Antimicrobial activity and Characterization of Marine bacteria

*Corresponding Author: P.Jeganathan, Centre for Botanical Research , The Madura College, Madurai. T.N. India. E-Mail:

[email protected] 38

Indian J.Pharm.Biol.Res. 2013; 1(4):38-44

Original Research Article

Antimicrobial activity and Characterization of Marine bacteria P.Jeganathan

1*, K.M.Rajasekaran

2, N.K. Asha Devi

3 and S.Karuppusamy

4

1, 2, 4Centre for Botanical Research , The Madura College, Madurai. T.N., India.

3Department of Zoology, Thiagarajar College, Madurai. T.N., India.

ARTICLE INFO:

Article history:

Received: 3August 2013

Received in revised form:

14 September 2013

Accepted: 25 September 2013

Available online: 7 December 2013

Keywords:

Marine bacteria,

Ethyl acetate,

Antimicrobial activity,

Agar diffusion and TLC.

Abstract

Marine bacteria were isolated from seawater was collected from different coastal areas of the

Tamilnadu Sea. The antimicrobial activities of these bacteria were investigated. Ethyl acetate

extracts of marine bacterial fermentation were screened for antimicrobial activities using the

method of agar diffusion. The results showed that 25 strains of the isolates have antimicrobial

activity. The proportion of active bacteria associated with isolated from seawater. The active

marine bacteria were assigned to the genera Alteromonas, Pseudomonas, Bacillus and

Marinobacter. The TLC autobiographic overlay assay implied that the antimicrobial

metabolites produced by four strains with wide antimicrobial spectrum were different. These

marine bacteria were expected to be potential resources of natural antibiotic products. It can

be concluded that isolation of Marine bacterial samples can offer a numbers of microbial

strains for sources of new biomolecules from Marine sources.

1. Introduction

Compared with terrestrial organisms, the secondary metabolites

produced by marine organisms have more novel and unique

structures owing to the complex living circumstance and diversity

of species, and the bioactivities are much stronger [5, 24, 26].

Furthermore, along with the deep studies of marine natural

products biosynthesis, some evidence indicates that many bioactive

compounds previously found in marine animals and plants were in

fact produced or metabolised by associated microorganisms [6,

18, 23, 25, 29]. Because of the low content of active compounds

in marine animals and plants, as well as limitation of bioresource

supply, more and more researches have been focused on marine

microorganisms as sustainable resources [3, 11, 14, 16].

Competition among microbes for space and nutrient in marine

environment is a powerful selection pressure that endows marine

microorganisms to produce many natural products possessing

medical and industrial values [1]. Many antimicrobial,

antifouling substances have been found among these kinds of

bacteria due to the specialized role they play in their respective

hosts [4, 12, 13]. It is suggested that the primary role of these

antibiotic substances could be related to ecological competition. If

this were true, we would expect the antibiotic producing bacteria

associated with some particular hosts to be proportionally higher

than others. However, few investigations have been conducted to

study and compare the antibiotic activities of marine bacteria

isolated from different origins [4, 15, 18]. In the present work,

different coastal areas of Tamilnadu. These bacteria were then

compared with marine bacteria isolated from seawater producing

secondary metabolites with antimicrobial activity. We have also

investigated whether the antibiotic compounds produced by

different active bacteria were the same. In this study, the

distribution, Morphological characteristics and antimicrobial

activities of marine bacterial species in coastal seawater was

collected.

2. Materials and Methods

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Jeganathan. et al. / Indian J. Pharm. Biol. Res., 2013; 1(4):38-44

Original Research Article 39

2.1 Collection of samples

The 25 samples were collected in the intertidal zone during low tide

at three coastal locations near Rameswaram and Pudhumadam. To

study the morphological characteristics and antimicrobial

properties of the marine bacterial species, coastal water was

collected. The Sample water was collected in clean, sanitized and

sterilized glass water bottles.

2.2 Isolation of Marine bacteria

The samples (0.5 g) were triturated, suspended with sterile

seawater and spread on the entire surface of 1/10 Marine Agar

(peptone 0.5 g, yeast extract 0.1 g, FePO4 0.1 g, agar 15 g, dissolved

in 1 L seawater, pH 7.2-7.6). After incubation at 250

C for 20 days

all colonies with different morphology were chosen for bacteria

isolation.

2.3 Preparation of bacterial cultures and crude extracts

The marine bacteria were cultured in 300 ml Marine Broth

(peptone 5 g, yeast extract 1 g, FePO4 0.1 g, dissolved in 1 L sea

water, pH 7.2-7.6) in 500 ml Erlenmeyer flasks for the production

of secondary metabolites. Flasks were incubated on a rotatory

shaker at 220 rpm for 7 days at 25 °C. The broth was first

centrifuged at 5000 x g for 30 min to remove the cells, and then

extracted 3 times with 100 ml ethyl acetate (EtOAc). After solvent

removal under reduced pressure at 37 °C, the extracts were used as

the crude samples for bioactivity assay.

2.4 Bioassay of antimicrobial activity from marine bacteria

All the isolated marine bacteria were screened for antimicrobial

activity, using terrestrial microbe including Salmonella typhi

(National Center for Medical Culture Collections), Staphylococcus

aureus, Escherichia coli, Enterobacter aerogenes and

Streptococcus mutans (Agricultural Culture Collection of

Tamilnadu) as the test microorganisms. Antimicrobial activity was

assayed in duplicate using a standard paper disc assay [20]. The

dried crude extracts were dissolved in EtOAc to a concentration of

100 mg ml-1. The samples (20 µl) were used to saturate the

antimicrobial assay paper disks (6 mm) with a period of drying

between each application. The disks were placed onto the agar

surface containing the test microorganisms, and incubated at 37 °C

for 24 h after a diffusion process for 10 h at 8°C. The diameters of

any inhibition zones formed around the paper disks were then

measured.

2.5 TLC autobiography overlay assay

The crude extracts of four marine bacteria (MB 14,MB 16,MB 18

and MB 20 with wide antimicrobial spectrum were used in TLC

autobiography overlay assay [9]. Each crude extract was

dissolved in EtOAc and made up to a concentration of 100 mg

ml-1. The solution (2 µl) was submitted to TLC analysis on a 3.5

x 5 cm silicagel plate (TLC aluminium sheets, 20 x 20 cm, Silica

Gel 60F 254 Merck Co, USA) using to some types of chemicals

are(DCM and MeOH) dichloromethane (DCM):EtOAc:Methanol

(MeOH) 5:5:1, v/v) as the mobile phase. UV/Vis absorption was

used for detection at wavelengths of 254 nm and 365 nm. The

developed TLC plates were sterilized by UV lamp for 30 min

before enchased in the base nutrient agar in a Petridish (9 mm). It

was then covered by melting nutrient agar (46 °C) containing test

microorganism Staphylococcus aureus. After 10 h diffusion

process at 8 °C, the plate was then incubated at 37°C for 24 h and

the upper agar was sprayed with 5 mg ml-1 of

methylthiazoletetrazolium (MTT, Sigma, M5655, USA) to

convert to a formazan dye by the test microorganism. Inhibition

zones were observed as clear spots against purple background

and their Rf values were calculated.

2.6 Identification of bacterial strains The isolated bacteria with antimicrobial activity were first

identified to the genus level by observing their morphology and

biochemical metabolism characteristics according to the schemes

[21, 27]. The bacteria with wide antimicrobial spectrum were

identified to the species level by PCR amplification of the 16S

rRNA gene, BLAST analysis, and comparison with sequences in

the GenBank nucleotide database. Specifically, the 16S rRNA gene

from the strain was amplified using universal primers 27f (5’-

AGAGTTTGATCCTGGCTCAG-3’) and 1492r (5’-

GGTTACCTTGTTACGACTT-3’). The PCR conditions used

were the same as those described previously [2]. The PCR

products were purified and sequenced by the Dingan Bio-

company. The sequences were compared with known sequences in

the GenBank nucleotide database and the species level was

identified as the nearest phylogenetic neighborwith >99%

sequencesimilarity[10]



3. Results

3.1 Antimicrobial activity and identification

Twenty five marine bacteria were isolated from seawater samples.

The antimicrobial assay showed that 25 strains inhibited at least

one test microorganism (Table 1). After taxonomic study it was

concluded that the bacteria with antimicrobial activity belong

mainly to the genera Alteromonas (10 strains), Pseudomonas (8

strains), Bacillus (3 strains) and Marinobacter (4 strains). Four

strains were able to inhibit all test microorganisms and they include

MB 14 isolated from sea water around Rameswaram and MB 18 isolated

from around Pudhumadam. Six organisms were then preserved in

a slant and broth cultures, and were used in studying the colony,

morphology, microscopical characteristics, gram staining,

motility and other biochemical assays. (Table 2) shows the

organisms colony morphology shape and motility in medium.

(Table 3) shows IMVIC, oxidase and catalase reaction in MB 14

and MB 18 bacterial organisms. Based on their 16S rRNA

sequence analysis, the two strains MB 14 and MB 18 were identified as

Marinobacter hydrocarbonoclasticus and

Pseudomonas

bromoutilis respectively. The separation and identification of

bioactive compounds with wide antimicrobial spectrum from these

marine bacteria were undergoing.

3.2 Antimicrobial metabolites of different strains

Crude extracts of two strains MB 14 and MB 18 with wide

antimicrobial spectrum were subjected to autobiographic overlay

assay, and the results were presented in Figure 1. Each extract of

different strains showed one or several inhibition spots under the

TLC development system (DCM:EtOAc:MeOH, 5:5:1 v/v), and

the Rf values of these spots were all different. For extracts of

strain MB 14 and MB 18 the Rf values of inhibition spots were

0.70 and 0.62respectively.

Table 1 - Results of antimicrobial activity of marine bacteria using agar diffusion assay and the taxonomy of the active

bacteria

Strain No Genus Antimicrobial activity

ST SA EC EA SM

MB 1 Alteromonas sp. + ++ − ++ +

MB 2 Alteromonas sp. − − − + −

MB 3 Alteromonas sp. + − − − −

MB 4 Pseudomonas sp. − + − + −

MB 5 Pseudomonas sp. + + − + −

MB 6 Alteromonas sp. − + − + −

MB 7 Alteromonas sp. + − − + −

MB 8 Marinobacter sp. + + − + −

MB 9 Pseudomonas sp. + − − + −

MB 10 Alteromonas sp. + − − − −

MB 11 Pseudomonas sp. + − − + −


MB 13 Pseudomonas sp. + ++ − − −

MB 14 Marinobacter sp. +++ +++ + ++ ++

MB 15 Bacillus sp. + − − + −

MB 16 Bacillus sp. +++ ++ + ++ ++


MB 18 Pseudomonas sp. +++ ++ + ++ ++


MB 20 Alteromonas sp. + + + - +

MB 21 Marinobacter sp. − − − + −

MB 22 Bacillus sp. + − − + −

MB 23 Marinobacter sp. − − − + −

MB 24 Pseudomonas sp. − ++ − − −

MB 25 Pseudomonas sp. + + − + −



The test microorganisms are: ST, Salmonella typhi; SA, Staphylococcus aureus; EC, Escherichia coli; EA, Enterobacter

aerogenes; SM, Streptococcus mutans. −: no inhibition; +: inhibition zone was 0~3 mm; ++: inhibition zone was 3~5

mm;+++: inhibition zone was ≥ 5 mm.

Table 2 Isolated organisms bases on colony Morphology and other Characteristics

Table 3 Biochemical Characteristics

S.No

Organisms

Oxidase

Catalase

IMVIC

I M V C

1 MB 14 + + - - + +

2 MB 18 + + - - - +

+ Indicates Positive - Indicates Negative

A B C

S.No. Organisms Colony

morphology

Cell shape Motility Gram Staining

1 MB 14 White coloured,

smooth edged

Rod + -

2 MB 18 Fluorescent

colony

Rod + -



Figure 1 TLC autobiographic overlay assay

TLC autobiographic overlay assay for wide antimicrobial

spectrum strains against Staphylococcus aureus. The samples

are extracted from the strains: A, MB 16; B, MB 14 and C, MB

18. The inhibition spot was observed.

4.Discussion

In the present work, we have isolated 25 strains from seawater

and a large number of bacteria could live on it [28]. These

bacteria species are generally not real symbiotic to the host but

can instead be regarded as associated bacteria [3] with

consanguineous relationship with their hosts. On one hand,

these bacteria could acquire the necessary nutrition such as

vitamin, polysaccharide and fatty acid from their animal or plant

hosts; while on the other, they could excrete products such as

amino acid, antibiotic and toxin propitious for the development

and metabolism of the hosts, or to improve the chemical defense

capability of the hosts [1]. Our results are quite consistent with the

reported previous investigations.

The antimicrobial activity of marine bacteria of coastal sea water

had been reported previously [18], 17% of these marine bacteria

showed antibacterial activity to Staphylococcus aureus. Selected

bacteria to testing were pigment colonies and all antibiotic

producing bacteria belonged to the Pseudomonas-Alteromonas

group. In our work, the proportion of antimicrobial activity

producing bacteria isolated from sea water was 11%, including

both pigment and no pigment producing colonies. They were

identified as Bacillus, Marinobacter besides Pseudomonas

and Alteromonas group. In fact, the results of our molecular

identification study indicated that the three Alteromonas sp.

strains with wide antimicrobial spectrum were

Pseudoalteromonas sp., a new genus separated from the genus

Alteromonas [8]. These results are similar to those observed by

other researchers [4, 15]. It is evident that the antimicrobial spectra

of the active marine bacteria were different. In all 25 strains with

antimicrobial activity, 21 strains (84%) inhibited Enterobacter

aerogenes, 16 strains (64%) inhibited Salmonella typhi, 12 strains

(48%) inhibited Staphylococcus aureus, 5 strains (20%)

inhibited Streptococcus mutans, and 4 strains (16%) inhibited

Escherichia coli. To our surprise only four strains, which showed

inhibition activity on all tested microorganisms, have inhibition

effect to E. coli. [15] also observed this result. Lower outer

membrane permeability was therefore expected to contribute

greatly to the intrinsic resistance of Gram negative bacteria to a

wide range of antibiotics [7, 11].

Although there were many studies on marine medical screening

and ecological investigation pertaining to sponge associated

bacteria and fungi [3, 10, 11, 22, 30,31] this study is the first to

report bacteria with antimicrobial activity. The results of TLC

autobiographic overlay assay demonstrated that different species

could produce different antimicrobial metabolites, and some had

more than one antimicrobial substance. The results implied that

some marine bacteria could probably release various antibiotic

compounds to provide themselves the survival competition

superiority. Marine microorganisms as model systems offer the

potential to understand and develop treatments for disease based

on the normal physiological role of their secondary metabolites

[10] and are currently being applied to the development of new

drugs. In order to find more novel structures, new ways of

screening of these compounds should be applied.



5. Conclusion

It can be concluded that isolation of Marine bacterial samples can

offer a numbers of microbial strains for sources of new

biomolecules from Marine sources. This study indicated that

certain strains of bacterial it could be induced to produce

antibiotics.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgement Our sincere thanks are due to Dr. K.M. Rajesekaran, Dr.

N.K.Ashadevi,Dr.S.Karuppusamy,Mrs.K.Rajeswari,Mr.P.N.Raja

rajan and Mr.Thirugnanadass for constant encouragement for

bringing out best in us and the Principal and The Madura College

for moral support Management.

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All © 2013 are reserved by Indian Journal of Pharmaceutical and Biological Research

Cite this article as: P.Jeganathan, K.M.Rajasekaran, N.K.Asha Devi and S.Karuppusamy. Antimicrobial activity and

Characterization of Marine bacteria. Indian J. Pharm. Biol. Res.2013; 1(4):38-44.

Antimicrobial activity and Characterization of Marine bacteria

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