chapter-6 antimicrobial studies of newer...
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Chapter-6
ANTIMICROBIAL STUDIES OF NEWER COMPOUNDS
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Overview
This Chapter includes the antimicrobial evaluation of all the compounds mentioned
in Chapters 3 to 5.
7.1 INTRODUCTION
Infection is a major category of human disease and antimicrobial agents are the prime
need of the society. 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 harming the host. Great attention has been paid for
curing diseases caused by microorganisms. Many infectious pathogenic
microorganisms develop resistance against the prevailing drugs, and this situation has
necessitated a search for advanced and novel antimicrobial drugs from generation to
generation. Heterocyclic compounds, particularly five- and six-membered ring
compounds have occupied a prominent place among the organic compounds in view
of their diverse biological activities.
Many substances that we now know 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
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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 coined the term ‘chemotherapy’ in 1906 and 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 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 perspectives on drugs.
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, Florey and Chain undertook an investigation of antibiotics, that is, 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.
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.
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A few antimicrobials have useful activity across several of these groups. Few
examples, metronidazole inhibits obligate anaerobic bacteria (such as 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 agents: act primarily by arresting bacterial multiplication, such
as sulphonamides, tetracyclines and chloramphenicol.
Bactericidal agents: 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 of per iodic acid,
Gram strains, and
Zeil-Nelsonm acid fast strains.
7.6 EVALUATION METHODS
The following conditions 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.
A septic/sterile environment should be maintained.
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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 of three types:
i. Agar cup method,
ii. Agar ditch method, and
iii. Paper disc Method.
In present study Agar cup diffusion method is used.
7.7 FACTORS AFFECTING ZONE OF INHIBITION
7.7.1 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.
7.7.2 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.
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7.7.3 Effect of pH
The activity of amino glycosides is enhanced in alkaline media and reduced in acidic
media, the reverses is shown by tetracycline.
7.7.4 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
7.8.1 The culture medium preparation
Nutrient agar medium was used. Chemical composition of the medium used in the
present study is as follow:
Peptone 1.0 g
NaCl 0.5 g
Meat extract 0.3 g
Distilled water 100 ml
pH 7.6
Agar 2.0 g
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
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different test-tubes. The test-tubes were plugged by cotton-wool and sterilized at
121.5C and 15 pounds per square inch (psi) pressure for 15 min.
7.8.2 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 45C with gentle
shaking to bring out uniform cooling. It was inoculated with 0.5-0.6 ml of 24 h 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 h) and there after
the “cups” were made by punching into the agar surface with a sterile cork borer and
sooping out the punched part of agar. Into these “cups,” 0.1 ml of test solution
(prepared by dissolving 10 g of a sample in 10 ml 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-6.1.
The results shown by compounds and standards are corrected for DMF.
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Table 6.1 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 6 6 6 6
2 Ampicillin 18 13 20 20
3 Tetracyclin 20 16 17 23
4 Gentamycin 20 20 20 20
5 Chloramphenicol 19 25 17 22
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Table 6.2 Antimicrobial activity of [Arylidine-3-(3-(isoindol-1,3-dione
methyl)-6-hydroxy- benzoyl amine)] (3a-3h)
Compound
(Designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
3a 10 14 14 11
3b 11 14 09 09
3c 13 14 13 11
3d 10 12 12 08
3e 16 18 13 17
3f 12 11 09 14
3g 19 20 13 16
3h 11 14 16 13
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Table 6.3 Antimicrobial activity of 3-[3-(isoindol-1,3-dione methyl)-
6-hydroxy- benzoyl amino]-2-aryl-thiazolidine-4- ones (4a-4h).
Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
4a 09 09 16 08
4b 10 10 14 10
4c 11 10 11 16
4d 17 14 19 14
4e 07 09 10 08
4f 10 12 10 11
4g 08 07 10 12
4h 10 15 11 13
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Table 6.4 Antimicrobial activity of 3-[3-(isoindol-1,3-dione methyl)-
6- hydroxy- benzoyl amino]-2-aryl-5-(phenyl arylidine)
thiazolidine-4-ones (5a-5h)
Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
5a 09 13 10 11
5b 10 10 11 08
5c 11 09 07 08
5d 13 11 18 12
5e 11 13 14 09
5f 19 16 16 21
5g 14 12 14 13
5h 17 15 17 22
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Table 6.5 Antimicrobial activity of 2,3-di phenyl-5-substituted phenyl-
6-[3-[3-(isoindol-1,3-dione methyl)-6- hydroxy-benzoyl-amino]-
1-yl]-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d]thiazole derivatives
(6a-6h)
Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
6a 11 11 10 18
6b 17 14 13 13
6c 18 15 14 11
6d 24 19 17 17
6e 14 17 16 09
6f 14 12 10 11
6g 13 14 09 18
6h 12 16 15 14
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Table 6.6 Antimicrobial activity 3-chloro-1-[3-(isoindol-1,3-dione methyl)-
6-hydroxy- benzoyl amino]-4-aryl- azetidin-2-ones (7a-7h)
Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
7a 11 10 11 15
7b 16 12 13 17
7c 15 11 08 18
7d 17 16 15 12
7e 10 08 10 14
7f 14 13 09 10
7g 08 08 05 12
7h 17 16 16 21
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Table 6.7 Antimicrobial activity of 1-[3-(isoindol-1,3-dione methyl)-
6-hydroxy- benzoyl amino]-2-oxo-5-aryl-3,5-dihydro-1H-
pyrrole-4-carboxylic acid (8a-8h)
Compound
(Designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
8a 12 14 14 12
8b 11 12 09 09
8c 14 14 09 11
8d 10 11 12 08
8e 15 18 13 18
8f 12 11 18 14
8g 15 10 13 16
8h 09 14 05 13
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Table 6.8 Antimicrobial activity of 1-[3-(isoindol-1,3-dione methyl)-
6-hydroxy- benzoyl amino]-2-oxo-5-aryl-pyrrolidinone-4-
carboxylic acid (9a-9h)
Compound
(designation)
Zone of Inhibition (in mm)
Gram positive Gram negative
B.Subtillis S.Aureus E.Coli Ps.Aeruginosa
9a 09 13 10 11
9b 12 10 11 18
9c 11 09 07 08
9d 13 11 17 12
9e 11 14 14 09
9f 13 16 16 22
9g 14 12 15 13
9h 18 15 17 22
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Fig. 6.1 Antimicrobial activity of compound 3a, 4c, 5e, 6a and 7c
(B.Subtillis)
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Fig. 6.2 Antimicrobial activity of compound 3a, 4c, 5e and 6h
(Ps.Aeruginosa)
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The compounds tested for antimicrobial activity are listed in Tables 6.2 –6.8, these
tables 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) show
moderate to good activity against all four bacterial strains.
Among (3a-3h) (Table 7.2), compounds 3a, 3c and 3e show good antimicrobial
activity.
Table 7.3 indicates that the compounds 4b, 4d and 4h have good antimicrobial
activity.
Among (5a-5h) (Table 7.4), compounds 5d, 5f and 5g exhibit good antimicrobial
activity.
Table 7.5 predicts that the compounds 6a, 6d and 6h have good anti-microbial
activity.
Among (7a-7h) (Table 7.6), compounds 7b, 7d and 7h exhibit good antimicrobial
activity.
Table 7.7 reveals that compounds 8a, 8e and 8f possess good antimicrobial activity.
Among (9a-9h) (Table 7.8), compounds 9b, 9f and 9h show good antimicrobial
activity.
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Other prepared compounds show moderate activity compared to standard drugs
against all four bacterial strains B.Subtillis, S.Aureus, E.Coli and Ps. Aeruginosa.
REFERENCES:
1. B.William, “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).