Control of Microorganisms:Principles and Physical Agents

Terms:

Antiseptic – a chemical substance that is applied to the skin or mucous membranes to prevent growth by either inhibiting or destroying microorganisms

Disinfectant- essentially the same but this term is usually reserved for use of inanimate objects.

Antimicrobial agents- substances that either kill microorganisms or prevent their growtheg. Antibacterial, antifungal or antiviral (depending on the kind of

microorganisms affected).Microbicidal agents- antimicrobial agents that kill microorganisms

eg. Bactericidal, virucidal and fungicidal (indicate the type of microorganismskilled).

Sterilization – Killing all microorganisms present in a material, including any spores.

Microbiostatic agents- agents that merely inhibit the growth of microorganismseg. Bacteriostatic or fungistatic

Sanitizer – any agent that reduces bacterial numbers to safe levels accdng to PH reqts

Conditions that affect Antimicrobial Activity -some important variables to consider when assessing the

effectiveness of a microbicidal agent are:

1. Size of microbial population2. Intensity or concentration of the microbicidal agent3. Time of exposure to the microbial agent4. Temperature at which the microorganisms are exposed to the

microbicidal agent5. Nature of the material containing the microorganisms6. Characteristics of the microorganisms which are present

Mechanisms of Microbial Damage- antimicrobial agents inhibit or kill microorganisms by

damaging certain structures of the cell, such as:1. the cell wall2. or the cytoplasmic membrane3. or substances within the cytoplasm such as:

a. enzymesb. ribosomesc. or nuclear material

Method Temperature Applications Limitations

Moist Heat

1. Autoclave

2. Boiling water

3. Pasteurization

121.6oC at 15 lb/in2 pressure, 15-30 min

100oC, 10 min

62.8oC for 30 min, or 71.7oC for 15 s

Sterilizing instruments, linens, utensils, and treatment trays, media and other liquids

Killing vegetative cells on instruments, containers

Killing vegetative cells of disease-causing microorganisms and of many other microorganisms in milk, fruit juices, and other beverages

Ineffective against organisms in materials impervious to steam; cannot be used for heat-sensitive articlesEndospores are not killed; cannot be relied upon to sterilizeDoes not sterilize

Table 6. The Use of Temperature to Control Microorganisms

Method Temperature Applications Limitations

Dry Heat 1. Hot-air oven 170-180oC for 1-2 h Sterilizing materials

impermeable to or damaged by moisture, e.g. Oils, glass, sharp instruments, metals

Destructive to materials that cannot withstand high temperatures

Incineration Hundreds of oC Sterilization of transfer loops and needles; disposal of carcasses of infected animals; disposal of contaminated objects that cannot be reused

Size of incinerator must be adequate to burn largest load promptly and completely; potential for air pollution exists

Low Temperatures 1. Freezers

2. Liquid-nitrogen refrigerators

Less than 0oC

-196oC

Preservation of foods and other materials

Preservation of microorganisms

Mainly microbiostatic instead of microbicidalHigh cost of liquid nitrogen

Contn of Table 6. Physical methods for control of microorganismsAgent Action Use

Radiation 1. Ultraviolet

2. X-rays

3. Cathode rays

Formation of thymine dimers

Ionization; peroxide formationIonization

Microbicidal effect, with limitations owing to lack of penetration; reduces airborne infections in hospitals, restaurants and schoolrooms.Destruction of organisms on surfaces, in water, etc.

Research; used to induce mutationsResearch; may be used for sterilizing effects in pharmaceutical houses and food industry in the future.

Filtration Separation of bacteria from the suspending fluid

Sterilization of certain liquids which can be damaged by heat or chemical treatment; separation of bacteria from toxins, enzymes, etc.; measurement of the approx size of some viruses

Dessication Removes water Effect chiefly bacteriostaticPreservation of various food

High Temperatures-the use of high temperatures is one of the most effective and widely utilized means of killing microorganisms

*moist heat is much more effective than dry heat for killing microorganisms becauseMH causes denaturation and coagulation of vital proteins like enzymes -denaturation of cell proteins occurs with lower T and shorter exposure times DH causes oxidation of the organic constituents of the cell -need longer time at high T to achieve the same result with MH

eg.1. Bacillus anthracis endospores destroyed in:MH – 2 to 15 min at 100oCDH - up to 180 mins at 140oC

2. vegetative cellsMH – 5-10 min at 60-70oC (bacteria) -5-10 min at 50-60oC (yeast and other fungi)

Steam - use of pure steam under pressure is the most practical and dependable way to apply moist heat - provides T higher than the possible steam (nonpressurized) and boiling -rapid heating and greater penetration -make used of autoclave (lab apparatus designed to sterilize w/ pressurized steam)

Container Mins of Exposure at 121-123oC (250-254oF)

Test tubes 18 x 150 mm 32 x 200 mm 38 x 200 mm

12-1413-1715-20

Erlenmeyer flasks 50 ml 500 ml 1000 ml 2000 ml

12-1417-2220-2530-35

Milk-dilution bottle, 100 ml 13-17

Serum bottle, 9000 ml 50-55

Table 6.1 Exposure Periods Required for Aqueous Solns or Liqs in various containers affordinga reasonable factor of safety for sterilization by autoclaving

Filtration - physical process - one way of sterilizing materials which can’t be sterilized by autoclaving and dry

heat in labs and industries - make used of membrane filters

*cellulose esters of thin disks (150um) with pores small enough to preventthe passage of the microorganisms*are superior to older types of filters bec:

1. pores of membrane filters are of a uniform known diameter 2. the filters can be manufactured with any desired pore size

3. they absorb very little of the fluid being filtered4. filtration through membrane filters is more rapid than that obtained with older filters

- also used for separating types of microorganisms and for collecting microbialsamples

High-Efficiency Particulate Air (HEPA) Filters-found in biological safety cabinet which traps particulate matter such as

microorganisms.-it captures 99 percent of the particulate matter from the exiting air.

Lyophilization -dehydration of microorganisms quickly at freezing temperatures and then sealedIn containers under vacuum

Osmotic pressure -by plasmolysis -microbicidal effect in the preservation of various food

eg. Matls w/ high concentrations of sugar and salt such as jelly and jams and salted fish (inhibit microbial growth)

Control of Microorganisms: Chemical Agents

Characteristics of an ideal chemical agent

1. Antimicrobial Activity –ability to inhibit or kill microbes-the chemical at low concns should have a broad spectrum of antimicrobial activity

2. Solubility - the substance should be soluble in water or other suitable solvents (alcohol) to the extent necessary for effective anti-microbial activity

3. Stability -storage for reasonable periods shld not result in significant loss of antimicrobial action

4. Lack of toxicity - it shld not harm humans or animals5. Homogeneity -the ingredients shld not settle to the bottom of the

container6. Minimum inactivation by extraneous material –some antimicrobial chemicals

combine readily with proteins and other organic matls found inthe substances being treated.this decreases the amount of the chemical available for action against microorganisms

7. Activity at ordinary T –it shld not be necessary to raise the temperature beyond that normally found in the environment wherethe agent is to be used

8. Ability to penetrate - unless the chemical can penetrate the surface, itsantimicrobial action is limited to the site of application

9. Material Safety - the compd. shld not rust or otherwise disfigure metals, nor should it stain or damage fabrics

10. Deodorizing ability -the agent shld be odorless or have a pleasant smell.

The ability to deodorize is a desirable attribute.11. Detergent ability -an antimicrobial agent that has the cleansing properties

has the advantage of being able to remove microorganisms mechanically from the

surfaces beingtreated

12. Availability and low cost- the product should be readily available and inexpensive

• Synthetic antimicrobial agents (Figure 20.13) are selective for Bacteria, viruses, and fungi.

• Figure 20.14 shows the mode of action of major antimicrobial chemotherapeutic agents.

• Antimicrobial chemotherapeutic agents each affect a limited group of microorganisms (Figure 20.15).

RESISTANCE TO ANTIMICROBIAL DRUGSA. Mechanisms of Drug Resistance. Microoganisms exhibit resistance to antimicrobial drugs by different mechanisms.

1. Microorganisms produce enzymes that destroy the active drug. Examples: P-lactamases (Penicillinases) produced by certain bacteria destroy penicillin. Staphylococci resistant to penicillin G produce a P-lactamase that destroys the drug. Other P-lactamases are produced by Gram-negative rods.

2. Microorganisms change their permeability to the drug, e.g. tetracycline accumulate in susceptible bacteria but not in resistant bacteria.

3. Microorganisms develop an altered metabolic pathway that bypasses the reaction inhibited by the drug. Sulphonamide resistant bacteria do not require extracellular PABA, but can utilize preformed folic acid.

4. Microorganisms develop an altered structural target for the drug: Erythromycin resistant organisms have on altered receptor on the 50S subunit of the ribosome. Aminoglycosides resistant is due to alteration or loss of a specific protein in the 30S subunit of the bacterial ribosome that serve as a binding site in susceptible organisms. Resistance to some penicillins and cephalosporins occurs due to alteration or loss of PBPs.

5. Microorganisms develop an altered enzyme that can perform its metabolic function but is much less affected by the drug

Penicillin-Binding Protein (PBP)

Penicillin-Binding Proteins (PBPs) are a family of essential bacterial enzymes involved in the synthesis of peptidoglycan, the major component of the bacterial cell wall. β-lactam antibiotics bind to PBPs, disrupting the cell wall and killing bacteria. Unfortunately, many bacteria have acquired enzymes, β-lactamases, which destroy the β-lactam and provide the bacteria with resistance to the antibiotic. The recent spread of these enzymes amongst gram negative bacteria (in particular,Enterobacteriaceae) is significantly compromising the clinical utility of β-lactam antibiotics.

PBPs normally catalyze the cross-linking of the bacterial cell wall, but they can be permanently inhibited by penicillin and other β-lactam antibiotics. (NAM = N-acetylmuramic acid; NAG = N-acetylglucosamine)

B. Origin of Drug Resistance. The origin of drug resistance may be genetic or nongenetic.

1. Genetic Origin of Drug Resistance. Most drug-resistant microorganisms emerge as a result of genetic change and subsequent selection processes by antimicrobial drugs. Genetic mechanism may be chromosomal or extra-chromosomal.

(i) Chromosomal Resistance. Chromosome-mediated resistance occurs by spontaneous mutation in a locus that controls susceptibility to the drug. The antimicrobial drug serves as a selecting mechanism to suppress susceptible organisms and favor the growth of drug-resistant mutants. Spontaneous mutation is not a frequent cause of the clinical drug resistance in a given patient. But it occurs with high frequency to rifampicin. Mutation can result in the loss of PBPs, making such mutants resistant to a-Iactam drugs. Examples Rifampicin, streptomycin, erythromycin. Resistance of M. tuberculosis to rifampicin is caused by mutation in RNA polymerase and that to isoniazid by mutation in catalase.

(ii)Extrachromosomal Resistance(a) Plasmid Resistance. This occurs by the extrachromosomal genetic elements called plasmid. Plasmid mediated drug resistance is more common than that of chromosome. R factors (drug resistance plasmids) are a class of plasmids that carry genes for resistance to antimicrobial drugs. A single plasmid can carry genes that code for resistance to several drugs (multi-drug resistance -MDR) such as streptomycin, chloramphenicol, tetracycline and sulphonamides. Plasmid genes control the formation of enzymes capable of destroying the antimicrobial drugs, e.g. P lactamases destroy P-lactam ring of penicillins and cephalosporins.

(b) Transposon Resistance.Genetic material responsible for antimicrobial resistance of a donor cell may be transferred to a sensitive recipient cell, and the recipient cell thus becomes resistant to the drug(s). The intercellular transfer of genetic material may occur by : (a) Conjugation, Conjugation is the most important of these mechanisms for the transfer of antimicrobial resistance. In most cases of conjugation the transferable DNA is plasmid, but chromosomal DNA may also be transferred, (b) Transduction. Transduction is the transfer of cell DNA by means of a bacterial virus (bacteriophage, phage). Transfer of gene for Beta lactamase production is mediated by bacteriophage, and (c) Transformation. It is a natural occurrence, and is a direct uptake of donor DNA by recipient cells.

Disinfectant or antiseptic Concentration Examples of uses Levels of activityPhenolic compounds Hexylresorcinol, o-phenylphenol, cresols

0.5-3.0%Aqueous solution

Disinfection of inanimate objects such as instruments, floor and table surfaces and (w/ cresol) rectal thermometers

Intermediate to low

Alcohols ethyl alcohol isopropyl alcohol alcohol plus iodine

70-90%

70+0.5-2.0%iodine

Disinfection of skin, delicate surgical instruments, thermometers

Intermediate

Iodine iodophor (polyvinylpyrrolidone) Tincture of iodine

1.0%

2% iodine +2% sodium iodide + 70% alcohol

Disinfection of skin, minor cuts, and abrasion; also used for disinfection of water and swimming pools

Intermediate

Chlorine compounds 0.5-5.0 g availableChlorine per liter

Disinfection of water, nonmetal surfaces, dairy equipment, restaurant utensils, household items

Low

Table 6.2 Some Commonly Used Disinfectants and Antiseptics

Quaternary compds 0.1-0.2% Environmental sanitation of surfaces

Low

Mercurial compds Merthiolate Mercurochrome

1.0%Disinfection of skin, instruments; also used asA preservative in some biological materials

Low

*Levels of microbial activity: high =kills all forms of microbial life including spores;intermediate = kills tubercle bacilli, fungi, and viruses but not bacterial spores;low = does not kill bacterial spores, tubercle bacilli, or nonlipid viruses within

reasonable time

• Antimicrobial activity is measured by determining the smallest amount of agent needed to inhibit the growth of a test organism, a value called the minimum inhibitory concentration (MIC) (Figure 20.11).

CELL STRUCTURES ANTIMICROBIAL CHEMICALS(THEIR SITES AND MODE OF ACTION)

Cell wall Phenol, sodium hypochlorite, and merthiolate(low concentrations) cause lysis

Cytoplasmic membrane Phenols, alcohols, and detergents affect CM, causing leakage

Nuclear material(DNA) Hypochlorites, iodine, ethylene oxide, glutaraldehyde and saltsof heavy metals combine with-SH

Ribosomes Ethylene oxide, and glutaraldehyde combine with –NH2 groups

Cytoplasm Mercury salts, glutaraldehyde and high concentrations of phenol coagulate proteins

Table 6.3Summary of the sites and modes of action of various antimicrobial chemicals

The Major Groups of Procaryotic Microorganisms: Bacteria

Bergey’s Manual of Systematic Bacteriology-It does not only contain descriptions of all established genera

and species but also provides a practical arrangement for differentiatingthese organisms, together with appropriate classification outlines andTables.

EUBACTERIACell wall present Cell wall absentGram-negative: Spirochetes Aerobic or microaerophilic curved rods Aerobic rods Facultative anaerobic rods Anaerobes Rickettsias and chlamydias Anoxygenic phototrophs Gliding bacteria Sheathed bacteria Buddingand/or appendaged bacteria ChemolithotrophsGram-positive: Cocci Endospore formers Regularly shaped rods Irregularly shaped rods Mycobacteria Actinomycetes

Mycoplasmas

Table 6.4. Major Groups of Bacteria

ARCHAEBACTERIA

Methane producersRed Extreme halophilesSulfur-dependent archaebacteria

Thermoplasmas

Ass. List down the characteristics of the different microorganisms and theirexamples

ArchaebacteriaSubgroups Characteristics Examples

Methanogens Anaerobic, produces large amounts of methane gas-occur in anaerobic habitats rich in organic matter-marshes, swamps, pond and lake mud sediments and the rumen of cattle-also thrive within anaerobic sludge digesters in sewage treatment plants

Methanosarcina –gram + cocci in clustersMethanobacterium –gram+ long rodsMethanospirillum –gram- wavy filaments

Red Extreme Halophiles Gram-negative aerobic bacteria; require an environment that provides 17 to 23% NaCl for growth-can’t grow in solns less than 15% NaCl nor in seawater (3%) but in saltlakes-colonies are red to orange, some contain purple pigment (bacteriorhodopsin)

Halobacteria (salt bacteria)

Sulfur-dependent Predominate in acidic hotsprings Sulfolobus –aerobic, obtain E

-grow in temperatures of 50oC or higher like 87oC-can’t grow above pH 4.0 to 5.5

By oxidizing elemental sulfur or organic compounds such as sugars or amino acids.Thermoproteus –obtain E by removing electrons from either Hydrogen gas or organic compounds and then using the electrons to reduce elemental Sulfur to hydrogen sulfide

Thermoplasmas -resemble the mycoplasmas in that they do not have cell wall and are bounded only by the CM-they differ in their ability to grow at high Ts under acidic conditions.-optimum growth T=55-59oC-optimum pH is 2.-disintegrate at pH 7-isolated from piles of burning coal refuse