In nature, all organisms need to compete in order to survive in their habitat.

This
biological task can be achieved by the development of competitive mechanisms such as
the production of toxins, enzymes and antimicrobial agents like antibiotics. Antibiotics
are natural or synthetic chemical compounds that can act by inhibiting growth or by
killing the cell (3, 9). These compounds have different modes of action. Generally they
interfere with biological processes such as replication, translation and cell wall synthesis
(8). Some antibiotics, like tetracycline, interfere with protein synthesis by associating
with the 30S ribosomal subunit, and others, like penicillin, produced by Penicillium
notatum, prevent transpeptidation of N-acetyl-muramic acid resulting in a weakened
peptidoglycan structure. Research has demonstrated that these metabolites are most likely
produced by fungi and bacteria. The actinomycetes are a group of prokaryotic
filamentous soil microorganims which are known to be the top producers of antimicrobial
agents, especially Streptomyces (17, 21). Some of the antibiotics produced by
Streptomyces are erithromycin, amphotericin, neomycin, streptomycin, and rifamycin.
Soil is a diverse medium composed of many minerals and substrates essential for
metabolic pathways of prokaryotic and eukaryotic inhabitants (6). The abiotic and biotic
diversity present in this medium makes it difficult for the isolation of all the microbial
community present. That is why research has demonstrated that not even 1% of the entire
soil microbial community has been identified (3, 11, 21). There is great opportunity for
discovering new groups of microorganisms with industrial and clinical importance in
soil. It is not possible to recreate all of the specific requirements that every soil
microorganism needs. That is why standard microbiological techniques and innovative

antibiotics because of mutations caused in their genome and by incorporating foreign

genomic material like plasmids. This is an event of great concern for the medical
community since pathogenic organisms are becoming resistant to a large quantity of
antibiotics.
Considerable research is being done in order to find new chemotherapeutic agents
isolated from soil (3, 4, 7, 9, 15, 14, 21). One percent of the entire microbial population in
soil is cultivable making it very difficult to access the entire microbial population by
traditional microbiological techniques, since one can not provide all the nutritional
requirements necessary for the entire soil population. In order to isolate antimicrobial
agent producing microorganisms, and detect chemotherapeutic encoding genes,
researchers have developed several techniques which combine the use of classic
microbiology and molecular genetics. One can study a vast percent of the microbial
population using functional genomic approaches such as the generation of metagenomic
libraries from soil. Our goal was to isolate and characterize organisms and encoding
genes that exhibit the capability of producing antimicrobial agents.

Soil Cultivable Microbial Communities

Soil microbial communities are among the most complex, diverse and important
assemblages of organisms in the Biosphere. They participate in various biological
activities such as mineralization and decomposition of biotic matter, biocontrol and
antagonism (22). It is said that bacteria that are found colonizing soil are ubiquos since
the chemical, physical and biotic characteristics present in such medium vary immensely.
Recently Horner et al. (23) postulated that microbial population can be various in a
particle of soil due to oxygen concentration. Sprusansky et al. (47) postulated that soil
bacteria display amazing versatility in their ability to use relatively poor sources of
carbon and nitrogen and that they thrive on a mixture of complex carbohydrates and
proteins that result from the degradation of organic material which demonstrates that soil
microorganisms are an important source for the search of novel antimicrobial agents and
molecules with biotechnological importance.
Some of the cultivable microbes that most commonly are isolated from soil
samples belong to the genera of Bacillus, Streptomyces and Pseudomonas (5, 46) The
Actinomycetes; in particular Streptomyces are responsable of the production of over 70%
of the antibiotics that have been isolated and reported (14,28). The genus Pseudomonas is
comprised of a gram-negative bacteria and is vastly involved in biological control of
many plant pathogens.

Soil Rhizosphere
One of the areas in soil where one can find abundance in microbial populations is
the rhizosphere. This area is comprised by the soil that is nearest to the root system of the
plant. The high nutritional content of this area promotes a high microbial colonization
which includes bacteria, fungi and nematodes. Even though this area has a high
nutritional content, the organisms that colonize it have to compete for space, water
availability, and other physical factors (24). Therefore, these communities exhibit and
maintain active their competition and survival mechanisms which include symbiotic
relations, parasitism and the production of antagonistic substances such as antimicrobial
agents and hydrolytically enzymes.
In nature, organisms need to survive in the environment where they are found.
One of the interactions that take place is natural control, which is accomplished when the
metabolic regulation of an antagonistic organism is used in nature for the biological
control of another organism. It is very important to state that this type of control excludes
any human manipulation that can be encountered, so this type of mechanism can be
found anywhere in nature. Natural control is obtained by the production and secretion of
enzymes, siderophores, antimicrobial agents and other compounds. Due to the
contamination that pesticides have provoked in the environment, the use of antagonistic
organisms can serve as an alternative for the control of plant pathogens (31,52).
Literature Review
1.1 Antimicrobial Agents
Antimicrobial agents are natural or synthetic chemical substances which have the
capacity of inhibiting or terminating total metabolic cell activity. These chemical
molecules are classified depending on their targets. They can also be referred to as broad
or narrow spectrum depending on its strength of action towards their targets. Key targets
include cell wall synthesis, protein synthesis and also DNA replication. The major class
of antibacterial agents are the β-lactams (including penicillins, cephalosporins,
monobactams, carbapenems), aminoglycosides, tetracyclines, sulfonamides, macrolides
(such as erythromycin), quinones and glycopeptides (vancomycin). These secondary
metabolites can affect many metabolic reactions in a cell in order to render effect.
Penicillins and cephalosporins mode of action is the biosynthesis of the
peptidoglycan present in the bacterial cell wall. They affect specifically the
transpeptidase that forms the peptide cross-linking. β-lactams also affect peptidoglycan
synthesis by forming covalent bonds with a specific group of proteins known as the
penicillin binding proteins. Both, gram-positive and gram-negative microorganisms,
posses these proteins.
Other antimicrobials, such as the quinolones, noviobiocin, inhibit DNA
replication by affecting enzymes such as the prokaryotic DNA gyrase and the eukaryotic
Topoisomerase II. Antimicrobials, known as 6-Aniniluracil, inhibits DNA polymerase
III. Classes of antimicrobials like tetracyclines, chloramphenicol and macrolides inhibit
protein synthesis.

1.2 Ecological interactions

In nature, multiple ecological interactions take place; which can be negative or
positive for the organisms involved. The organisms and the physical-chemical conditions
present in an ecological niche will delimit the type of interactions that can be observed.
Competition is an interaction encountered in all habitats since the organisms present need
to do so in order to survive. Also, it is known that when various communities in an
ecological niche utilize the same type of substrates they must compete (6). Both
theoretical and empirical studies suggest that in plant and animal communities,
competitive interaction is the key determinant of species abundance and diversity (25).
As part of physiological and metabolic processes, communities that are found
colonizing certain areas render the production of intracellular or extra-cellular low
molecular weight components such as alcohols, fatty acids, secondary metabolites and
some antimicrobial agents (21, 22). The substances secreted to the environment can be
harmful or toxic to the surrounding organisms acting as a competitive advantage for the
secretor. Amensalism or antagonism is the term used in the classification of ecological
interactions where one component has the competitive advantage of producing and
secreting substances that have inhibitory effects on other populations (29, 30). The
substances must alter the habitat in a detrimental manner so that the interaction may be
categorized as antagonism or amensalism.
1.3 Soil Microbial Interactions
An ecological niche is composed of many microhabitats; each microhabitat is
composed of a microscopic diversity which includes bacteria, protozoa, fungi,
nematodes, and a macroscopic diversity that includes plants and insects. Soil is a
complex medium in which one can encounter many kinds of microbial communities.
Application of nucleic acid-based methods to analyze soil microbial communities has
revealed high prokaryote diversity (27). The microbial diversity or communities present
in soil depend on the composition of the soil and many physical chemical properties that
the medium possesses. Also, the flora and decomposing organic mater on the surface of
the soil will influence vastly with the microbial diversity present. For example, the fallen
trees, barks and flowers provide nutrients both to the microbes and plants present,
through microbial degradation of carbohydrates, lipids and proteins to sugars, fatty acids,
glycerol and amino acids and respectively to mineralization. Besides providing these
nutrients, plant secondary metabolites that are generally toxic to microorganisms will
need to be degraded or detoxified by certain microbes. These degraders (microbes) are
selectively pressured and ultimately evolve to produce novel secondary metabolites
possibly to counteract the toxic plant secondary metabolites (34).
There are approximately 106-109 colony forming unites per gram of soil.
Microbes present in a medium posses advantages that will permit or facilitate their
survival in that medium. For example, Skujins (25) research has demonstrated that in
desert crusts or in soil that has a low water availability, gram-positive and spore forming
microbes are most abundant. The gram-positive bacteria posses a thicker layer of murein
in their cell wall which makes the cell less vulnerable to the limiting conditions present in
these habitats. Also, spore forming bacteria can resist long periods of desiccation and
limiting nutrient conditions since they compact and protect their genomic material in the
bacterial spore, until conditions are favorable for sporulation to occur (25).
1.4 The Soil Rhizosphere
Interactions that take place in the rhizosphere can be beneficial for the plant and
also for the microbial community present. The effect that takes place in the soil
rhizosphere is due to the influx of mineral nutrients to the plants roots through diffusion
alongside the efflux and accumulation of plant root exudates (21). Every metabolically
active system has the capability of secreting molecules as by-products (22). Exudates
released by plants have various effects in the surrounding ecosystem. For example, they
can alter the physical-chemical properties of soil by inhibiting the growth of other plants,
enhancing symbiotic relationships, and selecting the type of microbiota that can colonize
the area. Plant exudates can attract plant pathogens such as nematodes. The microflora

present can degrade these exudates in to smaller molecules so that the pathogens will not
be attracted to the plant tissue and therefore the plant will not be colonized by the
pathogens. Also, the microflora present in the rhizosphere can produce antagonistic
molecules that will inhibit or kill the pathogens present (9, 13, 21, 22). Certain
interactions may increase the immunological mechanisms of the plant making pathogen
colonization almost impossible.
In certain plants one could find nodules that are associated with Rhizobium, a
symbiotic soil microorganism, which produces a nodule in the root surface of the plant
that permits the microbe to receive nutrients directly from plant tissues. The plant
benefits by this interaction since the bacteria fix nitrogen for the plant, an important
molecule in the synthesis of molecular components (20, 35). One can also find colonizing
the soil rhizosphere microbes with the capability of increasing plant growth rate. These
populations are known as plant growth promoting rhizobacteria (PGPR). These microbes
can synthesize diverse growth regulators such as auxins, gibberellins, ethylene and
cytoquinins that increase the rate of seed germination. Growth regulators can also
improve the development of root hairs and cell elongation in stems. Some examples of
PGPR are Arthrobacter, Pseudomonas and Agrobacterium.
1.5 Soil Antimicrobial Agent Producing Microbes
Many groups of microorganisms like gram-positive, gram-negative bacteria and
fungi have the ability of synthesizing antimicrobial agents. Pandey et al. (19) states that
the top cultivable antimicrobial agent producers present in soils are the actinomycetes.
The actinomycetes are a group of gram-positive bacteria that exhibit characteristics of
both bacteria and fungi. These microbes produce filamentous structures which

agglomerate forming pseudo-mycelia. Actinomycetes are also spore forming microbes,

characteristic shared with fungi. Some of the characteristics that they share with bacteria
is the formation and composition of the cell wall, the flagella and the ribosome. About
10% - 33% of the total bacterial community present in soil is comprised by these bacteria,
being the genera Streptomyces and Nocardia the most abundant actinomycetes found in
soil (17).
The genus Streptomyces is the responsible of the synthesis of the majority of
antimicrobial agents with clinical importance like, for example, amphotericin,
erythromycin, streptomycin, tetracycline, and rifamycin (16). Protein synthesis is the
mode of action of all the above except for amphotericin which attacks the cell membrane.
Also the majority of these antibiotics are of broad spectrum. These microbes exhibit a
vast metabolic versatility. They can complete many physiological cycles that produce
intermediate molecules such as enzymes or secondary metabolites with antibacterial,
antifungal and antiviral capabilities.
Another group of gram-positive bacteria present in soil and responsible for the
production of antimicrobial agents with clinical and agricultural importance is the genus
Bacillus. This genera is characterized by being gram-positive, spore forming rods. It has
been demonstrated that these microbes produce antimicrobial agents in various stages of
their growth curve. For example, B. subtilis 168 can produce non ribosomal oligopeptides
with antifungal and antimicrobial properties such as surfactins, inturinics and bacilysin
(18, 32). Ribosomal antibiotics are also synthesized by this strain which include
subalancin and subtilosin. Previous research has demonstrated that the growth phase
interferes with the type of antimicrobial agent produced.
Materials and methods
2.1 Soil Sampling
Soil samples (about 5 cm from soil surface) were collected from the Guánica Dry
Forest, Guajataca Forest, and Toro Negro at Puerto Rico. All samples were collected
aseptically in Whirl pack® bags, and were placed on ice until processing (about 3 hrs.).
All the rocks and debris present in the samples were aseptically removed. Global
Positioning System (GPS) was used in order to obtaine the exact coordinates of sampling
sites which are listed in table 2.1.
2.2 Isolation of Antimicrobial Agent Producing Microbes by Crowded Plate
Technique
Serial dilutions were held out as follows: one gram of soil was diluted in 9 ml of
0.85% physiological saline solution 10-1. The soil suspensions were homogenized by
shaking at 200 rpm for 15 minutes at 30°C. Since we wanted to observe antagonism
present in the cultivable soil microbes, serial dilutions were done up to 10-3. Two
repetitions of each of the dilutions were inoculated in Tryptic Soy Agar (TSA) and the
cultures (or master plates) were incubated at 30°C for 48 hours. After the incubation
period, colonies that presented antagonism were designated as Antimicrobial Agent
Producing Microbes (AAPM) and were subcultured and purified by streaking them on a
TSA plate. After purification, the isolated AAPM’s were preserved and stored at -80°C
for further tests.

2.3 Antibiograms
2.3. A Susceptibility Test with Non-filtrated Supernatant
The Antimicrobial agent producing capability of the isolates was tested by a
modification of the Kirby-Bauer method described by Boyle (7). The target
microorganisms used were Escherichia coli ATCC 8739, Pseudomonas aeruginosa
ATCC 9721, Klebsiella pneumoniae ATCC 13882 and Staphylococcus epidermidis
ATCC 12228. AAPM’s were incubated 24 hours at 30°C and the targets were incubated
also for 24 hours at 37°C. For the construction of the antibiograms, 500 μl of the broth
culture of AAPM was transferred to a sterile 1.5 ml microtube; the microtubes were
centrifuged at 13,000 rpm for 20 minutes. The supernatants were transferred to a sterile
microtube.
Physiological saline solutions (0.85%) were used in order to prepare cellular
suspensions with a 0.08-0.1 absorbance of each target. Absorbance of the target cellular
suspensions was determined by using a spectrophotometer set at 625 nm, which is
equivalent to a McFarland standard of 0.5. In order to create a bacterial lawn of the
targets in TSA, the spread plate technique was employed by using 200 μl of each target.
Sterile paper discs were impregnated with 20 μl of the AAPM’s supernatant and placed
over the bacterial lawns. A negative control was included by using a sterile disk
impregnated with the sterile TSB culture media. Antibiograms were incubated 24-48
hours at 37°C. After this period, inhibition zones were measured with a ruler using a cm
scale (fig 2.1).

2.3.B Susceptibility Testing Using Filtered Supernatant

A modification of the procedure described in section 2.3A was used in order to
determine if the AAPM secreted an antimicrobial substance constitutively to the medium.
The same procedure was repeated but this time the centrifuged cultures of the AAPM’s
were filtered with a low binding protein cellulose filter with 0.2μm pore size (Fisher),
and this filtrate was used for the construction of the antibiograms. (fig. 2.1).
2.3.C Streak Susceptibility Testing
In vitro antimicrobial assays were also done in order to test if the antimicrobial
agent produced by the AAPM was secreted to the culture medium. Individual bacterial
lawn was inoculated by adding 100 μl of the AAPM TSB culture and dispersing the
culture in half of the TSA surface, these cultures were incubated at 30°C for 48 hours.

After the incubation period each target was inoculated in a perpendicular streak manner
(without touching the bacterial lawn) on the TSA surface. Cultures were incubated at
37°C for 24-48 hours (fig. 2.2).
2.3.D Radial Inhibition
The radial inhibition assay was also used in order to observe the antagonistic
potential of the AAPM isolated. For this part the AAPM were treated with the same
conditions described in section 2.3 A. An impregnated disc of each of the AAPM
supernatant were placed in the center of the TSA plates (individually). By using a
calibrated 10μl loop a linear streak was done with each of the targets (fig. 2.2). Radial
inhibition assays were incubated at 37°C and inhibition was monitored 24-48h after the
incubation period.

2.5 Macroscopic characterization of the AAPM

The AAPM’s were inoculated on the surface of TSA plates and morphological
characteristics such as margins, pigmentation, and texture were observed. Also
sedimentation, flocculation, and pellicle formation were observed by inoculating the
AAPM in Tryptic Soy Broth (TSB). The isolates were incubated in the respective media
at 30°C for 24h.
2.6 Microscopic characterization
Determination of the microscopic morphological characteristics of the isolates
was analyzed by light microscopy using an Olympus microscope. Gram stain reaction
was done as described by Cappuccino and Sherman (9) and all reagents used were
obtained from Fisher Scientific. All gram-positive AAPM’s were submitted to Schaffer –
Fulton spore staining method (9).
2.7 Physiological characterization
2.6.A Biochemical tests
To test the physiological requirements of the isolates, biochemical tests were done
which included: InVIC (indole, methyl red, Vogues Proskauer, and citrate utilization).
Other biochemical test such as oxidase and peroxidase production, nitrate reduction, and
motility were also performed to the AAPM. All tests were incubated at 30°C for 24-48