Abstract Natural products continue to play a very significant role in the search and discovery of antimicrobial drugs. Amongst the so far known antibiotics-producing microbes, the genus Streptomycete has been ranked the largest producers of antibiotics. In this study, we investigated the phenotypic diversity among soil-borne Streptomycetes, using soil samples from different sources. The antibiotic inhibition as well as resistance profile of Streptomycetes against each other, against a set of Gram-negative and Gram-positive bacteria, and against antibiotic discs was also investigated. A Gram-staining reaction was carried out to determine the spore pattern, hyphae formation, and Gram reaction of Streptomycetes. The result of this study shows that Streptomycetes were susceptible and in some cases resistant to antibiotics produced by other Streptomycetes. It was discovered that most of the time (over 50%), Gram negative bacteria were resistant to antibiotics produced by Streptomycetes, while in most cases, the Gram positive bacteria were susceptible to antibiotics produced by Streptomycetes. The Gram's reaction yielded positive results, confirming the nature and cell wall composition of Streptomycetes.
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Keywords Natural products, antimicrobial drugs, inhibition and resistance profile, and Gram's reaction.
The prevalence of pathogenic micro-organisms and their resistance to therapy, poses serious threats to public health. It has been shown from previous studies that resistance to antibiotics by most micro-organisms is evolutionary (Michael et al., 2007). In a recent documentary by the British Broadcasting Corporation (BBC) on bacteriophage therapy, concerns were expressed by stakeholders about the rate at which bacteria were increasingly developing resistance to chemotherapy. It is known that following an intensive therapy with an antibiotic, bacteria often become resistant to the drug. Bacterial resistance to antibiotics can also be transferred from resistant species to non-resistant species.
Despite the prevailing menace caused by resistant pathogenic bacteria to antibiotics, natural products continue to serve as the sources of a wide variety of antibiotics used for therapeutic purposes (Vincent et al., 2006). According to Watve et al (2001), "Of the 520 new drugs approved between 1983 and 1994, 39% were natural products or derived from natural products and 60-80% of antibacterial and anticancer drugs were derived from natural products. " Thus their role in the fight against disease causing microbes cannot be over-emphasized. This study determines the resistance and the inhibitory activities of Streptomycetes to the antibiotics produced by other Streptomycetes. It also investigates the resistance profile of Gram positive and Gram negative bacteria to antibiotics produced by Streptomycetes. Results show that Streptomycetes inhibited the growth of other bacteria consistent with it's ability to produce quite a huge amount of antibiotics. It was also observed that Streptomycetes were inhibited by conventional antibiotics used in this study. Although Streptomycetes play a pivotal role in drug search and discovery as the largest "reservoir" of antibiotics so far known, only a little fraction of these drugs have been isolated. There is a need to develop novel approaches for the discovery of microbial natural products.
Materials and Methods
Preparation of media
Oatmeal extract was prepared by mixing 20g of oatmeal (grocery store regular oats) with 1 liter of distilled water. Mixture was autoclaved for 30 minutes (flask was put in a tray filled with some water). Mixture was strained through cheesecloth. Oatmeal chunks were disposed and extract distributed into 500ml flasks (250 ml of extract each). Extracts were reautoclaved and stored until they were needed. A clean, empty 250ml flask was autoclaved and 74ml of 95% ethanol and 26 ml of sterile water were added. 1g of cycloheximide powder was then added and the mixture stored at 4Â°C. 500ml of distilled water was added to 500ml of extracted oatmeal in a 2liter flask. 15g of agar was weighed and added to the water-oatmeal mixture in the flask. Mixture was autoclaved and cooled slightly. 10ml of cycloheximide solution was added to the cooled mixture and flask twirled to mix.
Soil samples used for this experiments were obtained from different sources ranging from an unimproved grassland pasture to an improved (fertilized) grassland pasture. Other soil sources used are sand dune system, beech sand, unimproved pasture, improved pasture, and rhizosphere soil from beech tree. Soil were dried overnight in foil trays under sterile cheesecloth. The following morning the soils were placed in a drying over at 60Â°C for 90minutes. Soil samples were allowed to cool.
Isolation of bacteria from soil
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1g of soil from the improved (fertilised) grassland pasture was placed in 9ml of sterile distilled water. The mixture was vortexed for 1 minute. The sample was allowed to sit at room temperature for 30 minutes. A serial dilution of the sample mixture from 10-2 - 10-5 (dilution factor 1:10) was performed in four ml Ringer's solution tubes. Using micropipettes, 100ïl aliquots of each dilution were plated unto four (4) different oatmeal agar plates (pre-treated with cycloheximide). A sterile bent glass rod was used to spread the sample on the plates. Plates were incubated for five (5) days at 28Â°C. Plates were collected and observed after the incubation period. The population of total bacteria and Sreptomycetes was determined using the plate count methodology and results were noted.
Isolation and purification of individual Streptomycetes
Streptomycete spores were collected from incubation plates using a sterile toothpick. Spores were then streaked onto fresh oatmeal plates. A total of eight (8) isolates were collected and streaked onto two (2) OA plates by gently gliding the toothpick across the agar when streaking the OA plate. OA plates were labelled "Subcultures 1-4" and "Subcultures 5-8". Plates were incubated for 7 days at 28Â°C. After the incubation period, plates were collected and observed for colony characteristics of Streptomycetes. Observations were noted.
Purification of selected Streptomycete subcultures
Based on the Streptomycete's characteristics of being fast growers (i.e. of spreading the most on plates and producing the most powdery spores) and of showing diverse colours, four isolates that appeared pure were selected using a sterile toothpick to streak each isolate onto half a plate of OA. Two OA plates were used, each bearing two isolates. Plates were labelled "Spore collection 1-2" and "Spore collection 3-4". The plates were then incubated for 5-7 days at 28Â°C.
Spore suspension preparation
Using a micropipette, 0.75ml of sterile 20% (v/v) glycerol was aliquot into four 1.5ml sterile microfuge tubes. Tubes were labelled "Spore suspension 1,2,3 and 4". A moistened sterile cotton swab (by dipping into 20% v/v glycerol) was used to collect Streptomycete spores by gently rolling the moist swab across the spore collection plate, one collection plate at a time. Spores were liberated by twirling the cotton swab in the microfuge tube. The same swab was reswabbed three more times to make sure there was good spore harvest. The spore collection process was repeated for each of the four isolates using a fresh swab in each case. The swabbed collection plates were preserved for future use.
Four OA plates were labelled "Streptomycete 1,2,3, and 4" antibiotic assay plates respectively. Using a micropipette, 100ïl of spore suspension was added to the OA plate labelled "Streptomycete 1 antibiotic plate". This was repeated until four Streptomycete lawns were made each containing spore suspensions from different isolates. A sterile bent glass rod was used to spread the spores in order to make good, solid lawns. Lawns were allowed to dry for about 5 minutes. One antibiotic each was added to the antibiotic assay plate. The antibiotic disc used was neomycin. A pair of forceps was used to tap the antibiotic disc down so that it adheres to the agar. 10ïl spots of three other Streptomycete spore suspension were made on each antibiotic assay plate. A fresh tip was used each time to avoid a mixture of different spore suspension. Plates were incubated for 7 days at 28Â°C.
Gram negative and Gram positive antibiotic assay
Two (2) Mueller-Hinton (MH) plates were labelled. One of these plates was labelled "Gram-positive antibiotic assay" and the other "Gram-negative antibiotic assay". One MH plate was divided in half and with the use of two sterile swabs, two half lawns of Gram-negative bacteria were made. The Gram-negative bacteria were Pseudomonas flourescens and Escherichia coli. The second MH plate was also divided in half and with the use of two sterile swabs, two half lawns of Gram positive bacteria were made. The Gram-positive bacteria used were Bacillus subtilis and Micrococcus luteus. Each half lawn was labelled with the name of the swabbed bacteria. A cork borer was flame sterilized using ethanol after which it was used to punch four holes in the swabbed spore collection plate 1. This was repeated for plate 2,3 and 4. Each plug contained any antibiotic that was produced by the Streptomycete and that diffused into agar. Using a sterile forceps, plugs were carefully picked and placed onto each MH lawn. Plates were incubated for 7 days at 30Â°C.
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After the period of incubation, the antibiotic assay plates were collected for zones of inhibition. Measurements of the zones of inhibition was done to the nearest mm from the edge of the spotted Streptomycete spore isolate or disc or plug to the edge of each zone. Measurements were recorded on a data sheet.
Streptomycete spores and filaments were collected from plates containing Streptomycete isolates using a sterile toothpick. Spores were heat-fixed to a glass slide by mixing with a drop of water on the glass slide , air-drying and passing glass slide over Bunsen flame. The heat-fixed smear of cells were flooded with crystal violet staining reagent. The slide was washed in gentle stream of tap water for 2 minutes. Slide was flooded with mordant (Gram's iodine) for 1 minute. Drops of decolourising agent (ethanol) was added until the decolourising agent running from the slide ran clear. Slides were then flooded with counter stain, safranin , for 1 minute. The slide was washed in a gentle stream of water and blot dried with absorbent paper. The slide was observed under oil immersion using a Bright field microscope at 1000X magnification.
Isolation of soil bacteria
Results from previous experiments (data not shown) reveals that quantitative isolation of Streptomycetes from the soil vary according to the source and nature of the soil (Arai et al, 1976).To isolate and quantify pure Streptomycetes from the soil, one (1) gram of improved fertilised grassland pasture was diluted serially using Ringer's solution and dilutions were plated out on oatmeal plates. Plates were pre-treated with cyclohexamide to prevent the growth of spore-forming micro-organisms other than bacteria. Plates were incubated for 5 days at 28Â°C. The population of total bacteria and Streptomycetes were determined. Significant growths were observed at a dilution of 10-3 and 10-4. Results were collected from other groups also.
Table 1: Total bacteria and Streptomycete colony forming units from different soil samples.
Total bacteria (cfu/gm)
of total bacteria
Medium organic content from improved grassland pasture
215* 10* 10-3
Medium organic content from unimproved grassland pasture
67* 10* 10-3
Low organic content sand dune
Low organic content beech sand
High organic content unimproved pasture
High organic content improved pasture
High organic content rhizosphere soil from beech tree
The Streptomycete colony forming units per gram of soil sample formed over 50% of the total bacteria colonies in most cases. This may likely be due to the activity of the antibiotics produced by Streptomycetes (to which they are resistant). Soil samples from different sources were also discovered to contain varying numbers of colony forming units of Streptomycetes per gram of soil. The highest number of Streptomycete colonies were found in high organic content soils while lower numbers of colonies occurred in medium to low organic content soils.
Where MIC represents the medium organic content improved soils, MUC are soils from medium organic content unimproved sources, LCS are low organic content sand dunes, LCB are low organic content beech soils, HUC represents soil from high organic content unimproved sources, HIC are high organic content improved soils and HRC, high organic content rhizosphere soil from beech.
Colony forming characteristics of Streptomycetes
Streptomycetes are known to grow as vegetative mycelium on agar as well as aerial mycelium. These aerial mycelium have chains of sporophores which makes them appear powdery unlike shiny (moist) and soft bacterial colonies (Grace et al ,1989) .To observe the colony characteristics (phenotypes) of Streptomycetes, oatmeal agar (OA) plates containing dilutions of soil sample were incubated for 5 days at 28Â°C. Plates were collected after the incubation period and observations were made. Spores were formed and ornamented . In some cases, yellow colonies were observed while in most cases, Streptomycete colonies observed were white and powdery.
To assay the susceptibility and resistance of Streptomycetes and Gram-positive as well as Gram-negative bacteria to antibiotics produced by other Streptomycetes and to conventional antibiotics, four Streptomycete lawns were made. Streptomycete colonies were provided on oatmeal agar plates. Plates were labelled "Streptomycete 1","Streptomycete 2","Streptomycete 3" and "Streptomycete 4" respectively. Spore suspensions were made from each plate and from these suspensions, Streptomycetes were challenged against each other and against the antibiotic gentamycin. Plugs from the spore collection plates were also used to challenge the growth of Gram-positive and Gram-negative bacteria. Antibiotic assay plates were incubated for a period of 7 days at 28Â°C. After the incubation period, plates were collected and the zones of inhibition were measured to the nearest millimetre(mm).
Table 2: Zones of inhibition produced by spots, plugs and antibiotic disc.
In all, 19 zones of inhibition were observed. The zone of inhibition measurements from antibiotic assays using Streptomycetes isolated from medium organic content (improved) grassland pasture shows that Streptomycetes are resistant and in some cases susceptible to the antibiotics that they produce. Also, Streptomycetes isolated from this soil sample, were susceptible to the activity of the antibiotic disc, gentamicin. Generally, the sizes of the zones of inhibition of antibiotics produced by Streptomycetes against other Streptomycetes were smaller than those produced by the antibiotic disc against Streptomycetes. This implied that Streptomycetes were more susceptible to the conventional antibiotic disc used in this experiment than they were to the antibiotics that were self-made.
Fig 2: Representative plates showing zones of inhibition by antibiotics and Streptomycetes to lawns of Streptomycetes and Gram negative and Gram positive bacteria.
GN1=Gram-negative bacteria 1(Pseudomonas flourescens), GN2= Gram negative bacteria 2 (Escherichia coli), GP1= Gram-positive bacteria 1(Bacillus subtilis), GP2= Gram-positive bacteria 2 (Micrococcus luteus).
Antibiotic assays against Gram-positive and Gram-negative bacteria revealed a larger percentage of the Gram-negative bacteria being resistant to the antibiotics produced by the Streptomycetes and a smaller percentage of the Gram-positive bacteria being resistant to the antibiotics produced by Streptomycetes.
Results from antibiotic assays of other groups were also collected and values obtained were compared with the results of this experiment.
Table 3: Inhibition pattern of different soil Sreptomycete isolates to different antibiotic discs.
MICS= medium organic content improved soil Streptomycete isolates, LCBS= low organic content beech sand, MUCS= medium organic content unimproved soil Streptomycete isolates, LCSS= low organic content sand dune Streptomycete isolates, HRCS= high organic content rhizosphere soil isolates, HUCS= high organic content unimproved soil isolates, HICS= high organic content improved soil isolates, R= resistant and S= Susceptible.
The results of the antibiotics assay from other groups shows that Streptomycete isolates from different soil samples have different inhibition patterns to different antibiotics. Streptomycete isolated from medium organic content improved soils were susceptible to the antibiotic gentamicin. The low organic content beech soil Streptomycetes were resistant to daptomycin while Streptomycetes isolates from medium organic content unimproved soils were susceptible to neomycin. Low organic content sand dunes and high organic content improved soil Streptomycetes were resistant to tetracycline and synercid respectively while high organic content rhizosphere and high organic content unimproved soil Streptomycetes were susceptible to erythromycin and cindamycin respectively.
To investigate the Gram's reaction of Streptomycetes, a Gram's stain was carried out. Spores were heat-fixed to glass slide and the slide flooded in Gram's iodine. The stained slide was decolourised using a decolourising agent (ethanol) and then counterstained with safranin for 1 minute. The slide was washed in a gentle stream of water and blot dried. Using a bright field microscope at 1000X magnification, the slide was viewed in oil immersion. The result of the Gram's reaction showed that Streptomycetes are Gram-positive as they stained blue (results not shown) in the test.
Generally speaking, Streptomycetes are known to inhabit the soil. They constitute a large population within the soil microbial community (Williams,1983). According to Grace et al (1989), Streptomycetes are chemo-organotrophs in nature and so require organic sources (carbon) as well as inorganic nitrogen and mineral salts to grow. The results of this study (table 1) shows that in soil samples with high organic contents and in some cases improved with fertilizers, the number of streptomycete colony forming units were more than those found in low organic content or unimproved soils. This suggests that the presence of a suitable carbon source in some soil samples favoured the growth of Streptomycetes in such soils. Although nutrient availability is an important factor that determines the distribution of soil Streptomycetes as well as their activity, it has been reported that other factors such as pH, temperature and moisture also play important roles in determining Streptomycete distribution in the soil. (Arai ,1976). Streptomycetes are in most cases neutrophilic or alkilinophilc and are almost always found in such environments.
The antibiotic effects of Streptomycetes in soil samples were also observed (Table 1). In most of the soil samples from which Streptomycetes were isolated, over 50% of the total bacterial population were Streptomycetes. Documented reports shows that Streptomycetes are the largest known producers of antibiotics (Zhang and Demain,2005). Reports also shows that most Streptomycetes have the target site for the antibiotics they produce but at the same time possess mechanisms by which they protect themselves from their own antibiotics (Goodfellow et al,1988). The predominance of Streptomycetes in the soil may be correlated to their ability to defend themselves from the effects of their own antibiotics. In one instance, during this study when fungal contamination occurred on plates without cyclohexamide (results not shown), zones of inhibition were observed around Streptomycete colonies. Indicating the broad spectrum activity of antimicrobials produced by Streptomycetes.
Investigation of the resistance profile of Streptomycetes to conventional antibiotics revealed that Streptomycetes are susceptible to gentamicin (Table 2). Gentamicin, an aminoglycside aminocyclitol, is known to act by the inhibition of protein biosynthesis in bacteria. It is proposed that the antibiotic binds directly to the ribosomal RNA and inhibits translocation by preventing the interaction between the ribosome and the elongation factor-G (EF-G) (Russell and Chopra,1990). Development of resistance by bacteria to this group of antibiotics (aminoglycoside aminocyclitol) has been hypothesized to be by modification of the antibiotic within the cell of the bacteria (Russell and Chopra,1990). As the antibiotics are being taken up by the organism through the cell membrane, they undergo modification inside the cell. Although the rate of modification by modifying enzymes is not high, the degree of resistance to these drugs becomes dependent on a competition between the rate of uptake of the drugs and their rates of modification. If the rate of uptake of the drug becomes higher than the rate at which it is been modified, there will be little or no resistance by the bacteria. Conversely, with a higher rate of drug modification, an increased rate of resistance is observed (Bryan ,1989). Bacteria can also acquire resistance to antibiotics by undergoing mutation at some loci in a gene to which the antibiotics bind. Genetic resistance to antibiotics can also be by transfer of extrachromosomal materials such plasmids and transposons from resistant species to non resistant species (Mann et al, 1996). Other antibiotics used by other groups in this study showed different inhibition patterns against Streptomycetes isolated from different soil samples. For example Streptomycetes resisted the activity of the antibiotic, tetracycline (Table 3). Although the process of tetracycline resistance in bacteria is not fully understood, it has been demonstrated in some instances to be linked to a decline in the amount of tetracycline in some mutant strains (Bryan,1989). The antibiotic disk erythromycin prevented the growth of Streptomycetes from high organic content rhizosphere soil. Erythromycin has been widely used clinically since it's discovery in 1952. It is known to act by selectively binding to the 70S bacterial ribosome precisely on the 50S subunit and causes the peptidyl-tRNA to dissociate from the ribosome. This ultimately inhibit's the process of protein biosynthesis (Rogers et al, 1980).
Another important factor that determines the ability of bacteria to resist the activity of antibiotics
is their cell wall composition. Bacteria are generally classified into two (2) on the basis of their cell wall composition ; Gram-positive and Gram-negative bacteria. Gram-positive bacteria are characterised by the presence of a thick peptidoglycan layer in their cell wall and will stain blue to purple in a Gram's stain reaction while Gram-negative bacteria possess a thin layer of peptidoglycan in their cell walls and stain pink in a Gram's reaction (Sadava et al,2008). In a Gram stain, a crystal violet-iodine complex forms within the cell. In a Gram positive bacteria, due to the thick layer of peptidoglycan in the cell wall, alcohol (a decolourising agent) causes a dehydration of the cell wall, closing the pores in the cell wall and trapping the insoluble crystal violet-iodine complex. Thus when cells are counterstained with safranin (a red dye), they stain blue to purple. In contrast, Gram negative bacteria have a thin peptidoglycan layer which allows the entry of the decolourising agent and removal of the crystal violet-iodine complex. When counterstained with safranin, Gram-negative cells stain pink (Madigan and Martinko, 2006) In addition to the inner membrane and peptidoglycan layer found in Gram-positive bacteria, Gram-negative bacteria also have a periplasm and an outer membrane made up of lipopolysaccharides (Rogers et al, 1980). The outer membrane in Gram-negative bacteria is believed to play a significant role in determining the resistance of Gram-negative bacteria to antibiotics. It functions by acting as a selective permeability barrier preventing the entry of antibiotics into the bacterial cell.
The increasing rate at which pathogenic bacteria develop resistance to antibiotics is alarming. In a workshop report titled, "Antimicrobial Resistance: Issues and Options" it was stated that over 90% of Staphylococcus aureus, a major causative agent of nosocomial infections, have become resistant to a number of antibiotics (Harrison and Lederberg,1998). An increasing number of other pathogenic microorganisms are increasingly becoming resistant to available antibiotics. This raises a fresh and urgent need for novel antibiotics with novel modes of action. Although Streptomycetes are known to produce a large number of antibiotics, only a little percentage of these have been discovered so far. The decline in the discovery of antibiotics produced by Streptomycetes is thought to be as a result of a decrease in the screening efforts for antibiotics. Hence the need for novel screening techniques in the search for new antibiotics.