CONTROL OF MICROORGANISMS

POSITIVE control - you want to make them grow: Industrial Fermentations; beer, wine and bread making

NEGATIVE control - you want to destroy them by (1) physical or chemical means or (2) antibiotics

Usually we mean negative control and the rest of this discussion relates to the destruction or inhibition of microbes:

PHYSICAL AND CHEMICAL METHODS - see Tortora et. al. for terms.

When disinfecting, one must be concerned with the most resistant life forms present. The following list of organisms is arranged according to ease (or difficulty) of destruction:

When disinfecting, one must be concerned with INTERFERRING MATTER, such as organic matter (Pus, Blood, Feces, etc.) which inactivates certain disinfectants (halogens). Soap inactivates Quaternary Ammonium Compounds.

When disinfecting, one must remember that "Things Take Time." No disinfecting treatment is instantaneous.

HEAT - Denatures proteins; Moist heat is more effective than Dry heat.

1. Boiling - vegetative cells are killed within minutes. Spores can survive. What is Tyndallization?

2. Steam under pressure - AUTOCLAVE. Standard load = 121^oC (250^oF), 15 lbs. steam pressure for 15 min. This kills spores, therefore this is sterilizing. Things can go wrong therefore it is important to have quality control.: SPORE TEST also there are color change indicators.

3. Pasteurize - kills vegetative bacteria. Thermoduric bacteria will survive.

4. Dry Heat - Baking - 160^oC (320^oF) for 2 hours, or 170^oC (338^oF) are common sterilizing treatments.

5. Incinerate

FILTRATION - See the discussion and figure in Tortora et. al. on this. Membrane filters (Millipore^TM) are used to sterilize heat sensitive liquids. HEPA filters are used to sterilize air in biohazard hoods.

ULTRASONIC CLEANERS - Good for removing organic contaminants - "presterilize." Not bacteriocidal.

RADIATION:

Ultraviolet Light (See Tortora et.al., for a description of what UV light does to cells).

UVB = 280-320 nm; UVA = 320-400 nm

Germicidal lamps are used to disinfect air and surfaces. UV does not penetrate glass, plastic or water very well.

UV light does harm eyes and skin.

Ionizing Radiation = X-Rays and Gamma Rays - sporocidal and penetrate well.

CHEMICALS - many of these are called antiseptics or disinfectants or both.

Sterilizing Chemicals:

Ethylene Oxide

beta-Propriolactone

Phenol (spores may withstand)

Halogens (iodine, chlorine, bromine)

Formaldehyde

Glutaraldehyde

PHENOL - (Carbolic Acid) Works well in organic materials therefore good for blood and body fluid decontamination. Neurotoxic. Has residual effect (lasts a long time). Tuberculocidal, Not Sporocidal.

ALCOHOLS - (Ethyl and Isopropyl) Tuberculocidal but not sporocidal. Kills enveloped viruses but not naked viruses. 70% is commonly used concentration. Must make surfaces REALLY WET in order to achieve optimum killing.

HALOGENS

CHLORINE - Excellent disinfectant, sporocidal, however easily inactivated by organic material. Dilutions of household bleach between 1:10 - 1:100 are sporocidal after 10 minutes of treatment. 2-4 drops of bleach per liter of water can be used to treat drinking water (let stand 30 min.)

IODINE-Similar to chlorine. Often found as a tincture or alcohol solution. Also found as an iodophor (betadine^TM)where the iodine is complexed with an organic molecule. Iodophors are more stable than tinctures and they release iodine more slowly and steadily. Therefore they are less harmful to human tissue.

CATIONIC DETERGENTS - (Quaternary Ammonium Compounds) Benzalkonium chloride and Cetylpyridium chloride are some common QUATS found in mouth washes and sore throat remedies. These are weak antiseptics which are active against a wide variety of vegetative bacteria. They are not tuberculocidal. They are not sporocidal. They do not kill Pseudomonas. Many QUATS are inactivated by soaps and other detergents.

ALKYLATING AGENTS - These are very powerful disinfectants. They are sporocidal and therefore sterilizing. Formaldehyde and glutaraldehyde are used to fix and preserve infectious tissues. These are also known to be carcinogens. Ethylene oxide is used in gas sterilizers. This is a very noxious, explosive gas.

HEAVY METALS - Compounds made with mercury, silver, copper and tin have long been used for their antiseptic properties. In ancient times copper and silver containers were used to preserve water. Merthiolate, mercurochrome, silver nitrate and copper sulfate are all used as antiseptics. Silver nitrate has long been used to prevent neonatal gonorrheal opthalmia and in the treatment of burns.

CHLORHEXIDINE - active against vegetative bacteria and commonly used as a surgical scrub. This disinfectant is not sporocidal nor tuberculocidal.

HYDROGEN PEROXIDE - This is a strong oxidizing agent which is very effective against vegetative bacteria on inanimate surfaces. The catalase in human tissue neutralizes H₂O₂ however the generation of oxygen bubbles helps to clean out wounds and is strongly inhibitory to anaerobic bacteria.

 

Chemical Compounds Commonly Used as Antiseptics and Disinfectants

Compound	Type of Action	Applications
Hydrogen peroxide (3%)	Disinfectant/antiseptic	External surfaces, live tissue
Hypochlorites (0.5%) (Chlorox)	Disinfectant	External surfaces, non-living
Iodine (1% in 70% alcohol)	Disinfectant/antiseptic	External surfaces, live tissue
Iodophors (70 ppm available I2)	Disinfectant/antiseptic	External surfaces, live tissue, surgical scrub
Lysol (5%)	Disinfectant	External surfaces, non-living
Phenol (5%)	Disinfectant	External surfaces, non-living
Hexachlorophene (pHisohex)	Disinfectant/antiseptic	External surfaces, live tissue surgical scrub
Formaldehyde (4%)	Disinfectant	External surfaces, non-living
Zephrin and other quaternary ammonium compounds	Disinfectant	Exernal surfaces, non-living
Alcohol (ethyl or isopropyl at 70%)	Disinfectant/antiseptic	External surfaces, unbroken skin
Organic mercury (merthiolate, mercurochrome)	Disinfectant/antiseptic	External surfaces
Potassium permanganate	Antiseptic	superficial skin fungus infections
Silver nitrate (1%)	Antiseptic	prevent newborn eye infections
Ethylene oxide gas (12%)	Sterilizing disinfectant	Linens, heat labile plastics
Glutaraldehyde	Sterilizing disinfectant	metal instrument sterilization
Formaldehyde (20% in alcohol)	Sterilizing disinfectant	metal instrument sterilization

 

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CHEMOTHERAPEUTIC AGENTS - THE ANTIBIOTICS

Most are derived from compounds which are biosynthesized by other microorganisms. Streptomyces and Penicillium are two organisms which have given us antibiotics. The famous immunologist Paul Ehrlich devoted much of his career looking for the "Magic Bullet" or the chemical compound with SELECTIVE TOXICITY. That is, such a compound would be toxic for the infecting microbe but not for the human host. This dream was achieved by Flemming and Florey with the discovery and mass production of penicillin.

Antibiotics achieve selective toxicity by exploiting the differences between eucaryotes and procaryotes. The most profound differences are at the cell wall and at the ribosome level. It follows then that most of our antibiotics either inhibit bacterial cell wall synthesis or inhibit the synthesis of bacterial proteins. For instance: ampicillin and penicillin interfere with cell wall synthesis; chloramphenicol, gentamicin, streptomycin and tetracycline interfere with protein synthesis. How does triple sulfa work?

Antibiotic resistance is a growing problem which has at its source the indiscriminate and inappropriate use of antibiotics. Some of the current "Super Bugs" include methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococcus (VRE) and multidrug resistant Mycobacterium tuberculosis. It is important that physicians order sensitivities on bacteria isolated from infection sites so that appropriate antibiotics can be prescribed. Also, it is important that the infection be hit hard with high, optimum doses of the antibiotic and that tissue levels of the antibiotic remain high throughout therapy. Therapy must continue until all of the pathogens are dead - this means that the patient must continue to take the antibiotic even after the symptoms are gone.

Classes of Antibiotics:

• Antibiotics that inhibit cell wall synthesis
  • Beta-Lactams
    • Examples: penicillins, cephalosporins, carbapenems, monobactams
    • Usually bacteriocidal
    • Inhibit the last step in peptidoglycan synthesis (transpeptidation)
    • May trigger endogenous enzymes that degrade peptidoglycan
    • Spectrum of various beta-lactams varies

     

  • Glycopeptides
    • Examples: vancomycin, teichoplanin
    • Prevents peptidoglycan cross-linking
    • More effective against gram positive bacteria than gram negative bacteria
    • Important in treating multi-drug resistant bacteria

     

  • Phosphonomycin
    • Inhibits synthesis of peptidoglycan

     

  • Bacitracin
    • Inhibits synthesis of peptidoglycan
    • Used topically because of toxicity

     

• Antibiotics that inhibit protein synthesis
  • Aminoglycosides
    • Examples: kanamycin, gentamycin, streptomycin, tobramycin, amikacin, netilmicin, neomycin, spectinomycin
    • Bind 30s ribosomal subunit and prevent protein synthesis
    • Bacteriocidal
    • Potentially nephrotoxic and ototoxic

     

  • Tetracylines
    • Examples: tetracycline, doxycycline
    • Bind 30s ribosomal subunit and prevent protein synthesis
    • Generally bacteriostatic
    • Avoid use in pregnancy and in children under 8 because of interference with bone development and brown staining of teeth

     

  • Chloramphenicol
    • Example: chloramphenicol
    • Binds 50s ribosomal subunit and prevents protein synthesis. May also interfere with mitochondrial ribosomes leading to toxicity problems.
    • Used to treat typhoid fever

     

  • Macrolides
    • Examples: erythromycin, azithromycin
    • Bind 50s ribosomal subunit and prevents the elongation of peptide chain
    • Bacteriostatic for most bacteria; bacteriocidal for some gram-positive bacteria

     

  • Lincosamides
    • Examples: lincomycin, clindamycin
    • Bind 50s ribosomal subunit and prevents elongation of the peptide chain
    • Bacteriostatic for most bacteria; bacteriocidal for some gram-positive bacteria
    • Clindamycin penetrates bone well and also hits many gram positive and gram negative anaerobes -- except that Clostridium difficile is often resistant and this may lead to pseudomembranous colitis

     

  • Streptogramins (ketolides)
    • Example: Synercid (mixture of dalfopristin and quinupristin)
    • Binds 50s ribosomal subunit and prevents elongation of the peptide chain
    • Individual components are bacteriostatic, the mixture is bacteriocidal
    • Used for antibiotic resistant strains of Enterococcus and Staphylococcus

     

• Antibiotics that Inhibit Nucleic Acid Synthesis or DNA Replication
  • Quinolones
    • Examples: ciprofloxacin, norfloxacin, naladixic acid
    • Bind DNA gyrase and prevent DNA supercoiling
    • Broad bacteriocidal activity; effective against some intracellular bacteria
    • Doesn't hit Streptococcus nor anaerobes very hard, therefore tends to be benign to the oral an the intestinal flora

     

  • Metronidazole
    • Example: Flagyl
    • Interferes with DNA replication and creates breaks in the DNA
    • Must be activated by flavodoxin or ferredoxin of the host cell -- these are found in anaerobic bacteria.
    • Effective against anaerobic and microaerophilic bacteria. Also effective against some protozoa such as Trichomonas, Giardia, and Entamoeba.

     

  • Rifampin
    • Example: Rifadin
    • Prevents the synthesis of bacteria messenger RNA
    • Broad spectrum
    • Used in tuberculosis therapy and also to treat the contacts of bacterial meningitis

     

• Antibiotics that Interfere with Metabolic Pathways
  • Trimethoprim and sulfonomides
    • Examples: Bactrim, Septra
    • Inhibit enzymes in the tetrahydofolate biosynthetic pathway
    • Broad spectrum against bacteria, some fungi (Pneumocystis carinii), and protozoa