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Terminology. Decrease the microbial load by limiting growth" (inhibition) or reducing the load (killing)DecontaminationInanimate objects; inhibit growth, but not really reduce the initial load; not sterile, but safe to handleWiping a table after a mealDisinfectionInanimate object; use of a phy
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1. Microbial Growth Control Chapter 27
2. Terminology “Decrease the microbial load by limiting growth” (inhibition) or reducing the load (killing)
Decontamination
Inanimate objects; inhibit growth, but not really reduce the initial load; not sterile, but safe to handle
Wiping a table after a meal
Disinfection
Inanimate object; use of a physical agent to inhibit growth and reduce the initial load
Using bleach to clean a table
Antisepsis
Same as disinfection, but on external living tissue
Iodine on a wound
Antibiosis
In vivo killing or inhibition; internal tissues
Antibiotic given for an infection
3. More Terminology Sterilization
A completely different process than the rest
Removing all viable organisms and spores by killing or removing
Surgical instruments
Bacteriocidal versus Bacteriostatic versus bacteriolytic
Viri-
Fungi-
4. What to Attack (in general)? Plasma membrane
Consequences: PMF, permeability barrier
Proteins (enzymes)
Metabolism and transport disrupted
Nucleic acids
Cannot produce enzymes or make new membrane
Cell Wall
Lysis
5. Physical Methods: Heat Moist heat versus dry heat
Denaturation versus oxidation effects
Factors that effect sterilization by heat:
Time
Organism properties
Temperature
Concentration of microbe
Presence of organic material
Environmental
pH
Osmotic pressure
Type of heat
6. Heat is bacteriocidal
2 ways of describing
Decimal reduction time: time required for 10-fold reduction in population at a given temperature
Canning industry looks at 90% reduction
It increases for a population as temp. decreases
Thermal death time: time at which all cells are killed at a given temperature
Important for sterilization procedures Physical Methods: Heat
7. Moist Heat Method Autoclave
Bactericidal
Heat above boiling (high pressure)
Vegetative cells and spores: 121ş C (15 lbs/in2) for 10-15 min.
8. A Modern Research Autoclave
9. Moist Heat Method Pasteurization
For food
Bacteriocidal
Kill pathogens and increase shelf life
Flash: 71 ş C for 15 sec. and rapid cooling
Bulk: 63-66 ş C for 10-30 min. and slow cooling
Both effective, but flash is better for flavor
10. Physical Methods: Radiation Ionizing
Ex) gamma rays, X rays, high energy electrons
Short wavelength (<1nm) ?high energy
Produce ion (hydroxide, hydride, or electrons)
Damage to DNA or enzymes (bacteriocidal)
Food preservation, medical supply and some pharmaceutical sterilization
Some bacteria more resistant (Deinococcus radiodurans)
11. Nonionizing radiation
Longer wavelength (>100 nm)?less energy
Ex) UV light (best at 260nm)
UVC
Thymine dimers inhibit replication (bacteriostatic)
Initiate the SOS response!!!
Lower energy?Low penetration
Germicidal lamps and vaccine disinfection Physical Methods: Radiation
13. Physical Methods: Filtration Heat sensitive liquid materials (media, some antibiotics, etc.)
Separate microbes from fluid
Passage of liquid through a filter pore size of 0.2mm or less and collection in a sterile container
Viruses and spores can slip by so sterilization often difficult
14. Filtration
15. Other Physical Methods Low temperatures
Water in the cells crystallize below 0?C
Refrigeration slows the growth of most cells (not Listeria)
Osmotic pressure
Old method of preservation
Slows the growth of many non-halophilic cells
Salt or sugar
Molds better suited to these conditions
16. Chemical Methods Antiseptic versus disinfectant
Factors affecting efficiency of chemicals
Concentration
Initial load of microbes
Environmental: Temperature, pH, salt
Presence of organic matter
Time or exposure
Organism
Spores versus vegetative cells
Mycobacterium and lipid rich cell wall
Gram negative organisms and outer membrane
17. Chemical Methods Minimum inhibitory concentration (MIC)
Not constant (dependent on many factors)
Must standardize for comparison
Does not distinguish bacteriocidal versus bacteriostatic
Tube dilution technique
Smallest amount of an agent needed to inhibit the growth of a microbe
Agar diffusion method
Zone of inhibition
Diameter proportional to amount of agent, solubility, diffusion coefficient, and effectiveness
Cannot distinguish ability to diffuse from actual effectiveness
Does not distinguish bacteriocidal versus bacteriostatic
20. Chemotherapeutic Agents Used to decrease load in tissues
Antibiosis
Selective toxicity: injure bacteria and no harm to host
Synthetic versus antibiotic
Synthetic: growth factor analogs
Antibiotic: produced by microorganisms
21. Growth Factor Analog: Sulfa Drugs Microbes normally produce folic acid from a para-aminobenzoic acid (PABA) substrate and an enzyme
When sulfanilamide is present the enzyme binds to it instead
Why is it selectively toxic?
22. Other Analogs Nucleic Acid analogs
Similar to nucleic acids
DNA/RNA mutations
Ex: 5-bromouracil is a thymine analog
Isoniazid is a growth analog effective only against Mycobacteria
Interferes with synthesis of mycolic acid
23. Antibiotics Produced by microbes; kill or inhibit other microbes
Mostly produced by fungi or streptomycetes (a group of bacteria)
Spectrum of microbial activity
Broad spectrum: affect broad range of bacteria
Ex: Tetracyline
Also destroys normal flora (why is this a problem?)
Narrow spectrum: effective against single group
Superinfection: overgrowth of an organism that is either not sensitive to an antibiotic or is resistant
Ex: Candida albicans
24. Antibiotics: Cell Wall Penicillin G
Narrow (Gram +)
Inhibit cell wall synthesis
No transpeptidation
Cannot replace peptidoglycan lysed by autolysins during cell wall synthesis
25. Antibiotics: Proteins Inhibition of protein synthesis
Selective toxicity?difference in ribosomes
Bacteria= 70S ribosome (50S and 30S portions)
Ribosomes are key players in protein synthesis
Chloramphenicol (bacteriostatic and broad spectrum)
50S portion
Inhibits formation of peptide bonds
Tetracycline (bacteriostatic and broad spectrum)
30S portion
Interference with attachment of tRNA, which carries amino acid
Streptomycin (bacteriocidal and broad spectrum)
30S portion
Changes shape of 30S portion and genetic code read wrong
26. Antibiotics Injury to plasma membrane
Changes cell permeability
Loss of metabolites or cell bursting
Ex: Polymyxin B attaches to phospholipids (bacteriocidal and Gram neg.)
Inhibition of nucleic acid synthesis
More difficult to find b/c of lack of selective toxicity
Ex: Rifampin inhibits mRNA synthesis (bacteriocidal and Mycobacterium tuberculosis)
28. Antibiotic Resistance Organisms that produce them are resistant to them
Pass these resistance genes on to others
Resistance has always existed
Recent problems due to selection
Replacement of sensitive bacteria in a population by resistant bacteria*
Resistance is genetically coded
Plasmids*
Genomic
29. Plasmids: self replicating, extra chromosomal DNA, which can be passed on to others (typically between similar organisms).
30. Mechanisms of Drug Resistance Lack a target structure
Cell wall for Mycoplasma–penicillin resistance
Destruction or inactivation
Some Staphylococcus have enzymes that inactivate Penicillin
Impermeable
Gram – organisms and penicillin resistance
Modify the target site
Modify the ribosome to become erythromycin resistant
Rapid efflux
Able to pump out the antibiotics
Genetic change in metabolic pathway
Resistance to sulfonamides: take up folic acid rather than make it
31. Reasons for Resistance Problems Lack of prescriptions
Improper use (headaches and viruses)
CDC estimates percentage of wrong prescriptions
30% ear infections, 50% sore throats, 100% colds
Weak forms select for resistant bacteria
Not taken long enough
32. Drug Combinations In case the organism is resistant to one drug
Synergism: drug assistance
Ex: Penicillin and Streptomycin for bacterial endocarditis
Penicillin disrupts cell wall and Streptomycin gets in faster
Antagonism: One drug makes other ineffective
Ex: Penicillin and Tetracycline
Tetracycline interferes with Penicillin
33. Viral Control (HIV) Difficult to be selectively toxic
Nucleoside analog—AZT
Azidothymidine is analog of thymine
Inhibits elongation of viral nucleic acid chain
Host toxicity
Protease inhibitors
Viral protease enzyme used to cut up larger polypeptides to assemble new viruses
Binds to active site of protease enzyme
Inhibits viral maturation and processing of viral polypeptides
Interferons
Made by uninfected host cells to prevent viral infection
Host specific (not viral specific)
Clinical aspects: needs to be in high concentration in local area
Neuraminidase inhibitors--Tamiflu®
34. Protease Inhibitors and Neuraminidase Inhibitor
35. Chemotherapeutic Future Modify existing drugs
Extend spectrum
Prevent resistance
New drugs
New targets of activity
Novel organisms