chapter-6 antimicrobial studies of newer...

Chapter-6

ANTIMICROBIAL STUDIES OF NEWER COMPOUNDS

_____________________________________________________________________

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

Chapter-6

240

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.

Chapter-6

241

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”.

Chapter-6

242

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.

Chapter-6

243

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.

Chapter-6

244

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

Chapter-6

245

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.

Chapter-6

246

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

Chapter-6

247

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)



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

Chapter-6

248

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)




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

Chapter-6

249


6- hydroxy- benzoyl amino]-2-aryl-5-(phenyl arylidine)

thiazolidine-4-ones (5a-5h)

Compound

(designation)




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

Chapter-6

250

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)




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

Chapter-6

251

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)




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

Chapter-6

252


6-hydroxy- benzoyl amino]-2-oxo-5-aryl-3,5-dihydro-1H-

pyrrole-4-carboxylic acid (8a-8h)

Compound

(Designation)




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

Chapter-6

253


6-hydroxy- benzoyl amino]-2-oxo-5-aryl-pyrrolidinone-4-

carboxylic acid (9a-9h)

Compound

(designation)




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

Chapter-6

254

Fig. 6.1 Antimicrobial activity of compound 3a, 4c, 5e, 6a and 7c

(B.Subtillis)

Chapter-6

255

Fig. 6.2 Antimicrobial activity of compound 3a, 4c, 5e and 6h

(Ps.Aeruginosa)

Chapter-6

256

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.

Chapter-6

257

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).

chapter-6 antimicrobial studies of newer...

Documents