ww.sciencedirect.com

i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 4 9e1 5 2

Available online at w

journal homepage: www.elsevier .com/locate/ i jcas

Original Article

Antibacterial activity of pigment produced fromMicrococcus luteus KF532949

Kaliappan Umadevi, Marimuthu Krishnaveni*

Department of Biochemistry, Periyar University, Salem 636 011, India

a r t i c l e i n f o

Article history:

Received 5 August 2013

Accepted 29 August 2013

Available online 1 November 2013

Keywords:

Antibacterial

Beach water

Pigment

Sequencing

Wound isolate

* Corresponding author. Tel.: þ91 9894829823E-mail address: Krishnavenim2011@gma

0976-1209/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijcas.2013.08.008

a b s t r a c t

Aim: The problems of synthetic pigment causing toxicity and carcinogenicity in the human

body decrease its use. Therefore, interest in natural pigment production is increasing.

Therefore, the marine environment has recently become an attractive research subject for

many investigations, because of its rich biodiversity. Attempt has been initiated to isolate

Micrococcus luteus from marina beach water.

Methods: The sea water sample isolated was allowed to grow in a Zobell marine agar me-

dium and incubated at 37 �C for one week. The isolated strain from marine source was

confirmed by 16S rRNA sequencing. The GC content was found to be 58.6%. Phylogenetic

tree analysis was performed to know the sequences that are closely related. The strain was

assessed for pigment production in Zobell marine broth and incubated at 37 �C for two

days. The swab sticks collected from hospital were streaked directly on the labelled agar

plates such as eosin methylene blue, MacConkey, nutrient, blood, mannitol salt agar and

incubated at 37 �C for 24 h and characterized. The antibacterial activity of crude pigment

was tested against wound isolates.

Results: The individual yellow colour colonies obtained was sequenced, submitted to gene

bank under the accession number KF532949. Phylogenetic analysis revealed, that the

identified strain M. luteus is closely related to various Micrococcus sp. The crude pigment

showed promising results against Staphylococcus sp., Klebsiella sp., Pseudomonas sp. isolated

from wound.

Conclusion: From this result, we can conclude that the isolated strain M. luteus is able to act

against both gram positive and gram negative bacteria.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction only from fruits, vegetables, roots but also from microorgan-

Colour is a fundamental component and is probably one of the

first quality manifested by our senses. Since, the synthetic

colours are toxic, it is essential to produce coloured pigments

from natural resources. Natural pigments are extracted not

(mobile).il.com (M. Krishnaveni).2013, JPR Solutions; Publi

isms and are often called biocolours.1 Microbial pigments pose

no seasonal production problemsbut showshigh productivity.

Micrococcus luteuswas originally isolated byAlexander Fleming

in 1929 asMicrococcus lysodeikticus. It is very capable of survival

under stress conditions such as low temperature, starvation.

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

Fig. 1 e Phylogenetic tree showing relatedness.

Fig. 2 e SDS-PAGE analysis of crude pigment.

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 4 9e1 5 2150

2. Materials and methods

2.1. Sample collection, isolation, identification,extraction of crude pigment from Micrococcus luteus

Sea water sample collected from Marina beach, Chennai,

stored in a sterile glass container was transported to the lab-

oratory. The samples were plated on Zobell marine agar me-

dium2 and incubated at 37 �C for one week. After incubation,

the yellow colonies were picked out, purified by repeated

streaking. The pure cultures of the bacterial colonies were

inoculated into nutrient agar slants for further studies. The

DNA isolated was amplified using 16S rDNA universal primers

and sequenced for the identification of bacterial strain at

molecular level. The amplified 16S rDNA PCR product was

sequenced using automated sequencer (Synergy scientific,

Chennai). The sequence obtained was submitted to gene bank

for accession number. The sequence similarity search was

done for the 16S rDNA sequence using online search tool

BLAST. Phylogenetic analysis was performed with PHYLIP,

and a phylogenetic tree was constructed via the neighbour

joining method. The organism was inoculated in 5 ml Zobell

marine broth and allowed for incubation at 37 �C for two days.

The cultures were then centrifuged at 5000 rpm for 5 min and

the supernatant was discarded. 200 ml solubilizing buffer was

added to the cell pellet, resuspended and incubated for 24 h,

the fermented biomass wasmixed with 25 ml ethyl acetate by

using mortar and pestle. The crude pigment was collected,

concentrated by evaporation. After evaporation of the solvent,

theweight of the crude pigmentwasmeasured and stored in a

sterile vial.

2.2. Collection, isolation of bacterial isolate from woundand antimicrobial activity of crude pigment against woundpathogens

Purulent materials were borrowed from hospitals located at

Namakkal District and the samples were transported in cooler

boxes to laboratory, for bacteriological investigations within

4 h of collection. The swab sticks used for the sample collec-

tion were streaked directly on the labelled agar plates such as

eosin methylene blue, MacConkey, nutrient, blood and

mannitol salt agar and incubated at 37 �C for 24 h. After in-

cubation, cultures were examined for significant growth. The

primary identification of bacterial isolate was made based on

colonial appearance, pigmentation. The bacterial isolate was

assessed for b-lactamase, slime production. The crude

pigment was screened for biological activity against patho-

gens isolated fromwound by well diffusionmethod.3 The 18 h

old broth cultures of test bacterial pathogens were inoculated

by making a lawn on nutrient agar by using sterile cotton

swab. The crude pigment at 50, 75 and 100 ml concentration

was added on wells in Mueller Hinton agar plate.4 The diam-

eter of the inhibition zone was measured after 24 h of incu-

bation at 37 �C.

2.3. SDS-PAGE analysis

The test sample was prepared by diluting (1:1) in sample sol-

ubilizing buffer, which were placed for 10 min in a boiling

water bath. After cooling to room temperature, the samples

were spinned for 1 min. This step is not applicable to protein

marker, as it is a readymade one, add 3e5 ml of marker to well.

3. Results and discussion

3.1. Isolation, identification of Micrococcus luteus frommarine water isolate, crude pigment production

The marine water isolate showing yellow colour colonies was

subjected to preliminary screening, amplification of 16S rRNA

gene. The strain MKVKUD_2013 matched best with the genus

Micrococcus and showed 99% similarity toM. luteus (Fig. 1). The

crude pigment obtained by ethyl acetate evaporation was

used for antimicrobial studies.

3.2. Isolation, characterization of wound bacterialisolates

Different types of wound samples such as burn, accident, skin

infection and trauma were collected. All were found to be

biofilm, b-lactamase producers.

Plate 3 e Antimicrobial activity of crude pigment from

Micrococcus luteus against Escherichia sp.

Plate 1 e Antimicrobial activity of crude pigment from

Micrococcus luteus against Pseudomonas sp.

i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 4 9e1 5 2 151

3.3. Antibacterial activity of crude pigment fromM. luteus

The antibacterial activity of crude pigment fromM. luteuswas

assessed by disc diffusion assay. Zone of inhibition observed

was high with Staphylococcus sp., Klebsiella sp., Pseudomonas

sp., at 50, 100 ml concentration when compared to

Escherichia sp. The negative result was observed with Strepto-

coccus sp. The results are shown in Plates 1e5.

3.4. Molecular mass determination

Electrophoretic analysis of the crude pigment prepared using

a sample buffer containing the reducing agent 2-mercaptoe-

thanol showed a wide protein band occupying most of the gel.

The band ranged from 14.3 Kd to above 97.4 Kd was observed

(Fig. 2).

Plate 2 e Antimicrobial activity of crude pigment from

Micrococcus luteus against Streptococcus sp.

Plate 4 e Antimicrobial activity of crude pigment from

Micrococcus luteus against Klebsiella sp.

4. Conclusion

The identified isolate from seawater was found to beM. luteus,

able to produce pigment. The crude pigment produced from

the strain was found to contain antibacterial activity. Further,

purification may give better antibacterial compound.

Plate 5 e Antimicrobial activity of crude pigment from

Micrococcus luteus against Staphylococcus sp.

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 4 9e1 5 2152

Conflicts of interest

All authors have none to declare.

Acknowledgement

The author thank Honourable Vice Chancellor, Dr.

K. Muthuchelian Avl, Registrar, Dr. K. Angamuthu Avl, Periyar

University, Salem for their administrative support. The author

also thank Managing Director, Mr. D. Jagadeesh Kumar,

Chrompark Research Centre, Namakkal for providing lab fa-

cilities to carry out the research and Managing Director, Dr.

Sankarapandian Selvaraj, Helini Biomolecules, Chennai for

helping us in doing bioinformatics work.

r e f e r e n c e s

1. Pattnaik P, Roy U, Jain P. Biocolours: new generation additivesfor food. Indian Food Ind. 1997;16:21e32.

2. Baker FJ, Silverton RE. Introduction to Medical LaboratoryTechnology. 6th ed. Toronto: Butterworths; 1985.

3. Bauer AW, Kirby WMM, Sherris JC. Antibiotic susceptibilitytesting by a standard single disk method. Am J Clin Pathol.1966;45:493e496.

4. Cappuccino James G, Sherman Natalie.Microbiology A LaboratoryManual. 7th ed. Dorling Kindersley (India) Pvt. Ltd; 2009.