Introduction

The ever increasing knowledge in the area of pathogen`s drug resistance has evoked the discovery of new antibiotics by the screening of microbes. Last few decades has witnessed the production of novel antibiotics from different microorganisms. At present, aerobic Actinomycetes have attracted considerable attention of bacteriologist, geneticist and ecologist because of the production of novel antibiotics8. In the present study, Actinomycetes were isolated from soil samples collected form Senbagadaruvi, a natural water falls in Western Ghats, India. Due to it’s complexity in structure, heterogeneous landscape and high level of biodiversity5, the region has formed an ideal ground for screening of various types of Actinomycetes. The isolates were screened to identify their capacity to produce bioactive substances against microbes such as fungi and bacteria. A special attention was also given to Mycobacterium smegmatis, which is a rapidly growing analogue of Mycobacterium tuberculosis as well as one of the normal flora of human body3. In addition, an attempt was also made to identify the isolates at generic level.

Materials and Methods

In the present study the soil samples were collected from 20 different sites of Senbagadaruvi region using a clean spatula and polythene bags, labeled and placed in room temperature for 2 days. Actinomycetes were isolated from the soil samples by using Starch Casein Nitrate agar with cycloheximide (50µg/ml). The plates were incubated at room temperature for 5-7 days and the colonies of actinomycetes were identified based on the methods described by Williams and Cross10. The selected actinomycetes were labeled with SEN as prefix followed by numerical annotation. Microscopic structures like hyphae, surface mycelium, spores and their arrangement and etc of the Actinomycetes were determined from cover-slip culture and slide culture methods4. A simple qualitative analysis of cell wall amino acid DAPA (2-6 Diaminopimelic acid) was carried out9.

Antagonistic activities

Screening procedure was carried out by Giant Colony/ cross plate technique and well plate method2. Shake flask fermentation was carried out with 250ml of Antibiotic production medium (soluble starch 25g, glucose 10g, yeast extract 2g, CaCO33g, Trace salts solution 1ml, Distilled water 1 liter, pH 7.5±0.2.

Trace salt solution – FeSO4.7H2O – 0.5g; CuSO4.5H2O – 0.5g; ZnSO4.7H2O – 0.5g; MnCl2.4H2O – 0.5g in 100ml of Distilled water. The flask was incubated in a shaker for 6-7 days at 28ºC and 220rpm. The culture filtrate was tested for antimicrobial activities (IMTECH). Mycelial extract (1:1) was prepared with methanol, filtered and tested against microbes for it`s antimicrobial activity by the well plate method. In the present study solvent extraction was also done with selective Actinomycete`s spent medium using Ethyl acetate as solvent .The spent medium and the solvents were thoroughly mixed, the upper solvent layer was concentrated in vacuo to get extracted substances4.

Detection of type of Antibiotics produced

Experiments were carried to find out the presence of MLS (Macrolide-Lincosamide-Streptogramin) group of antibiotics, Glycopeptide / cell wall binding antibiotics and β-lactam antibiotics in the samples after fermentations (IMTECH).

Result and Discussion

A total of 104 different Actinomycetes strains were isolated from the soil samples collected from 20 different sites at Senbagadaruvi (Western Ghats) region. In the present investigation 14 isolates were chosen randomly and screened for antibiotic production. The photographic plate shows the isolated colonies of actinomycetes SEN1 on Starch Casein Nitrate Agar (Fig 1). The selected isolates were found to be Gram positive, amylase and lipase producers. The microscopic structures and the cell wall DAPA revealed that the isolates have characteristic features of Actinomycetes (Table 1). The photomicrograph of Actinomycetes SEN1 is shows in the Fig 2.

Fig. 1: Actinomycetes SEN1 colony on Strach Caesin Nitrate Agar.

Fig. 2: The Photomicrograph of Actinomycetes SEN1(X1000).

Table 1. DAPA and cover slip observation.

Based on the giant colony technique, the effective actinomycetes strains were chosen for further study. In giant colony, Actinomycetes showed antagonistic activity against B. thuringiensis and S.aureus and they were less effective against Gram negative bacteria like P. aeruginosa and E.coli. Fig 3. Strains SEN1 and SEN5 showed antimicrobial effect against Saccharomyces cerevisiae, Candida albicans and yeast isolated from fruit. Others strains were found to be ineffective. The results are shown in the Table 2 and it is clear that the strain SEN1 and SEN5 showed antifungal activity.

Fig. 3: Antibacterial Activity of Actinomycetes SEN12.

1. Bacillus thuringiensis 2. Staphylococcus aureus 3. Pseudomonas aeruginosa 4. E.coli.

Table 2. Giant colony method of antibacterial activity assay.

The spent medium of SEN5, SEN 9 and SEN12 were active against S. aureus and B. thuringiensis while SEN10 was effective against P. aeruginosa (Table 3). The antibacterial activity of strain SEN9 against S. aureus (MTCC2940) was shown in Fig 4. The mycelial extract of SEN9 and SEN12 were retained their antimicrobial activity where as SEN5 did not show similar activity. The secondary screening (well plate method) results differed from that of primary screening (Giant colony) method. Similarly1 observed that Actinomycetes isolates often show antibiotic activity on agar but not in liquid culture.

Fig. 4: Antibacterial activity of Actinomycetes SEN9 against S.aureus (MTCC2940).

Table 3. Well plate method assay for antibacterial compounds.

Selected strains like SEN 5, SEN 7, SEN 9 and SEN 12 (spent medium and mycelia extract) were tested against (Methicillin Resistant Staphylococcus aureus) MRSA, all strains showed potent antagonistic activity against MRSA. Strains SEN 5, SEN 9 and SEN 12 (spent medium and mycelia extract) were tested against M. smegmatis and showed antimycobacterial activity. The secondary metabolite recovered from whole fermentation broth of SEN5, SEN 9 and SEN 12 in Ethyl acetate was tested against B. thuringiensis and showed good activity. Similarly strains SEN2, SEN 5, SEN6 and SEN8 were active against yeast strains such as S. cerevisiae and C. albicans.

Our current study found that none of the isolates produced MLS type of antibiotics. Strains such as SEN5, SEN9 and SEN 12 produced both β-lactam antibiotics and Glycopeptide / cell wall binding antibiotics. SEN5, SEN 9 and SEN12 were identified as Streptomyces group based on the morphology and cell wall biochemistry. These strains were considered as the potent antibiotic producing strains. The results of primary and secondary screening revealed that most of the active isolates were active against Gram positive bacteria (B. thuringiensis and S. aureus). The observations are similar to that of Bhagabati1. Generally P. aeruginosa is considered to be highly resistant to antibiotics, but in our present study only one strain SEN10 is effective against P. aeruginosa.

The strains MRSA and Mycobacterium species are controlled by SEN5, SEN9 and SEN12 in our investigation. As these isolates may contain active bioactive substances. In all aspects, strain SEN5 showed broad spectrum of antimicrobial activity and it inhibits all groups of microbes used in our study.

Similarly, Deshmukh and Sridhar2 studied antimicrobial activity of mesophilic and thermophilic actinomycetes isolated from fresh water streams. They reported that the isolates like MS13, TC3, MM10 and MS19 showed inhibition against B. subtilis, E.coli, A. flavus and S. cerevisiae. Joseph7 isolated streptomycetes from the marine sponge Dendrilla nigra. The chosen isolate showed inhibitory interaction with Micrococcus luteus and the extracellular products contain potent antibacterial agents. Bhagabati 1 studied antibacterial activity of actinomycetes isolated from Lobuche area and Lukla area in Khumbu region. A total of 106 actinomycetes were subjected to primary screening by perpendicular streak method against Gram-positive (B. subtilis and S. aureus) and Gram-negative (Enterobacter aerogens, E.coli, Klebsiella sp, Proteus sp, Pseudomonas sp, Salmonella typhi and Shigella spe) bacteria. Gogoi4 screened Actinomycetes strains from different agro ecological niches of river island Majali, showed antimicrobial potential against Ralstonia sp, Xanthomonas sp, B. subtilis, Proteus mirabilis, E.coli, Rhizoctonia solani and Fasarium oxysporum.

Conclusion

In the present study, the actinomycetes isolated from soil samples of Senbagadaruvi, Western Ghats showed antibiotic activity against Gram positive and Gram negative bacteria, yeast, drug resistant MRSA and M. smegmatis. This analysis would be valuable to tackle drug resistant infections in the future. Further, attempts are being carried out to characterize the bioactive substances.