World Journal ofFish ISSN 2078-4589

and Marine Sciences 2 (4): 275-279,201 201 0

0

c IDOSI

Publications,

Control of Fish Bacterial Pathogens, by Antagonistic Marine Actinomycetes Isolated from Gulf of Mannar Coast
lR. Saravanakumar,
'Department 2Department Abstract: Actinomycetes of Zoology,

'tt. SyedMoomeen,
Kamaraj VHNSN College, College,

lJ. Ronald and 2M Kannan
Tuticorin 628 003, TN, India 626 001, TN, India

of Microbiology,

Virudhunagar

were isolated from different marine samples collected from various stations along the that the actinomycetes of GMC are strong antagonists of Representatives of the genera Streptomyces and Micromonospora inhibit the growth

Gulf of Mannar Coast (GMC). It was demonstrated other microorganisms. of fish pathogens, microorganisms antagonistic Key words:

V.harveyi, V. fischeri, A. hydrophila and A. sobria, as well as of antibiotic-resistant
display a wide range of active substances. and antibacterial activity inhibits

causing various fish diseases. Gulf of Mannar Coast actinomycetes

activity and are potential producers of new biologically

Vibrio harveyi . Antibiotic-resistant· INTRODUCTION

Micromonospora
resulting

from the effect of antibiotic substances,

the growth of microorganisms Actinomycetes biotechnologically responsible discovered antibiotics record amount isolation for drug Recently, terrestrial bioactive are the most economically They half and are of the interactions valuable secondary prokaryotes. of about metabolites

or kills them. Antimicrobial

influence, first, the structure of the microbial of microbial transformation censes; and rate of

community, i.e., the composition and diversity of bacterial species; second, the functioning overall, the nature and biogeochemical about two they 70% thirds produce

for the production [2], antitumor

[1], notably

agents [3], immunosuppressive in this regard, a significant sources from

substances in water bodies [8]. Of the ten are [9]. of actinomycetes origin; from of the them, for genus

agents [4] and enzymes [5]. Because of the excellent track of actinomycetes of effort has been focused of novel actinomycetes screening programs the rate of discovery actinomycetes on the successful fifty years.

thousand known antibiotics produced by microorganisms, representatives of the genus Streptomyces Actinomycetes 300 account

from terrestrial in the past

Micromonospora
substances [10].

occupy the second place in this list: over broad-spectrum of the antibiotic genera Representatives and frequently antagonistic other

of new compounds

has decreased, whereas the rate has increased. Thus, from as be pursued of actinomycetes habitats secondary

ofre-isolation it is crucial unexplored sources effective of

of known compounds that new groups or underexploited novel bioactive

Micromonospora

and Streptomyces are widely abundant prevail over other interrelations autochthonous of and [11, 12]. The goal of this work

in aquatic ecosystems groups of actinomycetes was to study the with actinomycetes allochthonous

metabolites. active

Searching for previously unknown microbial strains is an approach for obtaining new biologically substances. weaker aquatic bacteria It is known that the antimicrobial products of aquatic bacterial activity activity of

microorganisms

of Gulf of Mannar Coast.

the metabolic

strains is not and other of

MATERIALS AND METHODS
Overall, 84 actinomycetes 2008glass strains isolated from water, activity. with

than the corresponding organisms studied are potential display

of soil strains. bottom sediments and seaweeds of Gulf of Mannar from 2009 were tested for their antagonistic bottles. Bottom sediments were Water (250 ml) was sampled with a bathometer into sterile collected

Water, bottom sediments, these metabolites

as well as seaweeds

sources of producers activity

[6, 7]. Over one half of the marine antagonistic towards or bilateral antagonism,

other pelagic bacteria. Unilateral

Corresponding Author:

R. Saravanakumar, Department of Zoology, Kamaraj College, Tuticorin 628 003, TN, India Email: saravanakumar246@gmail.com 275

World J Fish & Marine Sci., 2 (4): 275-279, 2010

bucket bottom grab. Seaweeds from the bottom were taken using diving technique. Samples of bottom sediments were packed into sterile polyethylene bags to store at -12°C until used. Water and seaweeds samples were tested immediately (within 1 h) after sampling. Actinomycetes were isolated from water by direct submerged inoculation of the sample (1-2 ml) [13], samples from bottom sediments and seaweeds were isolated by limiting dilutions [14]. 0.5 ml aliquots of the samples were plated onto starch-casein agar medium (HiMedia, Mumbai, India) supplemented with nystatin and cyclohexamide at 25 ug/ml and 10 ug/ml, respectively, to minimize contamination with fungi and 10 J.lg nalidixic acid/ml to minimize bacterial growth [15]. The plates were incubated for one week at 30±0.2°C and actinomycetes colonies were characterized by their distinctive powdery appearance [16]. Cultivation was performed at room temperature for 2--4 weeks. The isolated actinomycete strains were identified according to morphological and cultivation characteristics with the help of Bergey's [17] and Hause's [18] keys. The structure of sporophores and the spore surface were examined using an Olympus (J apan) light microscope. The range of the antimicrobial activity of actinomycetes was tested by two methods, based on the ability of antibiotics produced by actinomycetes to diffuse into agar and either retard the growth of microorganisms within the diffusion zone of the active substance or inhibit it completely. The first method, the technique of perpendicular streaks, has been used for quite a long time and is quite demonstrative [19]. The second method involves concurrent cultivation of the producer and test cultures. Sterile petri dishes were filled with melted agar medium mixed with suspension of the culture tested. After solidification of the medium, the actinomycete strains tested were inoculated with a prick of a loop. This cultivation method provides equal growth conditions for the cultures; therefore, it is possible to detect the actually strongest antagonist strains that inhibit their competitors during development. If the diameter of the growth inhibition zone amounted to more than 5 mm, the corresponding strain was considered to be displaying antimicrobial activity. Each experiment was performed in three replicates and the diameter of the growth inhibition zone was determined according to the mean value. The following organisms were used as test cultures, Aeromonas sobria (MTCC 1608), Aeromonas hydrophila (MTCC 1739), V.fischeri (MTCC 1738) and Vibrio harveyi.(MTCC 3438). Sensitivities of the bacteria from the second group to the following antibiotics of various activity ranges were determined by disk diffusion

technique using Muller Hinton Agar (MHA) (Hi-Media, Mumbai, India) and MHA with 1% salt based on the test orgarusms; kanamycin, erythromycin, tetracycline, levomycetin, streptomycin, gentamicin, oxacillin, lincomycin, benzylpenicillin, ampicillin, cephazolin, cefuroxime, furadonin and carbenicillin. The strains tested were classified as sensitive, moderately sensitive, or resistant microorganisms according to the sizes of their growth inhibition zones. RESUL T AND DISCUSSION
It was demonstrated that Gulf of Mannar coast representatives of the genera Streptomyces and Micromonospora were strong antagonists microorganisms causing various fresh water and marine fish diseases. The first studies on determining antagonistic relationships involved microorganisms isolated from Gulf of Mannar (Table 1). Using the technique of perpendicular streaks, 27 strains from the genus Streptomyces and 13 Micromonospora strains were tested. As was observed, 13 Streptomyces strains inhibited the growth of V. harveyi; 10 strains suppressed V. fischeri; 9 strains, A. hydrophila; and 6 strains, A. sobria. On average, the diameter of the growth inhibition zone was 5-15 mm. Five Streptomyces strains were discovered that inhibited considerably the development of all four test cultures. One of these strains was Streptomyces sp. 8; the diameter of the zone of growth inhibition which it caused reached 23 mm for V. harveyi and 10-13 mm for other fish pathogens. The number of strains of the genus Micromonospora displaying antagonistic activity was considerably smaller. Only 16 of the 23 strains studied inhibited the growth of V. harveyi and V. fischeri test cultures. The average diameter of the growth inhibition zone was 3-5 mm. However, two of the strains belonging to this genus produced the zones of A. hydrophila were showed growth inhibition of 10-28 mm in diameter and one strain formed a zone of V. harveyi growth inhibition of about 21 mm. Studies of the antagonism of these Gulf of Mannar coast actinomycetes towards opportunistic microorganisms demonstrated that the number of Streptomyces strains that inhibited the growth of V. harveyi, 34.4% (21 strains); the growth of V. fischeri, 22.9% ( 14 strains); the growth of A. hydrophila, 14.7% (9 strains); and the growth of A. sobria, 9.8% (6 strains) (Fig. 1). Streptomycets exhibiting antibacterial activity have also been isolated from decaying material found in the littoral zone [20]. Three strains of the Genus M icromonospora inhibited the growth of A. sobria (13%);

276

World J. Fish & Marine Sci., 2 (4): 275-279, 2010
Table I, Showed the number of tested actinomycete strains isolated from Gulf ofMannar coast Genus Source of i sol ation Water Bottom Seaweeds Total S edi m ent s N urn ber of strain s

Streptomyces 9 43 9
61

Micromonospora

15 57 12
84

6 14 3 23

Table

2 Antibiogram analysis offish pathogens
A. hydrophil a V. harveyi A. sobria Vs fischeri

Anti hi oti c dis c Arnpi cillin Benzyl pen cilli n Car beni ci llin Cephazolin

+++

+++ +++

++
+++

+++ +++ +++ +++ +++

Cefur oxirne
Erythromycin Furadonin Gentamycin Kanamycin Levornycetin Lincomycin Oxacillin Streptomycin

++
+++

++

++

+++ +++
- = Sensitive';

++
+++
Sensitive

+++ +++

+++

T etracy din +++= Resistance;

++ = Moderately

35

" :~
" a
OJ)

c 30 .

"" 25 . "" .Ei
"-' c 20

~ ,,_, 10

e

15

V Harvey;

V Fischeri

V Hydrophila

A. Sabri a

Fi,h Pathogens

Fig. 1: Showed the antagonistic activity of actinomycetes collected from Gulf ofMannar seven strains, the growth of A. hydrophila (30%); four strains, V. harveyi (17.3%); and two strain, V. fische ri (0.8%;) (Fig. 1). As was demonstrated earlier, representatives of the genus Streptomyces are capable of producing several antibiotic substanc es concurrently [21]. Overall, 14 strains from the genus Streptomyces were discovered that inhibited V. harveyi and V. fischeri; two strains inhibited only, A. sobria and A. hydrophila. Compared with Streptomyces, members of the genus Micromonospora inhibited no more than two test

cultures. Six strains of this genus inhibited only V. fischeri; three strains, only A. hydrophila; and two strains inhibited both these species. Only one tested strain, Micromonospora sp. 12, inhibited V. harveyi and two strains of the same genus inhibited V. fischeri .. The disk diffusion test demonstrated that V. harveyi was resistant to tetracycline, erythromycin and ampicillin, being moderately sensitive to streptomycin and kanamycin. The V. fische ri strain was resistant to streptomycin and kanamycin and moderately sensitive to furadonin. A. hydrophile displayed resistance to streptomycin and tetracycline and was moderately sensitive to cephazolin and kanamycin. These three microorganisms were sensitive to the other antibiotics (Table 2). It is known that the majority of A. sob ria strains used are resistant to many antibiotics, antiseptics and disinfectants. Among the representatives of the actinomycete genera studied, we succeeded in finding strains capable of inhibiting the growth of this microorganism. Moreover, 15 strains of the genus Streptomyces and 3 strains of the genus Micromonospora produced growth inhibition zones with diam eters reaching 24--40 mm. The diam eter of the growth inhibition zone induced by 13.6% of all actinomycete strains tested amounted to 10--40 mm. The second method was found to be efficient and representative for studying antimicrobial activity. Of them, 8 strains inhibited

277

World J Fish & Marine Sci., 2 (4): 275-279, 2010

the growth of V harveyi; 6 strains, the growth of A. hydrophila; 4 strains, the growth of A. sobria; 3 strains, the growth of A. hydrophila. For all four fish pathogenic cultures, the diameter of growth inhibition zones amounted to 5 tolO nun. Although actinomycetes of the genera Streptomyces and Micromonospora form an insignificant fraction of the microbial community inhabiting Gulf of Mannar Coast and possess an additional disadvantage of very low growth and reproduction rates, they display very high antagonistic activities towards other microorganisms, particularly representatives of the genus Streptomyces. Presumably, this is an advantage that allows actinomycetes of the sampling area to occupy an appropriate position in the microbial community of the GMC, compete for nutrient sources and survive under the harsh conditions of this water body. Representatives of the genera Streptomyces and Micromonospora inhibit the vital functions not only of the bacteria inhabiting the same environment, but also of others, e.g., those causing various fish infectious diseases and exhibiting resistance to a number of antibiotics. We assume that actinomycetes inhibiting the growth of antibiotic-resistant microorganisms produce more active antimicrobial substances than the known antibacterial preparations. Both single targeted strains and strains with a wide range of antimicrobial activity were found among the tested representatives of the genera Streptomyces and Micromonospora. According to our opinion, the actinomycetes from Gulf ofMannar Coast are potential producers of new biologically active substances. REFERENCES
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