ANTIBACTERIAL ACTIVITY - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/2520/11/11_chapter...

Chapter - IV: Antimicrobial Activity

ANTIBACTERIAL ACTIVITY

INTRODUCTION

The science dealing with the study of the prevention and treatment of

diseases caused by micro-organisms is known as medical microbiology. Its sub-

disciplines are virology (study of viruses), bacteriology (study of bacteria),

mycology (study of fungi), phycology (study of algae) and protozoology (study of

protozoa). For the treatment of diseases inhibitory chemicals employed to kill

micro-organisms or prevent their growth, are called antimicrobial agents. These

are classified according to their application and spectrum of activity, as germicides

that kill micro-organisms, whereas micro-biostatic agents inhibit the growth of

pathogens and enable the leucocytes and other defense mechanism of the host to

cope up with static invaders. The germicides may exhibit selective toxicity

depending on their spectrum of activity. They may act as viricides (killing viruses),

bacteriocides (killing bacteria), algicides (killing algae) or fungicides (killing fungi).

The beginning of modern chemotherapy has largely been due to the efforts

of Dr. Paul Ehrlich (1910), who used salvarsan, as arsenic derivative effective

against syphilis. Paul Ehrlich used the term chemotherapy for curing the infectious

disease without injury to the host’s tissue, known as chemotherapeutic agents

such as antibacterial, antiprotosoal, antiviral, antineoplastic, antitubercular and

antifungal agents. Later on, Domagk (1953) prepared an important

chemotherapeutic agent sulfanilamide.

CLASSIFICATION OF ANTIBACTERIAL AGENTS

The antibacterial agents are classified in three categories:

197


(I) Antibiotics and chemically synthesized

chemotherapeutic agents.

(II) Non-antibiotic chemotherapeutic agents

(Disinfectants, antiseptics and preservatives)

(III) Immunological products.

(I) Antibiotics

They are produced by micro-organisms or they might be fully or partly

prepared by chemical synthesis. They inhibit the growth of micro-organisms in

minimal concentrations. Antibiotics may be of microbial origin or purely synthetic

or semisynthetic.1 They can be classified by manner of biosynthesis or chemical

structure. Structurally, they are classified into different classes as shown in the

following table.

CLASSIFICATION OF ANTIBIOTICS ACCORDING TO THEIR CHEMICAL

STRUCTURE (Berdy, 1974)2

No. Name of the group Example

1. Carbohydrate-containing antibiotics

Pure sugars

Aminoglycosides

Orthosymycins

N-Glycosides

C-Glycosides

Glycolipids

Nojirimycin

Streptomycin

Everninomicin

Streptothricin

Vancomycin

Moenomycin

2. Macrocyclic lactones

Macrolide antibiotics

Polyene antibiotics

Erythromycin

Candicidin

198


Ausamycins

Macrotetrolides

Rifamycin

Tetranactin

3. Quinones and related antibiotics

Tetracyclines

Anthracyclines

Naphthoquinones

Benzoquinones

Tetracycline

Adriamycin

Actinorhodin

Mitomycin

4. Amino acid and peptide antibiotics

Amino acid derivatives

β-Lactum antibiotics

Peptide antibiotics

Chromopeptides

Depsipeptides

Chelate forming peptides

Cycloserine

Penicillin

Bacteriacin

Actinomycins

Valinomycin

Bleomycins

5. Heterocyclic antibiotics containing oxygen

Polyether antibiotics Monensin

6. Heterocyclic antibiotics containing nitrogen

Nucleoside antibiotics Polyoxins

7. Aromatic antibiotics

Cycloalkane derivatives

Steriod antibiotics

Cycloheximide

Fusidic acid

8. Aromatic antibiotics

Benzene derivatives

Condensed aromatic antibiotics

Aromatic ether

Chloramphenicol

Griseofulvin

Novobiocin

9. Aliphatic antibiotics

Compounds containing phosphorous Fosfomycins

199


Synthetic antimicrobial agents include sulfonamides, diamino pyrimidine

derivatives, antitubercular compounds, nitrofuran compounds, 4-quinoline

antibacterials, imidazole derivatives, flucytosine etc.

(II) Non-antibiotics

The second category of antibacterial agents includes non-antibiotic

chemotherapeutic agents which are as follows:

1) Acids and their derivatives

Some organic acids such as sorbic, benzoic, lactic and propionic acids are

used for preserving food and pharmaceuticals. Salicyclic acid has strong antiseptic

and germicidal properties as it is a carboxylated phenol. The presence of –COOH

group appears to enhance the antiseptic property and to decrease the destructive

effect. Benzoic acid is used externally as an antiseptic and is employed in lotion

and ointment. Benzoic acid and salicylic acid are used to control fungi that cause

disease such as athlete’s foot. Benzoic acid and sodium benzoate are used as

antifungal preservatives. Mandolic acid possesses good bacteriostatic and

bactericidal properties.

2) Alcohols and related compounds

They are bactericidal and fungicidal, but are not effective against

endospores and some viruses. Various alcohols and their derivatives have been

used as antiseptics e.g. ethanol and propanol. The antibacterial value of straight

chain alcohols increases with an increase in the molecular weight and beyond C8-

the activity begins to fall off. The isomeric alcohol shows a drop in activity from

primary, secondary to tertiary. Ethanol has extremely numerous uses in

pharmacy.

200


3) Chlorination and compound containing chlorine

Chlorination is extensively used to disinfect drinking water, swimming pools

and for the treatment of effluent from industries. Robert Koch in 1981 first

referred to the bactericidal properties of hypochlorites. N-chloro compounds are

represented by amides, imides and amidines wherein one or more hydrogen

atoms are replaced by chlorine.

4) Iodine containing compounds

Iodine containing compounds are widely used as antiseptic, fungicide and

amoebicide. Iodophores are used as disinfectants and antiseptics. The soaps used

for surgical scrubs often contain iodophores.

5) Heavy metals

Heavy metals such as silver, copper, mercury and zinc have antimicrobial

properties and are used in disinfectant and antiseptic formulations.

Mercurochrome and merthiolate are applied to skin after minor wounds. Zinc is

used in antifungal antiseptics. Copper sulfate is used as algicides.

6) Oxidising agents

Their value as antiseptics depends on the liberation of oxygen and all are

organic compounds.

7) Dyes

Organic dyes have been extensively used as antibacterial agents. Their

medical significance was first recognized by Churchman3 in 1912. He reported

inhibitory effect of Crystal violet on Gram-positive organism. The acridines exert

bactericidal and bacteriostatic action against both Gram-positive and Gram-

negative organisms.

8) 8-Hydroxyquinolines

201


8-Hydroxyquinoline or oxine is unique among the isomeric hydroxyl-

quinolines, for it alone exhibits antimicrobial activity. This attributes to its ability

to chelate metals4, which the other isomers do not exhibit.

9) Surface active agents

Soaps and detergents are used to remove microbes mechanically from the

skin surface. Anionic detergents remove microbes mechanically; cationic

detergents have antimicrobial activities and can be used as disinfectants and

antiseptics.

(III) Immunological products

Certain immunological products such as vaccines and monoclonal

antibodies are used to control the diseases as a prophylactic measure.

MODE OF ACTION

Antimicrobial drugs interfere chemically with the synthesis of function of

vital components of micro organisms. The cellular structure and functions of

eukaryotic cells of the human body. These differences provide us with selective

toxicity of chemotherapeutic agents against bacteria.

Antimicrobial drugs may either kill microorganisms outright or simply

prevent their growth. There are various ways in which these agents exhibit their

antimicrobial activity.5 They may inhibit

(1) Cell-wall synthesis

(2) Protein synthesis

(3) Nucleic acid synthesis

(4) Enzymatic activity

(5) Folate metabolism or

(6) Damage cytoplasmic membrane

202


Bacteriostatic dyes

Stearn and Stearn6 attributed the bacteriostatic activity to

triphenylmethane dyes. Fischer and Munzo7 have found the relationship between

their structure and effectiveness of such dyes.

A number of drugs are metal-binding agents. The chelates are the active

form of drugs. The site of action within the cell or on the cell surface has not

been established. The site of action of oxine and its analogs has been suggested

inside the bacterial cell8 or on cell surface.9

Detoxification of antibacterials

P-Aminobenzoic acid is a growth factor for certain micro-organisms and

competitively inhibits the bacteriostatic action of sulfonamides. The metabolites

identified in man are p-amino-benzoylglucoronide; p-aminohippuric acid,

p-acetylaminobenzoic acid. 8-Hydroxyquinoline (oxine) and 4-hydroxyquinoline

are excerted as sulfate esters or glucorinides.

Bacteria

The bacteria are microscopic organisms with relatively simple and primitive

forms of prokaryotic type. Danish Physician Christian Grams, discovered the

differential staining technique known as Gram staining, which differentiates the

bacteria into two groups “Gram positive” and “Gram negative”, Gram positive

bacteria retain the crystal violet and resist decolorization with acetone or alcohol

and hence appear deep violet in colour; while Gram negative bacteria, which

loose the crystal violet, are counter-stained by saffranin and hence appear red in

colour.

203


These two groups of bacteria are recently classified into four different

categories as follows:

(1) The world of bacteria I: “Ordinary” Gram negative bacteria.

(2) The world of bacteria II: “Ordinary” Gram positive bacteria.

(3) The world of bacteria III: “Bacteria” with unusual properties.

(4) The world of bacteria IV: Gram positive filamantous bacteria of

complex morphology.

CLASSIFICATION OF IMPORTANT ORGANISMS

Class: Schizomycetes

Order Family Genus Species

Eubacterials Micrococeacea Staphylococcus

Micrococcus

Sarcina

S.Aureus

M.tetragenus

S.lutea

Lactobacill-aceae Streptococcus

Peptoatrepto-coccus

Lactobacillus

Diplococcus

Str.pygenes

Pep.putridis

L.Acidophilus

D.pneumoniae

Neisseriaceae Neisseria N.gonorrhoeae

N.meningitidi

N.catarrhalis

Corynebacte-riaceae

Corynebacterium

Listeria

Etysipeothrix

C.dipthheriae

L.monocytogene

L.rhusiopathiae

Achromobacte-riaceae

Alcaligenes Alc.faecalis

204


Enterobacte-riacea Eschercichia

Klebsiella

Citrobacter

Cloaca

Hafnia

Serratia

Salmonella

Shigella

Proteus

K.pneumoniae

K.pneumoniae

K.aerogenes

Cit.freundii

Cl.cloacae

Haf.alvei

Ser.marcescens

Sal.typhosa

Sh.dysenterise

Pr.vulgaris

Eubacterials Brucellaceae Pasteurella

Fancisella

Brucella

Haemophilus

Bordetella

Morarella

Actinubacillus

P.pestis

P.pseudotuber-culosis

F.tularensis

Br.meltitensis

Br.abortus

Br.suis

H.influenzae

H. duoreyi

Bord.pertussia

M.lacunata

A.mallei

A.lignieresii

Bacteriodacess Bacteriods

Fusobacterium

Strepto-bacillus

Sphaerophorous

Bact.fragilis

F.fusiforme

St.moniliformis

Sph.nacrophorous

Bacillaceae Bacillus B.anthracis

205


Clostridium

B.subtilis

Cl.tetani

Cl.welchii

Order Family Genus Species

Pseudo-

monadales

Pseudo-

monadaceae

Pseudomonas Ps.aeruginosa

Spirillaceae Vibrio

Spirillum

V.cholerae

Sp.minus

Mycoplasm-

atales

Mycoplasma-taceae Mycoplasma M.pneumoniae

M.mycoids

Actinomyce-

tates

Mycobacteri-aceae Mycobacterium M.tuberculosis

M.laprae

Actinomycet-aceae Actinomyces

Nocardia

A.israeli

A.bovis

N.modurae

Streptomycet

aceae

Streptomyces Strepto.griseus

Spirochaetel

-es

Spirochaet-aceae Spirochaeta

Saprospira

Nonpathogenic

Treponenat-

aceae

Borrelia Bor.duttoni

Bor.recurrentis

Bor.vincenti

206


Taeponema

Leptospira

Tr.pallidum

Tr.pertenue

L.icterohaemo-rrhagiae

(1) Staphylococcus aureus: Family (micrococcaceae)

In 1878, Koch observed micrococcus like organisms in pus; Pasteur (1880)

cultivated these cocci in liquid media. They are Gram-positive cocci, ovoid or

spheroidal, non-motile, arranged in group of clusters; they grow on nutrient agar

and produce colonies, which are golden yellow, white or lemon yellow in colour;

pathogenic strains produce, coagulated and ferment glucose lactose, mannitol

with production of acid, liquefy gelation and produce pus in the lesion.

Genus: Staphylococcus

The word staphylococcus is derived from the Greek language (Gr. Staphylo

= bunch of grapes; Gr. Coccus = a grain or berry), while the species name is

derived from Latin language (L. aureus = golden). Staphylococcus is

differentiated from micrococcus and another genus of the same family by its

ability to utilize glucose, mannitol and pyruvate anaerobically. Cells of

staphylococci, which are slightly smaller than those of Micrococci, are found on

the skin or mucus membrane of the animal body.

Species: Staphylococcus aureus

Basic habital of St. aureus is the anterior naves, though it is also a normal

flora of human skin, and of the respiratory and gastrointestinal tracts. The

individual cells are 0.8 to 0.9 μ in diameter. They are oval or spherical, non-

motile, non-capsulated, non-sporulating strains with ordinary aniline dyes and are

Gram-positive, typically arranged in groups or irregular clusters like branches of

groups in pus seen single or in pairs. They easily grow on nutrient agar; the

optimum temperature for the growth is 35 ºC. They are notorious as they cause

207


suppurative (pyogenic or pus forming) conditions, mostitis of women and cows,

boils and food poisioning. St. aureus grows rapidly and produce circular (1-2 mm)

endive edge, convex, soft, glistening colonies having a golden yellow pigment. St.

aureus can tolerate moderately high concentration of NaCl, hence they can be

selectively isolated on the nutrient medium containing 7.5 % sodium chloride. It is

also able to ferment mannitol to organic acid. St. aureus also produce the

coagulase which is able to clot citrated plasma. It also produces the enzymes

catalase, hyaluronidase as well as other virulent factors like hemolysins,

leucocidins, enterotoxins and exofoliatin.

(2) STREPTOCOCCUS PYOGENES

Genus: Streptococcus

The term Streptococcus was first introduced by Bilroth [1874] and the term

Streptococcus Pyogenes was used by Rosenbach [1884]. These are spherical or

ovoid cells; divide in one axis and form chains; nonmotile and nonsporing. The

growth is absence of native proteins in the medium; they produce characteristic

haemolytic changes in media containing blood; produce acid only by fermentation

of carbohydrates; often fail t liquefy gelatin; some strains produce exotoxin and

extracellular products; a few of them are Anaerobic.

Species: Streptococcus Pyogenes

Streptococcus Pyogenes is pathogenic to human and found in sore throat,

follicular tonsillitis, septicemia, acute or malignant ulcerative endocarditis etc.

These are spherical Cocci 0.5 to 0.75 micro in diameter, arranged in moderately

long chains of round Cocci and easily differentiated from Enterococci that from

short chains of 2 to 4 spheres. Streptococcus Pyogenes is recently isolated from

throat or other lesions; they show either mucoid or matt colonies. On keeping in

the laboratory, they undergo varation to a glossy type. Streptococci are

susceptible to destructive agents, and to penicillin and sulphomamides.

208


(3) Escherichia coli : Enterobacteriaceae

They are Gram-negative rods, motile with peritrichate flagella or non-

motile. They do not form spores. All are sometimes (i.e. from rarely to,

invariably ) found in intestinal treatment of man or lower animals.

Genus: Escherichia

This genus comprises Escherichia coli and several variants.

Species: Escherichia coli

Escherichia in 1885 discovered Escherichia coli which is a commensal of the

human intestine and is found in the sewage, water or soil contaminated by faecal

matters. These are Gram-negative rods, 2 to 4 μ, commonly seen in

coccobacillary form, which do not form any spore and have 4 to 8 paritrichate

flagella, are sluggishly motile, are facultative anaerobes and grow in laboratory

media. E. coli are generally non-pathogenic and are incriminated as pathogens,

because in certain instance some strains have been found to produce septicemia,

inflammation of liver and gall bladder, appendix and other infections and this

species is a recognized pathogen in the veterinary field.

(4) Pseudomonas aeruginosa

Genus: Pseudomonas

Pseudomonas is a Greek word (Gr. Pseudo = false, Gr. Monas = a unit)

while the word aeruginosa is of Latin origin (L. aeruginosa = full of copper rust

i.e. green).

Species: Pseudomonas aeruginosa

P.aeruginosa is Gram-negative short rod with variable length (1.5-3.0 x 0.5

μm). They are motile by means of one or two polar flagella. Organisms are non-

sporulating and non-capsulated, however, few strains possess slime layer up of

polysaccharide. Primary habitat of P.aeruginosa is human and animal gastro-

209


intestinal tract, water, sewage, soil and vegetation. It is physiologically versatile

and flourishes as a saprophyte in warm moist situations in the human

environment, including sinks, drains, respirators, humidifiers, etc. P.aeruginosa

produces several virulence factors, including exotoxin A., proteases, a leukocidin,

and phospholipase C. pseudomonas is an opportunistic pathogen which is able to

cause infections when the natural resistance of the body is low. They are mostly

related with hospital infections and post burn infections. They also cause

infections of middle ear, eyes and urinary tracts. It is also associated with

diarrhoea, pneumonia and osteomyelitis. Due to drug resistant nature of P.

aeruginosa it causes infection in patients receiving long term antibiotic therapy for

wounds, burns and cystic fibrosis and other illness. Approximately 25% of burn

victims develop infection which frequently leads to fatal septicemia.

EVALUTION TECHNIQUES

The following conditions must be met for the screening of antimicrobial

activity:

There should be intimate contact between the test organisms and

substance to be evaluated.

Required conditions should be provided for the growth of

microorganisms.

Conditions should be same through the study.

Aseptic / sterile environment should be maintained.

Various methods have been used from time to time by several workers to

evaluate the antimicrobial activity. The evaluation can be done by the following

methods:

• Turbidometric method.

• Agar streak dilution method.

• Serial dilution method.

210


• Agar diffusion method.

Following Techniques are used as agar diffusion method:

• Agar Cup method.

• Agar Ditch method.

• Paper Disc method.

We have used the Agar cup Method to evaluate the antibacterial activity. It is

one of the non automated in vitro bacterial susceptibility tests. This classic

method yields a zone of inhibition in mm result for the amount of antimicrobial

agents that is needed to inhibit growth of specific microorganisms. It is carried

out in Petri plates.

EXPERIMENTAL

MATERIALS AND METHODS

211


The bacteriostatic property of the compounds was tested by disc diffusion

method as described by Bauer kirby’s method.

[A] Preparation of Mueller-Hinton agar

(1) Beef infusion : 300 g

(2) Acid hydrolysate of casein : 17.5 g

(3) Starch : 1.5 g

(4) Agar : 17 g

(5) Distilled water : 1 Lit.

The above constituents were weighed and dissolved in water. The mixture

was warmed on water bath till agar dissolved. This was then sterilized in an

autoclave at 15 lbs pressure and 121 oC for fifteen minutes. The sterilized medium

(20 ml) was poured in sterilized Petri dishes under aseptic condition, allowing

them to solidify on a plane table.

[B] Preparation of Antibacterial Solution

All the compounds were dissolved in dimethyl formamide (DMF). Proper

drug controls were used. Fig. 1 and Fig. 2 represent the zone of inhibition of the

control and the compound.

Compound was taken at concentration of 100 µg/ml for testing

antibacterial activity. The compound diffused into the medium produced a

concentration gradient. After the incubation period, the zones of inhibition were

measured in mm. The tabulated results represent the actual readings control.

[C] Test cultures

212


Following common standard strains were used for screening of antibacterial

and antifungal activities:

• Escherichia coli [Gram negative] MTCC – 443

• Pseudomonas aeruginosa [Gram negative] MTCC – 424

• Staphylococcus aureus [Gram positive] MTCC – 96

• Streptococcus Pyogenes [Gram positive] MTCC – 442

• Candida albicans [Fungus] MTCC – 227

• Aspergillus Niger [Fungus] MTCC – 282

[D] Inoculum’s preparation

The inoculum was standardized at 1* 106 CFU/ml comparing with turbidity

standard (0.5 MacFarland tube)

[E] Swabs preparation

A supply of cotton wool swabs on wooden applicator sticks was prepared.

They were sterilized in tins, culture tubes, or on paper, either in the autoclave or

by dry heat.

[F] Experimental procedure

213


1) The plates were inoculated by dipping a sterile swab into inoculums.

Excess inoculum was removed by pressing and rotating the swab firmly

against the side of the tube, above the level of the liquid.

2) The swab was streaked all over the surface of the medium three times,

rotating the plate through an angle of 60 oC after each application. Finally

the swab was passed round the edge of the agar surface. The inoculation

was dried for a few minutes, at room temperature, with the lid closed.

3) Ditch the bore in plate. Add compounds solution in bore.

4) The plates were placed in an incubator at 37 oC within 30 minutes of

preparation for bacteria and 22 oC for fungal.

5) After 48 hrs incubation for bacteria and 7-days for fungal, the diameter of

zone (including the diameter disc) was measured and recorded in mm. The

measurements were taken with a ruler, from the bottom of the plate,

without opening the lid.

Results are presented in Table-36 to 42.

214


Fig-1

Showing cylinder cup method (Agar Diffusion Technique) with essential

arrangement (diagrammatic)

• Fig-2

215

Petridis

Heavy growth

Zone of inhibition

Cylinder cup


TABLE-36

Antibacterial activity of Standard drugs

Antifungal activity of Standard drugs

Standard

Zone of inhibition* (mm) (activity index)std

C. albicans A. niger

Nystatin 22 25

Standard

Zone of inhibition* (mm) (activity index)std

Gram positive Gram negative

S. aureus S. pyogenes E. coli P. aeruginosa

Chloramphenicol 20 20 23 19

Ciprofloxacin 22 21 28 26

216


Greseofulvin 27 29

*= average zone of inhibition in mm,

Activity index = Inhibition zone of the sample / Inhibition zone of the standard

TABLE-37

Antibacterial activity: Section-II

N N

NH2

R R'

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

Compound

Zone of Inhibition* (mm) (Activity Index) std.


S. aureus S. pyogenus E. coli P. aeruginosa

A-17 10 09 10 08

A -18 18 15 19 16

A -19 16 15 18 14

217


A -20 11 10 13 10

A-21 17 16 20 15

A-22 10 11 12 11

A-23 15 16 15 14

A-24 17 17 20 18

A-25 14 12 14 13

A-26 10 10 12 11

A-27 07 09 11 08

A-28 11 11 13 10

A-29 08 10 11 09

A-30 15 14 16 14

A-31 15 16 17 15

A-32 10 11 08 07

TABLE-38

Antibacterial activity: Section-IV (Part-A)

N N

NH

R R'

NCl

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

218


TABLE-39

Antibacterial activity: Section-IV (Part-B)

N N

NH

R R'

N

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

CH3

H3C

Compound




A-37 11 13 12 11

A-38 19 19 21 17

A-39 17 18 20 18

A-40 15 14 15 14

A-41 18 17 21 17

A-42 15 14 18 15

A-43 14 11 12 11

A-44 19 17 19 17

A-45 12 14 13 10

A-46 16 15 18 17

A-47 10 08 12 09

A-48 15 16 19 16

A-49 11 12 14 11

A-50 17 18 18 16

A-51 11 13 12 13

A-52 09 10 09 10

219


TABLE-40

Antibacterial activity: Section-IV (Part-C)

N N

NH

R R'

N

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

CH3

Cl

Compound




A-53 12 11 13 10

A-54 19 18 20 18

A-55 18 17 19 16

A-56 15 13 14 13

A-57 18 16 19 17

A-58 14 12 17 13

A-59 16 15 17 13

A-60 19 18 21 16

A-61 16 14 17 13

A-62 15 14 15 15

A-63 12 13 14 12

A-64 13 13 16 12

A-65 09 11 07 10

A-66 19 17 21 17

A-67 14 15 16 14

A-68 11 08 10 09

220


TABLE-41

Antibacterial activity: Section-IV (Part-D)

N N

NH

R R'

N

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

CH3

H3CO

Compound




A-69 11 10 13 10

A-70 17 18 20 17

A-71 18 17 19 16

A-72 15 13 14 15

A-73 18 17 19 17

A-74 14 13 17 14

A-75 12 11 14 13

A-76 18 18 20 17

A-77 11 13 10 11

A-78 15 14 15 14

A-79 09 10 13 08

A-80 13 14 16 14

A-81 10 09 13 11

A-82 14 13 13 11

A-83 12 11 16 13

A-84 11 09 07 10

221


TABLE-42

Antibacterial activity: Section-V

N N

NH

Br

H3CO

Cold brand reactive dye

Compound




A-85 12 14 16 14

A-86 18 19 21 18

A-87 19 18 20 17

A-88 16 14 15 16

A-89 19 18 20 18

A-90 15 14 18 15

A-91 14 12 14 11

A-92 17 19 18 17

A-93 13 08 11 08

A-94 16 15 16 14

A-95 13 14 15 12

A-96 14 15 17 15

A-97 12 11 13 10

A-98 15 14 15 13

A-99 12 11 09 12

A-100 08 12 13 10

222


ANTIFUNGAL ACTIVITY

INTRODUCTON

There are perhaps over 10,000 species of fungi, but less than 100 cause

diseases in human.10 Fungi may cause benign, but unsightly infections of the skin,

nail or hair, relatively trivial infection of mucous membranes (thrush) or systemic

infection causing progressive often fatal disease.

Compound




A-101 Nil Nil Nil Nil












223


CLASSIFICATION OF MEDICALLY IMPORTANT FUNGI 11

1. True yeasts (e.g. Cryptococcus neoformans)

2. Yeast like fungi that produce a pseudomycelium (e.g. Candida albicans)

3. Filamentous fungi that produce a true mycelium (e.g. Aspergillus fumigatus)

4. Dimorphic fungi that grow as yeast or filamentous fungi depending on the

cultural conditions (e.g. Histoplasma capsulatum)

Mycotic infectious diseases of humans

Diseases Etiolgical agents Main tissue affected

Contagious, Superficial agent

Dermatophytoses Epidermophyton, Microsporum,

skin, hair, nail

Non contagious, Systemic diseases

Aspergillosis Aspergillus spp. external ear, lungs, eye, brain

Blastomycosis Biastomyces dermatitis lungs, skin, bone, testes

Candidiasis Candida respiratory, gastrointestinal and urogentital tracks, skin

Choromomycosis Cladosporum fonsecaea and Phidlophora spp.

skin

Coccidioidomycosis Coccidioides immitis lungs, skin, joints, meninges

Cryptoccosis Cryptococcus neoformans

lungs, meninges

224


Histoplasmosis Hipstoplasma capsulatum

lungs, spleen, liver, adrenals, lymph nodes

Mucormycosis Absidia, Mucor, Rhizopus spp

nasal mucosa, lungs, blood vessels, brain

Paracoccidioidomycosis Paracoccidioides

brasilienses

skin, nasal

mucosa,

lungs, liver,

adrenals,

lymph modes

Pneumocystosis Pneumocystis

carinii

lungs

Pseudallescheriasis Pseudallescheria

boydii

external ear, lungs, eye

Sporodrichosis Sporothrix scnenkii skin, joints, lungs

POTENTIALLY EFFECTIVE ANTIFUNGAL COMPOUNDS

Diseases Compounds

Dermatophytosis Azoles (Itraconazole, Miconazole, Clotrimazole), Griseofulvin, Tolnaftate, Naftifine, Turbinatine

Aspergillosis Amphotericin B±5-Fluorocytosine, Itraconazole

Blastomycosis Amphotericin B, Itraconazole, Ketoconazole

225


Candidiasis Amphotericin B ± 5-Fluorocytosine, Nystatin, Azoles (Fluconazole, Ibaconazole, Ketoconazole, Clotrimazole, Miconazole, Econazole etc)

Chromomycoaia 5-Fluorocytosine, Itraconazole, Ketoconazole

Coccidioidomycosis Amphotericin B, Fluconazole, Ketoconazole, Itraconazole

Cryptococcosis Amphotricin B ± 5-Fluorocytosine, Fluconazole

Histoplasmosis Amphotericin B, Itraconazole, Ketoconazole

Mucormycosis Amphotericin B

Paracoccidioidomycosis Itraconazole, Ketoconazole

Pneumocytosis Trimethoprim / Sulfamethoxazole,

LY-303,366, Deferoxamine

Pseudallescheriasis Amphotericin B, Miconazole

Sporotrichosis Amphotericin B, Itraconazole,

Potassium iodide

CANDIDA ALBICANS

Genus: Candida

Candida species reproduce by yeast like budding cells but they also show

formation of pseudomycellum. These pseudomycellum are chains of elongated

cells formed from buds and the buds elongated without breaking of the mother

cell. They are very fragile and separate easily. Mycelia also form by the elongation

of the germ tube produced by a mother cell.

Species: Candida albicans

Candida albicans may remain as a commensal of the mucous membrane

with or without causing any pathologic changes to the deeper tissues of the same

fungus may cause pathological lesion of the skin. Such a fungus under favorable

226


conditions can cause superficial, intermediate of deep mycoses depending on the

condition of the host.

ASPERGILLUS NIGER

Genus: Aspergillus

The Aspergilli are widespread in nature, being found on fruits, vegetables

and other substrates, which may provide nutriment. Some species are involved in

food spoilage. They are important economically because they are used in a

number of industrial fermentations, including the production of citric acid gluconic

acid. Aspergilli grow in high concentrations of sugar and salt, indicating that they

can extract water required for their growth from relatively dry substances.

Derivatives of N-methyl piperazine are therapeutically useful having

antibacterial and antiprotozoal activity. These are active against Gram positive

and Gram negative organisms. In addition, they are active against Candida

albicans and other mycetes. They are also cytostatic.12

Keto oximes esters, i.e. 1,3-Dichloro-2-propan-1-o-(benzoyl)-oxime, are

useful in inhibiting the growth of bacteria and fungi. They also find use as

herbicides and acaricides.13 Substituted 5-nitro-2-furylaminoximes and their

herbicides are active antibacterial and antifungal agents and can be used in

disinfectant compositions to control a variety of micro-organisms.14 O-(N-

methylcarbamolyl)-carbethoxy-chloroform-aldoxime shows biocidal activity against

Aerobacter derogenes in paper pulp and fungicidal activity against Septoria in

weight grains.15

Substituted glyoxal dithiosemicarbazones possess anaplasmicidal activity,

being effective in the control of Anaplasmamarginale.16 Alloxan-5-

thiosemicarbazone possesses bacteriostatic, bactericidal, and fungicidal and

defoliant activities. It is especially useful for the control of SPP of Erwinia,

Staphylococcus and Salmonella.17 Aryl-5-fluoro-2-methoxyphenyl ketoximes and

their thiosemicarbazones were found to be active against Aspergillus Niger and

227


Aspergillus flavus.18 Fluorinated diaryl ketoximes and their thiosemicarbazones

possess antifungal activity.19

1-Propyl-1,2,4-triazolyl derivatives and their salts have a very broad

spectrum of fungicidal action and can be used in particular against parasitic fungi

which attack above ground parts of plants, such as Erysiphe, Podosphrea,

Piricularia and Pellicularia SPP and also against fungi attaking plants through the

soil and against seed-borne fungi.20

Chemical complexes of urea or its derivatives with a completely

halogenated acetone may be used as active components in fungicidal and

herbicidal compositions. They are particularly useful as systemic toxicants for

protecting food crops against harmful fungi, blights and similar pesticidal micro-

organisms.21 N2 -ethylhexyl-N1 -aryl ureas have a broad antibacterial activity

spectrum, including both Gram positive and Gram negative bacteria. They may be

used on a very broad basis, particularly for protecting organic substrates from

infection by destructive and pathogenic bacteria. They are suitable for use as

preservatives and disinfectants for textile and in cosmetics.22 The 2-Oxazolyl

thiourea compounds are useful antifungal agents, being effective in the control of

phytopathogenic fungi.23 N-(4 –bromo-3-halophenyl)-N1 -lower alkoxy ureas are

useful as fungicides and nematocides24. N-(1-cycloalken-1-yl)-urea and thiourea

are useful as phytotoxicants, fungicides, insecticides, nematocides, algaecides,

bactericides, bacteriostats and fungistats.25 The fungicidal effectiveness of

acylthiourea was demonstrated by the sore germination test on plasmparaviticola

sphaerotheca fuligiea26 etc. Uredophenylthiourea derivatives have a broad

spectrum and are particularly effective against parasitic fungi, pathogenic agents

and powdery mildew and rice diseases.27

Essential oils were extracted from members of the myrtaceae family (Bay,

Pumenta and Clove), with eugenol as the main constituent. Their antimicrobial

activity was investigated using S. aureus, B. subtilis, E. coli, P. vulgaris,

Pseudomonas auruginosa, Mycobacterium smegmatis and Candida albicans as

test organisms. Mono-, di- and tri-pyrimidine complexes of organotin compound

are active in the control of insects, fungi, weeds and their microorganisms such as

228


bacteria and viruses.28 Pyridine carbamates are useful as insecticides, fungicides

and nematocides.29 Dialkyltin salts of substituted pyridine-1-oxides are highly

effective bactericides and fungicides and have especially low toxicity to higher

animals. The compounds are useful as preservatives e.g. for protecting leather,

paint, paper, plastics, etc. against attack by mildew and other fungi. They are also

useful for sterilization and disinfecting purposes. Pyrimidine-organo copper

products are useful as catalyst, fungicides, pesticides and anthelmintics and as

intermediates in organic synthesis.30

Cynocarbamates are useful as fungicides, being effective in the control of

phytopathogenic fungi. Thiocyanoacetamides possess fungicidal activity and are

useful in the control of phyto-pathogenic fungi.31 Phosphoric acid esters are

primarily insecticides but fungicidal action has been found against the following

SPP: Alternaria tenuis, Botrytis cinerea, Clastero sporium, Coniothrium, Fusarium,

Mucor, Penicilium, Stemphylium and Botrytis fabae.32

Aryl and cycloalkyl substituted naphthols are useful as antimicrobial agents

and are used in injectable suspension, oral liquid suspension, tablets and capsules

to inhibit the growth of micro-organisms viz. Staphylococcus aureus,

Streptococcus faecalis, Bacillus subtilis, E. coli, proteus vulgaris, Histoplasma

capsulatum, Candida albicans, Aspergillus niger etc.33

Harendra Singh, L. S. Yadav, Kripa Sukla and Rajesh Dwivedi have

synthesized 6,7-dihydro-5H-thiazole[3,2-a]pyrimidin-5-ones. These compounds

displayed an antifungal activity against Aspergillus Flavus and Fusarium solani.34

During the past quarter century, various azoles, i.e. imidazoles, triazoles

have been screened for antifungal action and exhibited broad spectrum of activity

against pathogenic fungi. Azoles can be used both topically and systemically.35

Resistance to imidazoles or triazoles is very rare.36

The two most important drugs belonging to allylamines group are Naftifine

and Terbinafine. Naftifine shows broad spectrum fungicidal activity in vitro.

Griseofulvin, a product of Penicillium griseofulvum was discovered in 1939. But its

229


antifungal activity became known in 1951. Tolnaftate was found to be active

against dermatophytes when applied topically.

C. Mehwala37, J. Machhi38, A. Joshi39, V. Bhatt40 and A. Desai41 have very

recently worked on synthesis of quinoline based compounds and evaluated their

antifungal activity.

The synthesized compounds were screened for their antifungal activity

against Candida albicans (MTCC227), Aspergillus niger (MTCC282) using the agar

cup plate diffusion method by dissolving in DMF at a concentration of 100 µg/mL.

The zone of inhibition was measured after 7 days at 20 oC and it was compared

with the standard drugs Griseofulvin and Nystatin as shown in Table-43 to 48.

TABLE-43

Antifungal activity: Section-II

230


N N

NH2

R R'

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

TABLE-44

CompoundZone of inhibition (mm) (activity index)std


A-17 08 07

A-18 17 21

A -19 17 20

A-20 19 19

A-21 19 20

A-22 18 19

A-23 11 13

A-24 20 22

A-25 13 10

A-26 17 18

A-27 07 09

A-28 18 18

A-29 12 14

A-30 11 10

A-31 14 15

A-32 06 09

231


Antifungal activity: Section-IV (Part-A)

N N

NH

R R'

NCl

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

TABLE-

45



A-37 09 10

A-38 19 22

A-39 20 23

A-40 19 20

A-41 20 23

A-42 19 20

A-43 12 11

A-44 21 23

A-45 12 13

A-46 18 20

A-47 07 10

A-48 19 21

A-49 13 11

A-50 14 13

A-51 15 14

A-52 08 12

232


Antifungal activity: Section-IV (Part-B)

N N

NH

R R'

N

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

CH3

H3C

TABLE-

46



A-53 11 10

A-54 20 23

A-55 19 22

A-56 17 19

A-57 21 21

A-58 17 20

A-59 17 19

A-60 20 22

A-61 10 09

A-62 18 19

A-63 11 09

A-64 21 20

A-65 11 11

A-66 14 17

A-67 17 17

A-68 09 10

233


Antifungal activity: Section-IV (Part-C)

N N

NH

R R'

N

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

CH3

Cl

TABLE-47



A-69 10 11

A-70 20 23

A-71 19 22

A-72 17 19

A-73 20 22

A-74 18 20

A-75 10 13

A-76 20 22

A-77 12 09

A-78 18 19

A-79 09 10

A-80 19 21

A-81 11 13

A-82 13 12

A-83 13 12

A-84 11 14

234


Antifungal activity: Section-IV (Part-D)

N N

NH

R R'

N

R= 2,4-(Cl)2-5-F, 4-Cl, 4-OCH3, 4-CH3

R'= 4'-F, 4'-Cl, 3'-NO2, 3'-Br

CH3

H3CO

TABLE-

48



A-85 11 10

A-86 21 24

A-87 20 23

A-88 18 20

A-89 21 23

A-90 19 21

A-91 16 14

A-92 19 17

A-93 10 09

A-94 19 20

A-95 16 15

A-96 20 22

A-97 12 14

A-98 10 13

A-99 15 13

A-100 09 11

235


Antifungal activity: Section-V

N N

NH

Br

H3CO

Cold brand reactive dye



A-101 Nil Nil

A-102 Nil Nil

A-103 Nil Nil

A-104 Nil Nil

A-105 Nil Nil

A-106 Nil Nil

A-107 Nil Nil

A-108 Nil Nil

A-109 Nil Nil

A-110 Nil Nil

A-111 Nil Nil

A-112 Nil Nil

236

References

[1] Robert Cruickshank, Hand Book of Bacteriology, 394 (1962)

[2] K. D. Tripathi, Essentials of medical pharmacology, 625 (1994)

[3] J. W. Churchman, J. Exptl. Med., 16, 221 (1912)

[4] A. Albert, Brit. J. Exptl. Pathol., 34, 119 (1958)

[5] L. D. Gebbharadt, J. G. Bachtold, Proc. Soc. Exptl. Biol. Med., 88, 103 (1955)

[6] E. W. Stearn, A. E. Stearn, J. Bacteriol., 9, 463-479 (1924)

[7] E. Fischer, R. Muazo, J. Bacteriol., 53, 381 (1947)

[8] A. Albert, Brit. J. Expt. Pathol., 35, 75 (1954)

[9] A. H. Bakett, J. Pharm. Pharmacol., 10, 160 (1958)

[10] P. H. Jacobs, Fungal Diseases, 1, (1997)

[11] D. Greenwood, Antimicrob. Hemotherapy, Third Edition, 62, (1995)

[12] US 3580914, Soc. d’ Etudes de Rech., d’ Application Sci., Med.,

Microbiology Abstr., Vol. 9, No. 2, 9A, 1003 (1974)

[13] US 3592920, Stauffer Chem. Co.; Microbiology Abstr., Vol. 9, No. 2, 9A,

1009 (1974)

[14] US 3660390, Abbott Lab.; Microbiology Abstr., Vol. 9, No. 12, 9A, 7579

(1974)

[15] US 3742036, Roussel-Uclaf.; Microbiology Abstr., Vol. 10, 10A, 7281 (1975)

[16] US 3709935, Barrett P. A.; Microbiology Abstr., Vol. 10, No. 4, 10A, 2589

(1975)

236

References

[17] US 3773952; Microbiology Abstr., Vol. 11, No. 5, 11A, 3278 (1976)

[18] R. H. Khan, S. C. Bahel, Agric Biol. Chem., 40 (9), 1881-1883; Microbiology

Abstr., Vol. 12, No. 5, 12A, 3701 (1977)

[19] A. K. Srivastava, S. C. Bahel, Agric Biol. Chem., 40(4), 801-803 (1976);

Microbiology Abstr., Vol. 12, No. 4, 12A, 2955 (1977)

[20] GB 1291417, Bayer AG.; Microbiology Abstr., Vol. 12, No. 3, 12A, 2018

(1977)

[21] US 3966951, Stauffer Chem. Co.; Microbiology Abstr., Vol. 9, No. 2, 9A, 902

(1974)

[22] US 3592932, Ciba Ltd.; Microbiology Abstr., Vol. 9, No. 2, 9A, 977 (1974)

[23] US 3705903, Lilly Ind. Ltd.; Microbiology Abstr., Vol. 10, No. 3, 10A, 1636

(1975)

[24] US 3702363, Ciba-Geigy AG.; Microbiology Abstr., Vol. 10, No. 3, 10A, 1771

(1975)

[25] US 3701807, Monsanto Co.; Microbiology Abstr., Vol. 10, No. 3, 10A, 1816

(1975)

[26] GB 1313236, Merck Patent GmbH; Microbiology Abstr., Vol. 10, No. 6, 10A,

4214 (1975)

[27] GB 1313236, Bayer AG.; Microbiology Abstr., Vol. 10, No. 6, 10A, 4215

(1975)

[28] US 3661911, Monsanto Co.; Microbiology Abstr., Vol. 9, No. 12, 9A, 7577

(1974)

[29] US 3701779, Shell Oil Co.; Microbiology Abstr., Vol. 10, No. 3, 10A, 1777

(1975)

237

References

[30] US 3712894, Philips Petroleum Co.; Microbiology Abstr., Vol. 10, No. 4,

10A, 2608 (1975)

[31] US 3723439, Velsicol Chem. Corp.; Microbiology Abstr., Vol. 10, No. 6, 10A,

4065 (1975)

[32] GB 1314186, Ciba-Geigy AG.; Microbiology Abstr., Vol. 10, No. 6, 10A, 4198

(1975)

[33] US 3761526, Sandoz-Wander Inc.; Microbiology Abstr., Vol. 11, No. 3, 11A,

1616 (1976)

[34] H. Singh, L. Dhar, S. Yadav, K. N. Shukla, R. Dwivedi, J. Agric. Food. Chem.,

38, 1962-1964 (1990)

[35] Eugene D. & Weinberg, Burger’s Medicinal Chemistry & Drug Diseases, Fifth

Edition, 2, Therapeutic agents, 637 (1997)

[36] J. E. Bennet, In Goodman Gilman’s the Pharmacological Basis of

Therapeutics, Ninth Edition, 1175, (1996)

[37] C. K. Mehwala; Ph. D. Thesis, Veer Narmad South Gujarat University, Surat

(1999)

[38] J. K. Machhi; Ph. D. Thesis, Veer Narmad South Gujarat University, Surat

(2000)

[39] A. M. Joshi; Ph. D. Thesis, Saurashtra University, Rajkot (2002)

[40] V. S. Bhatt; Ph. D. Thesis, Veer Narmad South Gujarat University, Surat

(2005)

[41] A. V. Desai, Ph. D. Thesis, Veer Narmad South Gujarat University, Surat

(2007)

238

ANTIBACTERIAL ACTIVITY - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/2520/11/11_chapter...

Documents

Transcript of ANTIBACTERIAL ACTIVITY - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/2520/11/11_chapter...