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ORIGINAL ARTICLE  
Year : 2011  |  Volume : 3  |  Issue : 3  |  Page : 241-245
Anti-infective potential of hot-spring bacteria


1 Department of Biotechnology, The KET's V.G.Vaze College, Mumbai University, India
2 Natural Products, Piramal Life Sciences Limited, Mumbai, India

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Date of Web Publication6-Aug-2011
 

   Abstract 

Aim and Background: Antibiotic resistance currently spans most of the known classes of natural and synthetic antibiotics; limiting our options for treatment of infections and demanding discovery of new classes of antibiotics. Much effort is being directed towards developing new antibiotics to overcome this problem. Success in getting novel chemical entities from microbial sources depends essentially on novelty of its habitat. The diversity of geographical location decides the type of micro-flora. In the past various terrestrial and aqueous microorganisms have provided several novel bioactive secondary metabolites of pharmaceutical importance. Hot-springs have not been as extensively exploited as other terrestrial resources. However, perseverance with such microbes augment the probability of getting novel bioactive compounds. Materials and Methods: Hot-springs soil samples were collected from Hot-springs in Maharashtra. Actinomycetes and other eubacteria were isolated from these soil samples by selective methods and purified. They were classified based on gram's nature and morphology. Six representative morphological strains were screened for their anti-infective potential by agar well diffusion method as reported by Nathan P. et al (1974). The bioactivity of the active microbes was confirmed. Results: Seventy three strains of bacteria encompassing eight actinomycetes, and 65 eubacteria were isolated and purified. Among the actives eubacteria PPVWK106001 showed broad spectrum antibacterial activity encompassing both gram positive and gram negative bacterial test models. The extract was active against resistant bacteria such as MRSA and VREs. Activity was very specific as there was no activity against fungi even at 100 fold concentration. The active principle was extractable in butanol. Conclusions: The study showed that Hot-springs exhibit diverse bacteria and it serves as potential reservoirs for bacteria of antimicrobial importance with diverse facet of activities. Thus Hot-springs microbes have ability to address issue of resistant bugs.

Keywords: Antibacterial activity, Drug-resistant strains, Hot-springs

How to cite this article:
Pednekar P, Jain R, Mahajan G. Anti-infective potential of hot-spring bacteria. J Global Infect Dis 2011;3:241-5

How to cite this URL:
Pednekar P, Jain R, Mahajan G. Anti-infective potential of hot-spring bacteria. J Global Infect Dis [serial online] 2011 [cited 2019 Nov 19];3:241-5. Available from: http://www.jgid.org/text.asp?2011/3/3/241/83529



   Introduction Top


There is a growing awareness of public health concerns associated with the emergence of drug-resistant strains of bacteria. [1] The occurrence of multiple antibiotic-resistant bacteria has become a major challenge in the treatment of infectious diseases. [2],[3] This has created an urgent need for antibiotics with novel scaffolds and mechanism of actions. To achieve metabolite structural diversity, it is generally agreed that a diverse and novel repertoire of microbes is needed. [4] This can be accomplished by isolating microbes from diverse natural ecological units. Hot-springs have been less explored ecological sects for discovery of novel microbial bioactive compounds as compared to other terrestrial samples. [5]

It demonstrates extreme environmental conditions (higher temperature and alkaline pH). [6] Many microbial strains of following major groups/genera have been reported from Hot-springs, viz. sporulating and non-sporulating bacilli, actinomycetes and cyanobacteria, within it thermophilic actinomycetes comprise of the genera Streptomyces, Micromonospora, Actinomadura, Saccharomonospora and Thermoactinomyces. [7] A new strain,  Exiguobacterium aurantiacum Scientific Name Search Road (BAA -333), has been reported from Yellowstone National Park. [8] Bacilli like Thermus aquaticus, Thermus brockianus, from Hot-springs have gained commercial significance as source of thermostable enzymes. [9],[10] On the other hand, there is hardly any report of bioactive compounds from the micro-flora of Hot-springs till date, which opens a window for exploring this resource as potential cache of bioactive compounds. In this paper, we report on the microbial profiling of and antibiotic production potential of microbes isolated from Vajreshwari-Ganeshpuri Hot-Springs, in Western part of India as part of our exploration for new antinfective agents.


   Materials and Methods Top


Sampling

The Hot-springs stretching about 7-km in the bed of the river Tansa are mainly situated at Akoli, Vajreshwari, Ganeshpuri, and Satvalli, in the Western part of Maharashtra state in India. The temperature of the water in the springs ranged from 40°C to 65°C. During the month of May 2006, water from Hot-springs vase, inner wall scrapings of the vents, bottom sediment samples from four sampling points at Vajreshwari and Ganeshpuri Hot-springs were collected. The samples were collected from origin (referred to as face) of the Hot-springs, which are the area known to be not tampered by human activities as such. The samples were collected in sterile polycarbate containers. All the samples were transported to Piramal Life Sciences Limited for further work. They were processed within 24 hours after collection.

Isolation of micro-organisms

The Hot-springs source samples were suitably diluted with N-saline (0.85% sodium chloride in demineralised water) and 100 μl of diluted samples were spread on agarified isolation media. Initially optimization studies were carried out to decide a suitable media for isolation of Hot-spring bacteria (data not published). However, Actinomycetes Isolation Agar (AIA) and Soybean Casein Digest Agar (SCDA) were found to be most suitable, but with enhanced incubation period and incubation temperature (37−39°C instead of 28−30°C). For isolation of actinomycetes, AIA [11] and for other Eubacterial isolation SCDA [12],[13] were used. Amphotercine B was incorporated in both media at 20μg/ml concentration to limit the fungal growth, which often overgrow the isolation plates. The media were incubated at 37−39°C and observed regularly up to 30 days. Colonies of bacteria or actinomycetes growing on isolation plates were purified and the pure cultures were streaked on corresponding isolation media and incubated at 37−39°C. Well grown culture slants were used for shake flask fermentation.

Fermentation conditions and sample preparation

LPM-1 [Glucose 1.5%, Corn Steep Liquor; (CSL) 0.5%, Peptone 0.75%, Yeast extract 0.75%, CaCO 3 0.2%, NaCl 0.5%, pH 7.0] was used as seed medium for all isolates. Slant cultures were inoculated into LPM-1 medium and incubated at 30−32°C, 200 rpm for 72 hrs. The production medium LPM-2 [20 ml 5X M9 salts (6.4% Na 2 HPO 4 .7H 2 O, 1.5% KH 2 PO 4 , 0.25% NaCl), 0.2 ml 1M MgSO 4 , 10 μl 1M CaCl 2 , NH 4 Cl 0.1%, Glucose 0.4%, pH 7.3] was inoculated with 2% (v/v) seed and grown 30−32°C, 200 rpm for 7 days. Acetone extract of the whole broth was prepared using acetone and whole broth at 1:1 (v/v). The lyophilized extract was used further for anti-infective testings. The extracts were dissolved in sterile demineralized water and methanol (1:1, v/v) and used for anti-infective screening assays at 0.01, 0.1, 1.0 mg/ml concentration.

In vitro anti-infective screening

Bacterial test organisms

Staphylococcus aureus 209P (Methicillin Sensitive Staphylococcus aureus "MSSA"), Staphylococcus aureus E710 (Methicillin-resistant Staphylococcus aureus "MRSA"), Enterococcus faecium R-2 (Vancomycin Resistant Enterococci "VRE"), Enterococcus faecium (Vancomycin Sensitive Enterococci "VSE"), and  Escherichia More Details coli-2231-SS.

Test culture from the slant was inoculated into sterile 4.0 ml Soybean casein dextrose broth (SCDB). The tubes were incubated at 35−37°C for 18 hrs. About 100 μl of the culture suspension [~5 × 108 colony forming units (CFU)/ml] was added to sterile 4.0 ml SCDB and used as stock suspension.

Fungal test organisms

Candida albicans ATCC 14503, Candida krusei, Candida glabrata, and Aspergillus fumigatus.

The fungal culture from the slant was inoculated into sterile 4.0 ml Sabouraud dextrose broth (SDB). The tubes were incubated at 35−37°C for 18 hrs. 100 μl of this suspension [~10 8 colony forming units (CFU)/ml] was added to sterile 4.0 ml SDB and used as stock suspension.

The assay was performed by the method followed by Nathan et al. [14],[15] Predefined volume of stock test culture was put into 40 ml melted (maintained at 40°C) medium. The seeded butt was poured into the petriplate. It was allowed to set for about 30 mins. Required numbers of wells (of diameter 6 mm) were punched into the set medium. 50 μl of test sample was added in each well. The bioassay plates were pre-incubated at 4°C for 30 mins to allow diffusion of the compounds around the agar well. Later the plates were incubated at 35−37°C for 24 hrs.

The results were recorded as diameter of zone (expressed in mm) of clearance around each well. The values in the results are average of values obtained in duplicate testings.


   Results Top


Seventy three strains of bacteria encompassing eight actinomycetes, and 65 eubacteria were isolated and purified from the four sampling points of Hot-springs. The isolates reflected the diversity with respect to macroscopic and microscopic characteristics. Twenty four were Gram-positive and 49 were of Gram-negative bacteria. About 11% of the isolates were actinomycetes. Within 24 Gram-negative bacilli, nine were coccobacilli [Table 1]. Six representative morphological strains were screened for their anti-bacterial or anti-fungal potential. These six strains included five Gram-positive and one Gram-negative bacteria. All were distinct with respect to their colony characteristics [Table 2].
Table 1: Distribution of cultures isolated from Hot-springs

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Table 2: Colony characteristics of six strains isolated from Hot-springs of Vajreshwari-ganeshpuri

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The extracts were tested at concentration of 0.01, 0.1, 1.0 mg/ml. Among the six strains, the culture PPV-WK106001 showed better spectrum of inhibition at 1.0 mg/ml. It showed activity in both Gram-positive and Gram-negative cultures (a) [Table 3] and [Table 4]. Strain PPV-WK106003, at 1.0mg/ml showed moderate activity against only Enterococci faecium R-II 323 (VRE). Strain PPV-SK206032; an actinomycete also showed broad-spectrum antibacterial activity. None of these three isolate showed antifungal activity. It indicated the specific antibacterial activity [Table 3].
Table 3: Anti-infective activity of the six selected isolates determined by agar the well diffusion assay

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Table 4: Anti-infective activity of extracts of PPV-WK106001

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Based on the results of the screening the active extract of PPV-WK106001 was processed so as to find the polarity of the active compound. The active component was extractable in butanol (Refer Fraction (c) ) as it showed inhibition zones on 12 mm or greater against all four bacterial strains [Table 4]. There is no activity in EA extracts (b) or spent. It indicated that the active component in PPV-WK106001 was having intermediate polarity.


   Discussion Top


When the antibacterial activity of six bacteria was observed against test bacteria, Gram-negative test bacteria showed limited susceptibility to the extracts while the Gram-positive bacteria were more susceptible. All six isolates used were Gram-positive aerobic rods and some among these were filamentous. Our findings confirm that majority of antibacterial agents reported from bacilli, paenibacillus and streptomycetes, which are major representatives of antibiotic producing Gram-positive aerobic rods, are anti-gram positive. [16] Isolate PPV-WK106001 a Gram-positive, non-motile, non-spore bearing bacillus was distinct among the Hot-Spring isolates with respect to its activity. It showed a broad-spectrum activity encompassing Gram-positive bacteria such as Staphylococcus aureus 209P, Methicillin-resistant Staphylococcus aureus E710, E. faecium R-II (VRE), and Gram-negative bacteria such as Escherichia coli-2231 (Super Sensitive Strain). Its anti-gram positive activity is better than anti-gram negative activity. Moreover the strain PPV-WK106001 had shown no antifungal activity, which indicates that the antibacterial activity shown by this culture, was specific. The activity is maintained after 35 days at 4−6°C, reflecting its stability. This active compound being butanol extractable, indicates that it is more polar compound.

The potential of Hot-springs microbes as source of antibiotic drugs seems to be promising. Within three actives two had shown broad spectrum antibacterial activity including infections caused by pharmaceutical bad bugs like VREs [17],[18] and the MRSA. [19] This study has shown that Hot-springs exhibits diverse bacteria and it serves as potential reservoirs for bacteria of antimicrobial importance with diverse facet of activities. A detailed characterization of the active principle of the antibacterial extract is the subject of ongoing investigation in our team.

 
   References Top

1.Raghunath D. Emerging antibiotic resistance in bacteria with special reference to India. J Biosci 2008;33:593-603.  Back to cited text no. 1
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2.Davies J. Inactivation of antibiotics and the dissemination of resistance genes. Science 1994;264:375-82.  Back to cited text no. 2
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3.WiseR. The relentless rise of resistance? J Antimicrob Chemother 2004;54:306-10.  Back to cited text no. 3
    
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5.Auguet JC, Barberán A, Casamayor EO. Global ecological patterns in uncultured Archaea. ISME J 2010;4:182-90.  Back to cited text no. 5
    
6.Farmer JD. Hydrothermal systems: Doorways to early biosphere evolution. GSA Today 2000;10:1-9.   Back to cited text no. 6
    
7.Jiang C, Xu L. Actinomycete diversity in unusual habitats. Actinomycetes 1993;4:47-57. ( http://tspace.library.utoronto.ca/html/1807/21366/ac93007.html )  Back to cited text no. 7
    
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9.Brock TD, Freeze H. Thermus aquaticus gen. n. and sp. n., a non-sporulating extreme thermophile. J Bacteriol 1969;98:289-97.  Back to cited text no. 9
    
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11.Jayashree S, Vyas V, Bhatnagar GP, Syed S. On selection of methods and media for isolation of freshwater actinomycetes. Environ Technol 1991;12:1183-6.  Back to cited text no. 11
    
12.Balakrishnan S, John KR, George MR. Antibiotic susceptibility of Bacillus spp. isolated from shrimp (Penaeus monodon) culture ponds. Indian J Marine Sci 2003;32:81-4.  Back to cited text no. 12
    
13.Rifai S, Fassouane A, Kijjoa A, Soest RV. Antimicrobial activity of Untenospongin B, a metabolite from the marine sponge Hippospongia communis collected from the Atlantic coast of Morocco. Marine Drugs 2004;2:147-53.  Back to cited text no. 13
    
14.Nathan P, Law EJ, Murphy F, MacMillan BG. A laboratory method for selection of topical antimicrobial agents to treat infected burn wounds. Burns 1974;4:177-87.  Back to cited text no. 14
    
15.Hangngu Vu, McCoy LF, Carino E, Washington J, Dang T, Villarreal C, et al. Burn wound infection susceptibilities to topical agents: The Nathan′s Agar Well Diffusion Technique. Pand. T. 2003;27:390-6.  Back to cited text no. 15
    
16.Available from: http://www.antibiotics.or.jp/journal/database/database-top.htm [Last cited on 2010, Apr 13].  Back to cited text no. 16
    
17.Murray BE. Vancomycin-resistant enterococcus infections. N Engl J Med 2000;342:710-21.  Back to cited text no. 17
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19.Healy CM, Hulten KG, Palazzi DL, Campbell JR, Baker CJ. Emergence of new strains of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit. Clin Infect Dis 2004;39:1460-6.  Back to cited text no. 19
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Correspondence Address:
Roopesh Jain
Natural Products, Piramal Life Sciences Limited, Mumbai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-777X.83529

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]

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