The search for new natural products, aided by the discovery of novel actinomycetes, is urgently needed due to the increase of antibiotic resistance among pathogens (Coats & Hu., 2007); most notably members of the Mycobacterium tuberculosis complex responsible for tuberculosis (TB). An estimated third of the world's population is infected with the latent form of TB which provides an enormous reservoir for future active disease. Moreover, according to Dye (2006), 9 million cases of active TB were reported in 2004. TB is regarded as a leading cause of death from infectious disease making it a major global health concern, which is further exemplified by the emergence of resistant strains classed as multi-drug resistant TB (MDR-TB) and extensively-drug resistant TB (XDR-TB), making treatment complex and almost unattainable using current lines of drugs and antibiotics (Dover et al., 2008). TB is currently a worldwide pandemic, which has been highlighted recently by the emergence of Mycobacterium tuberculosis strains that are resistant to all front-line treatments, including second-line drugs and increased resistance towards second-line antibiotics such as fluoroquinolones (Dalton et al., 2012). This increased level of drug resistance is due to the extended use of second-line drugs in patients with MDR-TB, making TB infections virtually untreatable.
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The rediscovery of bioactive compounds from known actinobacteria isolated from common habitats is currently estimated to be over 90% (Busti et al., 2006; Lam, 2007). Furthermore, the problem of antibiotic resistance involves the majority of known antibiotic compounds and pertains to all of the significant bacterial pathogens, including Staphylococcus aureus, Streptococcus pneumoniae and Mycobacterium tuberculosis; therefore, there is a clear need for novel antimicrobial agents. It is well known that actinobacteria, particularly the genus Streptomyces, are prolific producers of antibiotics and have an unparalleled capacity to produce useful bioactive compounds (Kurtböke, 2012; Solecka et al., 2012), such as streptomycin derived from Streptomyces griseus and rifampicin from Amycolatopsis rifamycinica, which are both bactericidal antibiotics used to treat TB. However, the increase in antibiotic resistance and the vast number of global cases of TB, combined with the rediscovery rate of bioactive compounds from known actinomycetes, emphasises the need to develop strategies that enhances the discovery of novel taxa. Novel members of actinobacteria can potentially provide a rich source of new bioactive compounds which, in turn, could combat the increase of antibiotic resistance. Large numbers of actinomycetes have been isolated from soil over the past several decades and their derivatives account for most of the relevant secondary metabolites available in industry and academia (Baltz, 2008).
According to Busti et al., (2006), extensive screening programmes have led to an increased rediscovery of known actinomycetes leading to a reduced rate of discovery of novel bioactive compounds, creating a bottleneck, due to the limited supply of new natural products; therefore, the discovery of novel actinomycetes can be improved when new screening methods are incorporated or when samples derived from extreme unexplored habitats are examined (Goodfellow & Fielder, 2010). In this context, many untapped extreme natural environments are considered a rich source of novel or rare actinomycetes which have the capacity to produce new bioactive compounds (Liu et al., 2012), as demonstrated by the production of abyssomicins derived from Verrucosispora maris, a novel actinomycete isolated from marine sediment collected from the Sea of Japan (Goodfellow et al., 2012). It is becoming increasingly difficult to discover commercially useful metabolites from common actinomycetes; hence, exploring extreme environments is important for discovering novel bacteria as the extreme conditions of such ecosystems results in microbial adaptation, giving rise to uncharacterised strains.
Recently, the marine environment has become an essential source of useful secondary metabolites and is considered an untapped source of chemical diversity. Deep-sea environments exhibit conditions that are extremely different from the terrestrial environment and have been found to harbour diverse marine actinobacterial communities that have differing characteristics from microorganisms present in the terrestrial environment, revealing novel secondary metabolites (Pathom-aree et al., 2006). This is further demonstrated by the production of the dermacozines derived from Dermacoccus abyssi sp. nov., strains MT1.1 and MT1.2 isolated from the Challenger Deep of the Mariana Trench, which has been shown to have antitumour and antiprotozoal activity (Abdel-Mageed et al., 2010), and Benzoxacystol, an enzyme inhibitor derived from Streptomyces sp. NTK935, isolated from deep-sea sediment of the Canary Basin (Nachtigall et al., 2011).
Currently, most attention has been applied to deep sea environments as a rich source of novel actinomycetes and has successfully facilitated the discovery of a range of new metabolites; however, this extensive screening has led to these resources being somewhat exhausted; hence, screening alternative extreme environments, such as hyper-arid deserts, is necessary to further the discovery of novel actinobacterial communities.
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In addition to the marine ecosystem, the desert biome is regarded as a unique, under-explored source of novel actinobacterial diversity, with a large number of novel bacteria found in soil samples derived from hyper-arid regions of the Atacama Desert (Neilson et al., 2012). Neilson and her colleagues collected soil samples from three sites across the hyper-arid margin of the Atacama, with the aim of determining the bacterial communities and evaluating the potential functional diversity of these communities within the soils. Bacterial phylogenetic profiles were generated using a combination of pyrosequencing and 16S rRNA analyses, which indicated a range of novel bacteria, dominated by an abundance of novel actinobacteria and Chloroflexi.
The Atacama Desert, located in northern Chile, is widely regarded as the world's oldest and driest desert, consisting mainly of hyper-arid regions presenting with annual rainfall as low as 0.5 and 0.6 mm (Clarke, 2006). The Atacama is a coastal temperate desert (20Â°C to 30Â°C) and is a plateau located along the north western coast of Chile, South America, between 10Â°S to 35Â°S latitude and 70Â°W to 72Â°W longitude (Valdivia-Silva et al., 2012). Isolation of actinomycetes from the Atacama Desert is of great interest as the extreme levels of aridity provides a unique complex environmental setting for microbial adaptation, which increases the likelihood of undiscovered actinobacterial communities. Furthermore, studies on the Atacama Desert provide insight into how these microorganisms are physiologically adapted to such extreme environmental conditions and are vital to understanding how microbial life can survive in conditions without water (Azúa-Bustos et al., 2012). Different locations within the Atacama Desert present with varying levels of aridity (Figure 1).
C:\Users\Josh\Pictures\Atacama desert map drees 2006.jpg
Figure 1: Map of the Atacama Desert, Chile. The green square indicates the location of the hyper-arid (Salar de Atacama) region and the red square indicates the location of the extreme-hyper arid (Yungay) region of the Atacama Desert, from which the soil samples were collected (Drees et al., 2006).
The Laguna de Chaxa of the Salar de Atacama is the largest salt flat in Chile, located 160 km east of Antofagasta and is rich in halite (NaCl). Salar de Atacama is a giant evaporate basin and the Laguna de Chaxa area is important for water accumulation within salt flats of the Atacama as it consists of rich vegetation adapted to desert conditions, including grama grass (Boschetti et al., 2007). It is categorised as hyper-arid based on a mean annual rainfall (MAR) and mean annual evaporation (MAE) of less than 0.05; whereas, the extreme hyper-arid Yungay region presents with an MAR of less than 0.002 (Houston, 2006) (Figure 1.1.).
A)http://www.geotimes.org/may03/chile.jpgSoil of Laguna de Chaxa
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Figure 1.1: Comparison of: (A) Laguna de Chaxa, Salar de Atacama hyper-arid region (Google Images, n.d) to (B) Yungay extreme hyper-arid region (Geotimes, 2003).
The Yungay region is located within the core of the Atacama Desert and, based on MAR data, is considered the driest part of the Atacama. According to Navarro-Gonzalez et al., (2003), due to high levels of oxidation, the soil within the extreme hyper-arid region is depleted in organic material and consists of very low levels of culturable bacteria; therefore, the Yungay region of the Atacama Desert provides a promising setting to investigate the survival of microorganisms in conditions of extreme aridity. Based on regional settings and soil composition it is hypothesised that greater numbers of culturable bacteria will be isolated in soils derived from the hyper-arid region, Laguna de Chaxa of the Salar de Atacama compared to the extreme hyper-arid Yungay region.
Given this backdrop of extreme aridity, the harsh conditions of the Atacama Desert appear unfavourable for microbial life, particularly in the extreme arid core of the Atacama, which has been suggested as the dry limit of microbial survival in extreme environments (Navarro-Gonzalez et al., 2003). Furthermore, the level of aridity has had a profound impact on soil composition, with high concentrations of nitrate and sulphate (Connon et al., 2007). This is further exemplified by a combination of adverse conditions including high levels of UV radiation, inorganic oxidants, high salinity and extremely low levels of organic carbon ranging between 560 - 765 Âµg g-1 in the Yungay region (Lester et al., 2007).
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Despite the Atacama Desert being considered inhospitable to sustain microbial life, due to extremely desiccated soils and depleted organic matter, microorganisms are present (Connon et al., 2007; Lester et al., 2007). However, Navarro-Gonzalez and his colleagues (2003) initially found that the Yungay region contained extremely low levels of culturable bacteria, and in most cases, no bacterial colonies were detected from the lowest dilution inoculated on nutrient media. Additionally, the bacterial diversity within the soils was investigated by DNA extraction, PCR amplification and 16S rRNA sequencing; however, no DNA was recovered from the Yungay soils. The findings from this study demonstrated that as the conditions became drier, particularly near the extreme arid region, biological activity decreased, suggesting the dry limit of microbial survival. In contrast, Maier et al., (2004) managed to isolate culturable bacteria and successfully extract bacterial DNA from each soil sample taken from the Yungay region, which signifies the existence of microbial life within the most arid desert on Earth.
Recent studies have supported the existence of large actinobacterial diversity within these soils, as exemplified by Okoro et al., (2009) who isolated novel strains from different regions of the Atacama. A relatively high number of actinomycetes were isolated from arid, hyper-arid and extreme hyper-arid regions of the Atacama Desert using a range of selective isolation methods. Soils from the arid region (El Tatio) had a total colony count of 1.38 x 104 cfu g-1, whilst the hyper-arid regions (Salar de Atacama and Valle de la Luna) had counts of 2.51 x 104 cfu g-1 and 2.10 x 103 cfu g-1, respectively. Subsequent 16S rRNA phylogenetic analysis and phenotypic characterisation indicated that the majority of the isolates were novel members of the genera: Amycolatopsis, Lechevalieria and Streptomyces.
Okoro et al., (2010) subsequently assigned the putative Lechevalieria isolates to three novel species, classified as: Lechevalieria atacamensis sp. nov., Lechevalieria deserti sp. nov. and Lechevalieria roselyniae sp. nov., based on genotypic and phenotypic characteristics. Further studies followed involving the Streptomyces isolates, including the assignment of Streptomyces strain C63T, isolated from hyper-arid soil, to the novel species Streptomyces deserti sp. nov. (Santhanam et al., 2012). The novel strain presented with chemical and morphological characteristics consistent with its classification in the genus Streptomyces but could be distinguished from closely related type strains based on phenotypic properties. Furthermore, Streptomyces strain C60T, isolated from extreme hyper-arid soil, had been described as Streptomyces atacamensis sp. nov., using a polyphasic approach (Santhanam et al., 2011). Moreover, Streptomyces strain C34T, isolated from Laguna de Chaxa, Salar de Atacama, had been shown to produce new ansamycins with strong bactericidal activity against Gram-positive bacteria (Rateb et al., 2011), and strain C38T produced new macrolactones showing positive antitumor activity (Nachtigall etÂ al., 2011).
Prior to these studies, Drees et al., (2006) previously isolated bacteria and revealed unique communities, dominated by Gemmatimonadetes and Planctomycetes, between the hyper-arid and arid regions of the Atacama. Soil samples were collected along an east-west elavational transect and presented with significantly different bacterial community structures between the hyper-arid and arid regions, based on denaturing gradient gel electrophoresis (DGGE) analysis. Research has shown that culturable bacteria can be isolated from the Atacama Desert and suggests that these microorganisms can tolerate the extreme environments. Actinomycetes are one of the most widely distributed microorganisms as they have the ability to produce spores which increases their survival rate in extreme environments (Tian et al., 2009). Recent studies have indicated that a relatively high number of actinobacteria can be isolated from arid desert soils, and thus, potentially provides a prolific source of novel strains producing new bioactive compounds. A range of novel species have been described following the isolation of putatively novel isolates from the Salar de Atacama and the Valle de la Luna regions of the Atacama Desert by Okoro and her colleagues (2009). The findings from this extensive study subsequently led to the production new ansamycins derived from Streptomyces strain C34T and new macrolactones from Streptomyces strain C38T, which highlights the point that the Atacama Desert is a rich resource of untapped novel actinomycetes that have the capacity to provide new useful bioactive compounds which could have significant impact in industry and medicine.
Although these untapped ecological niches provide a rich source of novel actinomycetes, the limiting step of selective isolation is the use of regimented culture-dependent approaches that are somewhat empirical (Goodfellow & Fielder, 2010), regardless of exploring unique natural habitats. The overreliance on classical isolation procedures may inhibit the growth and recovery rate of novel or rare actinomycetes, whilst favouring the growth of known taxa (Khanna et al., 2011). Moreover, the successful recovery of members of novel actinobacterial taxa is dependent on the use of improved selective isolation strategies (Maldonado et al., 2008); therefore, various innovative selective isolation approaches are essential for isolating novel strains (Genilloud et al., 2011). These approaches could incorporate soil sample pre-treatments enriched with carbon sources; agar media known to support actinobacterial growth supplemented with antibiotics and antifungals, or combining alternative culture-based strategies for selective isolation. The use of selective media aims to favour the growth of specific microorganisms whilst inhibiting others; however, this may limit the isolation of novel actinomycete strains. Therefore, an alternative strategy could employ nutritious media instead selective media to prevent limiting the number and types of organisms isolated from extreme desert environments.
In order to facilitate the discovery of novel actinomycetes, the choice of selective media for selective isolation is of great importance. Starch-casein agar (SCA) and glucose-yeast extract agar (GYE) are both commonly used media for the isolation of actinomycetes. Hozzein et al., (2008) evaluated the effectiveness of different media for the isolation of actinomycetes from hyper-arid desert soils in Egypt, and stated that GYE provides a high ratio of actinomycetes and is simple to prepare. Furthermore, Maldonado et al., (2009) investigated actinobacterial diversity from marine sediments in Mexico using a range of selective media for the isolation of marine actinomycetes. A total of 300 actinomycetes were recovered using 17 different media, including SCA and GYE. Similarly, Okoro et al., (2009), who extensively studied the actinobacterial flora of the Atacama Desert, also incorporated SCA and GYE for their selective isolation procedures. As stated, relatively high numbers of actinobacteria were isolated, including the SCA plates which presented with a total of 1.42 x 104 cfu g-1 viable bacteria from all three soil sample locations.
As well as the choice of selective media and the various improvements on traditional selective isolation, methods for the extraction and recovery of actinomycetes from soils also have to be considered. In order to successfully recover microorganisms from the environment, disruption of the ecological integrity may be required (Bull, 2004); thus, representative sampling of actinomycetes by conventional isolation methods is limited by actinomycete-soil interactions; therefore, a dispersion and differential centrifugation technique is beneficial for disrupting such interactions.
The dispersion and differential centrifugation technique (DDC) is a multistage procedure first described by Hopkins et al., (1991) that combines several physicochemical treatments which are effective in increasing the yield and diversity of actinobacteria from natural habitats. The aim of the technique is to enhance the efficiency of sampling by dispersing soil aggregates and disassociating microorganisms from soil particles, which is achieved in four successive stages, resulting in four distinct fractions and sample residue. The method is effective in breaking down microorganism-soil associations by the use of sodium cholate, Tris buffer, physical disruption via mild ultrasonication and ionic shock using distilled water. The properties of this method results in different fractions of supernatant which helps pull out different microorganisms at each fraction, thus enhancing recovery and isolation of bacteria. DDC has greater recovery efficiency regarding bacterial counts and, according to Gomes et al., (1999), is approximately nine times more efficient than the conventional dilution plate technique. Furthermore, previous studies have reported that higher numbers of actinomycetes were isolated from marine sediments using the DDC procedure (Maldonado et al., 2005).
The few extensive bacteriological studies of the Atacama Desert have incorporated standard selective isolation procedures using actinomycete-selective isolation media which subsequently led to the successful isolation of microorganisms including novel strains, albeit at relatively low numbers using conventional dilution plating and heat shock regimes (Connon et al., 2007). Connon and her colleagues investigated the bacterial diversity within the Yungay region using the 16S rRNA gene and phospholipid fatty acid profiles (PFLA); moreover, microbial selective isolation techniques were applied such as the dilution plate technique and heat shock regimes which subsequently yielded low microbial growth, and in most cases no growth. Furthermore, Okoro et al., (2009) used standard isolation approaches, which incorporated physical heat pre-treatments by heating samples at 55Â°C for 6 minutes prior to dilution plating, and subsequently isolated a total of 5.10 x 104 cfu g-1 viable bacteria; similarly, Lester et al., (2007) isolated a total of 1.55 x 104 cfu g-1 viable bacteria.
Based on these observations, it is hypothesised that greater numbers of bacteria can be cultured, as well as the isolation of novel actinomycetes, using innovative selective isolation procedures involving sample pre-treatments with chitin and yeast nitrogen base carbon sources, grinding of soil samples which may aid in dispersing soil aggregates, inoculation of soil samples using sprinkling techniques and the application of DDC to help enhance recovery of microorganisms from soil.
Hypothesis 1: Novel actinomycetes and greater numbers of bacteria will be isolated using a combination of innovative culture-dependent selective isolation techniques using treated and ground soil.
Hypothesis 2: Higher numbers of culturable bacteria will be isolated from the hyper-arid region (Salar de Atacama) than the extreme hyper-arid region (Yungay).
The previous studies outlined here-in support the view that Atacama Desert soils contain a largely unexplored community of novel actinomycetes which can be effectively isolated using a combination of innovative culture-dependent approaches, involving sample pre-treatments and grinding of the Atacama soils.
The present investigation aims to isolate novel and undescribed non-pathogenic actinomycetes from hyper-arid and extreme-hyper arid regions of the Atacama Desert using selective microbial isolation. The search for new actinomycetes is directed by the need for new antibiotics, in which actinomycetes are notorious for producing bioactive compounds. Using different culture-based techniques in the isolation of the bacteria will help uncover the microbial diversity of the regions of the Atacama Desert; moreover, it is hypothesised that novel actinomycetes will be isolated in abundance using a combination of culture based selective isolation techniques. The main aim of the study can be expanded into a set of objectives:
1.2.1. Aims and Objectives
To isolate actinomycetes from an extreme habitat.
To establish the actinobacterial community and determine the extent of actinobacterial diversity in samples of hyper-arid and extreme hyper-arid Atacama Desert soils using culture-dependent methods.
Comparison of total viable bacteria between Salar de Atacama and the Yungay region of the Atacama Desert.
Comparison of the numbers of total viable bacteria and viable actinomycetes with the amounts isolated from previous studies.
Comparison of total viable bacteria and the numbers of putative actinomycetes isolated between treated and untreated soil samples from Salar de Atacama and Yungay region.
Comparison of total viable bacteria and the numbers of putative actinomycetes isolated between ground and unground soil samples from Salar de Atacama and Yungay region.
To characterise isolated actinomycetes from an extreme habitat
Classification of putatively novel isolates using 16S rRNA phylogenetic analysis