Isolation and Screening of Antibiotic Producing Psychrophilic Actinomycetes and its Nature from Rothang Hill Soil Against Viridans Streptococcus sp.

MATERIALS AND METHODS

The study was carried out at Department of Microbiology, Jmal Mohamed College during January 2009 to may 2009.

Isolation of Pathogenic Bacteria from the Decayed Tooth Sample
About fifty decayed tooth samples were collected from dental clinic, used for the isolation of test pathogen by routine microbiological laboratory procedure and identified by routine clinical tests.

Isolation of Psychrophillic Actinomycetes
The specimens (actinomycetes) used in this study were isolated from the soil of Rothang hill, Himachal Prethesh, India. The soil sample was collected from the ice point of Manali during October 2008 at a distance of 4061 km from the sea level were brought to the laboratory in aseptic condition. Actinomycetes from the soil had been isolated by pour plate technique on Starch-casein agar and Glycerol-arginine agar and incubated at 15°C for 15 days.

Screening of Psychrophilic Actinomycetes for Antimicrobial Activity
The screening method consists of two steps; Primary screening and secondary screening. In primary screening the antimicrobial activity of crude culture filtrate were used to determine the effect of isolate by agar well diffusion method on Muller Hinton agar per National Committee for Clinical Laboratory Standards (NCCLS, 1999). The medium is slightly modified with the addition of anti coagulated blood for enhance the growth of Haemolytic bacteria. Secondary screening was performed with purified protein extract by ammonium sulphate precipitation and dialysis. The test organisms used were Streptococcus mutans and Streptococcus oralis.

Characterization of Psychrophilic Actinomycetes
The potent actinomycetes selected from secondary screening were characterized by morphological and biochemical method described by Nakazawa et al. (2006). Morphological methods consist of macroscopic and microscopic methods. The microscopic characterization was done by cover slip culture method. The mycelium structure, color and arrangement of conidiospore and arthrospore on the mycelium was observed through the oil immersion (Kawato and Shinobu, 1959). The biochemical tests used in this study for the identification of the potent isolates are casein hydrolysis, starch hydrolysis, urea hydrolysis, acid production from sugar, utilization of sugar and cell wall analysis for Diaminopimelic acid (DAP) (Staneck and Roberts, 1974).

Fermentation Process
Fermentation was carried out in a 1L Erlenmeyer flask containing 500 mL of Balanced Salt Medium (BSM). The process carried out for 7 days at 15°C with 75 rpm agitation.

Protein Purification
The crude extract was mixed with saturated ammonium sulphate and kept over night at 4⁰C then centrifuged. The precipitate dialyzed in phosphate buffer overnight to purify the protein. The sample is characterized by SDS Page electrophoresis.

Isolation of Antibacterial Metabolites
Antibacterial compound was recovered from the filtrate by solvent extraction method following the process. Ethyl acetate was added to the filtrate in the ratio of 1:1(v/v) and shaken vigorously for 1 h for complete extraction. The ethyl acetate phase that contains antibiotic was separated from the aqueous phase. It was evaporated to dryness in water bath at 8-90°C and the residue obtained was weighed. Thus, obtained compound was used to determine antimicrobial activity.

Determination of the Antimicrobial Activity
The antimicrobial activity was determined by agar well method (Zamanian et al., 2005).The partially purified extract obtained by the evaporation of the ethyl acetate extract was dissolved in 1 mL 0.2 M phosphate buffer (pH 7.0). Then 100 μL of sample was loaded into well bored against test organism prepared as per 0.5 McFarland turbidity standards as follows:

The test plates were incubated at 37°C for 18-24 h and examined. The diameter of the zones of complete inhibition was measured to the nearest whole millimeter with the help of zone scale.

RESULT

Nearly six different Actinomycetes were isolated from the soil. The isolated Actinomycetes are identified as A: Intrasporangium sp., B: Dactylsporangium sp., C: Micromonospora sp., D: Streptoverticilium sp., D1: Streptomyces sp. and D2: Streptomyces sp. Their morphological characters are listed on Table 1. This identification is based on their mycelia, spores, starch and nitrate utilization probability described by Shirling and Göttlieb (1966). The morphology and biochemical properties are listed on Table 2. Four of isolates having L-DAP in their cell wall and two are meso- DAP. The isolated actinomycetes having ability to utilize sucrose, mannitol, dextrose and xylose. However, they differ in their utilization of lactose, arabinose, meso-inositol and maltose (Table 3).

The test organism used in this study where isolated from fifty of decayed tooth sample is S. mutans and S. oralis. Their biochemical characteristics are listed on the (Table 1). The results shows these two organisms are predominant and primary bacteria’s responsible for dental carries.

The antibiotic production of isolated Actinomycetes and its activity against the test organism such as S. mutans and S. oralis was listed in the Table 4. Among the six psychrophilic actinomycetes Dactylsporangium roseum shows its maximum activity against S. mutans (33 mm) and its molecular weight of is 66 kDa.

Table 1:	Morphological characteristics of actinomycete isolates
A: Intrasporangium sp., B: Dactyl sporangium sp., C: Micromonospora sp., D: Streptoverticilium sp., D1: Streptomyces sp., D2: Streptomyces sp, +: Positive, -: Negative

Table 2:	Biochemical characters
A: Intrasporangium sp., B: Dactylsporangium sp., C: Micromonospora sp., D: Streptro verticillium sp., D1: Streptomyces purpures, D2: Streptomyces microflavus

Table 3:	Utilization of sugar and acid production
A: Intrasporangium sp., B: Dactylsporangium sp., C: Micromonospora sp., D: Streptro verticillium sp., D1: Streptomyces purpures, D2: Streptomyces microflavus

Table 4:	Zone of psychrophilic actinomycetes produced against pathogenic bacteria (mm)
A:Intrasporangium sp., B: Dactylsporangium sp., C: Micromonospora sp., D: Streptro verticillium sp., D1: Streptomyces purpures, D2: Streptomyces microflavus

The isolate Streptomyces sp. more active against S. oralis than other strains. The inhibition activity is 36 mm of zone. Intrasporangium sp. is not effective against S.mutans similarly Dactylsporangium roseum is ineffective towards S. oralis the rest of the strain are produced metabolites which are effective towards S. mutans and S. oralis.

DISCUSSION

The test organism isolated from decayed tooth is identified as Gram positive cocci in chain. The isolated two strains are differing in their haemolytic activity. The S mutans are α-haemolytic and S. oralis is β-haemolytic in nature. Both are comes under viridians group of streptococci. The inhibitory effect of six isolated Actinomycetes against S. mutans shows five are effective except intrasporangium among those five Dactylsporangium sp. shows its maximum activity against S. mutans (33 mm). The strain capable to produceTetracyclin derivative active against Gram positive organism (Wells et al., 1992). Streptomyces sp. also effective towards S. mutans (Wanbanjob et al., 2008) followed by Dactylsporangium. Micromonospora sp. and Streptoverticillium sp. are moderatively active against S. mutans, they produce 24 and 26 mm zone of inhibition the least activity 18 mm zone of inhibition is expressed by S. microflavus (Kubo et al., 1993). In this study the genus Micromonospora also exhibit antimicrobial activity against test organism (Charan et al., 2004). The activity of inhibitory effect against S. oralis is not produced by Dactylsporangium roseum, more efficient activity is observed on the S. purpurens which is 36 mm. It was also found that the crude extract shows maximum inhibitory against Streptococcus oralis and S. mutans. The only pertinacious substance active against S. mutans is reported by Dactylsporangium roseum and no one protein active against S. oralis. In this study Streptoverticillium is moderatively active against S. oralis (24 mm) and Intrasporangium, Micromonospora sp. and S. microflavus are less effective strain.