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Physical and Chemical Control of Microorganisms

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Title: Physical and Chemical Control of Microorganisms


1
Physical and Chemical Control of Microorganisms
2
Controlling Microorganisms
  • Reduce or destroy undesirable microbes in a given
    area
  • Physical
  • Chemical
  • Mechanical

3
Primary Targets of Control
  • Microorganisms capable of causing infection or
    spoilage
  • vegetative bacterial cells
  • endospores
  • fungal hyphae and spores
  • yeast
  • protozoan trophozoites and cysts
  • worms
  • viruses
  • prions

4
Relative Resistance of Microbes
  • Highest resistance
  • Bacterial endospores
  • Moderate resistance
  • Pseudomonas sp.
  • Mycobacterium tuberculosis
  • Staphylococcus aureus
  • Protozoan cysts
  • Least resistance
  • most vegetative cells
  • fungal spores
  • enveloped viruses
  • yeast
  • protozoan trophozoites

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Terms for Microbial Control
  • Sterilization
  • destroys all viable microbes
  • including viruses endospores
  • Disinfection
  • destroy vegetative pathogens
  • not endospores
  • Sanitization
  • cleansing technique that mechanically removes
    microbes
  • Degermation
  • reduces the number of microbes
  • Microbial agents
  • Bactericide
  • Sporocide
  • Fungicide
  • Viricide
  • Microbistasis
  • Bacteriostatic
  • Fungistatic

7
Microbial Death
  • Involves permanent loss of reproductive
    capability
  • even under optimum growth conditions
  • Still alive, but cant reproduce!!
  • Not viable in environment
  • Hard to detect
  • Loss of movement??

8
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

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

11
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?

12
Methods of Physical Control
  • Heat Moist verse Dry
  • Cold temperatures
  • Desiccation
  • Radiation
  • Filtration

13
1. Heat
  • Moist heat use of hot water or steam
  • mode of action
  • denaturation of proteins
  • destruction of membranes
  • destruction of DNA
  • Sterilization
  • Steam under pressue
  • Autoclave
  • Intermittent sterilization
  • unpressurized steam
  • 100oC 30-60 min for 3 days
  • Disinfection
  • boiling at 100oC for 30 minutes
  • destroy non-spore-forming pathogens
  • Pasteurization

14
Pasteurization
  • Pasteurization
  • heat applied to kill potential agents of
    infection and spoilage
  • without destroying the food flavor or value
  • 63C - 66C for 30 minutes
  • batch method
  • 71.6C 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

15
1. Heat
  • Dry heat - higher temperatures than moist heat
  • incineration
  • 600-1200oC
  • combusts dehydrates cells
  • dry ovens
  • 150-180oC
  • coagulate proteins
  • Dehydrates cells and removes water
  • Can also sterilize

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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

18
2. Cold Temperatures
  • Microbistatic
  • slows the growth of microbes
  • refrigeration 0-15oC
  • freezing lt0oC
  • used to preserve food, media and cultures

19
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

20
4. Radiation
  • Ionizing 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

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5. Filtration
  • physical removal of microbes by passing a gas or
    liquid through filter
  • used to sterilize heat sensitive liquids air in
    hospital isolation units industrial clean rooms

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

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

27
1. Halogens
  • Chlorine
  • Cl2, hypochlorites (chlorine bleach), chloramines
  • Denaturation of proteins by disrupting disulfide
    bonds
  • Can be sporicidal
  • Iodine
  • I2, iodophors (betadine)
  • Denature proteins
  • Can be sporicidal
  • 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 (25)
  • 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
  • Amonia compounds act as surfactants
  • 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
Antimicrobial Therapy
37
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

38
Chemotherapy
  • Antimicrobial
  • 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
  • Narrow 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

39
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

40
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

41
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

42
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

43
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

44
3. Drugs that Block Protein Synthesis
  • Ribosomes
  • eukaryotes differ in size and structure from
    prokaryotes
  • Antimicrobics usually have a selective action
    against prokaryotes
  • can also damage the eukaryotic mitochondria
  • Aminoglycosides (streptomycin, gentamicin)
  • insert on sites cause misreading of mRNA
  • Tetracyclines
  • block attachment of tRNA and stop further
    synthesis

45
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

46
Drug Resistance
  • Microorganisms begin to tolerate an amount of
    drug that would ordinarily be inhibitory
  • due to genetic versatility or variation
  • intrinsic and acquired
  • 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

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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

49
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

50
Considerations in Selecting an Antimicrobial Drug
  • Identify the microorganism causing the infection
  • Specimens should be taken before antimicrobials
    initiated
  • Test the microorganisms susceptibility
    (sensitivity) to various drugs in vitro when
    indicated
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  • Overall medical condition of the patient

51
Testing for Drug Susceptibility
  • Essential for groups of bacteria commonly showing
    resistance
  • Kirby-Bauer disk diffusion test

52
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

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Major Antimicrobial Drug Groups
  • Antibacterial drugs
  • Antibiotics
  • Synthetic drugs
  • Antifungal drugs
  • Antiparasitic drugs
  • Antiviral drugs
  • About 260 different antimicrobial drugs are
    classified in 20 drug families

55
Antimicrobial Drug Groups
  • Act on cell wall
  • Penicillin
  • Cephalosporin
  • Non-beta-lactam
  • Damage cell membranes
  • Polymixins
  • Act on DNA and RNA
  • Fluoroquinolones
  • Interfere with protein synthesis
  • Aminoglycosides
  • Tetracycline
  • Chloramphenicol
  • Block metabolic pathways
  • Sulfonamides

56
Penicillins
  • More economical to obtain natural penicillin
    through microbial fermentation
  • Then modify it to semi-synthetic forms
  • Penicillium chrysogenum
  • major source
  • Differ in side chains

57
Penicillins
  • Penicillins G and V most important natural forms
  • Drug of choice for gram-positive cocci
    (streptococci)
  • Some gram-negative bacteria (meningococci and
    syphilis spirochete)
  • Semisynthetic penicillins
  • ampicillin, carbenicillin amoxicillin have
    broader spectra
  • gram negative enterics rods
  • Penicillinase-resistant
  • methicillin, nafcillin, cloxacillin
  • Primary problems
  • allergies and resistant strains of bacteria

58
Cephalosporins
  • Account for majority of all antibiotics
    administered
  • Isolated from Cephalosporium acremonium mold
  • Beta-lactam ring that can be altered
  • Relatively broad-spectrum
  • resistant to most penicillinases
  • cause fewer allergic reactions
  • Some are given orally

59
Four Generations of Cephalosporins
  • First generation
  • cephalothin, cefazolin
  • most effective against gram-positive cocci
  • Second generation
  • cefaclor, cefonacid
  • more effective against gram-negative bacteria
  • Third generation
  • cephalexin, cefotaxime
  • broad-spectrum activity against enteric bacteria
    with beta-lactamases
  • Fourth generation
  • Cefepime
  • Effective against gram positive and negative

60
Polymixins
  • Source is Bacillus polymyxa
  • Damages cell membrane
  • Can treat drug-resistant organisms
  • Pseudomonas
  • Severe urinary tract infections

61
Aminoglycosides
  • composed of 2 or more amino sugars
  • and an aminocyclitol (6C) ring
  • products of various species of soil actinomycetes
    in genera Streptomyces Micromonospora
  • Uses
  • broad-spectrum
  • inhibit protein synthesis
  • especially useful against aerobic gram-negative
    rods certain gram-positive bacteria
  • Streptomycin bubonic plague, tularemia, TB
  • Gentamicin less toxic, used against
    gram-negative rods

62
Fluoroquinolones
  • Acts on DNA or RNA
  • Readily absorbed from intestines
  • Binds gyrase
  • Quinolones, ciprofloxacin

63
Tetracycline Antibiotics
  • Broad-spectrum, block protein synthesis
  • Doxycycline minocycline
  • oral drugs taken for STDs, Rocky Mountain spotted
    fever, Lyme disease, typhus, acne protozoa

64
Chloramphenicol
  • Isolated from Streptomyces venezuelae
  • Blocks peptide bond formation
  • No longer derived from natural source
  • Very toxic, restricted uses, can cause
    irreversible damage to bone marrow
  • Typhoid fever, brain abscesses, rickettsial
    chlamydial infections

65
Antifungal Drugs
  • Macrolide polyene
  • Amphotericin B
  • mimic lipids, most versatile effective, topical
    systemic treatments
  • Nystatin
  • topical treatment
  • Griseofulvin
  • stubborn cases of dermatophyte infections,
    nephrotoxic
  • Synthetic azoles
  • broad-spectrum
  • ketoconazole, clotrimazole, miconazole
  • Flucytosine
  • analog of cytosine cutaneous mycoses or in
    combination with amphotericin B for systemic
    mycoses
  • Echinocandins
  • Damage cell walls

66
Antiparasitic Drugs
  • Antimalarial drugs
  • quinine, chloroquinine, primaquine, mefloquine
  • Antiprotozoan drugs
  • Metronidazole (Flagyl), quinicrine, sulfonamides,
    tetracyclines
  • Antihelminthic drugs
  • immobilize, disintegrate, or inhibit metabolism
  • mebendazole, thiabendazole
  • broad-spectrum
  • inhibit function of microtubules
  • interfers with glucose utilization disables
    them
  • pyrantel, piperazine
  • paralyze muscles
  • niclosamide
  • destroys scolex

67
Antiviral Drugs
  • Block penetration into host cell
  • Block transcription or translation
  • Nucleotide analogs
  • Acyclovir herpesviruses
  • Ribavirin- a guanine analog RSV, hemorrhagic
    fevers
  • AZT thymine analog - HIV
  • Prevent maturation of viral particles
  • Protease inhibitors HIV

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