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.

Chapter 7

Page 252

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

Chapter 7

Page 253

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”.

Chapter 7

Page 254

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.

Chapter 7

Page 255

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

Chapter 7

Page 256

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

Chapter 7

Page 257

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.

Chapter 7

Page 258

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

Chapter 7

Page 259

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

Chapter 7

Page 260

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

Chapter 7

Page 261

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

Chapter 7

Page 262

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

Chapter 7

Page 263

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

Chapter 7

Page 264

Gra

ph

1:

An

tim

icro

bia

l a

ctiv

ity o

f C

om

po

un

ds

(6a

-h)

Chapter 7

Page 265

Gra

ph

2:

An

tim

icro

bia

l a

ctiv

ity o

f C

om

po

un

ds

(7a

-h)

Chapter 7

Page 266

Gra

ph

3:

An

tim

icro

bia

l a

ctiv

ity o

f C

om

po

un

ds

(8a

-h)

Chapter 7

Page 267

Gra

ph

4:

An

tim

icro

bia

l a

ctiv

ity o

f C

om

po

un

ds

(9a

-h)

Chapter 7

Page 268

Gra

ph

5:

An

tim

icro

bia

l a

ctiv

ity o

f C

om

po

un

ds

(10

a-h

)

Chapter 7

Page 269

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.

Chapter 7

Page 270

Other prepared compounds shows moderate activity compared to

standard drugs against all four bacterial strains B.Subtillis, S.Aureus, E.Coli

and Ps. Aeruginosa.

Chapter 7

Page 271

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).