirby Bauer method: determines the susceptibility of various bacterial isolates to specific antibiotic drugs by placing antibiotic discs on solid media inoculated with bacteria. The inhibition zones created around the discs are then evaluated. E-test MIC and diffusion: uses gradient of antibiotic concentration to determine the MIC (minimal inhibitory concentration).

Dilution Susceptibility Tests

• Tubes showing no growth determine the MIC (minimal inhibitory concentration).

Disk Diffusion Tests

• Disks with specific drugs are placed on inoculated bacterial plates.
• Drug diffusion, zone of inhibition.

Kirby-Bauer method

• Standardized method for carrying out disk diffusion test.
• Antibiotic discs are placed on plates containing the bacteria of interest.

The E-test

• Convenient for use with anaerobic pathogens.
• Similar to disc diffusion method but uses strips rather than discs.
• Strips contain a gradient of antibiotic.
• Intersection of zone of inhibition with strip indicates MIC.

Microbial Control Methods (overview)

• A chart showing various methods.

How do antibiotics work?

• Antibiotic targets the most fundamental processes in bacteria.
• Examples are inhibitors of cell wall synthesis, protein synthesis inhibitors, nucleic acid synthesis inhibitors, metabolic antagonists.

Mechanisms of Action

• Antibiotic target the most fundamental processes in bacterial cells.
• Examples are inhibitors of cell wall synthesis, protein synthesis inhibitors, nucleic acid synthesis inhibitors, metabolic antagonists

Major Targets of Common Antibiotics

• Cell Wall: Penicillins, Glycopeptides, Ethambutol, Isoniazid
• DNA: Fluoroquinolones, Novobiocin, Nitrofurans
• Ribosomes: Tetracyclines, Aminoglycosides, Macrolides, Chloramphenicol

Inhibitors of Cell Wall Synthesis

• Penicillins (β-lactams): Most important antibiotic class; 6-aminopenicillanic acid derivatives; β-lactam ring is essential for bioactivity.

Penicillins

• Mode of action: Blocks the enzyme that catalyzes transpeptidation.
• Prevents the synthesis of complete cell walls leading to lysis of cells and only acts on growing bacteria that are synthesizing new peptidoglycans.

Strands are cross-linked

• Amino acids serve to link glycan chains together in several ways.

Protein Synthesis Inhibitors

• Many antibiotics bind bacterial ribosome subunits (30S or 50S).

Aminoglycoside antibiotics

• Large family containing a cyclohexane ring and amino sugars.
• Binds to 30S ribosomal subunit interfering with protein synthesis and causing messenger RNA misreading.

Antibiotic Resistance

Drug Resistance

• An increasing problem.
• Resistance originates in population, transmitted to other bacteria.
• A mechanism can provide resistance to multiple antibiotics.
• Bacteria develop resistance spontaneously.

Mechanisms of Drug Resistance

• Modification of target enzyme.
• Inactivation of the antibiotic.
• Antibiotic-altering enzymes.
• Drug efflux (pumping the drug out of the cell).
• Use of alternative pathways/enzymes

The origin and transmission of drug resistance

• Resistance genes can be found on bacterial chromosomes, plasmids or transposons.
• Antibiotic-producing microbes contain resistance genes.
• Resistance genes are transferred to non-producing microbes.

Drug-resistant "superbug"

• Methicillin-resistant Staphylococcus aureus (MRSA).
• Vancomycin-resistant Staphylococcus aureus (VRSA).
• Vancomycin-resistant enterococci (VRE)

Summary

• There is significant motivation to control microbes for human health and industry.
• Understanding the mechanisms of action of antimicrobials allows for targeted drug design methods.
• Selection pressure for resistance to antimicrobials is very high.
• Overuse and misuse of antimicrobials often increases selection for drug resistance

Test your knowledge on microbial control methods, including antisepsis, sanitation, and chemotherapy. Explore concepts such as microbial reduction rates, biofilm resistance, and factors affecting antimicrobial efficacy. This quiz is essential for understanding public health strategies related to infection control.