Ann Microbiol DOI 10.1007/s13213-014-1028-3
ORIGINAL ARTICLE
Culturable bioactive actinomycetes from the Great Indian Thar Desert Kavita Tiwari & Dilip J. Upadhyay & Eva Mösker & Roderich Süssmuth & Rajinder K. Gupta
Received: 4 June 2014 / Accepted: 25 December 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015
Abstract The present study was attempted to determine the antimicrobial potential of actinomycetes from the Great Indian Thar Desert. A total of 100 different morphotypes based on phenotypic characterization were isolated from desert ecosystems located in the northwest of India and tested for their antimicrobial activity by the cross-streak method. Among the strains tested, 13 actinomycetes exhibiting strong antimicrobial activities against several test organisms, including multidrug resistant bacteria (MRSA) were chosen for a phylogenetic diversity study. The results of 16S rRNA gene sequencing showed their affiliation to actinobacterial genera: Actinomadura, Nocardia, Nonomuraea, Spirillispora, and Streptomyces. Three of these isolates were considered to be new members of the Streptomyces genus and another strain also seemed to be a new species of the genus Spirillispora. Among these strains, five were chosen to study the bioactive products using Q-Tof-MS because of their broad spectrum activity against the panel of test pathogens used. The results showed that they produce many known compounds and might produce few unknown compounds as well. This is the first such report on the selective isolation of actinomycetes from the Great Indian Thar desert, and their screening for antibacterial potential. This ecosystem has never before been explored to this extent. K. Tiwari : R. K. Gupta (*) School of Biotechnology, Guru Gobind Singh Indraprastha University, Dwarka, New Delhi 110078, India e-mail:
[email protected] D. J. Upadhyay New Drug Discovery Research, Department of Infectious Diseases, Ranbaxy Laboratories, Gurgaon, India E. Mösker : R. Süssmuth Institut für Chemie / OC / Biologische Chemie, Fakultät II Mathematik und Naturwissenschaften, Technische Universität Berlin, Sekr. TC 2, Straße des 17. Juni 124, 10623 Berlin, Germany
Keywords Great Indian Thar desert . Actinomycetes . Selective isolation . Antimicrobial activity . 16S rRNA . Bioactive compounds
Introduction Because of the growing problem of antibiotic resistance, pharmaceutical companies are now looking for novel molecules whose specificity is paralleled by decreased side effects. Natural products remain the most propitious source of novel antibiotics. Bio-diversity can provide us with an overwhelming reservoir of potentially active secondary metabolites. Actinomycetes are one of the most prolific producers of natural bioactive compounds. To date, actinomycetes have been isolated primarily from soil. However, recently, the rate of novel compound discovery from the widely explored terrestrial strains has decreased significantly (Lam 2006). To overcome this problem, actinomycetes have been isolated from other resources such as a desert ecosystem and marine sediments (Bull et al. 2005; Bull and Stach 2007). The likelihood of finding new molecules could be increased by switching the search away from explored environments to unexplored ones (Clardy et al. 2009). This is a response to the realization that microbial diversity has not been efficiently explored before. Most importantly, major environmental habitats are yet to be sampled for their unprecedented chemical diversity potential (Tiwari and Gupta 2013). Many natural environments are still either unexplored or underexplored and thus can be considered as a prolific resource for the isolation of lesser studied microorganisms including rare actinomycetes (Tiwari and Gupta 2012). These unexplored and under-explored niche habitats are regarded as bio-diversity hotspots, where it is believed that the effect of
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the local environment might result in the evolution of novel secondary metabolic pathways. It is assumed that rare actinomycetes undoubtedly represent an important source of novel secondary metabolites exhibiting a greater structural diversity than standard combinatorial chemistry (Tiwari and Gupta 2014). Screening actinomycetes from such environments in natural product screening collections could be one way of achieving this daunting task. More and different ecological niches need to be studied as sources of a greater diversity of novel microorganisms. Indeed, habitats such as desert biomes and marine ecosystems are now being described on a regular basis (Bull et al. 2005; Bull and Stach 2007; Bredholt et al. 2008; Okoro et al. 2009). The saline soils of deserts could be prime targets for discovering novel microbial biodiversity producing new bio-active compounds (Okoro et al. 2009). Most recently, three new 22-membered macrolactone antibiotics, atacamycins A–C, were found to be produced by Streptomyces sp. C38, a strain isolated from a hyper-arid soil collected from the Atacama Desert in the north of Chile (Nachtigall et al. 2011) and a new angucyclinone compound, antibiotic R2, was found to be produced by a Streptosporangium sp. Sg3, isolated from an Algerian Saharan soil (Boudjella et al. 2010). Numerous works on the biodiversity of actinomycetes that produce novel antibiotic molecules in India have been conducted and published to date, particularly from marine sources (Balagurunathan and Subramanian 1993; Balagurunathan and Subramanian 2001; Kokare et al. 2004). However, this type of research has not yet been reported from Indian hot deserts from this part of the Indian sub-continent apart from a single study conducted where a yellow colored antibiotic pigment was isolated from an actinomycete strain, Streptomyces hygroscopicus subsp. ossamyceticus (strain D10), previously isolated from Thar Desert soil, Rajasthan by (Selvameenal et al. 2009). The Great Indian Desert or Thar Desert is biogeographically the easternmost edge of the Saharan-Arabian desert zone. It is the desert region with the highest human population density in the world and is one of the most heavily populated (in terms of both people and cattle) deserts of the world (Arora et al. 2010). Indeed, this arid ecosystem may support a wide variety of microorganisms. Therefore, we chose to study the potential for biodiversity of microorganisms (particularly actinomycetes) in Indian hot deserts for industrial applications. Moreover, no studies have been conducted and published so far on the biodiversity of microorganisms of this ecosystem or on their biodiscovery potential. In the current study, we investigated the diversity of bioactive actinomycetes from “The Thar desert” (India). Our aim was to isolate actinomycetes from the soil of this hot desert and to screen them for antimicrobial activity against relevant bacterial pathogens. Novel antibiotic molecules might be secreted by microorganisms living in ecosystems with few competitors. Keeping
all this in mind we chose 20 separate sites in the sandy desert of Jaisalmer and Jodhpur districts of Rajasthan, India and tried to isolate species of actinomycetes that might prove to be sources for the discovery of novel antimicrobial agents. Through this study, we wish to update our understanding of the potential of actinomycetes from Indian hot deserts by focusing on the ways and means of enhancing their biodiscovery potential. Our results shed light on the potential of the biodiversity of actinomycetes from this harsh environment. The study of actinomycetes from this vast Indian hot desert may give an insight into their diversity, which, in turn, may guide better management of their bioactive potential. This work is the first report of the selective isolation of rare actinomycetes from the Great Indian Thar Desert and their antimicrobial activities against MDR pathogens. The findings might later be investigated further for their bio-discovery potential.
Materials and methods Sampling locations and collection of samples Soil samples were collected from various locations near the Jaisalmer and Jodhpur region of “The Great Indian Thar Desert”, northwest India. Sampled habitats included the rhizosphere of plants and trees growing in the desert, agricultural soil, and arid soil samples from preserved areas near the Sam and Khuri sand dunes. A total of 14 samples of arid soil were collected randomly. Soil samples were collected aseptically according to the technique of Pochon and Tardieux (1962). Using a large sterile spatula, the first 5 cm of the surface layer of the soil was removed, then, with a small sterile spatula in the layer subjacent (between 5 and 15 cm of depth) 100–150 g soil was collected and deposited on a sterile aluminum sheet. Stones and roots, etc., were removed manually and then only the soil was used for further isolation purpose. The samples were placed in sterile polyethylene bags, closed tightly and stored at 4 °C until required for further use. Isolation of actinomycete strains Various pretreatment procedures and selective isolation media were used to assess the best conditions to detect microbial diversity in the soil samples and for isolating novel and rare actinomycetes. All soil samples collected were subjected to various pretreatment procedures prior to serial dilution. Pretreatment of soil samples, by both drying (Hayakawa et al. 1991; Tamura et al. 1997) and moist heating (Matsukawa et al. 2007a, b) was employed. Chemical compounds like chloramine-T (Hayakawa et al. 1997), SDS and yeast extract (Hayakawa and Nonomura 1989) and
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enrichment with calcium carbonate (Tsao et al. 1960) were used to selectively isolate actinomycetes. Actinomycete colonies were isolated and enumerated by the soil dilution plate technique described by Johnson et al. (1959) using eight different selective isolation media: starch casein nitrate agar (Kuster and Williams 1964), actinomycetes isolation agar (AIA; Difco 212168), humic acid-vitamin agar (Hayakawa and Nonomura 1987; Cho et al. 1994), minimal medium (Hozzein et al. 2008) supplemented with 1 mL microelement stock solution (Labeda and Shearer 1990), Gause (No.1) mineral medium (Gause et al. 1957), glucose–yeast extract medium (Gordon and Mihm 1962), Bennett’s medium (Jones 1949), and soil extract agar (Atlas 1997). All media were prepared at pH 7.0±0.2 and sterilized at 121 °C for 15 min before adding amphotericin B (50 μg/mL) and tetracycline (20 μg/mL) (Himedia, Mumbai, India) to prevent fungal and bacterial growth, respectively. The collected soil samples were pretreated and diluted to 10−6, and 0.1 mL of each diluted sample was spread over the agar plates; triplicates of each dilution were maintained. The inoculated plates were incubated at 30 °C for 7–30 days. Following incubation, the appearance and growth of microorganisms were observed daily on all the isolation plates; actinomycete colonies were recognized by their characteristic tough and chalky-to-leathery appearance. Consecutive transfers and technical purification steps were carried out for selected randomly picked single isolated colonies, which were transferred from the mixed culture of the isolation plates onto ISP-2 agar plates. Pure cultures of all the isolates were subcultured on ISP-2 slants; incubated at 30 °C for 7–25 days to achieve good sporulation, and then preserved at 4 °C and also in 20 % glycerol at −80 °C as stock cultures (Williams and Cross 1971).
measuring the respective zones of inhibition produced by the actinomycete against the pathogenic strains. Nutrient agarglucose medium plates with simultaneously streaked test organisms but without inoculation of the actinomycete isolate were maintained as controls.
Secondary screening and production of bioactive compounds A few selected isolates were tested for their bioactivity spectrum against relevant clinical bacterial isolates comprising Gram-positive (including anaerobes) and Gram-negative pathogens obtained from the Department of Infectious Diseases Culture Collection, New Drug Discovery Research, Ranbaxy Laboratories Limited, Gurgaon, India (Table 1). Here, we also used “emergent” strains of bacteria that have recently been widely publicized, largely in relation to hospitalacquired infections, namely methicillin resistant Staphylococcus aureus (MRSA), Clostridium difficile and ESBL-producing E. coli, etc., in order to evaluate the antibacterial potential of these actinomycete isolates. The isolated strains were first inoculated onto ISP-2 agar plates and incubated at 30 °C for their respective incubation days for mass preparation of inoculum. After growth, the actinomycete mycelium was inoculated aseptically into 10 mL seed medium (soybean meal 1 %; glucose 1 %; NaCl 0.5 %; CaCO3 0.5 %; pH 7.0±0.2) prepared in 150-mL Erlenmeyer flasks and incubated in a rotary shaker at 180 rpm for 3–7 days (depending on their respective growth period at 30 °C). Then, 5 % of actinomycete inoculum was transferred aseptically into 25 mL soybean meal production medium (soybean meal 1.5 %; glucose 1.5 %; NaCl 0.01 %; CaCO3 0.2 %; pH 7.0±0.2) prepared in 250-mL Erlenmeyer flasks and incubated in rotary shaker at 180 rpm for 3–4 days at 30 °C.
Primary screening of isolates producing antimicrobial compounds The actinomycetes were initially screened to determine their ability to produce antimicrobial compounds by the crossstreak method (Egorov 1985). In this method, all the isolates were streaked as a single straight line in the middle of a Petri plate containing modified nutrient agar (NA+1 % glucose) medium. After inoculation, plates were incubated at 30 °C for 7–10 days for growth of the actinomycete strains. Following the appearance of a well-defined ribbon of growth marking the original streak on the plates, 24-h-old pathogenic test bacterial strains viz., S. aureus MTCC 96, Bacillus subtilis MTCC 121, Micrococcus luteus MTCC 106, Escherichia coli MTCC 729, Shigella flexneri MTCC 1457, and Proteus mirabilis MTCC 425 were streaked perpendicular to the original growth line of the actinomycete strain in the same plate. The cross-streaked plates were incubated at 37 °C for 24–48 h. Strains producing antimicrobial compounds were selected by
Table 1
Pathogenic bacterial strains used in secondary screening
Test organisms Gram positive
Gram negative
Staphylococcus epidermidis 12228 Staphylococcus aureus 25923
Escherichia coli ACR E. coli 25922
S. aureus 29213 MRSA 562 (Methicillin-resistant S. aureus) MRSA MU 50 Enterococcus faecelis 29212 Anaerobes Clostridium difficile Respiratory pathogens Streptococcus pneumoniae
Pseudomonas aeruginosa
Bacteroides fragilis Haemophilus influenzae ACR
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Preparation of crude extracts After fermentation, the production medium was centrifuged at 3,500 rpm for 10–20 min to separate the supernatant and mycelium. After centrifugation, about 5 mL culture supernatant was collected in sterile vials to study its antimicrobial activity. Extracellular metabolites present in the remaining supernatant were extracted by liquid– liquid extraction using ethyl acetate in a separating funnel. Ethyl acetate was added to the filtrate at a ratio of 1:2 (v/v) and shaken vigorously for 1 h for complete extraction of the metabolites. Then, the ethyl acetate phase was separated from the aqueous phase and both phases were kept to determine their antimicrobial activity. For extraction of intracellular metabolites, the pellet obtained by centrifugation was extracted with methanol. The biomass was soaked in 10 mL methanol for 12–18 h and then centrifuged to obtain methanol extract from it. All four fractions (culture supernatant, ethyl acetate extract along with its aqueous phase, and methanol extract) were kept to study their antimicrobial activity. Determination of the antimicrobial activities of crude extracts The antimicrobial activity of all the crude extracts was determined using the well diffusion method described by Magaldi and Camero (1997) against a panel of 13 different pathogenic bacterial strains. The crude extract (50 μL) was loaded into wells bored in Muller Hinton agar plates spread with test organisms (0.5 McFarland turbidity standards) using a 5-mm diameter sterile cork borer. The plates were incubated at 37 °C for 24–48 h and examined. After incubation, the diameter of the zone of inhibition for each strain was measured and recorded to evaluate the anti-microbial activity of the isolated actinomycete strain. The most active strains were selected for further analysis of active compounds. Chromatographic analysis of active extracts The cell-free supernatant of 5-L shake cultures of the five most active isolates was divided into three parts and one part each was extracted by ethyl acetate and n-butanol. These were further evaporated to dryness under reduced pressure on a rotary evaporator. The third part was lyophilized and used as is. These extracts were constituted in a small amount of methanol, then filtered and first analyzed by quadrupole time-of-flight (Q-Tof) mass spectrometry. Several spectra were obtained. The gradient was developed to separate most of the peaks of these very complex extracts. The gradient used for analysis was 5 % acetonitrile, which was kept for 5 min, than the acetonitrile level was increased up to 100 % within the next 15 min
followed by another 5 min of isocratic 100 % acetonitrile flow. Finally, the recondition step was added to the gradient, going back to starting condition at 5 % acetonitrile and keeping that for another 5 min. Finally, it was possible to obtain a gradient that suited our needs. A reversed phase column with hydrophilic end capping of 2 mm× 50 mm was used at a flow rate of 300 μL/min. Morphological characterization of the isolates All the isolates were grown on ISP-2 medium at 30 °C and the growth rate was monitored every day for 30 days. Isolates showing good growth after 7 days were considered as fast growers; those that showed good growth between 7 and 15 days were classified as moderate growers; slow growers took more than 15 days to grow. Preliminary morphological characterization of the isolates was made on the basis of growth characteristics and various colony characteristics such as color of the spore mass, reverse side color, and diffusible pigment production observed after incubation. These isolates were characterized by the methods described by Shirling and Gottileb (1966). Molecular taxonomical characterization of selected antagonistic actinomycetes Strains showing potent anti-bacterial activity after secondary screening of all the isolates were first identified on the basis of micromorphology; presumably representing diverse actinomycete genera, these isolates were then assessed using molecular taxonomical studies and the phylogenetic relationship between them determined by 16S rRNA sequence analysis. Genomic DNA (PCR template preparation) of the bio-active actinomycetes was extracted for 16S rRNA gene analysis by the phenolchloroform method (Sambrook et al. 1989). The 16S rRNA gene was PCR amplified from genomic DNA using the actinomycete-specific primers 8 F and 907R as previously described by Lane (1991) and Turner et al. (1999), respectively. The amplification products were analyzed by agarose gel electrophoresis, and PCR purified by the PEG-NaCl method. All sequencing reactions were performed using a Big Dye® Terminator v1.1 (Applied Biosystems, Foster City, CA) containing all the necessary components. Since sequencing requires only a single primer, one of the above mentioned primers was taken as a sequencing primer for each reaction. The primers were obtained from IDT (Integrated DNA technologies, Coralville, IA). Good quality, partial sequences (700–800 bp) were obtained for most of the strains by a cycle sequencing reaction setup using these primers in an ABI 3730XL automated
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DNA sequencing machine. Post-cycle sequencing clean up was performed using the sodium acetate-ethanol precipitation method. The 16S rRNA gene sequences of these isolates have been deposited with GenBank (NCBI; http:// www.ncbi.nlm.nih.gov/genebank) under the accession numbers KC333875, JX431292, JX467563, JX467565, JX474743, JX474744, JX474745, JX474746, JX474747, JX474748, JX474749, JX474750, and JX847140. DNA sequences obtained with the different sequencing primers, depending on the isolate, were compared with the data available in the RDP Data: release 10.29 (Ribosomal Database Project) using the sequence match tool (Seqmatch: version 3), to determine their relative phylogenetic positions. More than 150,000 sequences were included in the search and the screening was based on 7-base oligomers. The 16S rRNA gene sequences of the isolates were used to search the NCBI GenBank database using the BLASTN algorithm to determine relative their phylogenetic positions. An entry with the highest score was downloaded and alignment was performed using CLUSTALW software (version 1.8.1) with representative actinomycete 16S rRNA gene sequences. In addition, the sequences were aligned with the most closely related homologous actinomycete 16S rRNA sequences from GenBank and a phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) software version 5.05 (Tamura et al. 2011). The bootstrap values (5,000 replicates) were obtained using the neighbour-joining and minimal evolution methods provided by the software.
Results Selective isolation of desert actinomycetes Actinomycetes, especially streptomycetes, have been reported from nearly all habitats. The present study was designed specifically to investigate soil-derived actinomycetes from the unusual habitats of ‘The Great Indian Desert or Thar Desert’ as a source of novel molecular scaffolds for natural product screening programs. This desert remains completely unexplored and could be a potential storehouse of novel microorganisms, particularly actinomycetes, exhibiting varied biological and chemical properties. Several selective isolation techniques were employed in order to isolate actinomycetes from 14 different soil samples collected across different locations in the desert. Cultures that exhibited growth morphology indicative of actinomycetes were selected for further study. Colonies of presumptive actinomycetes were recognized by their ability to form leathery colonies and produce an
earthy odor. The aerial spore mass was counted and expressed as the number of colony forming units (CFU) per gram dry weight sand. The number of cultured actinomycete isolates in different sediments ranged from 15× 103 to 3×106 CFU per gram dry weight sand. A total of 100 actinomycete strains was recovered from this less explored arid ecosystem of India using a combination of five different pre-treatment techniques and eight different selective isolation media. Antimicrobial properties of the isolates It is a well established fact that most actinomycetes exhibit antimicrobial activity. In this line, all 100 isolates were tested for their ability to produce inhibitory substances against six test bacterial pathogens. Only 44 isolates (44 %) showed antimicrobial activity against at least one of the test pathogens. These were screened out after in vitro primary screening against a panel of six test organisms (three Gram-positive and three Gram-negative bacteria pathogens). All these strains were found to be potential antagonists against the bacterial pathogens used and exhibited good inhibitory activity against the majority of these test organisms. Hence, these strains were selected for further evaluation of their antimicrobial efficacy against several clinical and drug-resistant bacterial pathogens during secondary screening to confirm their activity in culture broth. The antibacterial activity of all 44 isolates was tested against relevant clinical bacterial pathogens comprising a panel of eight Gram-positive (including anaerobes) and five Gram-negative pathogens (Table 1). Each isolate was able to confer robust growth inhibition on at least one test pathogen. Among these isolates, 31 (70 %) produced active substances against Gram-positive bacterial pathogens and 61 % of the strains (27 strains) showed activity against Gram-negative pathogens. A large fraction of the antibiotic compounds i.e., 42.61 %, exhibited activity exclusively against gram-positive bacteria (some 75 extracts) and only 34.375 % of compounds (some 55 metabolites) showed activity only against gram-negative bacteria. Of the 100 actinomycetes isolated, only 13 were found to be potential antagonists against human bacterial pathogens after in vitro secondary screening (Tables 2 and 3). Some isolates showed exceptionally good activity against the panel of bacterial pathogens used. All the extracts from strains viz., RK2_54, RK66, and RK71 were found to be highly active against the Gramnegative pathogens; whereas all the extracts from RK53, RK2_54, RK57, RK61, RK66, RK71, and RK75 showed good activity against Gram-positive pathogens. This indicates the production of both extracellular and intracellular bioactive compounds by all these strains. Isolates RK2_54, RK66, and RK71 exhibited a broad spectrum activity against and across
Ann Microbiol Table 2 Antibacterial activity of culturable actinomycetes isolated from the Great Indian Thar Desert against Gram-positive bacterial pathogens. Data presented are from isolates possessing significant activity against three or more pathogens. EA Ethyl acetate extract, ME methanol extract Strain no.
Extract
Test organisms (Inhibition zone measured as diameter in mm) SA25923
RK1 RK6 RK43 RK53 RK2_54 RK57 RK59 RK61 RK2_63 RK66 RK71 RK2_75 RK79
SA29213
MRSA562
MRSA MU50
SE12228
EF29212
CF
SP
–
18
EA
14
–
17
12
22
14
ME EA ME EA ME EA ME EA ME EA ME EA ME EA ME EA ME EA
15 20 18 – – 15 20 17 13 14.5 17 – – 17 22 18 17 30
10 19 21 12 10 17 20 15 15 15 15.5 – – 15 13 – – 14
18 15 – – – 20 17 16 15 13 12 10 – 20 17 – – 22
10 23 27 15 – 28 21 22 18 14 15 15 17 12 10 11 – 24
24 27 30 – – 32.5 32.5 25 27 27 23 – 22 30 27 15 14 23
16 21 24 – – 19 18.5 19 12 14 15 10 – 17 12 22 24 12
– – – – – 18 24 22 – 15 20 12 10 16 19 25 23 13
21 13 15 20 18 18.5 22 20 19 14 – – – 12 10 25 23 15
ME EA ME EA ME EA ME
28 29 28 18 15 – –
11 17 17 17 18 – –
20 19 19 20 19 10 12
23 19 21 24 21 14 12
21 30 31 30 30 22 –
13 27 25 15 13 10 10
– 29 30 14 10 – –
17 31 32 17 21 – –
the largest panel of test pathogens. RK59 inhibited the lowest number of test pathogens; however, it was the only isolate that inhibited the growth of Pseudomonas aeruginosa—an opportunistic Gram-negative pathogen. Methanol extracts were also equally active against the majority of test pathogens, clearly indicating the potential of the intracellular compounds produced by these isolates. Especially, methanol extracts of strains RK53 and RK57 inhibited the growth of all Gram-positive pathogens while methanol extract of the strain RK471 showed inhibitory activity against all the Gram-negative pathogens. Some isolates failed to show any activity against the set of pathogens used in this study. A total of five isolates viz., RK53, RK2_54, RK57, RK66, and RK71 exhibited what appeared to be robust inhibition of both Gram-positive as well as Gram-negative test organisms. All ethyl acetate extracts were found to possess a more potent inhibitory activity effect against MRSA MU 50
(Gram-positive) and Haemophilus influenzae ACR (Gram-negative) pathogens. Highest inhibition zones were observed against Staphylococcus epidermidis 12228 (Fig. 1) in the case of Gram-positive pathogens while the same results were obtained for Haemophilus influenzae ACR in case of Gram-negative pathogens (Fig. 1). The ethyl acetate extract of strain RK53 showed the highest zone of inhibition (32.56 mm) measured against Staphylococcus epidermidis 12228, whereas ethyl acetate extract of strain RK71 showed the highest zone of inhibition (37.53 mm) against Haemophilus influenzae ACR. Chromatographic analysis of active extracts Only isolates with promising or potent activity against multi drug resistant bacterial pathogens were chosen for study of their bioactive compounds. These most active strains, inhibiting both Gram-positive as well as Gram-
Ann Microbiol Table 3 Antibacterial activity of culturable actinomycetes isolated from the Great Indian Thar Desert against Gram-negative bacterial pathogens. Data presented are from isolates possessing significant activity against three or more pathogens. EA Ethyl acetate extract, ME methanol extract Strain no.
RK1 RK6 RK43 RK53 RK2_54 RK57 RK59 RK61 RK2_63 RK66 RK71 RK2_75 RK79
Extract
Test organisms (Inhibition zone measured as diameter in mm) E. coli ACR
E. coli 25922
H. influenzae ACR
Bacteroides fragilis
EA ME EA ME EA ME EA ME EA
14 11 13 15 – – – 10 16
– – – – – – – 13 18
16 20 29 27 21 18 34 31 31
27 31 16 12 – – 22 29 32
– – – – – – – – –
ME EA ME EA ME EA ME EA ME EA ME EA ME EA ME EA ME
13 15 10 – – – – 19 16 21 19 22 24 – – 10 –
19 12 10 – 10 – – – – 18 15 16 15 – – – –
32 35 33 17 – – 17 30 27 35 36 37.5 35 – – 29 30
36 – – 13 10 18 – – – 16 14 25 27 – 17 – 10
– – – 17 19 – – – – – – – – – – – –
negative test organisms, were selected for further analysis of their active compounds. Extracts from isolates RK2_54, RK57, and RK66 presented very complex profiles, with more than 30 compounds identified. Purification of active compounds from all these isolates Fig. 1 Antimicrobial activity exhibited by RK53 against Staphylococcus aureus 25923 and Staphylococcus epidermidis 12228
P.aeruginosa
will be performed in future using various techniques, and the active compounds will be identified. Our results also suggest that the lyophilized extracts are no longer necessary, because all compounds from lyophilized extracts could be found in ethyl acetate or buthanol extracts.
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A “less crowded” compound pattern was observed for the extracts from RK53 and RK71. So, we tried to identify some compounds using the Chapman and Hall database (Figs. 2, 3). RK53 was found to produce some known compounds like
Strain
Metabolite
Source
RT
deferoxamine, ferrioxamine, and prodigiosin C25, whereas antimycin A4 and antimycin A5 were detected in extracts from RK71. Compounds similar to derivatives of some of the known antibiotics, e.g., daunomycin, kanamycin, and
Molecular Mass
UVmax
Compound suggestion
RK_53
1
Lyo
13.21
560
194
RK_53 RK_53
2 3
Lyo Lyo
14.22 17.44
600 654
194 218, 390, 560
Ferrioxamin No unique hit in DB
RK_53
4
Lyo
26.46
224
No unique hit in DB
RK_53 RK_53
5 6
BuOH EtOAc
25.05 16.96
224 214, 392, 530
Prodigiosin C25 No unique hit in DB
269, 353, 452, 682 393 654
Fig. 2 Mass spectra of extracts from RK53 and some of the compounds suggested by the Chapman & Hall/CRC Chemical database Dictionary of Natural Products (http://dnp.chemnetbase.com/tour/). The mass spectra were generated using the software Masslynx (Waters, Bedford, MA).
Deferoxamine
Every single peak from the total ion current (TIC) was extracted to generate the corresponding mass spectra. The database search is evaluated with UV and mass spectra
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Strain
Metabolite
Source
RT
Molecular Mass
UVmax
Compound suggestion
RK_71
1
Lyo
14.35
600
194
RK_71
2
Lyo
17.11
474, 492
218, 370
Antimycin A5
RK_71
3
Lyo
18.06
488, 506
242, 374
Antimycin A4
RK_71 RK_71
4 5
Lyo BuOH
18.37 15.14
270 484, 600
220, 318 196, 212
No unique hit in DB Kanamycin derivative
RK_71
6
EtOAc
16.72
479, 497
220
Daunomycin derivative
RK_71
7
EtOAc
17.64
493, 511
230, 374
RK_71
8
EtOAc
18.55
270
224, 318
Oxaunomycin derivative No unique hit in DB
Fig. 3 Mass spectra of the extracts from RK71 and some of the compounds suggested by the Chapman & Hall/CRC Chemical database Dictionary of Natural Products (see legend to Fig. 2 for details)
oxaunomycin, were found to be produced by isolate RK71. Interestingly, direct mass-based search did not give any hit from the database for a majority of the metabolites from both these isolates, suggesting the production of some novel
compounds. Although not quantitative enough, these results do give an estimate of abundance for bioactive secondary metabolites from these most likely novel isolates. Investigative searches on all these molecules are in progress.
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Morphological characterization of the isolates The cultural characteristics, including growth characteristics of colonies on the plate, presence of substrate and aerial mycelia, and pigments produced, confirmed the taxonomic diversity of the selected isolates as actinomycetes. Only the most bio-active strains amongst them were analyzed for additional morphological observations combined with rRNA analysis to group them. These 13 representative isolates can be assigned to the genera Streptomyces (8 strains), Actinomadura (2 isolates), Nocardia, Nonomuraea, and Spirillospora. All 13 isolates exhibited differences in rRNA sequence, morphology, and growth inhibition patterns. In addition, 16S partial ribosomal sequences of ten isolates did not show 100 % identity with any organisms existing in GenBank on 11 February 2014 (Table 4). Isolates RK2_54, RK66, and RK 71 showed 100 % 16S rRNA gene sequence similarity to Streptomyces albus, Streptomyces vinaceusdrappus, and Streptomyces flocculus respectively. Cultural characteristics and microscopic examination of each of the 13 most bio-active actinomycete strains are summarized in Table 4. The morphologies indicated in Table 4 refer to those seen when the isolates were grown for 2–3 weeks on ISP-2 medium. The majority showed robust growth on ISP-2 medium, with the exception of RK43, RK59, RK2_75, and RK79, all of which showed retarded growth. Molecular taxonomy of the bioactive actinomycetes A comparison of the 16S rRNA gene sequences of the bioactive strains tested here against sequences in the GenBank database revealed homologies of greater than 97 % to members of different genera, such as Actinomadura, Nocardia, Nonomuraea,
Spirillispora and Streptomyces. According to 16S rRNA identification procedures, this group was dominated by Streptomyces, followed by Actinomadura. It should be noted that this is the first report of these genera from the Great Indian Thar desert and this specific geographical origin. Examination of their sequences for signature nucleotides requires further study in order to validate the phylogenetic identity of these strains. The combined morphological and rRNA sequences suggest that all 13 organisms might be unique. Although RK1 (98 %) and RK71 (100 %) have partial rRNA sequences similar to Streptomyces flocculus, these two organisms have distinguishable growth morphologies and different growth inhibition profiles. When grown on ISP-2 medium, RK1 has a white colored surface and a faint-greenish-yellow colored reverse pigment; whereas RK7 has white-to-brownish colored colonies producing no diffusible pigment. But, unlike RK1, it secretes more potent bioactive metabolites into the ISP-2 medium. Two other organisms sharing such similarities to Streptomyces albus are RK6 (99 %) and RK2_54 (100 %). Their growth morphology, rRNA sequence data, and growth inhibition profile differ from each other. On ISP-2 medium, the reverse side of the RK6 colonies is faint-yellowish in color as opposed to the brownish color of RK2_54. Although RK6 and RK 2_54 show good anti-microbial activity, RK2_54 is the most bioactive isolate of the current research, showing extremely high broad spectrum activity. Phylogenetic analyses Standard methodologies were used to create a phylogenetic tree. The evolutionary history was inferred using the neighbor-
Table 4 Phenotypic characterization and closest BLASTN matches for the partial 16S rRNA sequences and their percentage similarity with the closest actinobacterial strains Strain code
GenBank accession number
Closest GenBank match (% identity)
RK61 RK1 RK59 RK66 RK79
KC333875 JX431292 JX467563 JX467565 JX474743
RK71 RK57 RK53 RK43 RK6 RK2_75 RK2_63 RK2_54
JX474744 JX474745 JX474746 JX474747 JX474748 JX474749 JX474750 JX847140
Color of aerial mycelium
Color of substrate mycelium
Diffusible pigment
Type of spore chain morphology and/or surface ornamentation
Streptomyces mutabilis (98 %) Ash grey Streptomyces flocculus (98 %) White Spirillospora albida (97 %) Ash grey Streptomyces vinaceusdrappus (100 %) Off white Actinomadura geliboluensis (99 %) White
Brown Light green Beige Light yellow Beige
Brown Yellow None None None
Spiral, smooth Spiral, smooth Warty Straight, smooth Spiral, warty
Streptomyces flocculus (100 %) Streptomyces chromofuscus (99 %) Streptomyces eurythermus (99 %) Nonomuraea roseoviolacea (98 %) Streptomyces albus (99 %) Actinomadura nitritigenes (99 %) Nocardia amamiensis (99 %) Streptomyces albus (100 %)
Brown Yellow black red Red Yellow Sand yellow Orange Light brown
None None Purple red None None None None None
Spiral, smooth Rectiflexibilis, warty Smooth Closed spirals, smooth Rectiflexibilis, rugose Warty Smooth Rectiflexibilis, smooth
White Grey Light ivory Ruby red White Light brown White White
Ann Microbiol
RK59, RK2_63, RK2_75, and RK79, the five nonStreptomyces species, fall into different clades. On the basis of phenotypic and genotypic properties, these isolates could be differentiated from each other and from their closest phylogenetic relatives.
joining method (Saitou and Nei 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (Felsenstein 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura 1980) and are in the units of the number of base substitutions per site. The analysis involved 26 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 718 positions in the final dataset. The 16S rRNA sequences were placed in MEGA (version 5.05), the relatedness of our isolates to each other and to their closest named relatives in GenBank was compared (Fig. 4) and evolutionary analyses conducted (Tamura et al. 2011). Three of our organisms matched known species. Interestingly, six isolates (RK43, RK 2_75, RK79, RK2_63, RK6, and RK71) belonging to both Streptomyces and non-Streptomyces species show higher identity in 16S rRNA sequence to each other than to any other named relative in GenBank. As expected, RK43,
Discussion Actinomycetes are a physiologically diverse group and are of great importance in biotechnological processes because of their ability to produce a large number of antibiotics, enzymes, and other therapeutically useful secondary metabolites with diverse biological activities (Tiwari and Gupta 2012, 2014). Moreover, such metabolites have original and unforeseen structures, and are selective inhibitors of their molecular targets. Nevertheless, during the past 50 years, only the “tip of the iceberg” of actinomycetes have been isolated from soil and their antibiotic products sampled (Watve et al. 2001; Baltz 91
90 90 99
RK61 (KC333875) Streptomyces mutabilis NRRL ISP-5169T (EF178679)
99
RK66 (JX467565) Streptomyces vinaceusdrappus NRRL 2363T (AY999929) Streptomyces eurythermus (D63870)
Streptomyces chromofuscus NBRC 12851T (AB184194) 60
59 99
RK57 (JX474745) RK53 (JX474746) RK71 (JX474744)
99
99
Streptomyces flocculus NBRC 13041T (AB184272) 100 67
RK2_54 (JX847140) RK6 (JX474748) Streptomyces albus subsp. albus DSM 40313T (AJ621602) Kitasatospora setae (U93332) Nocardia asteroides DSM 43757T (AF430019)
96
RK2_63 (JX474750)
100 99
Nonomuraea pusilla (D85491)
73 99 73
63
Nocardia amamiensis (AB275164)
RK43 (JX474747) Nonomuraea roseoviolacea IFO 14098T (AB039959)
Actinomadura madurae (X97889) RK2_75 (JX474749) 86
Actinomadura nitritigenes (AY035999) RK79 (JX474743)
58
Actinomadura geliboluensis A8036T (HQ157187) Spirillospora albida (D85498)
0.1
Fig. 4 Evolutionary relationships of taxa based on 16S rRNA sequence alignments. Organisms represented are the isolates from this study and their closest named GenBank matches. The optimal tree with the sum of branch length = 1.11264473 is shown
Ann Microbiol
2005). However, still many environments have not been fully explored and there is a tremendous potential to identify novel organisms with various biological properties from such environments. Moreover, despite continued efforts to isolate, characterize and exploit actinomycetes commercially, much of this natural and potential treasure is unexplored owing to the vastness of their diversity and wide distribution. The search for new bioactive metabolites of microbial origin is based largely on the isolation of novel microorganisms from diverse sources. Novel organisms from previously unexplored or under-explored areas are more likely to possess metabolic pathways that may lead to the production of novel secondary metabolites. Actinomycetes from several unexplored environments have been studied intensively in last few decades for novel and potent molecules (Mitra et al. 2008). Although a few reports exist on the actinomycetes from deserts (Barakate et al. 2002; Badji et al. 2005; Hozzein et al. 2008; Okoro et al. 2009; Selvameenal et al. 2009; Meklat et al. 2011; Nachtigall et al. 2011) to date, there has been no such extensive study on their antimicrobial potential against multi-drug-resistant pathogens. After reviewing the literature, it has also been found that the region we selected for sampling is comparatively unexplored and no substantial data could be found on the diversity and the bio-discovery potential of its micro-flora. We consider this as an added advantage over the fact that we also tried to selectively isolate actinomycetes from this unexplored habitat. Moreover, only little information is available on the actinomycetes of the Indian hot deserts, which are a very productive ecosystem with regard to the occurrence of novel micro-flora (Selvameenal et al. 2009). This study represents the first report of the diversity and bioactivity of actinomycetes isolated from the Great Indian Thar desert. By employing selective isolation techniques, a total of 100 different isolates of actinomycete were isolated from 14 soil samples collected from different locations in this hot desert in the northwest part of India, suggesting that actinomycetes are widespread throughout this arid region. As the main aim of this study was to assess the bio-active potential of actinobacterial diversity, we focused on screening all the isolated actinomycete strains for their antibacterial activity against a panel of six pathogenic strains on solid media during primary screening. Based on our results, of 100 isolates, 44 (44 %) strains demonstrated antimicrobial activity against at least one of the various pathogens tested during primary screening; this percentage is slightly higher than that reported by Hozzein et al. (2011; 42.67 %) and much higher than that described by Saadoun and Gharaibeh (2003; 30 %). However, all of these isolates failed to manifest activity in secondary screening, which is in line with results reported earlier by some researchers (Pickup et al. 1993; Singh et al. 2006) and only 13 isolates displayed antimicrobial activity against the panel of multi-drug resistant pathogenic organisms.
Reports of anti-microbial activity of actinomycetes isolated from Indian hot deserts against clinical multi drug resistant pathogens are very rare. Seventy five percent of isolates were active against one or more of the test organisms. This percentage is lower than that described by Barakate et al. (2002) studying the activity of soil actinomycetes from Moroccan habitats, and much higher than described by Meklat et al. (2011) studying the activity of actinomycetes in Saharan soils of Algeria. We also found that these isolates were comparatively more active than the other antagonistic isolates and interestingly showed a higher antimicrobial activity against Gram-positive bacteria than against Gram-negative bacteria. This is in line with a recently reported study performed by Ding et al. (2013). In general, most antibiotics are extracellular in nature (Augustine et al. 2005) and our results agree with the findings of this latter study. As a result, most of the strains in this study had the potential ability to produce active compounds. An unidentified complex mixture of compounds was observed for most of the isolates. However, both known and unknown compounds were produced by the two isolates RK53 and RK71, some of which were identified as antimycin A4 and A5, derivatives of kanamycin, daunomycin, and oxaunomycin produced by RK71. As a group, such a complex mixture of compounds along with some unknown components has so far never been reported from a single Streptomyces sp. Analyzing such complex mixtures forms the ultimate challenge of analytical chemistry. These isolates might produce some unknown compounds and thus are worthy of further study. We are very likely to identify a range of new compounds within such highly complex extracts. The magnitude of diversity harbored by the arid desert is quite significant. Streptomyces is shown to be the most dominant genera of actinomycetes isolated from desert ecosystems worldwide in numerous studies (Hozzein et al. 2008; Okoro et al. 2009; Selvameenal et al. 2009). Arid soil samples of alkaline pH tend to contain fewer Streptomyces and more “rare” genera such as Actinoplanes and Streptosporangium (Arifuzzaman et al. 2010). The isolates from our study need to be subjected to other precise methods of identification to verify species fidelity. It has been established earlier that solid media are more suitable for the growth and development of isolates producing antibiotics (Shomura et al. 1979; Iwai and Omura 1982; Badji et al. 2005). Our results correspond to these findings. Based on the results of our study, we conclude that sequencing of the 700–800 bp 16S rRNA gene does not provide enough information for the species-level identification of these actinomycetes isolates. However, the accurate assignment of a species to a particular taxa based on a rRNA sequence will depend largely on the generation of good quality, near full-length sequences from wellcharacterized isolates, and additional taxonomic studies to resolve the appropriate classification of these presently
Ann Microbiol
unnamed species. According to 16S rRNA sequencing, five genera were obtained from this environment: Streptomyces (8 strains) was the largest group followed by Actinomadura (2 strains), Nocardia (1 strain), Nonomuraea (1 strain), and Spirillospora (1 strain). Here, significant differences in species composition compared with other desert ecosystems can be found (Okoro et al. 2009). Based on the 16S rRNA analysis and morphological characteristics, isolates RK1, RK2_54, RK53, and RK59 might represent promising strains, and their appropriate classification is in progress. On the other hand, the remaining isolates showed high similarity to the closest type strains, which have also been reported as worthy strains. In this study, more than one-third of these strains were rare actinomycetes, and this shows that selective isolation techniques might be suitable for recovering worthwhile strains from this environment. In conclusion, the results suggest that the desert ecosystem in the northwest part of India is a source of bioactive actinomycetes, and indicate that the culturable actinomycetes in this environment are an interesting and promising topic for further study. The results of this study strongly support the idea that actinomycetes species isolated from this ecosystem possess a significant capacity to produce compounds having unique antibacterial activity. We believe that the results of the study will be crucial to the further study of the diversity of actinomycetes from the Indian deserts as well as to their evaluation as a potential storehouse for novel antibiotics. The potential of desert microorganisms as a source of novel antimicrobial agents seems to be promising. Such studies are also imperative to exploring the bioactive potential of microorganisms associated with this region and achieving their conservation. Among the isolates studied here, five showed broad spectrum antibacterial activity including against infections caused by bad bugs like VREs and the MRSA. In conclusion, the results obtained from this work are promising and hence merit further studies concerning the purification, characterization and identification of the active secondary metabolites as well as of the producer strains. A detailed characterization of the active isolates, as well as the active principle compounds of extracts showing good antibacterial activity, is the subject of ongoing investigation. Acknowledgments The authors thank the Department of Infectious Diseases Culture Collection, New Drug Discovery Research, Ranbaxy Laboratories Limited, Gurgaon, India for providing relevant clinical bacterial isolates comprising Gram-positive (including anaerobes) and Gramnegative (including anaerobes) pathogens and also for providing the necessary facilities for carrying out secondary screening experiment in their laboratories. Help received from Dr. Kamlesh Jangid, and Dr. Ashish Polkade [Microbial Culture Collection (MCC), National Center for Cell Science (NCCS), Pune, India] in providing the sequencing facility and in preparing the phlogenetic tree, is gratefully acknowledged. The authors are also very grateful to the University Grants Commission for financial
support under the Special Assistance Progamme (SAP) from 2011 to 2016. The authors report no declarations of interest.
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