Abstract: The present study was conducted with a view to isolate and identifies antibiotic producing microorganisms from pond sediments against fish pathogenic and human clinical bacterial isolates. During the course of study, a total of twenty actinomycetes isolates were recovered from 10 soil samples from different regions of Satkhira and Sylhet district using starch-casein-agar and glycerol-arginine-agar media supplemented with cyclohexamide (5 μg L-1). Selected isolates from the isolation media was carried out by multiple streak method to obtain the pure cultures of selected isolates. All of the 20 isolates exhibited a range of colors including brown, creamy, yellowish and greenish in the isolation media. All of the isolates were then screened for their antagonistic activity against 4 genera of fish pathogenic and 2 genera of human clinical bacterial isolates using agar streak method. The fish pathogenic isolates belonged to Pseudomonas sp. Aeromonas sp. Flavobacterium sp. and Edwardsiella sp. whereas human clinical samples included Klebsiella sp. and Salmonella sp. isolates. All of the isolates showed remarkable antimicrobial activities against the test organisms and isolate No. N4/10 and N4/4 appeared to be the most promising ones. Several morphological, physiological and biochemical characterization tests were performed to identify the antibiotic producing soil isolates up to genus level. All the isolates were identified as actinomycetes.
INTRODUCTION
Antibiotics are one of the pillars of modern medicine (Ball et al., 2004) but the rate of loss of efficacy of old antibiotics is outstripping their replacement with new ones for many species of pathogenic bacteria (Hancock, 2007). The emergence of antibiotic resistant bacteria is a problem of growing significance in dermatological and surgical wound infections (Colsky et al., 1998; Giacometti et al., 2000). In general, the most important resistance problems in the management of wounds have been observed with S. aureus and coagulase-negative Staphylococci among the Gram-positive species and with E. coli, Klebsiella pneumoniae and P. aeruginosa among the Gram-negative species (Filius and Gyssens, 2002).
The history of new drug discovery processes shows that skeletons have, in the majority of cases, come from natural sources (Bevan et al., 1995). Considerable research is being done in order to find new chemotherapeutic agents isolated from soil (Rondon et al., 2000; Crowe and Olsson, 2001; Courtois et al., 2003). Soil microbial communities are among the most complex, diverse and important assemblages of organisms in the biosphere and they participate in various biological activities. Accordingly, they are an important source for the search of novel antimicrobial agents and molecules with biotechnological importance (Hackl et al., 2004).
Many groups of microorganisms like Gram-positive, Gram-negative bacteria and fungi have the ability of synthesizing antimicrobial agents and the top cultivable antimicrobial agent producers present in soils are the actinomycetes (Pandey et al., 2002). On the whole, the last 55 years have seen the discovery of more than 12,000 antibiotics. The actinomycetes yielded about 70% of these and the remaining 30% are products of filamentous fungi and non-actinomycete bacteria (Nanjwade et al., 2010). Actinomycetes are responsible for the production of about half of the discovered bioactive secondary metabolites (Berdy, 2005), notably antibiotics (Strohl, 2004).
Actinomycetes encompass a wide range of bacteria. They have universal occurrence and play an active part in the cycle of nature. They are a well defined group of Gram positive, free-living, saprophytic bacteria with high G+C content in their DNA (Lo et al., 2002). They are predominant in dry alkaline soil. Many of the presently used antibiotics such as streptomycin, gentamicin, rifamycin and erythromycin are the product of actinomycetes. Keeping in mind the above mentioned ideas the present study was aimed to screen actinomycetes from pond sediments for antibacterial compounds against some fish pathogenic and human clinical bacterial isolates.
MATERIALS AND METHODS
Collection of soil samples: A total of 10 soil samples were collected from pond sediment of different localities of Satkhira and Sylhet district during February 2012 to April 2012. All the soil samples were collected using some clean, dry and new polythene bags along with sterile spatula and transported to the USDA (United States Department of Agriculture) Project Laboratory of the Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet for further studies.
Pretreatment of soil samples: All the soil samples were pretreated for inhibiting or eliminating unwanted microorganisms. Soil samples were dried at 70°C for 15 min to kill other competing bacteria and enabled the desired microbes to grow on the provided media.
Isolation of actinomycetes: Numerous media have been used for the isolation of actinomycetes from soil and other natural materials. In this investigation, starch-casein-agar (Kuster and Williams, 1964) and glycerol-arginine-agar (GAA) medium (Porter et al., 1960) were used for isolation of actinomycetes. Cyclohexamide (50 μg L-1) was added into the isolation media just before pouring into the plates.
The soil samples (1g) were added to a conical flask containing 100 mL of sterile water and few drops of Tween 80. The flasks were shaken for 30 min in an orbital shaker incubator at 27°C and their contents were designated as stock cultures. A series of culture tubes containing 9 mL of sterile water was taken. From the stock culture, 1 mL suspension was transferred aseptically to the 1st tube (10-1) and mixed well. From the 1st tube, 1 mL of suspension was transferred into 2nd tube (10-2), mixed well. Similarly, dilutions up to 10-5 were made (serial dilution technique). Suspension (0.1 mL) from each culture tube was spread on starch-casein-agar and glycerol-arginine-agar medium plates aseptically in a laminar-air low cabinet. The plates were incubated at 27±2°C for 84 h. The plates were observed intermittently during incubation. After 72 h, whitish pin-point colonies, characteristic of actinomycetes and with a clear zone of inhibition around them were seen. The pinpoint colonies with inhibitory or clear zone of inhibition were selected and purified into nutrient agar plates by multiple streaking methods. The stock cultures of each selected isolate was prepared and maintained in nutrient agar slants at 4°C. The pinpoint colonies isolated were selected for further studies.
Study of antibacterial activity of soil bacterial isolates: All the soil bacterial isolates were tested for their antibacterial activity against four genera of fish pathogenic namely pseudomonas sp., Aeromonas sp., Flavobacterium sp., Edwardsiella sp. and two genera of human clinical (Klebsiella sp. and Salmonella sp.) bacterial isolates. The microbial sensitivity of the soil isolates was analyzed by agar streak method. Each of the isolate was streaked as a straight line on nutrient agar plate and incubated at 27°C for 6 days (144 h). On 6th day of incubation, test microorganisms were streaked at right angle but not touching each other and then incubated at 30°C for 24 h. If the test organism is susceptible to the antibiotic produced by the selected strains, then it would not grow near that strain. The zone of inhibition against each test organism was noted. Based on their antimicrobial properties, isolates were chosen for further morphological and biochemical characterization.
Characterization of the selected isolates: The selected soil bacterial isolates were characterized by morphological and biochemical methods. Morphological characteristics such as colony size, shape, colour etc. were observed. Recovered soil bacterial isolates with supporting growth characteristics of actinomycetes were subjected to various biochemical tests named melanoid pigment formation, catalase, oxidase, Hydrogen Sulphide Production (H2S), Methyl Red (MR), Voges Proskauer (VP), casein hydrolysis, starch hydrolysis and indole reaction.
RESULTS
Isolation of actinomycetes: This study was undertaken with a view to screen actinomycetes having antimicrobial activity. In the present study, eighty one whitish pinpoint colonies bearing colony characteristics of actinomycetes and with a clear zone of inhibition were isolated using starch-casein agar and glycerol-arginine-agar media. Out of 81 actinomycetes isolates that were subjected for primary screening process, only 20 isolates showed inhibitory activities against test organisms. Pertinent details of the soil samples and the actinomycetes are shown in Table 1.
| Table 1: | Characteristics of soil samples and No. of isolated colonies |
HA: Heat not applied, AP: Heat applied, AGA: Glycerol-arginine-agar, SCA: Starch-casein-agar, Dilution of soil samples was done by two-fold serial dilution technique | |
The isolates were designated as N2/1-N2/10 and N4/2-N4/11. Among 20 isolates 6 isolates exhibited significant antibacterial activity against test microorganisms (Table 2-7). The test organisms used in this study were collected from USDA project Laboratory, Shahjalal University of Science and Technology, Sylhet and MAG Osmani Medical College, Sylhet, Bangladesh. Antimicrobial tests conducted showed that isolate N4/10, N2/9 and N4/5 were the most promising for Pseudomonas sp. isolates (Table 2 and Fig. 1, 2) whereas N4/4 and N4/10 for Aeromonas sp. isolates (Table 3).
| Table 2: | Antibacterial activity of soil bacterial isolates against fish pathogenic Pseudomonas sp. isolates |
+++: Very good inhibition, ++: Good inhibition, +: Moderate inhibition, -: No inhibition | |
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| Fig. 1: | Antibacterial activity of isolate N4/5 against Pseudomonas sp. isolates |
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| Fig. 2: | Antibacterial activity of isolate N4/10 against Pseudomonas sp. isolates |
| Table 3: | Antibacterial activity of soil bacterial isolates against fish pathogenic Aeromonas sp. isolates |
+++: Very good inhibition, ++: Good inhibition, +: Moderate inhibition, -: No inhibition | |
Besides this, isolate N2/9, N2/10 and N4/5 were found to be most effective against Salmonella (Table 7), Klebsiella (Table 6) and Edwardsiella sp. (Table 5).
Identification of soil bacterial isolates: Identification of the isolates was carried out using morphological and biochemical properties. Morphological studies were carried out by microscopic observation and studies on growth characteristics in Petri dishes.
| Table 4: | Antibacterial activity of soil bacterial isolates against fish pathogenic Flavobacterium sp. isolates |
+++: Very good inhibition, ++: Good inhibition, +: Moderate inhibition, -: No inhibition | |
| Table 5: | Antibacterial activity of soil bacterial isolates against fish pathogenic Edwardsiella sp. isolates |
+++: Very good inhibition, ++: Good inhibition, +: Moderate inhibition, -: No inhibition | |
| Table 6: | Antibacterial activity of soil bacterial isolates against human clinical Klebsiella sp. isolates |
+++: Very good inhibition, ++: Good inhibition, +: Moderate inhibition, -: No inhibition | |
| Table 7: | Antibacterial activity of soil bacterial isolates against human clinical Salmonella sp. isolates |
+++: Very good inhibition, ++: Good inhibition, +: Moderate inhibition, -: No inhibition | |
| Table 8: | Biochemical characterization of soil bacterial isolates |
G: Gram staining, C: Catalase, O: Oxidase, H2S: Hydrogen sulfide, MR: Methyl Red, VP: Voges-proskauer, CH: Casein hydrolysis, SH: Starch hydrolysis, I: Indole, LBP: Light brown pigmentation (+): Positive result, (-): Negative result | |
On the basis of morphological and biochemical characteristics (Table 8) all the isolates were belonged to the actinomycetes group.
DISCUSSION
The increase in the frequency of multi-resistant pathogenic bacteria is created an urgent demand in the pharmaceutical industry for more rational approaches and strategies to the screening of new antibiotics with a broad spectrum of activity, which resist the inactivation processes exploited by microbial enzymes (Saadoun and Gharaibeh, 2003; Motta et al., 2004).
Screening and isolation of promising actinomycetes with potential antibiotics is still a thrust area of research and it is suggested that the exploration of materials from different areas and habitats have a vital role to play in the search for new microbes and novel metabolites and is urgent to counter the threats posed by the fast emerging phenomenon of antibiotic resistance (Saadoun and Gharaibeh, 2003). The present work was conducted with a view to isolate and identifies actinomycetes from pond sediments of Sylhet and Satkhira district and to screen their potential to generate antimicrobial activities. Several researchers have also screened soil samples collected from different parts all over the world for antimicrobial agent producing microorganisms (Huddleston et al., 1997; Ouhdouch et al., 2001; Haque et al., 1992; Thakur et al., 2007; Yadav et al., 2009; El-Naggar et al., 2006).
In the present study, the soil samples were heated at 70°C for 15 min for the recovery of actinomycetes and it is found that, the temperature at 70°C for 15 min was suitable treatment method to isolate more actinomycetes colonies from soil samples. Seong et al. (2001) also reported that when the soil samples were cultured without pretreatment (heat at 70°C) the number of colonies recovered was in the order of other bacteria, Streptomyces fungi and non-streptomycete actinomycetes. During the present study, four soil samples were cultured without pretreatment and it was apperared that no actinomycetes colonies were present on the isolation media. The results of the present study gives clear picture about the significance of pre-treatment method for the isolation of actinomycetes. This type of pre-treatment methods for isolation of actinomycetes has also been suggested by several researchers (Hayakawa and Nonomura, 1987; Hayakawa et al., 1991; Jensen et al., 1991; Kim et al., 1994).
The isolation media used in this study was supplemented with cyclohexamide (50 μg L-1) to inhibit fungal growth because fungi often contaminate the isolation media. Saadoun et al. (2007) also supplemented the isolation medium with cyclohexamide (50 μg L-1) in succeeding experiments. The present study was carried out using Starch-casein-agar (SCA) and glycerol-arginine-agar media for the isolation of actinomycetes. In this study starch-casein-agar media was found more effective than glycerol-arginine-agar media for the isolation of actinomycetes. Similar studies were also carried out by Raja et al. (2010). Different types of bacterial colonies were developed on isolation medium. Colonies having characteristic features such as powdery appearance with convex, concave or flat surface and color ranging from white, gray to pinkish and yellowish were selected. Isolates were initially screened based on zone of clearance and they were then subjected to preliminary screening. From the preliminary screening, twenty bacterial isolates were collected having antibacterial activities. However, out of these twenty bacterial isolates six isolates showed remarkable antibacterial activities against test organisms.
All of the 20 isolates showed antibacterial activity with at least one test bacteria. The test microorganisms included 6 Gram negative fish pathogenic and human clinical bacteria as indicated earlier. Then the antibiotic producing soil bacterial isolates were screened against the laboratory isolates belonged to six genera viz. Pseudomonas sp., Aeromonas sp., Flavobacterium sp., Edwardsiella sp., Klebsiella sp., Salmonella sp., Isolates N4/5, N4/10 and N2/9 showed antibacterial activities against all six isolates of Pseudomonas sp., having N4/10 was the most promising (Table 2). Isolate No. N4/10 showed very good inhibition against Pseudomonas sp., isolates P2F4, Cla287, Cla1B/8 and PFN3 (Table 2). Besides thzs, isolate no. N4/4 and N4/10 showed 100% antibacterial activities against all fifteen isolates of Aeromonas sp. and N4/10 was the most promising (Table 3). None of the antibiotic producing isolates showed 100% antibacterial activity against all of the isolates of fish pathogenic Flavobacterium sp. However, the isolates N4/4 and N4/10 showed 90% antibacterial activity against Flavobacterium sp. isolates (Table 4). None of the soil bacterial isolates showed promising result against the fish pathogenic Edwardsiella sp. isolates except N4/10 (Table 5). On the other hand isolates N2/9 and N2/10 exhibited promising result against all human clinical Klebsiella sp. isolates (Table 6). Above all isolate no. N4/10 appeared to be the most potent antibiotic producing isolate.
The soil bacterial isolates that were selected through microbial sensitivity test were further taken for physico-biochemical characterization. All the isolates were gram positive, catalase positive, oxidase positive, H2S positive and negative for MR, VP and indole test. Besides they were able to hydrolyze casein and starch. Based on the biochemical and morphological characteristics the isolates were identified as actinomycetes. It is expected that the current attempt for the isolation, characterization and the study on actinomycetes from pond sediments will be useful for the identification of new antibiotics effective against challenging pathogens.
ACKNOWLEDGMENT
The authors gratefully acknowledged to the authority of USDA Project Laboratory of the Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet, Bangladesh for providing facilities to carry out this research work.