Conditions that affect Antimicrobial Activity Mechanisms of Microbial Damage and etc.

1. Control of Microorganisms:
Principles and Physical Agents

2. 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
growth
eg. 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 microorganisms
killed).
Sterilization – Killing all microorganisms present in a material, including any spores.
Microbiostatic agents- agents that merely inhibit the growth of microorganisms
eg. Bacteriostatic or fungistatic
Sanitizer – any agent that reduces bacterial numbers to safe levels accdng to PH reqts

3. Conditions that affect Antimicrobial Activity
-some important variables to consider when assessing the
effectiveness of a microbicidal agent are:
1. Size of microbial population
2. Intensity or concentration of the microbicidal agent
3. Time of exposure to the microbial agent
4. Temperature at which the microorganisms are exposed to the
microbicidal agent
5. Nature of the material containing the microorganisms
6. Characteristics of the microorganisms which are present

4. Mechanisms of Microbial Damage
- antimicrobial agents inhibit or kill microorganisms by
damaging certain structures of the cell, such as:
1. the cell wall
2. or the cytoplasmic membrane
3. or substances within the cytoplasm such as:
a. enzymes
b. ribosomes
c. or nuclear material

5. 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 articles
Endospores are not
killed; cannot be
relied upon to
sterilize
Does not sterilize
Table 6. The Use of Temperature to Control Microorganisms

8. 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
microbicidal
High cost of liquid
nitrogen

9. Contn of Table 6. Physical methods for control of microorganisms
Agent Action Use
Radiation
1. Ultraviolet
2. X-rays
3. Cathode rays
Formation of thymine dimers
Ionization; peroxide formation
Ionization
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
mutations
Research; 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 bacteriostatic

11. 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 because
MH 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 100oC
DH - up to 180 mins at 140oC
2. vegetative cells
MH – 5-10 min at 60-70oC (bacteria)
-5-10 min at 50-60oC (yeast and other fungi)

12. 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
12-14
13-17
15-20
Erlenmeyer flasks
50 ml
500 ml
1000 ml
2000 ml
12-14
17-22
20-25
30-35
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 affording
a reasonable factor of safety for sterilization by autoclaving

13. 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 prevent
the 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 filtered
4. filtration through membrane filters is more rapid than that
obtained with older filters
- also used for separating types of microorganisms and for collecting microbial
samples
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.

15. Lyophilization
-dehydration of microorganisms quickly at freezing temperatures and then sealed
In 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)

16. Control of Microorganisms: Chemical Agents

17. 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 animals
5. Homogeneity -the ingredients shld not settle to the bottom of the
container
6. Minimum inactivation by extraneous material –some antimicrobial chemicals
combine readily with proteins and other organic matls found in
the 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 where
the agent is to be used

18. 8. Ability to penetrate - unless the chemical can penetrate the surface, its
antimicrobial 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 being
treated
12. Availability and low cost- the product should be readily available and
inexpensive

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

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

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

56. RESISTANCE TO ANTIMICROBIAL DRUGS
A. 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.
Aminoglycosidesresistant 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

57. 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.

58. 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)

61. 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.

62. (ii)Extrachromosomal Resistance
(a) Plasmid Resistance. This occurs by the extrachromosomal genetic elements
called plasmid. Plasmidmediated 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. Plactamases 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.

70. Disinfectant or antiseptic Concentration Examples of uses Levels of activity
Phenolic 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 available
Chlorine per liter
Disinfection of water,
nonmetal surfaces, dairy
equipment, restaurant
utensils, household
items
Low
Table 6.2 Some Commonly Used Disinfectants and Antiseptics

71. Quaternary compds 0.1-0.2% Environmental
sanitation of surfaces
Low
Mercurial compds
Merthiolate
Mercurochrome
1.0%
Disinfection of skin,
instruments; also used as
A 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

72. • 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).

75. 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

78. 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 differentiating
these organisms, together with appropriate classification outlines and
Tables.

79. EUBACTERIA
Cell wall present Cell wall absent
Gram-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
Chemolithotrophs
Gram-positive:
Cocci
Endospore formers
Regularly shaped rods
Irregularly shaped rods
Mycobacteria
Actinomycetes
Mycoplasmas
Table 6.4. Major Groups of Bacteria

80. ARCHAEBACTERIA
Methane producers
Red Extreme halophiles
Sulfur-dependent archaebacteria
Thermoplasmas
Ass. List down the characteristics of the different microorganisms and their
examples

81. Archaebacteria
Subgroups 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 clusters
Methanobacterium –gram+
long rods
Methanospirillum –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

82. -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