CLARITHROMYCIN: IN VITRO EVALUATION OF

CLARITHROMYCIN: IN VITRO EVALUATION OF ANTIMICROBIAL ACTIVITY AND ITS NEUTRALIZATION R.C.Pantola* and Rakesh Bahuguna Department of Quality Control, Ind-Swift Laboratories Ltd. Vill. Bhagwanpur, Near Derabassi, Mohali 140507, Punjab. E- mail: [email protected] This study was conducted for the evaluation of antimicrobial activity and its neutralization. Five bacterial strains and two yeast and mould strains were used. The antimicrobial activity was caused against all the microorganisms .The neutralization of antimicrobial activity of clarithromycin was done by using the concentration of

1 gm in 100 ml

lecithin

over a

and recovery was done by using subjected

microorganisms, the recovery status after neutralization was different for intended microorganism and some of them could not recovered.

Introduction

clarithromycin may enhance its clinical

The of use antibiotics for controlling of

efficacy. It has been demonstrated that

infections is from the time of immemorial,

clarithromycin inhibits the production of

either they were performed form plant

interleukin-1 (IL-1) by murine peritoneal

extracts or in the form of chemotherapeutics.

macrophages, lymophocyte proliferation and

The control of microbial contamination

lymphocyte transformation of murine spleen

requires a strict and contentious vigil of

cells at low concentrations (Dautzenberg et

healthcare

present

al., 1993) and is 2 to 10 times more active

scenario of chemotherapeutic measures is an

than erythromycin in several experimental

out

and

animal infection models (Loza et al., 1992).

development by Bio-scientists and is still

Clarithromycin is effective in sinusitis and

continuous.

otitis media (Karma et al., 1991; Marchi

The site of action of clarithromycin is same

1990; Aspin et al., 1994; Gooch et al.,

as that of erythromycin (Najma Sultana

1999), lung lesions (Hajime et al., 1994),

et.al.2002).The

anti-

ventricular dysrhythmias and other chest

by

infections (Kundu et al., 1997; Aldons 1991;

professionals.

come

inflammatory

of

The

continuous

more effects

February 2012 Vol.2(2)

efforts

potent exhibited

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R. C. Pantola et al

ISSN: 2277–1220

Neu & Chick 1993; Anderson et al., 1991;

Bacillus subtilis subsp.spizizenii (ATCC

Chien et al., 1993; Bradbury 1993; de Widel

6633), Escherichia coli (ATCC 8739),

etal., 1985; Hamedani et al., 1991; Still

Pseudomonas

1993).

,Staphylococcus

There are many examples of technologies

(ATCC

and quality parameters that have created

subsp.enterica serovar typhimurium (ATCC

competitive

increased

14023), Aspergillus brasilliensis (ATCC

quality of chemotherapeutics in global

16404) , and Candida albicans (ATCC

market. The present quality control system

10231).The microorganisms were procured

for

from

the

advantages

and

pharmaceutical

products

is

aeruginosa

(ATCC9027)

aureus

6538),

subsp.aureus

Salmonella

enterica

Microbiologics

Inc.USA.

continuously increasing in order to meet the

Clarithromycin (6 –O-Methyl-erythromycin

high standards of quality, safety and efficacy

A) is (2R, 3S, 4S, 5R, 6R, 8R, 10R, 11R, 12S,

of drugs.

13R)-3-(2,6-Dideoxy-3-C, 3-o-dimethyl--

The

bio-burden

monitoring

of

drug

L-ribo-hexo-pyranosyloxy)

-11,12-

substances which are administered orally is

dihydroxy-6-methoxy-2,4,6,8,10,12-

the requirement of regulatory authorities and

hexamethyl-9-oxo-5-(3,4,6-trideoxy-3-

there has become a general consensus

dimethylamino--D-xylo-

among United pharmacopoeia 2010 (USP),

hexopyranosyloxy)

European Pharmacopoeia (Ph.Eu) 2010,

(Florey 1996; Jame and Reynold 1996;

Japanese Pharmacopoeia (JP) 2006 and

Dollery1999).Molecular

Australian health regulatory authorities and

C38H69NO13, Molecular weight

accepted

bio-burden

and CAS No. [81103-11-9], having the

testing of pharmaceutical products having

molecular structure as Figure-1 was taken

the antimicrobial property is tedious job,

for the study.

unanimously.

The

HO

OCH3

H3C

OH

CH3

H3C O

and second to test for total viable count

O N(CH3)2

O

CH3 O

objectionable

CH3

O

HO

OCH3

O

CH3

HO

CH3

microorganisms.

CH3

Material and methods

Figure-1

The microorganisms used in the study were

Clarithromycin


747.95

CH3

H3C

first to nullify the antibiotic characteristics

and

formula

O

there testing is accomplished in two parts,

(Bio-burden)

pentadecan-13-olide

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Molecular

structure

of

9

R. C. Pantola et al

ISSN: 2277–1220

The microbiological media soyabean casein

digest

digest agar, soyabean casein digest broth

neutralization, dilution was kept as it is for

and sabouraud dextrose and lecithin make

one hour. The efficacy and absence of

of

and

toxicity for microorganisms of lecithin was

Phosphate buffer make of Merck Germany

demonstrated by carrying out a blank with

were used.

neutralizer and without product.

Preparation of inoculums

Preparation, inoculation, incubation and

The standardized suspension of all the test

recovery

strains was used; the suspension was

The

prepared

subsp.spizizenii (ATCC 6633), Escherichia

Himedia Laboratories, India

through

seed-lot

culture

broth.

To

recovery

of

the

Bacillus

subtilis

coli

than five passages removed from the

aeruginosa (ATCC9027) ,Staphylococcus

original master seed-lot. Test suspension

aureus

was diluted by sterile phosphate buffer

Salmonella enterica subsp.enterica serovar

solution pH 7.2 in order to make the strength

typhimurium (ATCC 14023), Aspergillus

of inoculums (suspension) not more than

brasilliensis (ATCC 16404) , and Candida

100 cfu/ml.

albicans (ATCC 10231).was done by using

Preparation of clarithromycin solution

the pour plate method , one ml of each

The sample was prepared by adding 1 gm of

culture having not more than 100 cfu with

clarithromycin in soyabean casein digest

clarithromycin and without clarithromycin

broth and final volume was made 100 ml.

before and after neutralization was plated

Neutralization of antibiotic activity of

by using the soyabean casein digest agar

clarithromycin solution

media for bacterial strains and sabouraud

The sample was prepared by adding 1 gm of

dextrose agar for fungal strains, in duplicate.

clarithromycin in soyabean casein digest

The bacterial plates were incubated at 30° to

broth having 1 percent lecithin (as a

35°C for 3 to 5 days and fungal plates at 20°

neutralizer) and final volume was made

to 25°C for 5 to 7 days. The recovery and

100ml. The neutralization of antibiotic

percentage recovery was calculated against

activity of Clarithromycin was done by

the

using 1 percent lecithin; the lecithin was

microorganisms. The test for absence of

added before sterilization of soyabean casein

toxicity of lecithin was also done by

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8739)

proper

maintenance technique and was not more


(ATCC

have

subsp.aureus

recovery of

,Pseudomonas

(ATCC

respective

6538),

challenged

10

R. C. Pantola et al

ISSN: 2277–1220

performing the test with lecithin + diluent

shown in Table 1-9. The percentage

and lecithin + diluent + clarithromycin.

recovery

Results

canaliculated

The results of negative control were

microorganisms after subtracting the count

negative and the tabulated results of

which was found as total aerobic microbial

clarithromycin sample dilution as it is

count (TAMC) has been shown in Table 10-

without neutralizer and with neutralizer and

11.

of

each

test

against

strains

the

was

challenged

recovery results after neutralization has been Table -1 Negative control CFU/Plate Average Plate-I

Plate-II

Nil

Nil

Nil

Table-2 Total aerobic microbial count in sample as it is without neutralizer. CFU/Plate Plate-I

Plate-II

00

00

Average

00

Table-3 Recovery of bacterial strains in sample as it is without neutralizer Name of organism

Standard

Counts in product having

inoculums

standards inoculums (cfu)

count (cfu)

Plate-I

Plate-II

Average

51

00

00

00

Staphylococcus aureus

69

00

00

00

Escherichia coli

58

00

00

00

Pseudomonas aeruginosa

53

00

00

00

Salmonella entirica subsp.

47

00

00

00

Bacillus subtilis subsp. spizizenii

entiritica,abony Table-4 Recovery of yeast and mould strains in sample as it is without neutralizer February 2012 Vol.2(2)

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Name of organism

Standard


inoculums count


(cfu)

Plate-I

Plate-II

Average

Candida albicans

60

00

00

00

Aspergillus brasiliensis

47

00

00

00

Table-5 Results of 1.0 percent lecithin and lecithin + clarithromycin Name of

Standard

Lecithin

microorganism

inoculums

Plate

Plate

count (cfu)

I

II

51

48

56

69

59

Escherichia coli

58

Pseudomonas

Lecithin +product

Average

Plate Plate

Average

I

II

52

00

00

00

64

62

00

00

00

61

55

58

49

53

51

53

65

60

63

50

55

53

47

53

57

55

51

46

49

Candida albicans

60

57

52

55

47

52

50

Aspergillus

47

54

50

52

00

00

00

Bacillus subtilis subsp. spizizenii Staphylococcus aureus

aeruginosa Salmonella entirica subsp. entiritica,abony

brasiliensis Table-6 Total aerobic microbial count (TAMC) in sample after neutralizer CFU/Plate


Plate-I

Plate-II

01

01

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Average

01

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Table-7 Total aerobic yeast and mould count (TYMC) in sample after neutralizer CFU/Plate Plate-I

Plate-II

0

0

Average

0

Table-8 Recovery of bacterial strains Name of organism

Standard


inoculums count


(cfu)

Plate-I

Plate-II

Average

51

00

00

00


69

00

00

00

Escherichia coli

58

56

44

50


53

45

48

47


47

40

34

37


entiritica,abony Table-9 Recovery of fungal strains Name of organism

Standard


inoculums count


(cfu)

Plate-I

Plate-II

Average

Candida albicans

60

51

44

48


47

00

00

00

Table-10 Percentage recovery of bacterial strains Name of microorganism

Percentage recovery


00


00

Escherichia coli

85


87


77

entiritica,abony


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ISSN: 2277–1220

Table-11 Percentage recovery of fungal strains Name of microorganism

Percentage recovery

Candida albicans

80


00

Discussion

14023), and Candida albicans (ATCC

In this study it was found that the

10231) was found more than 70 percent but

clarithromycin is effective again against all

the Bacillus subtilis subsp.spizizenii (ATCC

the subjected bacterial and fungal strains,

6633), Staphylococcus aureus subsp.aureus

when the clarithromycin sample was tested

(ATCC) and

without neutralization for total aerobic

(ATCC 16404) could not recovered.

microbial count. The total aerobic microbial

Conclusions

count was nil. The neutralization was done

We conclude that (i) although clarithomycin

by using 1 percent lecithin in order to check

is a well known broad spectrum antibiotics

the total bio-burden of clarithromycin after

which acts against bacteria and fungus, its

neutralization the results for total aerobic

antimicrobial activity can be neutralized in

microbial count (TAMC) was 1 cfu and total

vitro for of Escherichia coli,Pseudomonas

yeast and mould count (TYMC) was nil. The

aeruginosa,

absence

was

subsp.enterica serovar typhimurium, and

comparing the results of

Candida albicans but can not be neutralized

of

toxicity

evaluated by

of

lecithin

Salmonella

for

+sample which proofs that the lecithin it self

Staphylococcus aureus subsp.aureus

has no impacts on the findings .The recovery

Aspergillus brasilliensis it proves the these

of

challenge

microorganisms shall not be found in the

microorganisms was done separately by

product clarithromycin as atotal aerobic

adding

cfu

microbial count (TAMC) and total yeast and

with

mould count (TYMC).(ii) the methodology

sample .The recovery of Escherichia coli

which has been used for the testing of total

(ATCC 8739) ,Pseudomonas aeruginosa

aerobic microbial count (TAMC) and total

(ATCC9027)

yeast and mould count (TYMC) can be

the

the

subjected

not

more

than

100

standardized respective inoculums

,

Salmonella

enterica

subsp.enterica serovar typhimurium (ATCC February 2012 Vol.2(2)

IJPI

adopted for the

subtilis

enterica

lecithin + diluent and lecithin+ diluent

all

Bacillus

Aspergillus brasilliensis

subsp.spizizenii, and

method suitability or 14

R. C. Pantola et al

ISSN: 2277–1220

validity for the bio-burden monitoring of

acquired pneumonia. A multicentre,

clarithromycin.

double-blind,

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