THE CONTROL

OF
MICROBIAL GROWTH

1
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
-Factors influencing the effectiveness of antimicrobial treatments
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents

2
 The control of microbial growth

Control of growth = to prevent growth of microorganisms :

Inhibiting the growth

Killing microorganisms
of microorganisms

Control of growth usually involves the use of physical or chemical agents which either kill
or prevent the growth of microorganisms.

3
s
s

4
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
-Factors influencing the effectiveness of antimicrobial treatments
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents

5
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
-Factors influencing the effectiveness of antimicrobial treatments
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents

6
 The rate of microbial death

106

Logarithm of number of microbial survivors

105
Deaths per Number of
Time One log decrease =
(min)
minute survivors 104 90 % of population killed

0 0 1,000,000
103
1 900,000 100,000
2 90,000 10,000
102
3 9,000 1,000
4 900 100
101
5 90 10
6 9 1
100
0 1 2 3 4 5 6
Time (min)

Decimal reduction time (DRT, or D value) =

the time (min) in which 90% of a population
of bacteria at a given temperature will be killed.
7
 Factors influencing the effectiveness of antimicrobial treatments

1- Population size

106

Logarithm of number of microbial survivors

105
High population load

104

103
Low
population
102 load

101

100
0 1 2 3 4 5 6
Time (min)
2- Population composition: bacterial endospores >> vegetative cells and,
younger cells (log phase) <<mature organisms.
Some species are able to withstand adverse conditions better than others (ex: Mycobacterium
8
tuberculosis).
3- Concentration or intensity of antimicrobial agent
Exceptions!.

4- Duration of exposure

5- Temperature

6- Environmental influences

9
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
-Factors influencing the effectiveness of antimicrobial treatments
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents

10
 Actions of microbial control agents

Alteration of membrane permeability (passage of nutrients & elimination of waste)

Damage to the lipids or proteins of the plasma membrane by antimicrobial agents causes
cellular contents to leak into the surrounding medium and interferes with the growth
of the cell

Damage to proteins
Proteins (enzymes) are functional in their three-dimensional shape.
This shape is maintained by chemical bonds:

Breakage
Hydrogen Covalent bonds Denaturation
(-SH) (cellular death)

Damage to nucleic acids is frequently lethal to the cell

The cell can no longer replicate, nor can it carry out normal metabolic functions
such as the synthesis of enzymes

11
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
-Factors influencing the effectiveness of antimicrobial treatments
4- Actions of microbial control agents
5- Physical methods of microbial control:
-Heat (incineration, boiling, autoclaving, pasteurization, …)
-Filtration
-Low temperatures
-High pressure
-Desiccation
-Osmotic pressure
-Radiation

6- Chemical methods of microbial control 12

7- Antibiotics and other chemotherapeutic agents
 Physical methods of microbial control

Sterilization (boiling, autoclaving, hot air oven) kills all microorganisms with heat; commonly
employed in canning, bottling, and other sterile packaging procedures.

1- Heat

Thermal death point (TDP)

Thermal death time (TDT)

13
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
-Factors influencing the effectiveness of antimicrobial treatments
4- Actions of microbial control agents
5- Physical methods of microbial control:
-Heat (incineration, boiling, autoclaving, pasteurization, …)
-Filtration
-Low temperatures
-High pressure
-Desiccation
-Osmotic pressure
-Radiation

6- Chemical methods of microbial control

14
7- Antibiotics and other chemotherapeutic agents
 1-Heat

Incineration: burns organisms and physically destroys them. Used for needles, inoculating
wires, glassware, etc. and objects not destroyed in the incineration process.

Boiling: 100° for 30 minutes. Kills everything except some endospores. To kill endospores, and
therefore sterilize the solution, very long or intermittent boiling is required.

Autoclaving (steam under pressure or pressure cooker): 121° for 15 minutes

(15pounds/in2 pressure). Good for sterilizing almost anything, but heat-labile substances
will be denatured or destroyed.

15
Pasteurization is the use of mild heat to reduce the number of microorganisms in a product or
food (without damaging the taste).

For pasteurization of milk:

batch method: 63°/30 minutes (low-temperature long-time = LTLT).
flash method : 71°/15 seconds (high-temperature short-time = HTST).
The milk keeps well under refrigeration.

Ultra-high-temperature (UHT) : 74° 140°/3 sec 74°

so that it can be stored without refrigeration.

16
Dry heat (hot air oven): 160°/2 hours or 170°/1 hour. Used for glassware, metal, and objects
that won't melt.

17
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
-Factors influencing the effectiveness of antimicrobial treatments
4- Actions of microbial control agents
5- Physical methods of microbial control:
-Heat (incineration, boiling, autoclaving, pasteurization, …)
-Filtration
-Low temperatures
-High pressure
-Desiccation
-Osmotic pressure
-Radiation

6- Chemical methods of microbial control 18

7- Antibiotics and other chemotherapeutic agents
2- Filtration sterilizes solutions which would be denatured by heat
(e.g. antibiotics, injectable drugs, amino acids, vitamins, enzymes, etc.)
HEPA > 0.3 µm in diameter

19
Small volume solutions
Diameter: 0.45 µm (bacterial elimination)
0.22 µm (enzymatic filtration)

20
21
3-Low temperatures (refrigeration and freezing

1 microbe reproducing 3 times/day  ~2 millions microbes/week!!!!

App: food, drug and culture preservation

4-High pressure

Barophile endospores!!

This technique preserves the flavors, colors, and nutrient values of the products (ex:
treatment of fruit juices).

22
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
-Factors influencing the effectiveness of antimicrobial treatments
4- Actions of microbial control agents
5- Physical methods of microbial control:
-Heat (incineration, boiling, autoclaving, pasteurization, …)
-Filtration
-Low temperatures
-High pressure
-Desiccation
-Osmotic pressure
-Radiation

6- Chemical methods of microbial control 23

7- Antibiotics and other chemotherapeutic agents
5-Dessication (drying): microbes and certain foods (coffee, fruit additives) are
preserved by lyophilization or freeze-drying.

24
6-Osmotic pressure (drying)

Often used to preserve foods (e.g. fruits, grains, cure meats, etc.).

-addition of salt or sugar (hypertonic environment).

25
7- Radiation (ionizing and nonionizing)

Ionizing radiation: The principal effect of ionizing radiation is the ionization of water, which
forms highly reactive hydroxyl radicals, which react with organic cellular components, especially
DNA. Effective against both vegetative cells and endospores, but not as effective against viruses.

Ionizing Nonionizing

10-5 nm 10-3 nm 1 nm 103 nm 106 nm 1m 103 m

Gamma Radio
X rays UV Infrared Microwaves
rays waves

UV light Visible light

UV in sunlight

Tanning
Bactericidal

200 nm 400 nm 750 nm

280 nm 295 nm 330 nm

Wavelength increase 26
Energy increase
27
Nonionizing radiation (IR, UV): the best ex is UV light.
They are used to control microbes in the air, to disinfect vaccines and other medical products.
A major disadvantage of UV light as a disinfectant is that the radiation is not very penetrating,
so the organism to be killed must be directly exposed to the rays.
Organisms protected by solids are not affected.
The UV can damage human eyes, and prolonged exposure can cause burns and skin cancer in
humans.

28
Microwaves! Sterilization?!

Moisture containing food are heated by microwave action, and the heat will kill the most
vegetative pathogens.
Solid foods heat unevenly because of the uneven distribution of moisture.

29
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
-Phenol and Phenolics
-Bisphenols
-Biguanides
-Halogens
-Alcohols
-Heavy Metals and their compounds
-Surface-Active Agents
-Chemical food preservatives
-Aldehydes
-Gaseous chemosterilizers
-Peroxygens (Oxidizing agents)
30
7- Antibiotics and other chemotherapeutic agents
31
 Chemical methods of microbial control

The effectiveness of an antimicrobial chemical agent depends on:

-its concentration
-the nature of the material being disinfected (presence or organic materials)
-the pH of the medium
-the contact of the disinfectant with the microbes (rinse the surface before disinfection)

Phenol and phenolics

Chemical
Mechanism of action Preferred use Comment
agent
Disruption of plasma Rarely used, except as Seldom used as a disinfectant or
Phenol membrane, denaturation of a standard of antiseptic because of its irritating
enzymes comparison qualities and disagreeable odor
Environmental
Derivative of phenol that are reactive
surfaces, instruments,
Disruption of plasma membrane even in the presence of organic material
skin surfaces, and
Phenolics (& CW of some bacteria), (ex: O-phenylphenol), stable and persist
mucous membranes,
denaturation of enzymes for long period after application, less
pus, saliva & feces
irritating.

Disinfectant hand Derivative of phenol, (ex: triclosan*),

Probably disruption of plasma soaps and skin lotions, broad spectrum but most effective
Bisphenols membrane (*inhibition of an incorporated to against gram-positives.
(2 phenols) enzyme needed for the (*kitchen cutting Hexachlorophene: skin infections in
biosynthesis of lipids) boards, handles of newborns (excessive use can lead to 32
knives), … neurological damage)
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
-Phenol and Phenolics
-Bisphenols
-Biguanides
-Halogens
-Alcohols
-Heavy Metals and their compounds
-Surface-Active Agents
-Chemical food preservatives
-Aldehydes
-Gaseous chemosterilizers
-Peroxygens (Oxidizing agents)
7- Antibiotics and other chemotherapeutic agents 33
 Biguanides
Chemical
Mechanism of action Preferred use Comment
agent
Skin disinfection,
Bactericidal to gram-positives and
Biguanides Disruption of plasma membrane especially for surgical
gram-negatives; non toxic, persistent
scrubs

Halogens
Chemical
Mechanism of action Preferred use Comment
agent
Iodine is an effective antiseptic available
Iodine inhibits protein function
as a tincture (allergy problem!!) and an Iodine and chlorine
and is a strong oxidizing agent;
Halogens iodophor; chlorine gas is used to may act alone or as
chlorine forms the strong
(Cl2, Br2, I2, disinfect water; chlorine compounds are components of
oxidizing agent hypochlorous
As2, F2) used to disinfect dairy equipment, eating inorganic or organic
acid, which alters cellular
utensils, household items, and glassware compounds
components
(carcinogen!!)

34
35
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
-Phenol and Phenolics
-Bisphenols
-Biguanides
-Halogens
-Alcohols
-Heavy Metals and their compounds
-Surface-Active Agents
-Chemical food preservatives
-Aldehydes
-Gaseous chemosterilizers
-Peroxygens (Oxidizing agents)
7- Antibiotics and other chemotherapeutic agents 36
 Alcohols
Chemical Mechanism
Preferred use Comment
agent of action
Thermometers and other instruments; in
Bactericidal and fungicidal, but not
swabbing the skin with alcohol before
Protein effective against endospores or
an injection, lost of the disinfecting
Alcohols denaturation and nonenveloped viruses; commonly
action probably comes from a sample
lipid dissolution used alcohols are ethanol and
wiping away (degerming) of dirt and
isopropanol
some microbes

Heavy metals and their compounds

Chemical Mechanism
Preferred use Comment
agent of action
Heavy metals such
Denaturation of Silver nitrate may be used to prevent gonorrheal
as silver and
Heavy metals enzymes and other ophtalmia neonatorum; mercurochrome disinfects skin
mercury are
essential proteins and mucous membranes; copper sulfate is an algicide
biocidal

37
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
-Phenol and Phenolics
-Bisphenols
-Biguanides
-Halogens
-Alcohols
-Heavy Metals and their compounds
-Surface-Active Agents
-Chemical food preservatives
-Aldehydes
-Gaseous chemosterilizers
-Peroxygens (Oxidizing agents)
7- Antibiotics and other chemotherapeutic agents 38
 Surface-Active Agents
Chemical agent Mechanism of action Preferred use Comment
Mechanical remove of Skin degerming and Many antibacterial soaps contain
Soaps
microbes through scrubbing removal of debris antimicrobials
Not certain; may involve Sanitizers in dairy and
Acid-anionic Wide spectrum of activity; nontoxic,
enzyme inactivation or food-processing
detergents noncorrosive, fast-acting
disruption industries
Cationic
Bactericidal, bacteriostatic,
detergents Enzyme inhibition, protein
Antiseptic for skin, fungicidal, virucidal against
(quaternary denaturation, and disruption
instruments, utensils enveloped viruses; (quats: Zephiran,
ammonium of plasma membranes
Cepacol)
compounds)

Organic acids
Chemical Mechanism
Preferred use Comment
agent of action
Metabolic inhibition, Widely used to
mostly affecting Sorbic acid and benzoic acid effective at low pH; control molds and
Organic
molds; action not parabens much used in cosmetics, shampoos; calcium some bacteria in
acids
related to their propionate used in bread. foods and
acidity cosmetics.
39
 Quats

40
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
-Phenol and Phenolics
-Bisphenols
-Biguanides
-Halogens
-Alcohols
-Heavy Metals and their compounds
-Surface-Active Agents
-Chemical food preservatives
-Aldehydes
-Gaseous chemosterilizers
-Peroxygens (Oxidizing agents)
7- Antibiotics and other chemotherapeutic agents 41
 Aldehydes
Chemical Mechanism
Preferred use Comment
agent of action
Protein &
Glutaraldehyde (Cidex) is less irritating than formaldehyde Very effective
Aldehydes nucleic acid
(HCHO) and is used for disinfection of medical equipment antimicrobials
denaturation

Gaseous sterilants
Chemical Mechanism
Preferred use Comment
agent of action
Gaseous Protein Excellent sterilizing agent, especially for objects that
Ethylene oxide
sterilants denaturation would be damaged by heat

Peroxygens (oxidizing agents)

Mechanism
Chemical agent Preferred use Comment
of action
Peroxygens
Contaminated surfaces; some deep wounds, in which they
(oxidizing Oxidation
are very effective against oxygen-sensitive anaerobes
H202
agents)
42
 Chemical agents

(Oxidizing agents)
chemosterilizers
Surface-Active

Chemical food
Heavy Metals

preservatives

Peroxygens
Biguanides
Bisphenols

Aldehydes
Phenolics

Halogens
Alcohols

Gaseous
Phenol

Agents
Site of action

Cell wall +
Cytoplasmic + + + + + +quats
membrane
Proteins (denaturation) + + + + + + + +
Nucleic acids +gld
Enzymes with SH + + +quats, +gld +
groups acid
anionic

Amino acids +for, +

gld
43
44
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents
-Definitions (chemotherapy, chemotherapeutic agent and antibiotics…)
-Historical highlights of chemotherapy
-Properties of a satisfactory antibiotic
-classification of antibiotics and CA depending on their chemical structure
-classification of antibiotics and CA depending on their mode of action
-Measuring antimicrobial activity (MIC & MBC)
-Bacterial resistance to antibiotics

45
46
47
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents
-Definitions (chemotherapy, chemotherapeutic agent and antibiotics…)
-Historical highlights of chemotherapy
-Properties of a satisfactory antibiotic
-classification of antibiotics and CA depending on their chemical structure
-classification of antibiotics and CA depending on their mode of action
-inhibition of the cell wall synthesis
-damage to the cytoplasmic membrane
-inhibition of nucleic acid
-inhibition of protein synthesis
-competitive inhibitors
-Measuring antimicrobial activity (MIC & MBC) 48
-Bacterial resistance to antibiotics
Synthesis of Peptidoglycan

49
50
51
Inhibition of cell-wall synthesis by penicillins and cephalosporins (β-lactams)

52
53
 Inhibition of cell-wall synthesis by Glycopeptides

Cycloserine inhibits the enzymes involved in the synthesis of the pentapeptide side chains.
54
Inhibition of cell-wall synthesis by polypeptides

55
Beta lactam antibiotics are normally bactericidal and require that cells be actively growing in
order to exert their toxicity.

Although nontoxic, penicillins occasionally cause death when administered to persons who are
allergic to them.

56
INH (isoniazid) block the incorporation of mycolic acid into acid-fast cell walls while
ethambutol interferes with the incorporation of arabinoglactan. Both inhibit synthesis of the
acid-fast cell wall. Ex: treatment of M. tuberculosis

57
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents
-Definitions (chemotherapy, chemotherapeutic agent and antibiotics…)
-Historical highlights of chemotherapy
-Properties of a satisfactory antibiotic
-classification of antibiotics and CA depending on their chemical structure
-classification of antibiotics and CA depending on their mode of action
-inhibition of the cell wall synthesis
-damage to the cytoplasmic membrane
-inhibition of nucleic acid
-inhibition of protein synthesis
-competitive inhibitors
-Measuring antimicrobial activity (MIC & MBC) 58
-Bacterial resistance to antibiotics
 Damage to the cytoplasmic membrane

A very few antibiotics, such as polymyxins and colistins alter the microbial cytoplasmic
membranes and cause leakage of cellular needs. Polymyxins and colistins act as
detergents and alter membrane permeability in gram-negative bacteria. They cannot
effectively diffuse through the thick peptidoglycan layer in gram-positives.

59
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents
-Definitions (chemotherapy, chemotherapeutic agent and antibiotics…)
-Historical highlights of chemotherapy
-Properties of a satisfactory antibiotic
-classification of antibiotics and CA depending on their chemical structure
-classification of antibiotics and CA depending on their mode of action
-inhibition of the cell wall synthesis
-damage to the cytoplasmic membrane
-inhibition of nucleic acid
-inhibition of protein synthesis
-competitive inhibitors
-Measuring antimicrobial activity (MIC & MBC) 60
-Bacterial resistance to antibiotics
61
62
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents
-Definitions (chemotherapy, chemotherapeutic agent and antibiotics…)
-Historical highlights of chemotherapy
-Properties of a satisfactory antibiotic
-classification of antibiotics and CA depending on their chemical structure
-classification of antibiotics and CA depending on their mode of action
-inhibition of the cell wall synthesis
-damage to the cytoplasmic membrane
-inhibition of nucleic acid
-inhibition of protein synthesis
-competitive inhibitors
-Measuring antimicrobial activity (MIC & MBC) 63
-Bacterial resistance to antibiotics
64
Inhibition of protein synthesis by aminoglycosides

The aminoglycosides bind irreversibly

to the 30S subunit of bacterial
ribosomes.

There is evidence that some prevent the

transfer of the peptidyl tRNA from the
A-site to the P-site, thus preventing the
elongation of the polypeptide chain

65
66
Synergy - The aminoglycosides synergize with B-lactam antibiotics. The B-lactams inhibit
cell wall synthesis and thereby increase the permeability of the aminoglycosides.
67
Inhibition of protein synthesis by tetracyclines

The tetracyclines bind reversibly to the

30S subunit, distorting it in such a way
that the anticodons of charged tRNAs
cannot align properly with the codons
of the mRNA

68
Chloramphenicol has a broad spectrum of activity but it exerts a bacteriostatic effect.
It is effective against intracellular parasites such as the rickettsiae.

It inhibits the bacterial enzyme peptidyl transferase thereby preventing the growth of the
polypeptide chain during protein synthesis.
Chloramphenicol is entirely selective for 70S ribosomes and does not affect 80S
ribosomes.

69
Inhibition of protein synthesis by macrolides

The macrolides (erythromycin,

azithromycin, clarithromycin,
dirithromycin, troleandomycin, etc.)
bind reversibly to the 50S subunit.
They appear to inhibit elongation of
the protein by preventing the enzyme
peptidyltransferase from forming
peptide bonds between the amino acids

70
The Macrolides may also prevent the transfer of the peptidyl tRNA from the A-site to
the P-site

71
72
The oxazolidinones (linezolid) bind to the 50S ribosomal subunit and interfere with its
binding to the initiation complex

73
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents
-Definitions (chemotherapy, chemotherapeutic agent and antibiotics…)
-Historical highlights of chemotherapy
-Properties of a satisfactory antibiotic
-classification of antibiotics and CA depending on their chemical structure
-classification of antibiotics and CA depending on their mode of action
-inhibition of the cell wall synthesis
-damage to the cytoplasmic membrane
-inhibition of nucleic acid
-inhibition of protein synthesis
-competitive inhibitors
-Measuring antimicrobial activity (MIC & MBC) 74
-Bacterial resistance to antibiotics
Inhibitors of Folic Acid Synthesis
p-aminobenzoic acid + Pteridine
Pteridine
Sulfonamides
synthetase

Dihydropteroic acid

Dihydrofolate
synthetase

Dihydrofolic acid
Dihydrofolate
Trimethoprim reductase

Tetrahydrofolic acid

Thymidine Methionine
Purines
75
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents
-Definitions (chemotherapy, chemotherapeutic agent and antibiotics…)
-Historical highlights of chemotherapy
-Properties of a satisfactory antibiotic
-Classification of antibiotics and CA depending on their chemical structure
-Classification of antibiotics and CA depending on their mode of action
-Measuring antimicrobial activity (MIC & MBC)
-Bacterial resistance to antibiotics

76
77
78
Measuring antimicrobial activity
(broth dilution testing)

MBC MIC

Antimicrobial activity can be either

Bacteriostatic (100% survivors)
or bactericidal (0.01% survivors)

79
80
Ten tubes containing the same appropriate medium with an increased concentration of an antibiotic
(ATB) were inoculated with a constant inoculum of 103 bacteria/tube of the above isolated strain.
After 18 hours of incubation at 37°C, we enumerate the amount of bacteria in each tube using
the spectrophotometric method and we obtain the following results:

1 2 3 4 5 6 7 8 9 10
Tube
[ATB] 0 µg/ml 2 2.5 3 3.5 4 4.5 5 5.5 6
Incubation at 37°C during 18 hours
logN 8 8 7.7 7.5 7 6.5 3 2 ~0 ~0

2-Determine the MIC from the table above.

3-In which tubes does the antibiotic have a bactericidal effect? A bacteriostatic effect?

4-The tubes T9 and T10 were limpid. Cultures in plate agar were performed from these
two tubes and we obtain a positive culture in the case of the T9 while no growth was
observed in the Petri dish corresponding to the T10.

What represents T10? Give the full definition of the term representing the T10.

81
Ten tubes containing the same appropriate medium with an increased concentration of penicillin
were inoculated with 104 bacteria/tube of a strain X isolated and purified from a patient with
a nosocomial infection without any change in their concentration. After 18 hours of incubation at 37°C,
we enumerate the amount of bacteria in each tube using the spectrophotometric method and we
obtain the following results:

Tube 1 2 3 4 5 6 7 8 9 10
[Penicillin]
0 2 3 4 5 6 7 10 20 25
in µg/mL
Incubation at during 18 hours
logN 8 0.05 0.05 0.07 0.07 0.075 0.04 0.03 0.0 0.0

Determine the MIC from the table above and give its definition.

82
 The E (epsilometer) test

Bacterial culture

MIC =
= MIC

83
84
 Disk-diffusion method

Zone of inhibition
Zone of culture Absence of colonies
[ATB]<MIC [ATB]>MIC

Limit of the zone

of inhibition
[ATB]=MIC

C = n/V C = n/π . r2 . h
85
V = S . h V = π . r2 .h
As r increases C decreases
86
 Determination of LCC and HCC
Injected dose Detected dose in Treatment Side effects
(mcg/mL) the blood efficiency

1 0 - No
2 LCC 1 Minimum No
3 2 + No
4 3 + No

100 HCC 99 Maximum No

101 100 Maximum Yes

87
 LCC LCC

HCC HCC

Growth

If the diameter of the inhibitory zone > diameter of the LCC  the strain is sensible to the antibiotic

If the diameter of the inhibitory zone < diameter of the HCC  the strain is resistant to the antibiotic

If the diameter of the inhibitory zone is situated between 88

the 2 diameters of the CC  the strain is intermediate to the antibiotic
89
We have studied the susceptibility of the strain X to 4 antibiotics using the
Kirby-Bauer method. The results we have obtained are summarized in the table below.
a-Which antibiotic should be prescribed to treat an infection due to the strain X?
Justify your answer.

Diameter in mm
Antibiotic Zone of inhibition
LCC HCC
X
Penicillin 12 16 14
Gentamycin 18.5 22 19
Cephalosporin 13.5 18 14

Augmentin 18 22 19

90
 The control of microbial growth

1-Definition + Introduction
2-Terminology relating to the control of microbial growth
3- The rate of microbial death:
4- Actions of microbial control agents
5- Physical methods of microbial control:
6- Chemical methods of microbial control
7- Antibiotics and other chemotherapeutic agents
-Definitions (chemotherapy, chemotherapeutic agent and antibiotics…)
-Historical highlights of chemotherapy
-Properties of a satisfactory antibiotic
-Classification of antibiotics and CA depending on their chemical structure
-Classification of antibiotics and CA depending on their mode of action
-Measuring antimicrobial activity (MIC & MBC)
-Bacterial resistance to antibiotics

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 The basis of bacterial resistance to antibiotics

Inherent (Natural) Resistance. For example, a streptomycete has some gene that is
responsible for resistance to its own antibiotic; or a Gram-negative bacterium has an outer
membrane that establishes a permeability barrier against the antibiotic; or an organism lacks a
transport system for the antibiotic; or it lacks the target or reaction that is hit by the antibiotic.

Acquired Resistance Bacteria can develop resistance to antibiotics, e.g. bacterial populations
previously-sensitive to antibiotics become resistant. This type of resistance results from
changes in the bacterial genome. Acquired resistance is driven by two genetic processes in
bacteria: (1) mutation and selection; (2) exchange of genes between strains and species.

Vertical evolution is strictly a matter of Darwinian evolution driven by principles of natural

selection: a spontaneous mutation in the bacterial chromosome imparts resistance to a
member of the bacterial population. In the selective environment of the antibiotic, the wild
type (non mutants) is killed and the resistant mutant is allowed to grow and flourish.

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Horizontal evolution is the acquisition of genes for resistance from another organism. For
example, a streptomycete has a gene for resistance to streptomycin, but somehow that gene
escapes and gets into E. coli or Shigella. Or, more likely, some bacterium develops genetic
resistance through the process of mutation and selection and then donates these genes to some
other bacterium through one of several processes for genetic exchange that exist in bacteria
(conjugation, transduction or transformation).

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 Ways in which Bacteria May Resist Our Control Agents

Most bacteria, however, become resistant to antibiotics by way of one or more of the
following mechanisms coded for by genes in the nucleoid or in plasmids:
1. Producing an enzyme capable of destroying or inactivating the antibiotic;
2. Altering the target site receptor for the antibiotic to reduce or block its binding; and
3. Preventing the entry of the antibiotic into the bacterium and/or actively transporting the
antibiotic out of the bacterium.
4. Modulating gene expression to produce more of the bacterial enzyme that is being tied
up or altered by the antibiotic.

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Beta-lactamases break the beta-lactam ring of the antibiotic, thus destroying the drug..

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Altering the target site receptor for the antibiotic in the bacterium
to reduce or block its binding.

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Altering the membranes and transport systems to prevent the entry of the antibiotic
into the bacterium and/or actively transport the antibiotic out of the bacterium.

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