Microbial Control Flashcards
Sepsis
microbial contamination.
Asepsis:
absence of significant contamination. o Aseptic surgery techniques prevent microbial contamination of wounds. o Antimicrobial chemicals, expected to destroy pathogens but not to achieve sterilization § Disinfectant: the destruction of vegetative pathogens, used on objects § Antiseptic: used on living tissue
Nosocomial
hospital infection
Sterilization:
Removal of all microbial life (heat, filtration) o For food: Commercial sterilization to kill C. botulinum endospores
Sanitization
reduce microbe numbers to safe levels
Bacteriostatic:
Inhibits bacterial reproduction
bactericidal
kills bacteria
RATE OF MICROBIAL DEATH
Bacterial populations subjected to heat or antimicrobial chemicals die at a constant rate. • Microbial Death Curve, plotted logarithmically, shows this constant death rate as a straight line.
EFFECTIVENESS OF ANTIMICROBIAL TREATMENT
t depends on: • Time it takes to kill a microbial population is proportional to number of microbes. • Microbial species and life cycle phases (e.g.: endospores) have different susceptibilities to physical and chemical controls. • Organic matter may interfere with heat treatments and chemical control agents. • Exposure time: Longer exposure to lower heat produces same effect as shorter time at higher heat. • Environmental influences
ACTIONS OF MICROBIAL CONTROL AGENTS
• Alternation of membrane permeability • Damage to proteins • Damage to nucleic acids
PHYSICAL METHODS OF MICROBIAL CONTROL
Not for use on living organisms Somehow, alter membrane permeability and/or structure of proteins and nucleic acids Heat is very effective (fast and cheap).
Thermal death point (TDP):
The lowest temperature at which all cells in the culture are killed in 10 min.
Thermal death time (TDT):
Time to kill all cells in a culture
Decimal Reduction Time (DRT):
Minutes to kill 90% of a population at a given temperature
MOIST HEAT STERILIZATION
Denatures proteins
Autoclave
Steam under pressure Most dependable sterilization method • Steam must directly contact material to be sterilized. • Pressurized steam reaches higher temperatures. • Normal autoclave conditions: 121.5 OC for 15 min. • Prion destruction: 132 OC for 4.5 hours
PASTEURIZATION
Significant number reduction (esp. spoilage and pathogenic organisms) it does not sterilize!
The historical goal of pasteurization
the destruction of M. tuberculosis
Classic holding method: (pasteurization)
63 OC for 30 min
Flash pasteurization (HTST):
72 OC for 15 sec. o Most common in the US o Thermoduric organisms survive
Ultra-High Temperature (UHT):
140 OC for < 1 sec o Technically not pasteurization because it sterilizes
DRY HEAT STERILIZATION
• Kills by sterilization • Flaming of loop • Incineration of carcasses o Anthrax o Foot and mouth disease o Bird flu
FILTRATION
Air filtration using high efficiency particulate air (HEPA) filters. Effective to 0.3 µm Membrane filters for fluids. • Pore size for bacteria: 0.2 – 0.4 µm • Pore size for viruses: 0.01 µm
LOW TEMPERATURE
• Slows enzymatic reactions à inhibits microbial growth • Freezing forms ice crystals that damage microbial cells • Refrigeration (deep freezing, lyophilization)
High pressure in liquids
denatures bacterial proteins and preserves flavor
Desiccation
prevents metabolism
Osmotic pressure
causes plasmolysis
IONIZING RADIATION
• X-rays, gamma rays, electron beams
What element is most commonly used in Ionizing radiation
Commonly used Cobalt-60 radioisotope
bacteria that are sensitive to ionizing radiation
Salmonella and pseudomonas
UV Light
Most effective wavelength ~ 260 nm • Effect: thymine dimers • Actively dividing organisms are more sensitive because thymine dimers cause ____? • Used to limit air and surface contamination. Use at close range to directly exposed microorganisms E.g.: germicidal lamps in OR, cafeteria, and our lab
MICROWAVE
Wavelength: 1 mm - 1m • H2O quickly absorbs energy released as heat to environment • Indirect killing of bacteria through heat
CHEMICAL METHODS OF MICROBIAL CONTROL
• Few chemical agents achieve sterility. • Consider the presence of organic matter, degree of contact with microorganisms, and temperature • Disinfectants regulated by EPA • Antiseptics regulated by FDA
USE-DILUTION TEST
- Metal rings dipped in test bacteria are dried. 2. Dried cultures of S. choleraesuis, S. aureus, and P. aeruginosa are placed in disinfectant for 10 min at 20°C 3. Rings are transferred to culture media to determine whether bacteria survived treatment.
DISK-DIFFUSION METHOD
The disk of filter paper is soaked with a chemical and placed on an inoculated agar plate; a zone of inhibition indicates effectiveness.
PHENOL =
carbolic acid (historic importance) • Phenolics: Cresols (Lysol); disinfectant
BISPHENOLS
• Hexachlorophene (pHisoHex, prescription), hospitals, surgeries, nurseries • Triclosan (toothpaste, antibacterial soaps, etc).
CHLORINE
• Oxidizing agent • Widely used as a disinfectant • Forms bleach (hypochlorous acid) when added to water. • Broad spectrum, not sporicidal (pools, drinking water)
IODINE
• More reactive, more germicidal • Alters protein synthesis and membranes. • Tincture of iodine (solution with alcohol) à wound antiseptic • Iodophors combined with an organic molecule o iodine detergent complex (e.g. Betadine®). Occasional skin sensitivity, partially inactivated by organic debris, poor sporicidal activity
Ethyl (60 - 80% solutions) and isopropyl alcohol
• Denature proteins, dissolve lipids • No activity against spores and poorly effective against viruses and fungi
MERCURIC CHLORIDE
(HgCl2, Greeks & Romans for skin lesions); Thimerosa
COPPER
against chlorophyll-containing organisms à Algicides
SILVER NITRATE (AGNO3):
Antiseptic for eyes of newborns
ZINC (ZNCL2)
in mouthwashes, ZnO in antifungal In paint
SURFACE ACTING INGREDIENTS/SURFACTANTS
soaps and detergents
The major purpose of soap:
Mechanical removal and use as wetting agent
Acidic-
Anionic detergents: Anion reacts with the plasma membrane. Nontoxic, non-corrosive, and fast-acting. Laundry soap, dairy industry.
Cationic detergents
Quaternary ammonium compounds (Quats). Strongly bactericidal against a wide range, but especially Gram (+) bacteria
SULFUR DIOXIDE:
wine
ORGANIC ACIDS:
Sorbic acid, benzoic acid, and calcium propionate; Inhibit metabolism; Control molds and bacteria in foods and cosmetics
SODIUM NITRATE AND NITRITE:
prevents endospore germination; In meats; Conversion to nitrosamine (carcinogenic)
ALDEHYDES (alkylating agents)
• Inactivate proteins by cross-linking with functional groups (-NH2, -OH, -COOH, -SH)
Glutaraldehyde:
sterilant for delicate surgical instruments (Kills S. aureus in 5, M. tuberculosis in 10 min)
Formaldehyde:
Virus inactivation for vaccines
CHEMICAL STERILANTS
for heat sensitive material • Denature proteins • Ethylene oxide
PLASMA
• Luminous gas with free radicals that destroy microbes • Use: Tubular instruments, hands, etc
HYDROGEN PEROXIDE:
OXIDIZING AGENT • Inactivated by catalase à Not good for open wounds Esp. effective against anaerobic bacteria • Effervescent action may be useful for wound cleansing through the removal of tissue debris
Resistance order
Prions bacterial endospores Mycobacteria protozoa cyst g -ve bacterium Fungal spores virus without envelope gram +ve Virus with lipid
STERILIZATION
- The killing or removal of all viable organisms within a growth medium
INHIBITION
- Effectively limiting microbial growth.
DECONTAMINATION
- The treatment of an object to make it safe to handle.
DISINFECTION
- Directly targets the removal of all p
HEAT STERILIZATION
- Is the most widely used method of controlling microbial growth - Simplest means of sterilizing materials provided the material is itself resistant to heat.
Decimal reduction time
Amount of time required to reduce the viability tenfold
Antimicrobial agents can be classified as
bacteriostatic, bacteriocidal and bacteriolytic
MINIMUM INHIBITORY CONCENTRATION (MIC)
Is the smallest amount of an agent needed to inhibit the growth of a microorganism - Varies with the organism used, inoculum size, temp, pH, etc.
DISC DIFFUSION ASSAY
The antimicrobial agent added to the filter paper disc The antimicrobial agent added to the filter paper disc - MIC is reached at some distance § Zone of Inhibition – an area of no growth around the disc.
Antimicrobial drugs are classified on the basis of
- Molecular structure - Mechanism of action - Spectrum of antimicrobial activity
Characteristics of the ideal antimicrobial drug:
Selectively toxic to the microbe but nontoxic to host cells - Microbicidal rather than microbiostatic - Relatively soluble and function even when it is highly diluted in body fluids - Remains potent long enough to act and is not broken down or excreted prematurely - Not subject to the development of antimicrobial resistance - Complements or assists the activities of the host’s defences - Remains active in tissues and body fluids - Readily delivered to site of infection - Not excessive in cost - Does not disrupt the host’s health bu causing allergies of predisposing the host to other infections.
Inhibition of cell wall synthesis
Pencillin, Cephalosporins
Beta-Lactam drugs
selective inhibitors of bacterial cell wall synthesis
Inhibition/alteration of cell membrane function
Detergents disrupt cytoplasmic membrane o Ex. Valinomycin, Daptomycin, Telavancin
Inhibition of protein synthesis:
Bacteria have 70S ribosomes while mammalian cells have 80 S ribosomes, which explains why antimicrobials can inhibit protein synthesis in bacterial ribosomes without having a major effect on mammalian ribosomes Ex. aminoglycosides, Macrolides, Azalides and Ketolids, Lincosamides, tetracyclines, Glycylcyclines, Chloramphenicol, Streptogramins, Oxazolidinones
Inhibition of Nucleic acid synthesis:
on of Nucleic acid synthesis: o Inhibiting growth by binding strongly to DNA-Dependent RNA polymerase of bacteria and inhibits RNA synthesis. (Rifampicin) o Inhibit microbial DNA synthesis by clocking DNA gyrases (Quinilones and fluoroquinolones) o Sulfonamides enter into reaction and compete for active center of enzyme. o More examples: Trimethoprim and Timetrexate
SULFA DRUGS:
discovered by Gerhard Domagk in the 1930’s\Inhibit growth of bacteria (sulfanilamide is the simplest) - Isoniazid is a growth analog effective only against Mycobacterium.
NUCLEIC ACID-BASE ANALOGS:
formed by adding bromine or fluorine
QUINOLONES:
antibacterial compounds that interfere with DNA gyrase (e.g., ciprofloxacin)
Gram Negatives and positives
vary in their sensitivities to antibiotics but Broad spectrum antibiotics are effective against both groups.
B-lactam antibiotics are
one of the most important groups of antibiotics of all time and over half of all antibiotics used worldwide. Composed of Penicillins, Cephalosporins & Cephamycins.
Penicillin
Discovered by Alexander Fleming - Effective against Gram-positive, but some synthetic forms can act against gram-negative bacteria - Targets the cell wall synthesis - Natural: Benzylpenicillin (Penicillin G)
Methicillin
(acid-stable)
Oxacillin
(acid-stable)
Ampicillin
(broad spectrum & acid stable)
Carbenicillin
(broad spectrum & acid stable)
CEPHALOSPORINS
- Produced by fungus Cephalosporium - Targets cell wall synthesis - Treats Gonorrhoea
Daptomycin
produced by streptomyces- g+ve infection
Platensimycin
New antibiotic, BS, MRSA and vancomycin-resistant Enterococci
Aminoglycoside
Kanamycin, neomycin, amikacin
Macrolides
Erythromycin- lactone rings bonded to sugars
Tetracyclines
30S inhibition, BS, Used in Humans and animals, aureomycin, Terramycin
AZT-
block Reverse Transcriptase DNA
NNTRI
Bind directly to RT and inhibit reverse transcription
FUSION INHIBITORS
prevent viruses from successfully fusing with the host cell
Two categories of drugs successfully limit influenza infection:
- ADAMANTANES - NEURAMINIDASE INHIBITORS
Interferons
are small proteins that prevent viral multiplication by stimulating antiviral proteins in uninfected cells
Mechanism of drug resistance:
- Drug inactivated by Penicillinases - Decreased permeability to drug or increased elimination of drug from cell. - Change in metabolic patterns - Change in drug receptors
Methicillin-resistant S. aureus
(MRSA)
Origin of drug resistance:
Nongenetic origin of drug resistance o Microorganisms that are metabolically inactive may be phenotypically resistant to drugs, but their offspring are fully susceptible. Ex. Mycobacteria o Lose specific target structure for drug for several generations and may become resistant. Ex. Penicillin-susceptible organisms may change to cell wall deficient L forms o Microorganisms may infect host at sites where antimicrobials are excluded or not active. Ex. gentamycin not effective against Salmonella enteric fevers because they are found intracellularly and Gentamycin do not enter the cells.
The genetic origin of drug resistance
Result of change and subsequent selection process
Chromosomal Resistance
Result of spontaneous mutation
Cross-resistance
o Microorganisms resistant to a certain drugs may also be resistant to other drugs that share a mechanism of action.
Limitation of drug resistance:
- By maintaining high levels of drugs in tissues to inhibit both the original population and first-step mutants - Spontaneously administering two drugs that do not give cross-resistance - Avoiding exposure of microorganisms to a particularly valuable drug by limiting its use.
Bacteriophage therapy
- Use of bacteriophages to treat infections - A form of the virus that attacks bacteria
