Microbial Control and Preservation

Microbial Control

• People have been controlling microorganisms for hundreds of years through food and drinking water preservation and controlling disease.

Preservation of Perishable Products

• Physical methods include low temperature storage, freezing, and reducing available water.
• Weak organic acids like benzoic, ascorbic, and propionic acids are used as food preservatives in bread, cheese, and juice.
• Nitrates and nitrites are used in processed meats, inhibiting endospore germination and vegetative cell growth, but can be potent carcinogens.
• Numerous chemicals are used as preservatives, including formaldehyde and phenols for animal tissues.

Reducing Water Availability as a Preservation Method

• Decreasing water availability is achieved by salting or drying food which increases environmental solutes and causes cellular plasmolysis.
• Staphylococcus aureus can survive in high salt concentrations.
• Desiccation or drying supplements other methods like salting.
• Lyophilization (freeze drying) is widely used to preserve foods like coffee, milk, and meats

Understanding the Terms

• "-cide" means to kill; examples include bactericide, fungicide, virucide, sporicide, germicide, and microbicide.
• A bactericide is a chemical that destroys bacteria, but not endospores
• A fungicide is a chemical that kills fungal spores, hyphae, and yeasts
• A virucide is a chemical that inactivates viruses
• A sporicide can destroy bacterial endospores
• Germicides and microbicides are chemical agents that kill microorganisms
• "-static" means to stand still or prevent growth; examples include bacteriostatic, fungistatic, and microbiostatic.
• Bacteriostatic prevent the growth of bacteria
• Fungistatic inhibit fungal growth
• Microbiostatic materials used to control the growth of microorganisms

Different Types of Antimicrobial Agents

• Bacteriostatic agents inhibit growth, with the total cell count remaining constant but the viable cell count declining upon removal of the agent.
• Bactericidal agents kill bacteria, indicated by a decrease in viable cell count while the total cell count remains relatively stable.
• Bacteriolytic agents lyse bacterial cells, resulting in a sharp decline in both total and viable cell counts.

Principles of Control

• Sterilization removes and destroys all microbial life, using methods like autoclaving, incineration, and membrane filtration.
• Disinfection eliminates most pathogens; disinfectants are used on inanimate objects and surfaces; antiseptics are used on living tissues.
• Pasteurization involves brief heat treatment to reduce organisms that cause food spoilage
• Preservation is a process used to delay spoilage of perishable items, often including growth-inhibiting ingredients

Potency of Chemicals

• Antimicrobial formulations commonly contain more than one agent
• Formulations are regulated by the FDA for antiseptics on living/animate surfaces and by the EPA for disinfectants on non-living/inanimate surfaces.
• Germicidal agents, grouped by potency, do not destroy endospores
• Sterilants destroy all microorganisms

What is Microbial Death?

• Microbial death begins when cell structures become dysfunctional, leading to irreversible damage to the entire cell.
• A cell is considered dead when it can no longer reproduce under ideal environmental conditions.
• Death begins when an agent threshold is met, and proceeds in a logarithmic manner, not instantaneously.

The Rate of Microbial Death

• Decimal reduction time (DRT or D-value) measures the time to kill 90% of a specific bacteria population at a given temperature.
• Effectiveness of microbial treatment depends on the number of microbes, environment, time of exposure, and microbial characteristics

Understanding the Microbial Death Curve

• Logarithmic plotting reveals that the rate of killing being the same, it will take longer to kill all members of a larger population than a smaller one, whether using heat or chemical treatments.

Approaches to Control

• Control mechanisms are either physical, chemical, or a combination.
• Physical methods include heat, filtration, radiation, and mechanical removal
• Chemical methods use a variety of antimicrobial chemicals, depending on circumstances and degree of control required.

Microbial Control Methods are Highly Variable

• The applicability of these control methods are dependent on situation and the degree of control required
• Each method can be used for daily life, hospitals, microbiology laboratories, food and food production facilities, or water treatment

Choosing a Microbicidal Chemical

• Rapid action in low concentrations, solubility in water or alcohol, and long-term stability are needed
• Broad-spectrum microbicidal action is beneficial, but it has to be non-toxic to human and animal tissues
• It needs to penetrate inanimate surfaces and should be non-corrosive or non-staining, as well as having sanitizing and deodorizing properties.
• Affordability and availability is also a leading consideration.

Selection of an Antimicrobial Procedure

• Effective procedure selection is complicated; ideal methods don't exist and have process drawbacks
• Crucial factors include microbe type, contamination extent (number of organisms), environment, infection risk, and infected item composition

The Type of Microbe is Important

• Organism resistance or susceptibility is the most critical consideration.
• Resistant microbes include:
• Bacterial endospores, resistant to heat, drying, and chemicals.
• Protozoan cysts and oocysts, generally excreted in feces and cause diarrheal disease.
• Mycobacterium species, with cell wall structure initiating resistance.
• Pseudomonas species, which can grow in the presence of disinfectants.
• Naked viruses, lacking envelopes and being more chemically resistant.

Heat as Control

• Relatively convenient, dependable, and cheap
• Can be achieved faster if temperature is raised
• Destroys the majority of viruses and microorganisms
• Denaturing proteins and macromolecules is how heat works
• Boiling at 100°C can either sterilize or disinfect something
• However does not destroy endospores
• Autoclaving & Incineration destroy all life forms including endospores

Heat as Control - Pasteurization

• Pasteur developed it to avoid wine spoilage, it significantly reduces organisms but does not sterilize
• It’s used to increase food's shelf life
• The most protocols employ HTST method where it’s heated to 72°C and held for 15 seconds
• UHT (Ultra High Temperature) another protocol that heats to 140°C - 150°C, held for several seconds then is rapidly cooled
• Sterilized milk, creamer, and juice can be stored without refrigeration

Moist Heat as Control

• Can be achieved with an autoclave, which is a sealed heating device that uses steam under pressure
• It’s the preferred method of sterilization in health care environments
• The process involves heated water becoming steam which then increased pressure
• At 121°C and 15psi in 15 minutes, the sterilization kills vegetative cells and endospores but not prions
• Microbes are killed by temperature
• At 132°C and 15psi for 4.5 hours, prions are destroyed

Dry Heat as Control

• Not as effective as moist heat and sterilization requires longer times and higher temperatures
• 200°C for 1.5 hours (dry heat) VS. 121°C for 15 minutes (autoclave)
• Incineration method of dry heat sterilization oxidizes cell to ashes
• The method is used to destroy medical waste and animal carcasses and sterilizes inoculating loops

Other Physical Control Methods: Filtration

• Filtration used on heat-sensitive liquids and gases.
• Filters are used on pore sizes of 0.45 and 0.2 µm so that they are too small for parasites, fungi, and bacteria to pass, but bigger than most viruses.
• Depth filters are fibrous sheets made of overlapping paper that trap particles
• HEPA (High efficiency particulate air) filters remove >0.3 µm from air

Other Physical Methods of Control - Radiation

• Ionizing radiation ejects orbital electrons, forming ions.
• Nonionizing radiation excites atoms but doesn't ionize them
• Radiation can be used on diverse items like surgical supplies, plastic ware, drugs, fresh produce, and meat
• Germicidal ultraviolet C (UVC) lamps can be used in places like cafeterias, nurseries, and hospitals, and are very effective for surfaces but do not penetrate.
• Contact with eyes and skin need to be avoided

Non Ionizing Radiation (ultraviolet, 220-260 nm)

• Ultraviolet light can damage DNA by creating Thymine dimers.
• Created Thymine dimers induce a bond between bases on the same strand of DNA
• In UV damage, thymine dimers are formed, C-T or C-C bonds can also occur
• Effective for surfaces, does not penetrate eyes and skin must be avoided

Chemicals as Control

• Used to disinfect and sterilize
• Called germicidal chemicals or sterilants
• Reacts with vital cell sites, and can involve structures like the:
• Cytoplasmic membrane
• DNA
• Proteins

Alcohols

• Solutions of 60% - 80% isopropyl or ethyl alcohol kill vegetative bacteria and fungi, but are not effective against bacterial endospores and some naked viruses.
• Alcohols work by the coagulation of proteins and essential enzymes and/or damaging cell membranes.
• They’re frequently used as both antiseptics and disinfectants
• There is a limited range for how long they can take affect
• Plastic and rubber materials can be damaged

Halogens

• Include chlorine (sodium hypochlorite, NaOCI = Bleach) and iodine
• Have an oxidizing effect against the proteins and organic matter of the cell
• Chlorine destroys all types of organisms and viruses (including spores) and is a low-level disinfectant if 1:10 ratio
• Also replacing chlorine dioxide in drinking water for e.g.
• Iodine kills vegetative cells but is not reliable against endospores and is used in tincture or iodophore on skin

Peroxygens

• Hydrogen peroxide effectiveness is a function of the surface treated
• Living tissue produces catalase enzyme, breaking down hydrogen peroxide into oxygen and water
• Disifectant and cleaning agent
• More effective on organic matter than inanimate matter
• Leaves no residue on surfaces
• Hot solutions are used often for cleaning food industry surfaces

Heavy Metal Compounds

• High concentrations of many compound metals are harmful and toxic to human surfaces
• Disrupt normal protein functions by combining with sulfur-containing amino acids and interfere with the function of proteins
• Still used in bandages and wound care

Phenolics

• A phenol (aka carbolic acid) is one of the earliest disinfections
• Active ingredients in Lysol products
• Mode of action will destroy protein processes in membranes
• This kills vegetative cells and can kill mycobacterium at high concentrations but is not reliable on all viruses
• Triclosan and hexachlorophene are used in soaps and lotions

Antimicrobial Medications

• Chemotherapeutic drugs are used in the treatment, relief, or prophylaxis of a disease
• Prophylaxis is the use of a drug to prevent imminent infection of a person at risk
• Antimicrobial chemotherapy is the use of chemotherapeutic drugs to control infection, and are inclusive of all antimicrobial drugs regardless of the origin
• Antibiotics are substances produced by the processes of some microorganisms that inhibit/destroy other ones and its production
• Semisynthetic drugs are chemically modified in the laboratory after being isolated from natural sources
• Synthetic drugs synthesize antimicrobial compounds via a sequence of chemical reactions in the laboratory
• Antimicrobials effective against a limited array of microbial types are known as 'Narrow Spectrum'
• An example of a drug effective mainly on gram-positive bacteria.
• An example of a drug effective against both Gram (+ or -)
• Antimicrobials effective against a wide variety of microbial types known as 'Broad Spectrum', for example, a drug affecting both Gram (+ and -).

History and Development of Antibiotics - Alexander Flemming (1928)

• Alexander Fleming discovered that there were no Staphylococcus colonies growing near a mold
• That mold was identified as the penicillium which produces and effective bactericidal substance against a wide range of microbes

Penicillin - First Use

• Ernst Chain and Howard Florey successfully purified penicillin
• They shared the Noble prize in Physiology in medicine with Fleming (1945)
• in 1941, the substance was tested on a human who was suffering from a life threatening Staphylococcus aureus infection
• The treatment was initially effective but the supply ran out before the bacteria could be fully neutralized, and the patient died
• Once tested with an adequate amount of Penicillin, all patients fully recovered
• Mass production of penicillin was the focus during WWII in order to treat wounded soldiers

Features of Antimicrobial Drugs

• Most modern antibiotics come from bacteria and/or fungi inhabiting the soil
• bacterial species e.g. Streptomyces and Bacillus
• fungi e.g. Penicillium and Cephalosporium
• Antibiotics can be commercially produced by inoculating the strain into broth and incubating it until max concentration is reached
• once reached, the drug is extracted and purified
• Characteristics can be altered and are known as "semi-synthetic drugs"

Goal of Antimicrobial Chemotherapy

• Administer a drug to an infected patients which kills the infection without destroying the effects of the hosts cells
• This is difficult to accomplish.
• Chemotherapeutic agents are described with regard to origin, whether chemically synthesized, range of effectiveness, and/or whether they are naturally produced

Characteristics of The Ideal Antimicrobial Drug

• Exhibits selective toxicity to microbes without harming host cells; should be microbicidal rather than microbistatic
• Must exhibit a a high level of solubility and diluted functions
• Remains potent for an extended period of time before excretion prematurely
• Does not lead to either harm or antimicrobial resistance
• Helps and assists with the activities of the defense
• It must remain active in tissues and body fluids
• Easy and available to be delivered to sites of infection at a reasonably priced cost
• It cannot cause any disruptions to the host, health, allergies, or predispositions to infections

Features of Antimicrobial Drugs

• Differ depending on how they’re distributed metabolized and excreted, so this is an important factor for prescribing.
• The rate of elimination is expressed as half life; it's the time it takes to eliminate one half the original dose in serum which dictates the dosage frequency
• Patients who are suffering from liver and/or kidney damage excrete drugs more slowly.

Adverse Effects

• Allergic reactions can occur such as allergies to Penincillin Antibiotic colitis associated from the toxic side effects from some antimicrobial substances may establish and develop toxic organisms
• Toxic effect such as aplastic anemia can impair body's ability to make RBC or WBC production
• Antimicrobial resistances occurs when antibiotic is ineffective or harmless against infectious organism

Mechanisms of Action of Major Antibacterial Agents

• Antibiotics: Classified by method of action
• Each one has different aspects such as it's cell wall and folic acid metabolism
• Cytoplasmic aspects

Inhibition of cell wall synthesis

• The cell wall has peptidoglycan
• Can interfere with cell wall function but not impact eukaryotic
• Lack of cell wall in animal cells and differences in the cell wall in plant cells
• Also has drugs with a high therapeutic measure

Cephalosporins and Penicillins

• Can inhibit enzymes or functions to prevent glycan molecules Broad or narrow spectrum
• Resistance to drug can develop due to production of B-lactamase enzymes

The Cephalosporins

• broad spectrum drug or group
• majority of antibiotics

Cell Wall Synthesis Inhibition - Vancomycin and/or Bacitracin

• Vancomycin cannot cross the lipid membrane of gram negative bacterias, therefore they're resistant
• Usually provided intravenously and has poor absorbtion
• Acquired resistance is seen with VRSA

Inhibition of Protein Synthesis

• Structure of a prokaryotic ribosome
• Ribosomes have selective toxicity
• Can prevent tRNA from ribosome function
• Structure has a distinct stage

Aminoglycosides

• Block the initiation of translation and causes the misreading of mRNA
• Ineffective, or causes a reading of mRNA
• Also has the suffix of Gentamicin and streptomycin

Tetracyclines

• Cause the misreading of mRNA
• Has an effective component of Gram (+) and negative
• Can cause dislocation of teeth if taken as a you child

Bacterial Inhibition - Macrolids and/or Chloramphenicol

• Macrolids
• Has a variety of gram-positive bacteria
• often used for someone with penancillin
• has erythromycin, clarithromycin and azithromycin
• Chloramphenicol
• prevents peptide bonds
• causes anemia and infections

Antibacterial Drugs Used for Inhibition

• Quinolones: packaging of DNA, has broad specturm
• Rifamycins: Blocks infections of transciption polymerase

Reddish-Orange Urine From Rifampin Usage

• Sign of Mycobacterium and Neisseria

###Antibacterial Drugs that Inhibit Metabolic Pathways

• These are drugs that humans cannot make their on folic acid, it must come from diet.
• They inhibit production of folic acid
• Include both Sulfonamide and Trimethoprim

Antibacterial Drugs that effect the cell membrane

• Eukaryote membranes that are similar in structure to Polymyxin B
• Binds with the cell, limits usage of these agents to toical applications

###Other Antibacterial Drugs

• Growth factor analogs are structurally similar to growth factors but do not function in the cell • Analogs similar to vitamins, amino acids, and other compounds Sulfa drugs:
• Isonazid effective in only treating Mycobacterium from Domagh
• Used to treat membrane pore Gram postive material
• Platensimycin : for Mrsa and vancomycin

Mechanisms of Action of Antiviral Drugs

• They are only effective against specific types of virus
• Cannot eliminate anything latent
• Assembly with nucleic acids and release of entry

Targets of Some Antifungal Agents

• Medications in the plasma for most fungal infections can only be targeted with an ergosterol to inhibit it.
• Can be achieved through the use of Microtubules, polygenes and division of cells
• Remember a fungal infections come aresult of eukaryotes and its tough to kill

How to Determine Susceptibility of Bacteria to Antimicrobial Drug

• Suspectibiluty is not predictable
• Drug after drug is tried until a favorable response is found.
• A better way is to determine which drugs is the closest and most effective

Kirby-Bauer Disc Diffusion Test

• A common method that is regularly used to test if antimicrobial agents are effective against bacteria's
• Test strain is spread uniformly spread on standard media
• Specific concentrations is placed on plate and incubator

E Test and Antimicrobial Activity

• E- test uses strips impregnated with gradient concentration of antibiotic
• The higher the concentrate, the greater the organism and vice versa Minimum inhibitory concentration (MIC): smallest amount: Tube technique

Drug Resistance

• The use of adptive response is where microorganisms can and will start to accept ordinary drugs due the lack of their potency.
• This reaction can either acquired and/or intrinsic
• To be recognized, new spontaneous must begin mutations.

Examples Of Antimicrobial Resistance (Superbugs)

• Staphylococcus aureus is a common cause for nosocomial