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Chapter 7
Page 251
7.1 Introduction
Infection is a major category of human disease and skilled management
of antimicrobial drugs is of the first importance. The term chemotherapy is
used for the drug treatment of parasitic infections in which the parasites
(viruses, bacteria, protozoa, fungi, and worms) are destroyed or removed
without injuring the host.
Many substances that we now know to possess therapeutic efficacy were
first used in the distant past. The Ancient Greeks used male fern, and the
Aztecs chenopodium, as intestinal anthelmintics. The Ancient Hindus treated
leprosy with chaulmoogra. For hundreds of years moulds have been applied to
wounds, but, despite the introduction of mercury as a treatment for syphilis
(16th
century), and the use of cinchona bark against malaria (17th
century), the
history of modern rational chemotherapy did not begin until Paul Ehrlich
developed the idea from his observation that aniline dyes selectively stained
bacteria in tissue microscopic preparations and could selectively kill them. He
invented the word „chemotherapy‟ and in 1906 he wrote:
“In order to use chemotherapy successfully, we must search for
substances which have an affinity for the cells of the parasites and a power of
killing them greater than the damage such substances cause to the organism
itself… This means… we must learn to aim, learn to aim with chemical
substances.”
The antimalerials pamaquin and mepacrine were developed from dyes
and in 1935 the first sulphonamides, linked with a dye (Prontosil), was
introduced as a result of systematic studies by Domagk. The results obtained
with sulphonamides in puerperal sepsis, pneumonia and meningitis were
dramatic and caused a revolution in scientific and medical thinking.
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In 1928, Fleming accidentally rediscovered the long-known ability of
Penicillium fungi to suppress the growth of bacterial cultures but put the
finding aside as a curiosity.
In 1939, principally as an academic exercise, Florey and Chain
undertook an investigation of antibiotics, i.e. substances produced by
microorganisms that are antagonistic to the growth or life of other
microorganisms. They prepared penicillin and confirmed its remarkable lack of
toxicity.
When the preparation was administered to a policeman with combined
staphylococcal and streptococcal septicemia there was dramatic improvement;
unfortunately the manufacture of penicillin (in the local Pathology Laboratory)
could not keep pace with the requirements (it was also extracted from the
patient‟s urine and re-injected); it ran out and the patient later succumbed to
infection. Subsequent development amply demonstrated the remarkable
therapeutic efficacy of penicillin.
7.2 Classification of Antimicrobial Drugs
Antimicrobial agents may be classified according to the type of
organism against which they are active.
Antibacterial drugs
Antiviral drugs
Antifungal drugs
Antiprotozoal drugs
Anthelmintic drugs.
A few antimicrobials have useful activity across several of these groups.
Few examples, metronidazole inhibits obligate anaerobic bacteria (such as
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Clostridium perfringens) as well as some protozoa that rely on anarobolic
pathways (such as Trichomonas vaginalis).
Antimicrobial drugs have also been classified broadly into:
Bacteriostatic, i.e. those that act primarily by arresting bacterial
multiplication, such as sulphonamides, tetracyclines and
chloramphenicol.
Bactericidal, i.e. those which act primarily by killing bacteria, such as
penicillins, cephalosporins, aminoglycosides, isoniazide and rifampicin.
7.3 Bacteria
In 1928, a German scientist C.E. Ehrenberg used the term “bacterium”.
Bacteria are the microscopic organisms of plant kingdom and are devoid of
chlorophyll. They are relatively simple and primitive form of cellular
organisms known as “Prokaryotes”. Bacteriology is the science that deals with
the study of bacteria. The Danish physician Christian Gram in 1884, discovered
a strain known as Gram strain, which can divide all bacteria into two classes
“Gram positive” and “Gram negative”. The Gram-positive bacteria resist
decolouration with acetone, alcohol and remain strained (methyl violet) as dark
blue colour, while Gram-negative bacteria are decolorized.
Bacteria can be classified according to their morphological
characteristics as lower and higher bacteria. The lower bacteria have generally
unicellular structures, never in the form of mycelium or sheathed filaments,
e.g., cocci, bacilli, etc. The higher bacteria are filamentous organisms, few
being sheathed having certain cells specialized for producing diseases in animal
or human, are known as “Pathogens”.
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7.4 Classification of Organisms
Staphylococcus Aureus is species of schizomycetes class; having
Eubacterials order, micrococeaceac family and staphylococcus genus.
Escherichia Coli is species of schizomycetes class; having Eubacterial
order, Enterobacteriaceae family and Escherichia genus.
Bacillus Subtillis is species of schizomycetes class; having Eubacterials
order, Bacteriodaceac family and fusobacterium streptobacillus and
sphaerophorus genus.
Pseudomonas Aeruginosa is species of schizomycetes class; having
pseudominodales order, pseudominadaceac family and pseudomonas genus.
7.5 Identification Techniques of the Organisms
The organisms were identified by using the following strains [1,2].
Schiff technique periodic acid
Gram strains
Zeil Nelsonm acid fast strains
7.6 Evaluation Techniques
The following condition must be met for the screening of antimicrobial
activity.
There should be an intimate contact between test organisms and
substance to be evaluated.
Required conditions should be provided for the growth of
microorganisms.
Condition should be same throughout the study.
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Aseptic/sterile environment should be maintained.
Various methods have been used from time to time by several workers
to evaluate the antimicrobial activity [3,4]. The evaluation can be done by the
following methods [5].
1. Turbidometric method,
2. Agar streak dilution method,
3. Serial dilution method, and
4. Agar diffusion method.
Agar diffusion method is again of three types:
Agar cup method,
Agar ditch method, and
Paper disc Method.
In present work Agar cup method is used.
7.7 Factors Affecting Zone of Inhibition
Ingredient of culture media
Many substances are present in culture media, which may affect the
zone of inhibition. Common ingredients such as peptone, agar, etc. may vary in
their contents and many of these minerals may influence the activity of some
antimicrobials. It is well known that Ca, Mg, Fe, etc. ions affect the sensitivity
of zone produced by the tetracycline, gentamycin, NaCl reduce the activity of
amino glycosides and enhances the effect of fucidin
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Choice of media
Consistent and reproducible results are obtained in media prepared
especially for sensitivity testing; the plates must be poured flat with an even
depth.
Effect of pH
The activity of amino glycosides is enhanced in alkaline media and reduced
in acidic media, the reverses is shown by tetracycline.
Size of inoculums
Although large numbers of organisms do not markedly affect many
antibiotics, all inhibition zones are diminished by heavy inocula. The ideal
inoculum is one, which gives an even dense growth without being confluent.
Overnight broth cultures of organisms and suitable suspensions from solid
media can be diluted accurately to give optimum inocula for sensitivity testing.
7.8 Experimental
The culture medium preparation
Nutrient agar medium was used. Chemical composition of the medium
was
Peptone 1.0 gm
NaCl 0.5 gm
Meats extract 0.3 gm
Distilled water 100 ml
pH 7.6
Agar 2.0 gm
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The ingredient were weighed and dissolved in distilled water, pH was
adjusted to 7.6 and then agar power was added to it. The medium was
dispensed in 25 ml quantity in different test-tubes. The test-tubes were plugged
by cotton-wool and sterilized at 121.5OC and 15 pounds per square inch (psi)
pressure for 15 minutes.
Antibacterial susceptibility testing
The study has been conducted according to the method adopted by
Cruickshank et al [6]. Nutrient agar broth was melted in a water bath and
cooked to 45oC with gentle shaking to bring about uniform cooling. It was
inoculated with 0.5-0.6 ml of 24 hour old culture especially and mixed well by
gentle shaking before pouring on the sterilized Petri dish (25 ml each). The
poured material was allowed to set (1.5 hour) and there after the “cups” were
made by punching into the agar surface with a sterile cork borer and scooping
out the punched part of agar. Into this “cups” 0.1 ml of test solution (prepared
by dissolving 10gm of sample in 10ml DMF) was added by sterile
micropipette. The plates were noted.
7.9 Results and Discussion
Ampicillin, Tetracycline, Gentamycin, and Chloramphenicol were used
as standard drugs and a solvent control was also run to know the activity of
solvent.
Activity of standards and inhibition due to DMF (solvent) are given in
Table-7.1. The results shown by compounds and standards are corrected for
DMF. Typical specimens are shown in figures.
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Table-7.1: Antimicrobial activity of Standards and Solvent (DMF)
No.
Name of
Compound
Zone of inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
1 DMF 8 5 6 7
2 Ampicillin 15 12 20 20
3 Tetracyclin 21 22 15 18
4 Gentamycin 20 19 18 22
5 Chloramphenicol 21 23 17 24
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Table-7.2: Antimicrobial activity of 4-(arylideneamino)-N-(4-(2,4-dichloro
phenyl)-6-(6-methylnaphthalen-2-yl)pyrimidin-2-yl) benzenesulfonamide
(6a-h)
Compound
(Designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
6a (1) 12 13 08 12
6b (2) 10 12 10 10
6c (3) 14 14 15 10
6d (4) 10 10 08 09
6e (5) 06 16 12 20
6f (6) 13 11 10 14
6g (7) 21 19 14 16
6h (8) 14 14 18 17
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Table-7.3: Antimicrobial activity of 4-(3-chloro-2-oxo-4-phenylazetidin-1-
yl)-N-(4-(2,4-dichlorophenyl)-6-(6-methylnaphthalen-2-yl)pyrimidin-2-yl)
benzenesulfonamide (7a-h)
Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
7a (1) 08 07 15 07
7b (2) 10 10 14 10
7c (3) 16 14 10 19
7d (4) 15 19 22 14
7e (5) 08 09 10 04
7f (6) 20 13 08 13
7g (7) 09 09 10 13
7h (8) 08 16 12 13
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Table-7.4: Antimicrobial activity of N-(4-(2,4-dichlorophenyl)-6-(6-methyl
naphthalen-2-yl)pyrimidin-2-yl)-4-(4-oxo-2-phenylthiazolidin-3-yl)
benzenesulfonamide (8a-h)
Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
8a (1) 09 12 11 17
8b (2) 14 09 13 09
8c (3) 13 12 08 08
8d (4) 14 10 20 13
8e (5) 18 14 14 09
8f (6) 22 20 18 20
8g (7) 14 16 15 13
8h (8) 15 15 18 21
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Table-7.5: Antimicrobial activity of 1-(4-(N-(4-(2,4-dichlorophenyl)-6-(6-
methyl naphthalen-2-yl)pyrimidin-2-yl)sulfamoyl)phenyl)-5-oxo-2-phenyl-
2,5-dihydro-1H-pyrrole-3-carboxylic acid (9a-h)
Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
9a (1) 10 16 10 22
9b (2) 18 13 13 13
9c (3) 20 15 14 11
9d (4) 21 20 16 17
9e (5) 13 17 16 06
9f (6) 12 11 12 12
9g (7) 14 14 04 17
9h (8) 19 14 15 14
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Table-7.6: Antimicrobial activity 1-(4-(N-(4-(2,4-dichlorophenyl)-6-(6-methyl
naphthalen-2-yl)pyrimidin-2-yl)sulfamoyl)phenyl)-5-oxo-2-phenylpyrrolidine
-3-carboxylic acid (10a-h)
Section 1.01 Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
10a (1) 12 12 20 19
10b (2) 14 17 14 18
10c (3) 16 10 08 22
10d (4) 22 17 14 13
10e (5) 11 09 13 11
10f (6) 15 13 05 14
10g (7) 07 05 06 16
10h (8) 15 17 14 21
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Gra
ph
1:
An
tim
icro
bia
l a
ctiv
ity o
f C
om
po
un
ds
(6a
-h)
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Gra
ph
2:
An
tim
icro
bia
l a
ctiv
ity o
f C
om
po
un
ds
(7a
-h)
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Gra
ph
3:
An
tim
icro
bia
l a
ctiv
ity o
f C
om
po
un
ds
(8a
-h)
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Gra
ph
4:
An
tim
icro
bia
l a
ctiv
ity o
f C
om
po
un
ds
(9a
-h)
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Gra
ph
5:
An
tim
icro
bia
l a
ctiv
ity o
f C
om
po
un
ds
(10
a-h
)
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The compounds tested for antimicrobial activity are listed in Table:7.2 –
7.6 show size of zone of inhibition of bacterial growth procedure by test
compounds for broad range of antimicrobial activity inhibiting growth of
Gram-positive bacterial strains B.Subtillis and S.Aureus, and Gram-negative
bacterial strains E.Coli and Ps. Aeruginosa.
Comparison of antimicrobial activity of produced compounds with that
of standard antimicrobial drugs reveals that the produce compounds (Schiff
Bases, 2-Azetidinones, 4-Thiazolidinones, 2H-Pyrrole-2-ones and 2-
Pyrrolidinones) shows moderate to good activity against all four bacterial
strains.
Among 4-(3-chloro-2-oxo-4-phenylazetidin-1-yl)-N-(4-(2,4-dichlorophenyl)-6-
(6-methylnaphthalen-2-yl)pyrimidin-2-yl)benzenesulfonamide (7a-h)
(Table-7.3) compounds 7c, 7g and 7h shows good antimicrobial activity.
Among N-(4-(2,4-dichlorophenyl)-6-(6-methylnaphthalen-2-yl)pyrimidin-2-yl)-
4-(4-oxo-2-phenylthiazolidin-3-yl)benzenesulfonamide (8a-h).
(Table-7.4) compounds, 8e, 8d and 8f show good antimicrobial activity.
Among 1-(4-(N-(4-(2,4-dichlorophenyl)-6-(6-methylnaphthalen-2-yl)pyrimidin-2-
yl)sulfamoyl)phenyl)-5-oxo-2-phenyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid (9a-h)
(Table-7.5) compound , 9e, 9d and 9f show good antimicrobial activity.
Among 1-(4-(N-(4-(2,4-dichlorophenyl)-6-(6-methylnaphthalen-2-yl)pyrimidin-2-
yl)sulfamoyl)phenyl)-5-oxo-2-phenylpyrrolidine-3-carboxylic acid (10a-h). (Table-7.6)
compound 10b, 10c and 10d show good anti-microbial activity.
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Other prepared compounds shows moderate activity compared to
standard drugs against all four bacterial strains B.Subtillis, S.Aureus, E.Coli
and Ps. Aeruginosa.
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REFERENCES
1. B.Willium
“The textbook of Microbiology”, W.B. Saunders Co., London, 16th
edition, pp.12 and pp.145 (1945).
2. W.Robert and E.G.Scott
“Diagnostic Microbiology”, The C.V. Mosby Co., Saint Louis, 2nd
edition, pp. 318 (1966).
3. C.Robert
“Medical Microbiology”, ELBS, Livingston, 11th
edition, pp.815 and
901 (1970).
4. G.D.Sujatha et al.
Ind. J. Expt. Biol., 13, 286 (1975).
5. S.A.Walksman
“Microbial Antagonism and Antibiotic Substances”, Commonwealth
Fund, N.Y., 2nd
edition, pp. 72 (1947).
6. R.Cruickshank, J.P.Dugid, D.P.Marmion and R.H.A.Swain,
"Medical Microbiology”, Churchil-Livingstone, Edinburgh, London,
Vol. 2, 12th
edition (1975).