1. Physical and Chemical Control of Microorganisms

2. Controlling Microorganisms
Reduce or destroy undesirable microbes in a given area
Physical
Chemical
Mechanical

3. Relative Resistance of Microbes
Highest resistance
Bacterial endospores & prions
Moderate resistance
Pseudomonas sp.
Mycobacterium tuberculosis
Staphylococcus aureus
Protozoan cysts
Least resistance
most vegetative cells
fungal spores
enveloped viruses
yeast
protozoan trophozoites

5. Terms for Microbial Control
Sterile
Inanimate objects free of all life
Sterilization
destroys all viable microbes
including viruses & endospores
Disinfection
destroy vegetative pathogens
not endospores
Sanitization
cleansing technique that mechanically removes microbes to safe levels
Degerming
removing organisms from an object’s surface

6. Terms for Microbial Control
Microbicidal agents
Causes microbial death
Bactericide
Sporocide
Fungicide
Viricide
Microbistasis
Prevents microbial growth
Bacteriostatic
Fungistatic

7. Factors That Affect Death Rate
Number of microbes
Nature of microbes in the population
Temperature & pH of environment
Concentration or dosage of agent
Mode of action of the agent
Presence of solvents, organic matter, or inhibitors

9. Cellular Targets of Control
Mode of action of antimicrobials:
Cell wall
Cell membrane
Cellular synthetic processes (DNA, RNA)
Proteins

10. Practical Concerns for Microbial Control
Does the application require sterilization?
Is the item to be reused?
Can the item withstand heat, pressure, radiation, or chemicals?
Is the method suitable?
Will the agent penetrate to the necessary extent?
Is the method cost- and labor-efficient & is it safe?

11. Methods of Physical Control
Heat – Moist verse Dry
Cold temperatures
Desiccation
Radiation

12. 1. Heat (Moist) Moist heat – use of hot water or steam mode of action
denaturation of proteins
destruction of membranes
destruction of DNA
Sterilization
Steam under pressure
Autoclave
15psi/121oC/10-40 min
Steam must reach surface of item being sterilized!

13. Autoclave Tape

14. 1. Heat (Moist) Intermittent sterilization Disinfection Pasteurization
unpressurized steam
100oC min for 3 days
spores, which are unaffected, germinate during the intervening periods and are subsequently destroyed
Disinfection
boiling at 100oC for 30 minutes
destroy non-spore-forming pathogens
Pasteurization

15. 1. Heat (Moist) Pasteurization
heat applied to kill potential agents of infection and spoilage
without destroying the food flavor or value
63°C - 66°C for 30 minutes
batch method
71.6°C for 15 seconds
flash method
Not sterilization
kills non-spore-forming pathogens and lowers overall microbe count
does not kill endospores or many nonpathogenic microbes

16. 1. Heat (Dry) Dry heat - higher temperatures than moist heat
incineration oC
combusts & dehydrates cells
dry ovens oC
coagulate proteins
Bunsen burner
1870oC
Dehydrates cells and removes water
Can also sterilize

18. Thermal Death Measurements
Thermal death time (TDT)
shortest length of time required to kill all microbes at a specified temperature
Thermal death point (TDP)
lowest temperature required to kill all microbes in a sample in 10 minutes

19. 2. Cold Temperatures Microbistatic refrigeration 0-15oC
slows the growth of microbes
refrigeration 0-15oC
freezing <0oC
used to preserve food, media and cultures

20. 3. Desiccation gradual removal of water from cells
leads to metabolic inhibition
not effective microbial control
many cells retain ability to grow when water is reintroduced

21. 4. Radiation Ionizing radiation Nonionizing radiation
deep penetrating power breaks DNA
gamma rays, X-rays, cathode rays
used to sterilize medical supplies & food products
Nonionizing radiation
little penetrating power to sterilize air, water & solid surfaces
UV light creates thymine pyrmidines
interfere with replication

23. Chemical Agents in Microbial Control
Chemicals that sterilize
Disinfectants, antiseptics, sterilants
Chemicals that inhibit deterioration
Degermers, and preservatives
Desirable qualities of chemicals:
rapid action in low concentration
solubility in water or alcohol, stable
broad spectrum, low toxicity
penetrating
noncorrosive and nonstaining
affordable and readily available

24. Levels of Chemical Decontamination
High-level germicides
kill endospores
devices that are not heat sterilizable and intended to be used in sterile environments (body tissue)
Intermediate-level
kill fungal spores (not endospores), tubercle bacillus, and viruses
used to disinfect devices that will come in contact with mucous membranes but are not invasive
Low-level
eliminate only vegetative bacteria, vegetative fungal cells, and some viruses
clean surfaces that touch skin but not mucous membranes

25. Factors that Affect Germicidal Activity of Chemicals
Nature of the material being treated
Degree of contamination
Time of exposure
Strength and chemical action of the germicide
Dilutions
Volume of liquid chemical diluted in larger volume of solvent (water)

26. Chemical Control Of Microbial Agents
Halogens
Phenolics
Chlorhexidine
Alcohols
Hydrogen peroxide
Detergents & soaps
Heavy metals
Aldehydes
Gases and aerosols

27. 1. Halogens Chlorine Iodine
Cl2, hypochlorites (chlorine bleach), chloramines
Denaturation of proteins by disrupting disulfide bonds
Can be sporicidal
Iodine
I2, iodophors (betadine)
Denature proteins
Milder medical & dental degerming agents, disinfectants, ointments

28. 2. Phenolics Disrupt cell membranes & precipitating proteins
bactericidal, fungicidal, virucidal, not sporicidal
Lysol
Triclosan
antibacterial additive to soaps

29. 3. Chlorhexidine
A surfactant & protein denaturant with broad microbicidal properties
Not sporicidal
Used as skin degerming agents for preoperative scrubs, skin cleaning & burns

30. 4. Alcohols Ethyl, isopropyl in solutions of 50-90% Act as surfactants
dissolve membrane lipids and coagulating proteins of vegetative bacterial cells and fungi
Not sporicidal
Good for enveloped viruses

31. 5. Hydrogen Peroxide Weak (3%) to strong (35%)
Produce highly reactive hydroxyl-free radicals that damage protein & DNA while also decomposing to O2 gas
toxic to anaerobes
Strong solutions are sporicidal
in increasing concentrations

32. 6. Detergents & Soaps Ammonia compounds act as surfactants Soaps
alter membrane permeability of some bacteria & fungi
Not sporicidal
Soaps
mechanically remove soil and grease containing microbes
Low concentrations
Only have microbistatic effects

33. 7. Heavy Metals Solutions of silver & mercury
kill vegetative cells in low concentrations by inactivating proteins
Oligodynamic action
Not sporicidal

34. 8. Aldehydes
Glutaraldehyde & formaldehyde kill by alkylating protein & DNA
glutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments
formaldehyde
disinfectant, preservative, toxicity limits use

35. 9. Gases & Aerosols
Ethylene oxide, propylene oxide, betapropiolactone & chlorine dioxide
Strong alkylating agents, sporicidal

36. Mechanical Control Filtration physical removal of microbes
passing a gas or liquid through filter
organisms above a certain size trapped in the pores
used to sterilize heat sensitive liquids & air in hospital isolation units & industrial clean rooms
Air can be filtered using a high-efficiency particulate air (HEPA) filter

37. Antimicrobial Therapy

38. Origins of Antimicrobial Drugs
Antibiotics
Common metabolic products of aerobic spore-forming bacteria & fungi
bacteria in genera Streptomyces & Bacillus
molds in genera Penicillium & Cephalosporium
Inhibiting other microbes in the same habitat
antibiotic producers have less competition for nutrients & space

39. Ideal Antimicrobial Drug…..
Selectively toxic to microbe
Not host cells
Microbicidal, not microbistatic
Soluble
Potent
No antimicrobial resistance
Remains active
Readily delivered to site of infection
Expense
Not allergen

40. Chemotherapy Antimicrobial Antibiotic Semisynthetic Synthetic
Control infection
Antibiotic
Produced by the natural metabolic processes of microorganisms
Can inhibit or destroy other microorganisms
Semisynthetic
Chemically modified drugs in lab
Synthetic
Synthesized compounds in lab

41. Chemotherapy Narrow spectrum Broad spectrum
Effective against limited microbial types
Target a specific cell component that is found only in certain microbes
Broad spectrum
Effective against wide variety microbial types
Target cell components common to most pathogens

42. Selectively Toxic
Should kill or inhibit microbial cells without simultaneously damaging host tissues
Complete selective toxicity
Difficult to achieve
Characteristics of the infectious agent become more similar to the vertebrate host cell
More side effects are seen

43. Selective toxicity toxic dose of a drug therapeutic dose
The concentration causing harm to the host
therapeutic dose
the concentration eliminating pathogens in the host
Together, the toxic and therapeutic doses are used to formulate the chemotherapeutic index

44. Targets of Antimicrobial Drugs
Inhibition of cell wall synthesis
Inhibition of nucleic acid synthesis, structure or function
Inhibition of protein synthesis
Disruption of cell membrane structure or function

45. 1. Bacterial Cell Wall Most bacterial cell walls contain peptidoglycan
Penicillin and cephalosporin block synthesis of peptidoglycan
Causes the cell wall to lyse
Penicillins do not penetrate the outer membrane
less effective against gram-negative bacteria
Broad spectrum penicillins and cephalosporins
cross the cell walls of gram-negative bacteria

46. 2. Inhibit Nucleic Acid Synthesis
May block synthesis of nucleotides, inhibit replication, or stop transcription
sulfonamides and trimethoprim
block enzymes required for tetrahydrofolate synthesis
needed for DNA & RNA synthesis

47. 3. Drugs that Block Protein Synthesis
Ribosomes
eukaryotes differ in size and structure from prokaryotes
Aminoglycosides (streptomycin, gentamicin)
insert on sites cause misreading of mRNA
Tetracyclines
block attachment of tRNA and stop further synthesis

48. 4. Disrupt Cell Membrane Function
Damaged membrane
dies from disruption in metabolism or lysis
These drugs have specificity for a particular microbial group
based on differences in types of lipids in their cell membranes
Polymyxins
interact with phospholipids
cause leakage, particularly in gram-negative bacteria
Amphotericin B and nystatin
form complexes with sterols on fungal membranes
causes leakage

49. Drug Resistance
Microorganisms begin to tolerate an amount of drug that would ordinarily be inhibitory
due to genetic versatility or variation
intrinsic and acquired

50. Intrinsic verse Acquired
Intrinsic resistance
Microbe must be resistant to antibiotic that they produce
Acquired resistance:
1. spontaneous mutations in critical chromosomal genes
2. acquisition of new genes or sets of genes via transfer from another species
originates from resistance factors (plasmids) encoded with drug resistance, transposons

52. Mechanisms of Drug Resistance
Drug inactivation by acquired enzymatic activity
penicillinases
Decreased permeability to drug or increased elimination of drug from cell
acquired or mutation
Change in drug receptors
mutation or acquisition
Change in metabolic patterns
mutation of original enzyme

54. Antibiotic Resistance in Medical Community
Improper or excessive use of antibiotics causes antibiotic resistance
Unnecessarily large antibiotic doses
Allow resistant strains to overgrow susceptible ones
If resistant strains spread to other patients, a superinfection occurs
Antibiotics are available over the counter in developing countries
allows for overuse and incorrect use
Antibiotic use is widespread in livestock feeds
can be transmitted to humans through meat consumption

55. Side Effects of Drugs
5% of all persons taking antimicrobials will experience a serious adverse reaction to the drug
Toxicity to organs
Allergic responses
Suppression and alteration of microflora

56. Considerations in Selecting an Antimicrobial Drug
1. Identify the microorganism causing the infection
Specimens should be taken before antimicrobials initiated
2. Test the microorganism’s susceptibility (sensitivity) to various drugs in vitro when indicated
(Next slide)
3. Overall medical condition of the patient

57. Testing for Drug Susceptibility
Essential for groups of bacteria commonly showing resistance
Kirby-Bauer disk diffusion test

58. Dilution tests Minimum inhibitory concentration (MIC)
smallest concentration of drug that visibly inhibits growth
In vitro activity of a drug is not always correlated with in vivo effect
If therapy fails, a different drug, combination of drugs, or different administration must be considered
Best to chose a drug with highest level of selectivity but lowest level toxicity

59. What about viruses?!?!? Do not destroy their target pathogen
Instead they inhibit their development
Inhibit virus before enters cell
Viral-associated proteins
Stop it from entering the cell
Stop it from reproducing
Prevent from exiting the cell