ANTIBACTERIAL SCREENING OF SOIL BACTERIAL ISOLATES FROM SINAPSAPAN, JORDAN, GUIMARAS AGAINST Escherichia coli AND Staphylococcus aureus

West Visayas State University 1

COLLEGE OF ARTS AND SCIENCES

Iloilo City

Chapter 1

Introduction to the Study

Chapter one, consists of five parts, namely: (1)

Background and Theoretical Framework of the Study, (2)

Statement of the Problem and Hypothesis, (3) Significance

of the Study, (4) Definition of Terms, and (5) Delimitation

of the Study.

Part One, Background and Theoretical Framework of the

study, gives the reasons for the choice of the problem and

the variables considered in the conduct of the study.

Part Two, statement of the Problem and Hypothesis,

describes the problem and its purpose in conducting the

study and the hypothesis to be tested.

Part Three, Significance of the Study, enumerates the

benefits that may be derived from the results of the study

and persons who would benefit from them.

Part Four, Definition of Terms, deals with the

conceptual and the operational definitions of important

terms used in the study.

Part Five, Delimitation of the Study, sets the limit

and scope of the study.



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Background and Theoretical Framework of the Study

Soil is a natural reservoir for microorganism and their

antimicrobial products (Alanis, 2005).

A significant number of these organisms are undocumented

and some of them may have an effect on pathogenic bacteria.

Barangay Sinapsapan is located at Jordan, Guimaras Island,

on the lands bordering to Panay Gulf with approximate coordinates

of N 010 35’.00 E 122 30’.47. The distance from the sea is about

270 meters at an elevation of about 30 meters above sea level. The

terrain is a typical young coral island with the land rising

rapidly to about 30-40 meters elevation when proceeding

inland from the coastland some 100 meters. Going further inland

elevation varies up and down forming valleys and ridges

(Eckerwall, Personal Communication, October 10, 2013).

Barangay Sinapsapan has many unexplored territories and which

is most likely to yield purposeful results towards isolation of

new antibiotics. There are areas that are not inhabited by people.

Most of the soil in that area is rich in organic matter since

leaves and twigs cover most of the land. The texture of the soil

is mostly coarse. With regards to its structure, the arrangement

of the soil particles is in different aggregates. Soil granules



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clump or bind together (Española, Personal Communication, October

10, 2013).

In a single gram of soil, there can be billions of bacteria

(Reid and Wong, 2005). There are estimated 60,000 different

bacteria species, most which have yet to be even named, and each

has its own particular roles and capabilities (Reid and Wong,

2005). Most live in the top 10 cm of soil where organic matter

is present (Reid and Wong, 2005). Soil bacteria perform

important services related to disease suppression (Martin,

1976).

Pathogenic bacteria are capable of causing disease. Humans

are generally most interested in the species of pathogenic

bacteria which can cause disease in humans, although these

bacteria can also infect other animals and plants.

Some notable pathogenic bacteria include Staphylococcus

aureus and Escherichia coli. Staphylococcus aureus, often

referred to simply as “staph”, are gram-positive spherical

bacteria that occur in microscopic clusters resembling grapes.

Bacteriological culture of the nose and skin of normal humans

invariably yields staphylococci (Todar, 2008). Escherichia coli

is the head of the large bacterial family, Enterobacteriaceae,



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the enteric bacteria, which are facultatively anaerobic gram-

negative rods that live in the intestinal tracts of animals in

health and disease (Todar, 2008).

The science of antibiotics has remained and will

remain for many years, one of the most interesting natural

sciences, in both theoretical and practical aspects. Microbial

natural products still appear as the most promising source of

the future antibiotics that society is expecting (Pela´ez,

2006).

For the past years, pathogens underwent mutation which

enabled them to resist antimicrobials, thereby threatening

millions of people worldwide. Thus, it is imperative to screen

more and more bacteria from different soil samples for

antimicrobial activity in hope of getting some bacterial strains

that produce antibiotics that have not been discovered yet and

active against drug resistant pathogens.



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The relationship between the variables of the study is

presented in Figure 1.

Independent Variables: Dependent Variables:

Figure 1. Paradigm showing the relationship between the

independent and dependent variables.

Bacterial Isolates in

Brgy. Sinapsapan,

Jordan, Guimaras in

the high area


Brgy. Sinapsapan,

Jordan, Guimaras in

the intermediate area


Brgy. Sinapsapan,

Jordan, Guimaras in

the low area

Zone of inhibition

on Staphylococcus

aureus BIOTECH

1582

Zone of Inhibition

on Escherichia

coli BIOTECH 1634

Positive Control: 500

mg Ciprofloxacin / 10

ml distilled water

Negative Control:

distilled water



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Statement of the Problem and Hypotheses

This study was conducted to screen and determine the

antibacterial effects of soil bacterial isolates from three

sampling sites of Brgy. Sinapsapan, Jordan, Guimaras against S.

aureus and E. coli. Furthermore, soil bacterial isolates with

antibacterial properties against the test pathogenic bacteria

were characterized. Specifically, it sought to answer the

following questions:

1. How many bacterial isolates are present in the three

sampling sites of Brgy. Sinapsapan, Jordan, Guimaras?

2. What are the characteristics (colonial morphology and

gram staining reaction) of bacterial isolates that have

antibacterial properties against S. aureus and E. coli?

3. What are the effects of the bacterial isolates on S.

aureus and E. coli after 72 hours of incubation?

4. Are there significant differences on the zone of

inhibition of bacterial isolates from the three sampling

sites against S. aureus and E. coli?

Null Hypothesis:

1. There are no significant differences on the zone of

inhibition of bacterial isolates in the three sampling

sites against S. aureus and E. coli.



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Significance of the Study

The present study was undertaken to isolate bacteria from

the soil samples of Brgy. Sinapsapan, Jordan, Guimaras. Also, to

assess the soil bacteria for their anti-bacterial properties.

The resistance problem demands that to discover new

antibacterial agents effective against pathogenic bacteria

resistant to current antibiotics. Thus, it is imperative to

screen more and more bacteria from different soil samples for

antimicrobial activity in hope of getting some bacterial strains

that produce antibiotics that have not been discovered yet and

active against drug resistant pathogens.

Definition of Terms

The following terms are defined to ensure clarity and

understanding of the study:

Antibacterial effect- According to Mozo (2008), it is an

effect that is destructive to bacteria or suppressing their

reproduction or growth, effective against bacterial

infections.

In this study, “antibacterial effect” refers to the action

of screened bacterial isolates against S. aureus and E. coli.



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Barangay- is a village, especially when considered as the

smallest political unit (Department of Health, 2009).

In this study, “barangay” refers to Barangay Sinapsapan,

Jordan, Guimaras which is the sampling site of the experiment.

Escherichia coli- it is the head of the large bacterial

family, Enterobacteriaceae, the enteric bacteria, which are

facultatively anaerobic gram-negative rods that live in the

intestinal tracts of animals in health and disease (Todar,

2008).

In this study, “Escherichia coli” refers to one of the

bacterial pathogens tested.

Isolation - the process of separating, or the state of being

alone (O’Toole et al., 2003).

In this study, “isolation” refers to the process of

collecting the general bacterial populations in soil of Brgy.

Sinapsapan, Jordan, Guimaras.

High area sampling site- having an elevation of

approximately 21 meters- 30 meters above sea level (Eckerwall,

Personal Communication, October 10, 2013).



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In this study, “high area” refers to one of the sampling

areas of study from which the bacterial isolates were obtained.

Intermediate area sampling site- having an elevation of



In this study, “intermediate area” refers to one of the

sampling areas of study from which the bacterial isolates were

obtained.

Low area sampling site- having an elevation of



In this study, “low area” refers to one of the sampling

areas of study from which the bacterial isolates were obtained.

Soil- is a natural reservoir for microorganism and their

antimicrobial products (Alanis, 2005).

In this study, “soil” refers to the sample where the new

antibacterial agents are to be isolated.

Staphylococcus aureus- it is gram-positive spherical

bacteria that occur in microscopic clusters resembling grapes.



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Bacteriological culture of the nose and skin of normal humans

invariably yields staphylococci (Todar, 2008).

In this study, “Staphylococcus aureus” refers to one of

the bacterial pathogens tested.

Zone of inhibition- is the area of no bacterial growth

around an antimicrobial agent in the disk (Tortora, 2010).

In this study, “zone of inhibition” refers to the effect of

screened isolated antibacterial agents against S. aureus and E.

coli.

Scope and Limitation of the Study

This study aimed to determine the number of bacterial

isolates from the three sampling sites at Sinapsapan, Jordan,

Guimaras delineated by their elevation. The isolates are then

assessed for antibacterial effect using the agar disk diffusion

method against S. aureus and E. coli. All isolates with

antibacterial properties were then characterized based on their

colonial characteristics. Aseptic technique was employed in the

experiment. This study used both the experimental and the

descriptive mode of investigation. The experimental mode

involved the bioassay of the bacteria and the descriptive mode

involved the isolation and characterization of the obtained



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bacterial isolates. It was conducted on May 5 to 24, 2014 at

Rizal Hall of West Visayas State University, La Paz, Iloilo

City.



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Chapter 2

Review of Related Literature

This chapter presents the conceptual literature regarding

this study to support the background for greater understanding

of the research outcome. This is consisted of the following:

(1)Staphylococcus aureus’ morphology, (2) its pathogenicity,

(3)Escherichia coli’s morphology, (4) colony characteristics,

(5) its pathogenicity, (6) soil, and Barangay Sinapsapan, Jordan

Guimaras.

Staphylococcus aureus

Morphology. According to Brooks, Carroll, Butel, Morse and

Mietzner (2010) staphylocci are gram positive spherical cells,

usually arranged in grapelike irregular clusters. They grow

readily on many types of media and are active metabolically,

fermenting carbohydrates and producing pigments that vary from

white to deep yellow. Some are members of the normal flora of

the skin and mucous membranes of humans; others cause

suppuration, abcess formation, a variety of pyogenic infections

and even fatal septicemia. The pathogenic staphylococci often

hemolyze blood, coagulate plasma, and produce a variety of food



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poisoning is caused by a heat-stable staphylococcal enterotoxin.

Staphylococci rapidly develop resistance to many antimicrobial

agents and present difficult therapeutic problems. The genus

Staphylococcus has at least 40 species. The three most

frequently encountered species of clinical importance are

Staphylococcus aureus, Staphylococcus epidermis, and

Staphylococcus saprophyticus. Staphylococcus aureus is

coagulase-positive, which differentiates it from the other

species. Staphylococcus aureus is a major pathogen for humans.

Almost every person will have some type of Staphylococcus aureus

infection during a lifetime, ranging in severity from food

poisoning or minor skin infections to severe life-threatening

infections. Staphylococci are spherical cells about 1 um in

diameter arranged in irregular clusters. Single cocci, pairs,

tetrads, and chains are close also seen in liquid cultures.

Young cocci stain strongly gram-positive; on aging, many cells

become gram-negative. Staphylococci are non-motile and do not

form spores. Under the influence of drugs like penicillin,

staphylococci are lysed. The pathogenic capacity of a given

strain of a Staphylococcus aureus is the combined effect of

extracellular factors and toxins together with the invasive

properties of strain. At one end of the disease spectrum is



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staphylococcal food poisoning, attributable solely to the

ingestion of performed enterotoxin; at the other end are the

staphylococcal bacteremia and disseminated abscesses in all

organs. Pathogenic invasive of Staphylococcus aureus produces

coagulase and tend to produce a yellow pigment and to be

hemolytic. The prototype of Staphylococcal lesion is the

furuncle or other localized abscess. Group of Staphyloccus

aureus established in a hair follicle leads to tissue necrosis

(dermonecrotic factor). Coagulase is produce d and coagulates

fibrin around the lesion and within the lymphatics, resulting in

formation of a wall that limits the process and is reinforced by

the accumulation of inflammatory cells, and, later, fibrous

tissue.

Pathogenicity. According to Talaro (2008) it is surprising

that a bacterium with such great potential for virulence as

Staphylococcus aureus is a common, intimate human associate. The

microbe is present in most environments frequented by humans and

is readily isolated from fomites (Talaro, 2008).Colonization of

some infants begin within hours after birth and continues

throughout life. The carriage for normal healthy adults varies

anywhere from 20% to 60%, and the pathogen tends to be harbored

intermittently rather than chronically. Carriage occurs mostly



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in the anterior nares (nostrils) and, to a lesser extent, in the

skin, nasopharynx, and intestine. Usually this colonization is

not associated with symptoms, nor does it ordinarily lead to

disease in carriers or their contacts. Circumstances that

predispose an individual to infection include poor hygiene and

nutrition, tissue injury, preexisting primary infections,

diabetes mellitus, and immunodeficiency states. Staph infections

in the newborn nursery and surgical wards are the third most

common nosocomial infection. The so-called “hospital strains”

can readily spread in an epidemic pattern within and outside the

hospital. A serious concern has arisen from the increase in

community infections strains of Staphylococcus aureus called

MRSA (methicillin resistant S. aurues). Several outbreaks have

been reported in prison inmates, athletes, and schoolchildren.

The infections are spread by contact with skin lesions, and have

proved to be very difficult to treat or control (Talaro, 2008).

According to Roche (2003) Staphylococcus aureus is a

complex pathogen with numerous classes of virulence factors.

Protein secretion principally occurs via the Sec system and is

required to render many virulence factors functional. Compounds

which selectively block bacterial protein secretion while

leaving the host system unaffected may lead to novel



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antimicrobial therapies. Adherence to host tissues involves

MSCRAMMs (Microbial Surface Components Recognizing Adhesive

Matrix Molecules) which are redundant and overlapping. Microbial

Surface Components Recognizing Adhesive Matrix Molecules

(MSCRAMMs) continue to be targets of interest for vaccine

development, although no current immunization studies involve

children. The role of vaccines in combating S. aureus disease

and the identification of populations which should be targeted

for immunization strategies are questions yet to be resolved.

The importance S. aureus capability to invade and replicate in

nonprofessional phagocyte cells is unclear; intracellular

persistence with subsequent decreased antibiotic exposure could,

in the future, impact decisions of drug choice and therapy

duration for infections such as endocarditis and osteomyelitis.

An improved understanding of protein secretion, tissue adherence

and internalization in S. aureus pathogenesis carries the

promise of identification of new targets for novel therapies for

preventing and treating both acute and chronic S. aureus

infections.

According to Todar (2008) Staphylococcus aureus causes a

variety of suppurative (pus-forming) infections and toxinoses in

humans. It causes superficial skin lesions such as boils, styes



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and furuncles; more serious infections such

as pneumonia, mastitis, phlebitis, meningitis, and urinary tract

infections; and deep-seated infections, such

as osteomyelitis and endocarditis. Staphylococcus aureus is a

major cause of hospital acquired (nosocomial) infection of

surgical wounds and infections associated with indwelling

medical devices. S. aureus causes food poisoning by releasing

enterotoxins into food, and toxic shock syndrome by release of

superantigens into the blood stream.

According to Todar (2008) although methicillin-resistant

Staphylococcus aureus (MRSA) have been entrenched in hospital

settings for several decades, MRSA strains have recently emerged

outside the hospital becoming known as community associated-

MRSA( (CA-MRSA) or superbug strains of the organism, which now

account for the majority of staphylococcal infections seen in

the ER or clinic. For the majority of diseases caused by S.

aureus, pathogenesis is multifactorial, so it is difficult to

determine precisely the role of any given factor. However, there

are correlations between strains isolated from particular

diseases and expression of particular virulence determinants,

which suggests their role in particular diseases. The

application of molecular biology has led to advances in



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unraveling the pathogenesis of staphylococcal diseases. Genes

encoding potential virulence factors have been cloned and

sequenced, and many protein toxins have been purified. With some

staphylococcal toxins, symptoms of human disease can be

reproduced in animals with the purified protein toxins, lending

an understanding of their mechanism of action. Human

staphylococcal infections are frequent, but usually remain

localized at the portal of entry by the normal host defenses

(Todar, 2008). The portal may be a hair follicle, but usually it

is a break in the skin which may be a minute needle-stick or a

surgical wound. Foreign bodies, including sutures, are readily

colonized by staphylococci, which may make infections difficult

to control. Another portal of entry is the respiratory tract.

Staphylococcal pneumonia is a frequent complication of

influenza. The localized host response to staphylococcal

infection is inflammation, characterized by an elevated

temperature at the site, swelling, the accumulation of pus, and

necrosis of tissue. Around the inflamed area, a fibrin clot may

form, walling off the bacteria and leukocytes as a

characteristic pus-filled boil or abscess. More serious

infections of the skin may occur, such as furuncles or impetigo.

Localized infection of the bone is called osteomyelitis. Serious



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consequences of staphylococcal infections occur when the

bacteria invade the blood stream. A resulting septicemia may be

rapidly fatal; a bacteremia may result in seeding other internal

abscesses, other skin lesions, or infections in the lung,

kidney, heart, skeletal muscle or meninges (Todar 2008)

Escherichia coli

Morphology. According to Brooks et al. (2010), The

Enterobacteriaceae are short gram-negative rods. Typically

morphology is seen in growth on solid media in vitro, but

morphology is highly variable in clinical specimens. Capsules

are large and regular in Klebsiella, less so in Enterobacter,

and uncommon in the other species. Escherichia coli and most

other enteric bacteria form circular, convex, smooth colonies

with distinct edges. Enterobacter colonies are similar but

somewhat more mucoid. Klebsiella are large and very mucoid and

tend to coalesce with prolonged incubation. The salmonellae and

shigellae produce colony similar to E. coli but do not ferment

lactose. Some strains of E. coli produce hemolysis on blood

agar. According to Todar (2008) E. coli is facultatively

anaerobic Gram-negative rod that live in the intestinal tracts

of animals in health and disease. The Enterobacteriaceae are



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among the most important bacteria medically. A number of genera

within the family are human intestinal pathogens

(e.g. Salmonella, Shigella, Yersinia). Several others are normal

colonists of the human gastrointestinal tract (e.g.

Escherichia, Enterobacter, Klebsiella), but these bacteria, as

well, may occasionally be associated with diseases of humans.

Colonial characteristics. According to Todar (2008) E.

coli is versatile and well-adapted to its characteristic

habitats. It can grow in media with glucose as the sole organic

constituent. Wild-type E. coli has no growth factor

requirements, and metabolically it can transform glucose into

all of the macromolecular components that make up the cell. The

bacterium can grow in the presence or absence of O2. Under

anaerobic conditions it will grow by means of fermentation,

producing characteristic "mixed acids and gas" as end products.

However, it can also grow by means of anaerobic respiration,

since it is able to utilize NO3, NO2 or fumigate as final

electron acceptors for respiratory electron transport processes.

In part, this adapts E. coli to its intestinal (anaerobic) and

its extra intestinal (aerobic or anaerobic) habitats (Todar,

2008).E. coli can respond to environmental signals such as



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chemicals, pH, temperature, osmolarity, etc., in a number of

very remarkable ways considering it is a unicellular organism.

For example, it can sense the presence or absence of chemicals

and gases in its environment and swim towards or away from them.

Or it can stop swimming and grow fimbriae that will specifically

attach it to a cell or surface receptor. In response to change

in temperature and osmolarity, it can vary the pore diameter of

its outer membrane poring to accommodate larger molecules

(nutrients) or to exclude inhibitory substances. With its

complex mechanisms for regulation of metabolism the bacterium

can survey the chemical contents in its environment in advance

of synthesizing any enzymes that metabolize these compounds. It

does not wastefully produce enzymes for degradation of carbon

sources unless they are available, and it does not produce

enzymes for synthesis of metabolites if they are available as

nutrients in the environment.

Pathogenecity. According to Brooks et al. (2010),

Escherichia coli is the most common cause of urinary tract

infection and accounts approximately 90% of first urinary tract

infection in young women. The symptoms and signs include urinary

frequency, dysuria, hematuria, and pyuria. Flank pains are



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associated with upper tract infection. None of these symptoms or

signs is specific for E. coli infection. Urinary tract infection

can result in bacteremia with clinical signs of sepsis. Most of

the urinary tract infections that is involve the bladder or

kidney in an otherwise healthy host are caused by a small number

of O antigen types that have specifically elaborated virulence

factors that facilitate colonization and subsequent clinical

infections. These organisms are designated as uropathogenic E.

coli. Typically these organisms produce hemolysin, which is

cytotoxic and facilitates tissue invasion. Those strains that

cause pyelonephritis express K antigen and elaborate a specific

type of pilus, P fimbriae, which binds to the P blood group

antigen. However, the regular presence of E. coli in the human

intestine and feces has led to tracking the bacterium in nature

as an indicator of fecal pollution and water contamination. As

such, it is taken to mean that, wherever E. coli is found, there

may be fecal contamination by intestinal parasites of humans.

The commensally E. coli strains that inhabit the large intestine

of all humans and warm-blooded animals comprise no more than 1%

of the total bacterial biomass. The E. coli flora is apparently

in constant flux. One study on the distribution of different E.

coli strains colonizing the large intestine of women during a



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one year period (in a hospital setting) showed that 52.1%

yielded one serotype, 34.9% yielded two, 4.4% yielded three, and

0.6% yielded four. The most likely source of new serotypes

of E. coli is acquisition by the oral route. To study oral

acquisition, the carriage rate of E. coli carrying antibiotic-

resistance plasmids (R factors) was examined among vegetarians,

babies, and non vegetarians. It was assumed that non vegetarians

might carry more E. coli with R factors due to their presumed

high incidence in animals treated with growth-promoting

antimicrobial agents. However, omnivores had no higher an

incidence of R-factor-containing E. coli than vegetarians, and

babies had more resistant E. coli in their feces than non

vegetarians (Todar,2008). No suitable explanation could be

offered for these findings. Besides, investigation of the

microbial flora of the uninhabited Krakatau archipelago has

shown the presence of antibiotic-resistant E. coli associated

with plants. The bottom line seems to be that most of us have

more than one strain of E. coli in our gut, and intestinal

strains tend to displace one another about three or four times a

year (Todar,2008).



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Soil

According to Alanis (2005) serious infections caused by

bacteria that have become resistant to commonly used antibiotics

have become a major global healthcare problem in the 21st

century. Staphylococcus aureus, for instance, a virulent

pathogen that is responsible for a wide range of infections

including pimples, pneumonia, osteomyelitis, endocarditis and

bacteremia, has developed resistance to most classes of

antibiotics. For more than two decades, clinicians and public

health officials have faced hospital acquired methicillin-

resistant Staphylococcus aureus (MRSA), which also bears

resistance too many antibiotics. During much of this time,

vancomycin has been the therapeutic answer to MRSA, but that

paradigm has changed. Vancomycin resistant strains have emerged

clinically. Vancomycin-resistant S. aureus (VRSA) challenges

clinicians, not only because of vancomycin and methicillin

resistance, but also because of resistance to many other

antibiotics, including aminoglycosides, macrolides, and

fluoroquinolones. Fortunately, newer therapeutic agents,

daptomycin, linezolid, and a streptogramin combination

(quinupristin/dalfopristin) have entered the clinical arena in

the past few years. However, certain undesirable side effects



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and the spread of pathogens with this new antimicrobial drug

resistance emphasize the need for the development of other newer

antimicrobial agents with activity against such Gram positive

bacteria. Another cause of great concern is the Gram negative

antibiotic-resistant opportunistic pathogens. Gram negative

environmental and enteric organisms currently threaten patients

in hospitals and communities with multi-drug resistance,

including broad resistance to first, second, and third

generations of penicillin's and cephalosporin's. These bacteria,

like Pseudomonas aeruginosa, are common environmental organisms,

which act as opportunistic pathogens in clinical cases where the

defense system of the patient is compromised (Lyczak, Cannon and

Pier, 2000). In addition, other intrinsically antibiotic

resistant organisms such as Stenotrophomonas maltophilia

(Saiman, Chen, Gabriel & Knirsch, 2002) are emerging as

opportunistic pathogens. The end result of this phenomenon is

that many strains of bacteria have become resistant, and in many

cases multi-resistant to these therapeutic agents, thus

rendering these drugs ineffective as treatments of choice for

severe infections caused by these pathogens (Saiman et. al.,

2002). Rising numbers of antibiotic unresponsive infectious

disease agents confront patients worldwide and consensus has



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emerged that it is essential that novel antibiotic classes be

developed as part of the strategy to control the emerging drug-

resistant pathogens. In response, there is a renewed interest in

discovering novel classes of antibiotics that have different

mechanisms of action. Search for new antibiotics effective

against multi-drug resistant pathogenic bacteria is presently an

important area of antibiotic research. Natural products having

novel structures have been observed to possess useful biological

activities. Soil is a natural reservoir for microorganisms and

their antimicrobial products. Filamentous soil bacteria

belonging to the genus Streptomyces are widely recognized as

industrially important microorganisms because of their ability

to produce many kinds of novel secondary metabolites including

antibiotics. Of all known drugs 70% have been isolated from

Actinomycetes bacteria of which 75% and 60% are used in medicine

and agriculture respectively. Indeed, different Streptomyces

species produce about 75% of commercially and medically useful

antibiotics (Saiman et. al., 2002). They have provided more than

half of the naturally occurring antibiotics discovered to date

and continue to be screened for useful compounds. In the course

of screening for new antibiotics, several studies are oriented

towards isolation of Streptomyces from different habitats.



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Presently, there is little documented information of the

occurrence of Streptomyces spp. in the soil of Turkey with a

potential to produce antimicrobial compounds. In the present

study, the isolation and characterization as well as the

inhibitory effects of local Streptomycete isolates tested

against various multiple antibiotic resistant bacteria and yeast

were reported, along with some chemical properties of secondary

metabolites with high biological activities.

According to Prescott, Harley and Klein (2008) soil

scientists would describe soil as weathered rock combined with

organic matter and nutrients. An agronomist would point out that

soil supports plant life. However, a microbial ecologists knows

that the formation of organic matter and the growth of plants

depend on the microbial community within the soil. Historically,

the complexity of the soil as a habitat has been a challenge to

understanding soil microbial ecology. Soil is very dynamic and

is formed in a wide variety of environments. These environments

range from Arctic tundra regions, where approximately 11% of the

Earth’s soil carbon pool is stored, to Antarctic dry valleys,

where there are no vascular plants. In addition, deeper

subsurface zones, where plant roots and their product cannot

penetrate, also have surprisingly large microbial communities



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(Prescott et al., 2008). Microbial activities in these

environments can lead to the formation of minerals such as

dolomite, microbial activity also occurs in deep continental oil

reservoirs, in stones, and even in rocky outcrops. These

microbes are dependent on energy sources from photosynthetic

protists and nutrients in rainfall and dust. Most soils are

dominated by inorganic geological materials, which modified by

the biotic community, including microorganisms and plants, to

form soils. The spaces between soil particles are critical for

movement of water and gases. Total pore space, and thus gas

diffusion, is determined by the texture of the soil (Prescott

et. al., 2008).

Barangay Sinapsapan, Jordan, Guimaras

According to Eckerwall (Personal Communication, October 10,

2013) barangay Sinapsapan is located at Jordan, Guimaras Island,

on the lands bordering to Panay Gulf with approximate coordinates

of N 010 35’.00 E 122 30’.47.

The distance from the sea is about 270 meters at an elevation

of about 30 meters above sea level.

The terrain is a typical young coral island’s with the

land rising rapidly to about 30-40 meters elevation when

proceeding inland from the coastland some 100 meters. Going



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further inland elevation varies up and down forming valleys and

ridges.

According to Española (Personal Communication, October 10,

2013) many areas of the barangay are still untouched and

unexplored due to the accessibility. The barangay is composed of

different soil colors. It varies from dark brown and reddish-

yellowish brown. The texture of the soil is mostly coarse. With

regards to its structure, the arrangement of the soil particles is

in different aggregates. Soil granules clump or bind together.

According to Eckerwall (Personal Communication, October 10,

2013) in the valleys some 10 cm or topsoil or even more can be

found but the ridges offers only barren land with sharp

coral rocks and little soil between , where mainly fruit and

corn is planted. The spontaneous growth is bushes and a few

larger trees. The lots of holes and canals inside the Coral

Rocks give the roots a possibility to stretch very deep and

reach water, often salty. The proof of this statement is that so

much growth still survives in long periods of drought. The soil

quality is influenced by the strong winds with high salt content

blowing in from SW during Habagat.



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Chapter 3

Research Design and Methodology

This chapter consists of Three (3) parts: Part One –

Purpose of the Study and Research Design, Part Two – Method, and

Part Three – Data Analysis Procedure.

Part One, Purpose of the Study and Research Design,

elucidates on the purpose of the study and describes the

research design.

Part Two, Method, the instrument used, and the procedures

employed in gathering the data pertinent to the study.

Part Three, Statistical Data Analyses, explains the

statistical tools employed in the treatment of data and the

quantitative part which includes the measurement of the zone of

inhibition.



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Purpose of the Study and Research Design

The study employed both the experimental and descriptive

method of investigation. The main purpose of the study is to

screen and determine the antibacterial effects of soil bacterial

isolates from the three sampling sites of Brgy. Sinapsapan,

Jordan, Guimaras against S. aureus and E. coli. The independent

variables are the bacterial isolates from Brgy. Sinapsapan,

Guimaras in the high, intermediate, and lowareas. The dependent

variable is the zone of inhibitions exhibited by S. aureus and

E. Coli.

Materials

The research was conducted at the Central Science Laboratory

room at Rizal Hall of the Biological Science Department of West

Visayas State University, La Paz, Iloilo City.

Equipment and Apparatus

The following equipment and apparatuses were used: autoclave

machine, incubator, beakers, Erlenmeyer flask, culture tubes,

Pipette, test tubes, Petri dish and wire loop.



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Chemicals used

The following chemicals were prepared and used for this

study: Nutrient broth, Nutrient Agar, McFarland 0.5, Normal Saline

Solution, mineral oil, safranin, gram’s iodine, crystal violet,

Fluconazole and 70% Alcohol (as disinfectant).

Procedures

Sterilization of Materials. The glassware were washed,

dried and wrapped with a newspaper. They were placed inside the

autoclave at 15 psi for 15-20 minutes at 121°C. After

sterilization, they were dried in a drying oven. The materials

were stored for later use.

Sampling Site Description. The sampling sites are located

in Barangay Sinapsapan, Jordan, Guimaras Island, on the lands

bordering to Panay Gulf with approximate coordinates of N 010

35’.00 E 122 30’.47. The distance from the sea is about 270

meters at an elevation of about 30 meters above sea level

(Eckerwall, Personal Communication, October 10, 2013).

Preparation of Nutrient Agar Plates. In one liter of

distilled water, 23 grams of nutrient agar was dissolved and

thoroughly mixed in an Erlenmeyer flask. After complete

dissolution of the media, it was autoclaved for 15 minutes at



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121 0 C at 15psi.The culture media was allowed to cool and

aseptically transferred to sterile petri dish containing 10 ml

each. All NA plates were allowed to solidify and was

refrigerated for future use.

Preparation of Diluents. In one liter of distilled water, 9

grams of sodium chloride were completely dissolved in an

Erlenmeyer flask. After complete dissolution of salt, it was

autoclaved for 15 minutes at 121 0C at 15 psi. The solution was

allowed to cool and aseptically transferred to a 25 ml culture

tubes containing 9 ml each. All test tubes containing the

solution were tightly sealed and kept refrigerated for further

use.

Preparation of Nutrient Broth. In one liter of nutrient

broth solution, 13 grams of nutrient broth were dissolved and

thoroughly mixed in an Erlenmeyer flask. After complete

dissolution of the media, it was autoclaved for 15 minutes at

121 0 C at 15 psi. The culture media was allowed to cool and

aseptically transferred to sterile culture tubes containing 5 ml

each. All culture tubes were allowed to cool and kept

refrigerated for further use.

Soil Sample Collection. Twenty-seven soil samples were

collected from three sampling sites of Brgy. Sinapsapan, Jordan,



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Guimaras. The three sampling sites were based on there elevation

and labelled as high, intermediate and low. Each collection was

done from 10-15 cm depth of the soil. Approximately 10 g of soil

was scooped and placed into sterile plastic bags.

Isolation and culture condition. For each collected sample,

1g of the soil was suspended in 100 ml of normal saline

solution. It was incubated in an orbital shaker incubator at 280C

with shaking at 200 rpm for 30 min. Mixtures were allowed to

settle, and serial dilutions up to 10-6 were prepared using

sterile normal saline solution and were agitated normally. An

aliquot of 0.1 ml of each dilution specifically 10-2,10-4, and 10-

6 were taken and spread evenly over the surface of nutrient agar

medium. The medium was encircled with Fluconazole (75 mg/ml) to

inhibit fungal contamination. Plates were incubated at 350C, and

were monitored after 24 hours. Repeated streaking on NA agar

plates of isolated colonies was done to purify bacterial

colonies and then incubated for 18-24 hours. The procedure was

repeated three times to ensure purification and obtain well-

isolated bacterial colonies. The purified colonies were

described based on colony characteristics on agar media as seen

with the naked eye as described by Harley (2005). These were

described according to form, elevation, margin, pigmentation or



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color, appearance, optical property and texture. The isolated

strains were preserved at 40C overlaid with sterile mineral oil

for further use and were maintained for longer period by serial

subculture. Isolates with antibacterial properties in the

different sampling sites were provided with code as H1 to H6 for

high sampling site bacterial isolates, I1 to I6 for intermediate

sampling site bacterial isolates, and L1 to L6 for low sampling

site bacterial isolates.

Test organisms. The test organisms used in this study were

the gram-positive bacterium, Staphylococcus aureus BIOTECH 1582

and the gram-negative bacterium, Escherichia coli BIOTECH 1634.

All test bacteria were purchased from the Philippine National

Collection of Microorganisms (PNCM), University of the

Philippines Los Baños (UPLB) Biotech in Laguna. A letter for

requisition of purchase of the said bacteria was done ahead

prior to the microbial assay. Upon receiving the said culture of

bacteria, they were overlaid with sterile mineral oil and were

furthered sub-cultured for further use.

Broth Culture of Test Isolates and Test Pathogens. All sub-

cultured test pathogens and characterized test isolates were

inoculated using a sterile wire loop (loopful of each bacterium)

in each prepared broth culture using aseptic technique.



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Inoculation was done twice to ensure growth of bacterial culture

in broth solution. All bacterial broth cultures were incubated

at 370C for 24 hours.

Preparation of 0.5 McFarland Standard. The following

solutions were mixed, 0.5 ml of 0.048 M BaCl2 to 99.5 ml of 0.036

N H2SO4.About 5 ml of the solution of the same proportions as

those used in preparing the culture suspension were transferred

in a 25 ml screw-cap tube. The culture tube were tightly sealed

and stored in the dark at room temperature. Prior to use, the

turbidity standard was shaken thoroughly. The Mc Farland

standard was used to adjust the turbidity of the inoculum prior

to microbial assay. The turbidity standard may contain

approximately 1.5 x 108 CFU/ml of the cultured bacteria (Quinto &

Santos, 2005).

Preparation of Antibiotic Solution. An antibiotic

ciprofloxacin was purchased from a local drug store. The

preparation of the solution was based on the indicated

concentration i.e. 50 mg/ml (Allen & Ansel, 2013), wherein 500

mg of the antibiotic tablet was pulverized and dissolved in 10

ml sterile distilled water.

In vitro screening of Soil Bacterial Isolates for Anti-

bacterial Activity. Morphologically distinct colony of bacterial



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isolates was subjected for anti-bacterial activity screening

against the test organisms using the modified agar disk

diffusion method on agar medium. In this method, pure bacterial

isolate was inoculated in an agar disk with a thickness of 2mm

and a diameter of 10 mm on a plate of screening media spread

plated with the test pathogen. The plates were incubated for 24

h at 37 0C. Antagonism was measured by the size of the inhibition

zone (Madigan et al., 1997). The presence of zone of inhibition

of the test bacteria were derived using a formula and evaluated

according to the observed and corresponding modified inferences

from Quinto and Santos (2005):

𝑍𝑜𝑛𝑒 𝑜𝑓 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 =(𝑋 − 𝑌)

𝑍

Where

X = diameter of the petri dish used (90 mm)

Y = diameter of the punched hole on the agar plate (10 mm)

Z = number of descriptive scale used (4)



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Table 1. Descriptive Inferences of the Zone of Inhibition

Measurement Description

0-20 mm inactive;

21-40 mm partially active

41-60 mm Active

61-80 mm very active

Each bacterial isolate was tested on the test organism in

three replicates and in three trials. Zone of inhibition was

recorded every after 24 hours for three days.

Gram staining of Bacterial Isolates with Antibacterial

Properties. A smear of culture was taken in a clean glass slide

and was heated gently over a flame. The smear was covered with a

thin film of crystal violet for 1 min and was washed gently in

slow running tap water. Gram’s iodine solution was flooded over

the smear for 1 min and was washed with tap water. Alcohol was

used to decolorize the smear until the violet color ceased to

flow away. The slide was washed with water and counter stained

with safranin. The stain was flooded over the smear for 2 min,

then the slide was washed, drained, air dried, and viewed under

microscope. The culture retaining the violet color indicates a



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gram-positive organism. However, the culture with pink color

indicates a gram-negative organism.

Data Collection Procedure. The data were collected based on the

presence of zone of inhibition of bacterial isolates on S.

aureus and E. coli. The zone of inhibition was measured with a

ruler in millimeter units. Each bacterial isolate was tested in

three replicates and in three trials. The measurement of the

zone of inhibition on the test bacteria was noted.

Descriptive Statistics. The mean zone of inhibition on each

test bacterium was tabulated. Furthermore, the zone of

inhibition of each bacterial isolate was evaluated with the aid

of a descriptive scale on the zone of inhibition modified from

Quinto and Santos (2005).

Inferential Statistics. Repeated measures of Analysis of

Variance (rANOVA) was used to compare the average zone of

inhibition of the different bacterial isolates against E. coli

BIOTECH 1634 and S. aureus BIOTECH 1582over a seventy-two hours

incubation period.



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Chapter 4

Results and Discussion

Chapter Four has three parts: (1) Descriptive Data

Analysis, describes the results shown in the experiment; (2)

Inferential Data Analysis, shows the level of significance based

on the result; and (3) Discussion, discusses the reason behind

the effectiveness of the different bacterial isolates against

the test bacteria.

Descriptive Data Analysis

Bacterial Isolates in the Three Sampling Sites of Barangay

Sinapsapan, Jordan, Guimaras. Table 2 shows that there were six

bacterial isolates obtained from the three sampling sites of

Barangay Sinapsapan, Jordan, Guimaras.



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Table 2

Bacterial isolates obtained in the three sampling sites of Brgy.

Sinapsapan, Jordan, Guimaras.

Sampling Site Number of Isolates

High Area 6

Intermediate Area 6

Low Area 6

Characteristics of Bacterial Isolates in the Different

Sampling Sites in Barangay Sinapsapan, Jordan, Guimaras. Table 3

shows the characteristics of the six bacterial isolates in each

sampling site as characterized according to morphology which

include shape, elevation, margin, color, pigmentation, and

texture, cell arrangement, and Gram staining reaction. This is

further supported with the physical appearances of the colonies

and cell morphology in Appendices B, C, and D.



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Table 3

Characteristics of Bacterial Isolates in the High, Intermediate

and Low Area Sampling Sites of Brgy. Sinapsapan, Jordan,Guimaras

Isolate Number Sampling site Colony Characteristics Cell Shape & Cell

Arrangement

Gram Staining

Reaction

H1 High Irregular; Flat; Undulate; Dull;

Opaque; Non- pigmented; Smooth Bacilli occur singly Gram negative (-)

H2 High Circular; Flat; Entire; Dull; Opaque;

Non- pigmented; Smooth Cocci occur singly Gram negative (-)

H3 High

Punctiform; Flat; Entire; Dull;

Transluscent; Non- pigmented;

Smooth

Bacilli in clusters Gram positive (+)

H4 High

Rhizoid; Flat; Filamentous; Dull;


Smooth

Cocci in clusters Gram positive (+)

H5 High Circular; Flat; Entire; Dull; Opaque;

Yellow; Smooth Cocci in chain Gram positive (+)

H6 High Circular; Flat; Entire; Dull; Opaque,

Orange; Smooth Cocci in clusters Gram positive (+)

I1 Intermediate

Rhizoid; Flat;Filamentous; Dull;


Smooth

Diplobacilli Gram positive (+)

I2 Intermediate Circular; Flat; Entire; Dull; Opaque;

Non- pigmented; Smooth

Cocci occur singly Gram negative (-)

I3 Intermediate



Smooth

Cocci in clusters Gram negative (-)

I4 Intermediate Irregular; Flat; Undulate; Dull;

Opaque; Non- pigmented; Smooth Cocci in clusters Gram positive (+)

I5 Intermediate

Filamentous; Flat; Filamentous;

Dull; Opaque; Non- pigmented;

Smooth


I6 Intermediate Circular; Flat; Entire; Dull; Opaque;

Yellow; Smooth Cocci occur singly Gram negative (-)

L1 Low

Rhizoid; Flat;Filamentous;

Dull;Opaque; Non- pigmented;

Smooth

Bacilli occur singly Gram positive (+)

L2 Low

Filamentous; Flat; Filamentous;

Dull; Opaque; Non- pigmented;

Smooth


L3 Low



Smooth

Cocci in clusters Gram negative (-)

L4 Low

Irregular; Flat; Undulate; Dull;


Smooth

Bacilli occur singly Gram positive (+)

L5 Low Circular; Flat; Entire; Dull; Opaque;

Yellow; Smooth Cocci occur singly Gram negative (-)

L6 Low Circular; Flat; Entire; Dull; Opaque;

Orange; Smooth Cocci occur singly Gram negative (-)



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Zone of Inhibition of Bacterial Isolates in the Three

Sampling Sites Against Staphylococcus aureus BIOTECH 1582.

Figure 2 shows the mean zone of inhibition of highland soil

bacterial isolates against S. aureus BIOTECH 1582 after 24, 48

and 72 hours of incubation. H4 (M=25.89mm, SD=6.41)and H1

(M=34.22mm, SD=5.91; M=35.33mm, SD=4.97) have the highest zone

of inhibitions and described as partially active after 24, 48

and 72 hours of incubation among the isolates, respectively.

However the positive control antibiotic, ciprofloxacin has the

highest zone of inhibition after 24, 48 and 72 hours (M=28.55

mm, SD=10.33; M=49.67 mm, SD= 9.12; 54.67 mm, SD=10.78),

respectively.



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Figure 2. Zone of inhibition of highland soil bacterial isolates

against S. aureus BIOTECH 1582

Figure 3 shows the mean zone of inhibition of intermediate

land soil bacterial isolates against S. aureus BIOTECH 1582

after 24, 48 and 72 hours of incubation. I5 (M=28.67mm,

SD=10.31) and I2 (M=33.34mm, SD=4.21; M=29.44mm, SD=3.91) have

the highest zone of inhibitions and described as partially

active after 24, 48, and 72 hours of incubation among the

isolates, respectively. However, the positive control antibiotic

ciprofloxacin has the highest zone of inhibition after 24, 48

0

10

20

30

40

50

60

24 hours 48 hours 72 hours

isolate 1

isolate 2

isolate 3

isolate 4

isolate 5

isolate 6

Antibiotic

Negative Control

Inactive

Partially Active

Active



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and 72 hours (M=28.55 mm, SD=10.33; M=49.67 mm, SD=9.12; 54.67

mm, SD=10.78), respectively.

Figure 3. Zone of inhibition of intermediate land soil bacterial

isolates against S. aureus BIOTECH 1582

Figure 4 shows the mean zone of inhibition of lowland soil

bacterial isolates against S. aureus BIOTECH 1582 after 24, 48

and 72 hours of incubation. L4 (M=25.33, SD=2.92) and L1 (M=29,

SD=3.04; M=38, SD=5.79) have the highest zone of inhibitions and

described as partially active after 24, 48, and 72 hours of

incubation among the bacterial isolates, respectively. However

the positive control antibiotic ciprofloxacin has the highest

0

10

20

30

40

50

60


isolate 1

isolate 2

isolate 3

isolate 4

isolate 5

isolate 6

Antibiotic

NegativeControl

Inactive

Partially Active

Active



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zone of inhibition after 24, 48 and 72 hours (M=28.55 mm,

SD=10.33 M=49.67 mm, SD=9.12; 54.67 mm, SD=10.78), respectively.

Figure 4. Zone of inhibition of low land soil bacterial

isolates against S. aureus BIOTECH 1582

Zone of Inhibition of Bacterial Isolates in the Three

Sampling Sites Against Escherichia coli BIOTECH 1634. Figure 5

shows the mean zone of inhibition of highland soil bacterial

isolates against E. coli BIOTECH 1634 after 24, 48 and 72 hours

of incubation. H2 (M=20.11mm, SD=9.37)and H4 (M=26.22mm,

SD=3.67; M=34.22mm, SD=5.29) have the highest zones of

inhibitions and described as partially active after 24, 48, and

0

10

20

30

40

50

60


isolate 1

isolate 2

isolate 3

isolate 4

isolate 5

isolate 6

Antibiotic

NegativeControl

Inactive

Patially Active

Active



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72 hours of incubation among the isolates, respectively.

However, positive control antibiotic has the highest zone of

inhibition after 24, 48 and 72 hours of incubation (M=22.33 mm,

SD=3.39; 30.33 mm, SD=6.84; 36.65 mm, SD=5.66), respectively.

Figure 5. Zone of inhibition of highland soil bacterial isolates

against E. coli BIOTECH 1634

Figure 6 shows the mean zone of inhibition of intermediate

land soil bacterial isolates against E. coli BIOTECH 1634 after

24, 48 and 72 hours of incubation. I4 (M=19.44mm, SD=4.00), I1

(M=27mm, SD=3.50), and I6 (M=32.78mm, SD=6.65) have the highest

zones of inhibition and described as partially active after 24,

0

5

10

15

20

25

30

35

40


isolate 1

isolate 2

isolate 3

isolate 4

isolate 5

isolate 6

Antibiotic

NegativeControl

Inactive

Partially Active



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48, and 72 hours of incubation, respectively. However, positive

control antibiotic has the highest zone of inhibition after 24,

48 and 72 hours of incubation (M=22.33 mm, SD=3.39; 30.33 mm,

SD=6.84; 36.67mm, SD=5.66), respectively.

Figure 6. Zone of inhibition of intermediate land soil bacterial

isolates against E. coli BIOTECH 1634

Figure 7 shows the mean zone of inhibition of lowland soil

bacterial isolates against E. coli BIOTECH 1634 after 24, 48 and

72 hours of incubation. L1 (M=23mm, SD=10.77), L4 (M= 30.89mm,

SD=4.88), and L5 (M=34.78mm, SD=3.60) have the highest zones of

0

5

10

15

20

25

30

35

40


isolate 1

isolate 2

isolate 3

isolate 4

isolate 5

isolate 6

Antibiotic

Negative Control

Inactive

Partially Active



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inhibitions and described as partially active after 24, 48 and

72 hours of incubation, respectively. However, positive control

antibiotic has the highest zone of inhibition after 72 hours of

incubation (M=36.67 mm, SD=5.66).

Figure 7. Zone of inhibition of lowland soil bacterial isolates

against E. coli BIOTECH 1634

0

5

10

15

20

25

30

35

40


isolate 1

isolate 2

isolate 3

isolate 4

isolate 5

isolate 6

Antibiotic

Negative Control

InactiveInactive

Partially Active



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Inferential Data Analysis

Zone of Inhibition of Bacterial Isolates in the Different

Sampling Sites against Staphylococcus aureus BIOTECH 1582. Table

4 shows a no significant difference in the analysis of variance

with repeated measures for the zone of inhibition of bacterial

isolates in the different sampling sites against Staphylococcus

aureus BIOTECH 1582 after 72 hours of incubation, p < 0.05. This

may indicate that different soil bacterial isolates may have

comparable effects with the antibiotic ciprofloxacin.

Furthermore, this is supported by the Least Significant

Difference for pair wise comparison as indicated in Appendix F.

There were significant differences among the following: all

isolates and the antibiotic ciprofloxacin; and between isolate 6

and isolates 3, 4, and 5, p < 0.05.



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Table 4

Analysis of Variance with Repeated Measures for the Zone of

Inhibition of Bacterial Isolates in the Different Sampling Sites

against Staphylococcus aureus BIOTECH 1582.

Effect Value F Hypothesi

s df

Error

df

Signifi

cance

Partial

Eta

Squared

Time*Isolate

Wilk’s

lambda

0.825 1.868 4 74 0.125 0.092

P > 0.05 is not significant.

Zone of Inhibition of Bacterial Isolates in the Different

Sampling Sites against Escherichia coli BIOTECH 1634. Table 5

shows a significant difference in the analysis of variance with

repeated measures for the zone of inhibition and time of

incubation of bacterial isolates in the different sampling sites

against Escherichia coli BIOTECH 1634 after 72 hours of

incubation, p < 0.05. This may indicate that soil bacterial

isolates have varying effects on E. coli as indicated in Figure

7. Furthermore, this is supported by the Least Significant

Difference for pair wise comparison as indicated in Appendix G.

There were significant differences among the following: all



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isolates and the antibiotic ciprofloxacin; isolates 3 and 6;

isolates 4 and 5, and isolates 4 and 6, p < 0.05.

Table 5

Analysis of Variance with Repeated Measures for the Zone of

Inhibition of Bacterial Isolates in the Different Sampling Sites

against Escherichia coli BIOTECH 1634.

Effect Wilk’s

lambda

Value F Hypothes

is df

Error

df

Signifi

cance

Partial

Eta

Squared

Time*Isola

te

Wilk’s

lambda

0.681 3.915 4 74 0.006* 0.175

*P < 0.05 is significant.



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Discussion

A total of 18 bacterial isolates in the 3 sampling sites

with variable colony and cell characteristics in each sampling

site were collected. The bacterial isolates are all flat, dull,

non-pigmented and smooth. I1 is a diplobacilli while the rest

are either clustered or occur singly. Amongst the different

microorganisms inhabiting in the soil, bacteria are the most

abundant and predominant organisms (Sylvia, Fuhrmann, Hartel, &

Zuberer, 2005). Sylvia et al. (2005) added that morphologically,

soil bacteria are divided into three groups viz Cocci

(round/spherical), (rod-shaped) and Spirilla/ Spirillum (cells

with long wavy chains). Bacilli are most numerous followed by

Cocci and Spirilla in soil. The major soil factors which

influence the microbial population, distribution and their

activity in the soil are soil fertility, cultural practices,

soil moisture, soil temperature, soil aeration, light, soil pH,

organic matter, food and energy supply, nature of soil, and

microbial associations (Stephanie, Breznak, & Schmidt, 2007).

All these factors play a great role in determining not only the

number and type of organism but also their activities.

Fertilizers and manures applied to the soil for increased crop

production, supply food and nutrition not only to the crops but



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also to microorganisms in soil and thereby proliferate the

activity of microbes. Optimum soil moisture (range 20 to 60 %)

must be there for better population and activity of microbes in

soil. Seasonal changes in soil temperature affect microbial

population and their activity. The organic matter in soil being

the chief source of energy and food for most of the soil

organisms, it has great influence on the microbial population.

Organic matter influence directly or indirectly on the

population and activity of soil microorganisms. It influences

the structure and texture of soil and thereby activity of the

microorganisms. Ecological relationships among soil organisms

are influenced by soil structure. Changes in resource locations

may allow for microorganisms to colonize another area (Sylvia,

Fuhrmann, Hartel, & Zuberer, 2005).

Bacteria are usually the most abundant group in soils in

terms of numbers. The genus Bacillus is very common in soil.

Bacillus, a Gram positive, aerobic or facultative endospore

forming motile bacteria belongs to family Bacillaceae (Claus and

Berkeley 1986). Isolates H3, L1, and L4 reflect the cellular

characteristics of the Genus Bacillus. Thus, it may be implied

that these isolates belong to this genus. Bacillus can resist

and survive in a variety of environmental stresses and adverse



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conditions and considered as very important microbiota due to

its diverse ecophysiology, direct and indirect functions such as

N2 fixer (Liu et al., 2006), denitrifiers (Suharti et al.,

2004), antibiotic (Bacon and Hinton, 2002), and phytohormones

(Araujo et al., 2005) producers.

In general, the soil bacterial isolates exhibited an

antimicrobial activity after 24 hours of incubation. Most of the

zone of inhibition became even larger after 48 and 72 hours of

incubation. It can then be said that this study is likewise

comparable to the study of Das et al. (2006) that the soil

bacterial isolates may be producers of novel bioactive

compounds. Various antimicrobial substances from soil bacterial

isolates have been isolated and characterized including

aminoglycosides, anthracyclines, glycopeptides, betalactams,

macrolides, nucleosides, peptides, polyenes, polyester,

polyketides, actionomycins, and tetracyclines (PrashithKekuda,

Shobha, Onkarappa, Goutham & Raghavendra, 2012). Most of the

antibiotics are extracellular metabolites which are normally

secreted in culture media and have been used as herbicides,

anticancer agents, drugs, immunoregulators and antiparasitic

drugs (Kekuda, Shobha, & Onkarappa, 2010). Actinomycetes are

gram-positive rods grow as filaments, branching rods, and



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diphtheroidal rods (Bowden, 1996). One of the soil bacterial

isolates, I1 has almost parallel characteristics to

Actinomycetes as gram-positive, filamentous and diplobacilli.

Thus, I1 could probably be an Actinomycete isolate.

Bacillus spp. are considered to be the safe microorganisms

that hold remarkable abilities for synthesizing a vast array of

beneficial substances that may have antibacterial property

(Stein, 2005). They can produce IAA, siderophore, phytase,

organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate

oxidase, and solubilized various sources of organic and

inorganic phosphates as well as potassium and zinc.

According to Sharma et al. (2001), the ability of

microorganisms to produce enzymes that may have antibacterial

properties is influenced by environmental conditions such as

temperature, pH, and presence of inductors or repressors.

According to Hoagstron (2011), a common statement of the

competitive exclusion principle is as follows: species whose use

of resources is very similar cannot live in the same place for

an extended period of time. That is, if two or more species eat

the same things, use the same hiding places, occupy the same

habitats, etc. one species will be more efficient than the

others and will fill the niche with its offspring leaving no

http://www.sciencedirect.com/science/article/pii/S0944501312000602#bib0140



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resources for other species. In this way, the more efficient

species will “competitively exclude” the less efficient species.



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Chapter 5

Summary, Conclusions, Implications, and Recommendations

Chapter Five consists of four parts: (1) Summary of the

Findings, (2) Conclusions, (3) Implications, and (4)

Recommendations.

Part One, Summary of the findings; state the salient points

of the study.

Part Two, Conclusions, presents the interferences drawn

from the results of the study.

Part Three, Implications, clarifies the relationships

between the present investigation and the theories presented in

relation to the antibacterial screening of soil bacterial

isolates from Sinapsapan, Jordan, Guimaras against Escherichia

coli and Staphylococcus aureus.

Part Four, Recommendations, offer some suggestions in terms

of the findings, conclusions, and recommendations.



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Summary of the Problem

This study was designed to screen and determine the

antibacterial effects of soil bacterial isolates in three

sampling sites of Brgy. Sinapsapan, Jordan, Guimaras against S.

aureus and E. coli.

This study sought to answer the following questions:

1. How many bacterial isolates are obtained in the three

sampling sites of Brgy. Sinapsapan, Jordan, Guimaras?

2. What are the characteristics (colonial morphology and

gram staining reaction) of bacterial isolates that have

antibacterial properties against S. aureus and E. coli?

3. What are the effects of the bacterial isolates on S.

aureus and E. coli after 72 hours of incubation?

4. Are there significant differences on the zone of

inhibition of bacterial isolates in the three sampling sites

against S. aureus and E. coli?

Unexplored and untouched site in Brgy. Sinapsapan, Jordan,

Guimaras was selected and the soil sampling was done in three

different areas namely: high, intermediate and low. Experimental

method of investigation was employed to screen the antibacterial

properties of the different bacterial isolates from the sampling

site. Isolation and purification of the bacterial isolates were



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done using serial dilution, spread plate method, and streak

plate method. Characterization was done based on colonial

morphology and Gram staining for cellular morphology and

staining properties. Agar disc diffusion method was used to

assess the antibacterial properties of the isolates against S.

aureus and E. coli after 24 to 72 hours of incubation. Each was

done in three replicates and three trials.

For the antibacterial activity, the zone of inhibition was

measured in millimeter using a 30cm ruler. The zone of

inhibition was analyzed using ANOVA with repeated measures

together with the positive and the negative controls.

Conclusions

Based on the experimental study, the results showed the

following:

1. There were 6 soil bacterial isolates obtained in the

high, intermediate, and low sampling sites in Brgy. Sinapsapan,

Jordan, Guimaras.

2. Based on the characterization of the bacterial isolates

in Brgy. Sinapsapan, Jordan, Guimaras, the shared



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characteristics of the isolates in the three sampling sites are

elevation, appearance, pigmentation, and texture wherein they

are all flat, dull, no pigmentation and smooth, respectively.

The only characteristic that differs is the arrangement of

isolate 1 in the intermediate area which is diplobacilli while

the rest are either clustered or occur singly. Isolates 1 and 5

were the most effective soil bacterial isolate against S. aureus

and E. coli, respectively. This is may be due to the fact that

they can degrade and inhibit bacterial growth due to their

secretion of digestive enzymes.

3. After 72 hours of incubation, all bacterial isolates

exhibited variations on their antibacterial effect against S.

aureus and E. coli. For S. aureus, the following isolates had

the widest zone of inhibitions after 72 hours of incubation: in

the highland sampling site, isolate 4; in the intermediate

sampling site, isolate 2; and lowland sampling site, isolate 1.

For E. coli, the following isolates had the widest zone of

inhibitions after 72 hours of incubation: in the highland

sampling site, isolate 4; in the intermediate sampling site,

isolate 6; and lowland sampling site, isolate 5.

4. There was a significant difference on the zone of

inhibition of the different bacterial isolates in the three



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sampling sites due to varying antibacterial effects of the

isolates against S. aureus and E. coli. This may be due to

differences on the amount of substrates produced by the

different bacterial isolates which inhibit the growth of the

test pathogens, S. aureus and E. coli.

Implications

This study was conducted to screen the antibacterial

properties of soil bacterial isolates from Sinapsapan, Jordan,

Guimaras against S. aureus and E. coli. The findings have shown

that different isolates are more effective on S. aureus than on

E. coli. It is therefore possible for the isolates to be

considered as a prospective source of antibiotic against on S.

aureus than E. coli. Searching for new possible source of

antibiotics is necessary because it becomes a manner for

bacteria to be resistant to certain antibiotics. Antibiotic

resistant bacteria are increasing nowadays in number that makes

the treatment for some diseases to be difficult.

Bacteria become resistant more quickly when antibiotics are

used improperly. Since there are kinds of bacteria that are



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resistant to antibiotics and are untreatable, there is a greater

must to search for more possible sources of antibiotics like

from unexplored soil sources in the nearby locality.

As the number of antibiotic resistant bacteria increases,

the quantity of antibiotics they are resistant to also rises.

According to Collignon (2000), these bacteria are known as

superbugs. They have adapted to resist multiple antibiotics

including the “last-line” drugs. Different strains of bacteria

have become superbugs, even the bodies’ normal micro flora such

as S. aureus and E. coli are on the list. Due to resistance of

these bacteria to combinations of antibiotics, infection caused

by these multi-resistant bacteria becomes unstoppable. Thus, it

is imperative to screen more and more bacteria from different

soil samples for antimicrobial activity in hope of getting some

bacterial strains that produce antibiotics that have not been

discovered yet and active against drug resistant pathogens.

Since there were six isolates that were obtained from the

sampling sites, it could therefore be implied that there are

more isolates that could be obtained from the area that have

antimicrobial properties since the area is vast and due to



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limited sampling sites and time of sampling, the researchers

were not able to maximize the soil sample collection.

Since most of the characteristics shared by the bacterial

isolates were almost all the same but one isolate (Isolate 1)

from the intermediate site which is diplobacilli, it can be

implied that there might be more other isolates that have

different characteristics which might be a more effective

isolate against E. coli and S. aureus. This may also implied

that further molecular characterization of the different

bacterial isolates to differentiate the properties among the

isolate.

Recommendations

Based on the conclusions of the study, the following are

recommended:

1. The study was limited only on the characterization of

the colonies and gram staining technique, it is further

recommended to do special staining methods such as flagellar

staining to determine the arrangement of the flagella, endospore

staining to determine whether the isolates are spore-former and



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the position of their spores, capsule staining to determine

whether the isolates are virulent or not, and negative staining

to reveal the morphology of the cell.

2. The study did not determine the genera or the species of

the isolates with antibacterial properties, it is recommended to

do biochemical tests to identify the genus of each isolate and

confirm using Bergey’s Manual of Systematic Bacteriology for the

identification of the isolate. Furthermore, molecular

characterization of the isolates to reveal the identity of each

species of the isolate is also suggested. Phylogenetic analysis

should be done to determine the group of each isolate. This will

also help to determine if the bacterial isolates that were used

in this study are newly discovered or not.

3. The study was limited only on the antibacterial

screening of soil bacterial isolates on S. aureus and E. coli.

It is recommended that other pathogenic bacterial strains and

fungi be tested against this bacterial isolates to determine

their range of effectivity.

4. For further study, other microbiologist are advised to

explore more on the site for more bacterial isolates screening



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that have potential antibacterial properties on pathogenic

bacteria.

5. Microbiologists whose interest is on antibiotic-

producing bacteria are suggested to search further for isolates

in the same location but in different area as the unexplored

area is still vast.

6. The study was limited only to the use of modified agar

disc diffusion method to test the antibacterial potential of the

different isolates. It is recommended to use other antimicrobial

assays or techniques in the confirmation of the antibacterial

properties of the said isolates.



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References

Alanis A.J. (2005), Resistance to Antibiotics: Are We in the

Post-Antibiotic Era? Archives Med Research ,36, 697-705.

Araujo, F.F., Henning, A.A. and Hungria, M. (2005).

Phytohormones and antibiotics produced by Bacillus

subtilis and their effects on seed pathogenic fungi and on

soybean root development. World J. Microb. Biot., 21:

1639-1645.

Bacon, C.W. and Hinton, D.M. (2002). Endophytic and biological

control potential of Bacillus mojavensis and related

species. Biol. Control., 23: 274-284.

Boirivant, M. and Strober, W. (2007). The mechanism of action of

probiotics. Retrieved from http://journals.1ww.com/co-

gastroenterology/Abstract/2007/11000/The_mechanism_of_actio

n_of_probiotics.16.aspx

Brooks, G., Carroll, K.C., Butel , J., Morse, S. and Mietzner,

T. (2010). Jawetz, Melnick & Adelberg’s Medical

Microbiology, 25th Edition, USA: McGraw Hill Companies.

Bowden, G.H.W. (1996). Actinomyces, Propionibacterium

propionicus, and Streptomyces. In: Baron S, editor. Medical

Microbiology. 4th edition. Galveston (TX): University of

Texas Medical Branch at Galveston; Chapter 34. Retrieved



Iloilo City

from: http://www.ncbi.nlm.nih.gov/books/NBK8385

Claus, D. and Berkeley, R.C.W. (1986). Genus Bacillus Cohn 1872,

174AL. In: Bergey’s Manual of Systematic Bacteriology, 2,

1105-1139.

Collignon, P. (2000). Millenium Bugs. Retrieved from:

http://www. abc.net.au.com.htm

Das, S., Lyla, P. S. and Ajmal Khan, S. (2006). Marine microbial

diversity and ecology: present status and future

perspectives. Current Science,90(10): 1325-1335.

Department of Health (2009). National health data dictionary

Retrieved from:http://uhmis1.doh.gov.ph/standards/sdetails.

php?data_element=BARANGAY

Dong H., Gao, S., Han, S. & Cao, S. (1999). “Purification and

characterization of a Pseudomonas sp. lipase and its

properties in non-aqueous media,” Biotechnology and Applied

Biochemistry, vol. 30, no. 3, pp. 251–256.

Dotan, I. and Rachmilewitz, D. (2005). Probiotics in

inflammatory bowel disease: possible mechanisms of action.

Retrieved from:http://journals.lww.com/co-gastroenterology/

Abstract/2005/07000/Probiotics_in_inflammatory_bowel_diseas

e__possible.7.aspx.

http://www.ncbi.nlm.nih.gov/books/NBK8385

http://www/

http://journals.lww.com/co-gastroenterology/%20Abstract/2005/07000/Probiotics_in_inflammatory_bowel_disease__possible.7.aspx





Iloilo City

Enright, M.C. (2003). Structure of the MecI repressor from

Staphylococcus aureus in complex with the cognate DNA

operator of mec Curr Opinion Pharmacol, 3, 5, 474- 479.

Greg R. and Percy W. (2005). Soil Bacteria. State of New

South Wales Department of Primary Industries.

Harley, J.P. (2005). Laboratory exercises in Microbiology,

(6thed.) Boston: McGraw Hill Higher Education.

Hoagstrom, C. (2011). Competitive exclusion principle. Retrieved

from: http://www.eoearth.org/view/article/151404

Ingham, E.R. (2011). Soil Biology. Retrieved from:

http://soils.usda.gov/sqi/concepts/soil_biology/soil_food_w

eb.html

Kekuda, T.R.P., Shobha, K.S., & Onkarappa, R.(2010). Fascinating

diversity and potent biological activities of Actinomycete

metabolites. Journal of Pharmacy Research 3(2): 250- 256.

Liu, X., Zhao, H. and Chen, S. (2006). Colonization of maize and

rice plants by strain Bacillus megaterium C4. Curr.

Microbio., 52: 186-190.

Lyczak, J.B., Cannon, C.L. & Pier G.B. (2000). Establishment of

Pseudomonas aeruginosa infection: lessons from a versatile

opportunist, Microbes and Infection 2, 9, 1051-1060.

http://soils.usda.gov/sqi/concepts/soil_biology/soil_food_web.html

http://soils.usda.gov/sqi/concepts/soil_biology/soil_food_web.html



Iloilo City

Madigan, M. T., J.M., Stahl, D.A. & Clark, D.P. (2012). Block

biology of microorganism, (13thed.) Boston: Benjamin

Cummings.

Martin, J.P., (1976). Soil Microbiology and Biochemistry.

SSSA; Madison, WI.

Mozo, M.V. (2008). Bacteria. Retrieved from: http://www. biology

-online.org/dictionary/Bacteria

Oskay, M., Tamer, A.U. & Azeri, C. (2004). Isolation of soil

Streptomyces as source antibiotics active against

Antibiotic-resistant bacteria. African J Biotechnol, 3

(9), 441-446.

O’Toole, G.H., Kaplan, H.B., & Kolter, R. (2000).

Biofilm formation as microbial development. Retrieved

from: http://medicalmycology.org/biofilms.htm.

Pela´ ez, F. (2006). The historical delivery of

antibiotics from microbial natural products—Can

history repeat?. Biochemical Pharmacology, 71, 981-

990.

Pommier, Y., Leo, E., Zhang, H., Marchand, C. (2010). DNA

topoisomerases and their poisoning by anticancer and

antibacterial drugs" Chem. Biol 17: 421-433.

http://medicalmycology.org/biofilms.htm



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Prashith Kekuda, T.R.,Shobha, K.S., Onkarappa, R., Goutham,

S.A., Raghavendra, H.L. (2012). Screening biological

activities of a Streptomyces species isolated from soil of

Agumbe, Karnataka, India. Int. J. Drug Dev. & Res. 4 (3):

104-114.

Prescott, L.M., Harley, J.P. & Klein, D.A.(1993). Microbiology.

2nd ed. Wm. C. Brown Communications, Inc. USA.

Quinto, E. A. & Santos, M. A. G., (2005). A guidebook to

plant screening : Phytochemical and biological,

Microbiology section. España, Manila; University of

Santo Tomas Publishing House.

Reid, G. & Wong, P. (2005). Soil bacteria, State of New South

Wales Department of Primary Industries.

Roche F. (2003), The Staphylococcus aureus surface protein

SasG. Microbiology ,149, 2759–2767.

Saiman L., Chen, Y., Gabriel P.S., & Knirsch C. (2002).

Synergistic activities of macrolide antibiotic against

Pseudomonas aeruginosa, Burkholderia cepacia,

Stenotrophomonas maltophilia, and Alcaligenes

xylosoxidans isolated from patients with cystic

fibrosis. Antimicrobial Agents and Chemotherapy 46, 4,

1105-1107.



Iloilo City

Sharma, R., Chisti, Y., & Banerjee, U.C. (2001). “Production,

purification, characterization, and applications of

lipases,” Biotechnology Advances, vol. 19, no. 8, pp. 627–

662.

Stein, T. (2005). Bacillus subtilis antibiotics: structures,

syntheses and specific functions, MolMicrobiol, 56

(4)(2005), pp 1669-1679.

Stephanie, A., Breznak, E.J. A. & Schmidt, T. M. (2007).

Isolation and characterization of soil bacteria that

define ' Terriglobis ' gen. nov., in the Phyllum

'Acidobacteria' Appl. And Env. Microbiology Vol. 73, No. 8,

p.2708-2717.

Suharti, Heering, H.A. and de Vries, S. (2004). NO Reductase

from Bacillus azotoformans is a bifunctional enzyme

accepting electrons from menaquinol and a specific

endogenous membrane-bound cytochrome c551. Biochem.,

43: 13487-13495.

Sylvia, D. M., Fuhrmann, J. F., Hartel, P. G., & D.A Zuberer.

2005). Principles and applications of Soil Microbiology.

New Jersey, Pearson Education Inc.



Iloilo City

Talaro, K.P. (2008). Foundations in Microbiology, (7thed.)

Boston: Mc Graw Hill Co.

Todar, K. (2008). An Introduction to Bacteriology: University

of Wisconsin Inc.

Tortora, G.J., Funke, B.R., & Case, C.L. (2010). Microbiology:

an introduction, 10th ed. Pearson Education, Inc. USA.

Williams S.T., Sharmeemullah, M., Watson E.T., & Mayfield

C.I. (1972). Screening of Actinomycete Isolates from Niche

Habitats in Manipur for Antibiotic Activity. Retrieved

from: thescipub.com/pdf/10.3844/ajbbsp.2009.221.225.



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Appendix A

Sampling Site Map



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Appendix B

Characteristics of Bacterial Isolates in the High Area Sampling

Site of Brgy. Sinapsapan, Jordan Guimaras.

Figure 8.1. Colonial (left) and cell morphology

(right;600X magnification) of isolate 1. Figure 8.2. Colonial (left) and cell morphology (right;

600X magnification) of isolate 2.

Figure 8.3. Colonial (left) and cell morphology (right;

600X magnification) of isolate 3. Figure 8.4. Colonial (left) and cell morphology (right;







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Appendix C

Characteristics of Bacterial Isolates in the Intermediate Area

Sampling Site of Brgy. Sinapsapan, Jordan Guimaras.


600X magnification) of isolate 1. Figure 9.2. Colonial(left) and cell morphology (right;










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Appendix D

Characteristics of Bacterial Isolates in the Low Area Sampling

Site of Brgy. Sinapsapan, Jordan Guimaras.












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Appendix E

Antibacterial Data Sheet

Sampling Site: ____HIGH______ Test Pathogen:____E. coli______



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Sampling Site: ___INTERMEDIATE____ Test Pathogen:___E.coli______



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Sampling Site: __LOW______ Test Pathogen: ____E.coli____________



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Sampling Site: ___HIGH___ Test Pathogen: _____S. aureus________



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Sampling Site: _____INTERMEDIATE___ Test Pathogen: ___S. aureus_



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Sampling Site: _____LOW____ Test Pathogen: ___S. aureus_



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Appendix F

Least Significant Difference for Staphylococcus aureus Multiple

Comparisons

(I) Group (J) Group

Mean Difference

(I-J) Std. Error Sig.

95% Confidence Interval

Lower

Bound

Upper

Bound

Isolate 1 Isolate 2 -.2067 .91919 .823 -2.0675 1.6541

Isolate 3 -1.2574 .91919 .179 -3.1182 .6034

Isolate 4 -1.3422 .91919 .152 -3.2030 .5186

Isolate 5 -.5404 .91919 .560 -2.4012 1.3204

Isolate 6 1.6200 .91919 .086 -.2408 3.4808

Positive

Control -18.4530(*) 1.29993 .000 -21.0845 -15.8214

Isolate 2 Isolate 1 .2067 .91919 .823 -1.6541 2.0675

Isolate 3 -1.0507 .91919 .260 -2.9115 .8101

Isolate 4 -1.1356 .91919 .224 -2.9964 .7252

Isolate 5 -.3337 .91919 .719 -2.1945 1.5271

Isolate 6 1.8267 .91919 .054 -.0341 3.6875

Positive

Control -18.2463(*) 1.29993 .000 -20.8779 -15.6147

Isolate 3 Isolate 1 1.2574 .91919 .179 -.6034 3.1182

Isolate 2 1.0507 .91919 .260 -.8101 2.9115

Isolate 4 -.0848 .91919 .927 -1.9456 1.7760

Isolate 5 .7170 .91919 .440 -1.1438 2.5778

Isolate 6 2.8774(*) .91919 .003 1.0166 4.7382

Positive

Control -17.1956(*) 1.29993 .000 -19.8271 -14.5640

Isolate 4 Isolate 1 1.3422 .91919 .152 -.5186 3.2030

Isolate 2 1.1356 .91919 .224 -.7252 2.9964

Isolate 3 .0848 .91919 .927 -1.7760 1.9456

Isolate 5 .8019 .91919 .388 -1.0589 2.6627

Isolate 6 2.9622(*) .91919 .003 1.1014 4.8230

Positive

Control -17.1107(*) 1.29993 .000 -19.7423 -14.4792

Isolate 5 Isolate 1

.5404 .91919 .560 -1.3204 2.4012

Isolate 2 .3337 .91919 .719 -1.5271 2.1945

Isolate 3 -.7170 .91919 .440 -2.5778 1.1438

Isolate 4 -.8019 .91919 .388 -2.6627 1.0589

Isolate 6 2.1604(*) .91919 .024 .2996 4.0212

Positive

Control -17.9126(*) 1.29993 .000 -20.5442 -15.2810

Isolate 6 Isolate 1 -1.6200 .91919 .086 -3.4808 .2408

Isolate 2 -1.8267 .91919 .054 -3.6875 .0341

Isolate 3 -2.8774(*) .91919 .003 -4.7382 -1.0166

Isolate 4 -2.9622(*) .91919 .003 -4.8230 -1.1014

Isolate 5 -2.1604(*) .91919 .024 -4.0212 -.2996

Positive

Control -20.0730(*) 1.29993 .000 -22.7045 -17.4414

Positive

Control

Isolate 1 18.4530(*) 1.29993 .000 15.8214 21.0845

Isolate 2 18.2463(*) 1.29993 .000 15.6147 20.8779

Isolate 3 17.1956(*) 1.29993 .000 14.5640 19.8271

Isolate 4 17.1107(*) 1.29993 .000 14.4792 19.7423

Isolate 5 17.9126(*) 1.29993 .000 15.2810 20.5442

Isolate 6 20.0730(*) 1.29993 .000 17.4414 22.7045

Based on observed means.

* The mean difference is significant at the .05 level



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Appendix G

Least Significant Difference for Escherichia coli Multiple

Comparisons

(I) Group (J) Group

Mean

Difference

(I-J)

Std.

Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

Isolate 1 Isolate 2 .0863 .80336 .915 -1.5400 1.7126

isolate 3 -.7004 .80336 .389 -2.3267 .9260

isolate 4 -.7833 .80336 .336 -2.4097 .8430

isolate 5 .8626 .80336 .290 -.7637 2.4889

isolate 6 .9619 .80336 .239 -.6645 2.5882

Antibiotic -4.3559(*) 1.13613 .000 -6.6559 -2.0560

Isolate 2 Isolate 1 -.0863 .80336 .915 -1.7126 1.5400

isolate 3 -.7867 .80336 .334 -2.4130 .8397

isolate 4 -.8696 .80336 .286 -2.4960 .7567

isolate 5 .7763 .80336 .340 -.8500 2.4026

isolate 6 .8756 .80336 .283 -.7508 2.5019

Antibiotic -4.4422(*) 1.13613 .000 -6.7422 -2.1423

isolate 3 Isolate 1 .7004 .80336 .389 -.9260 2.3267

Isolate 2 .7867 .80336 .334 -.8397 2.4130

isolate 4 -.0830 .80336 .918 -1.7093 1.5434

isolate 5 1.5630 .80336 .059 -.0634 3.1893

isolate 6 1.6622(*) .80336 .045 .0359 3.2885

Antibiotic -3.6556(*) 1.13613 .003 -5.9555 -1.3556

isolate 4 Isolate 1 .7833 .80336 .336 -.8430 2.4097

Isolate 2 .8696 .80336 .286 -.7567 2.4960

isolate 3 .0830 .80336 .918 -1.5434 1.7093

isolate 5 1.6459(*) .80336 .047 .0196 3.2723

isolate 6 1.7452(*) .80336 .036 .1189 3.3715

Antibiotic -3.5726(*) 1.13613 .003 -5.8726 -1.2726

isolate 5 Isolate 1 -.8626 .80336 .290 -2.4889 .7637

Isolate 2 -.7763 .80336 .340 -2.4026 .8500

isolate 3 -1.5630 .80336 .059 -3.1893 .0634

isolate 4 -1.6459(*) .80336 .047 -3.2723 -.0196

isolate 6 .0993 .80336 .902 -1.5271 1.7256

Antibiotic -5.2185(*) 1.13613 .000 -7.5185 -2.9185

isolate 6 Isolate 1 -.9619 .80336 .239 -2.5882 .6645

Isolate 2 -.8756 .80336 .283 -2.5019 .7508

isolate 3 -1.6622(*) .80336 .045 -3.2885 -.0359

isolate 4 -1.7452(*) .80336 .036 -3.3715 -.1189

isolate 5 -.0993 .80336 .902 -1.7256 1.5271

Antibiotic -5.3178(*) 1.13613 .000 -7.6177 -3.0178

Antibiotic Isolate 1 4.3559(*) 1.13613 .000 2.0560 6.6559

Isolate 2 4.4422(*) 1.13613 .000 2.1423 6.7422

isolate 3 3.6556(*) 1.13613 .003 1.3556 5.9555

isolate 4 3.5726(*) 1.13613 .003 1.2726 5.8726

isolate 5 5.2185(*) 1.13613 .000 2.9185 7.5185

isolate 6 5.3178(*) 1.13613 .000 3.0178 7.6177

Based on observed means.

* The mean difference is significant at the .05 level.



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Appendix H

Zone of inhibition data of S. aureus

Number of

trials and its

corresponding

replicates

24 HOURS

(mm)

48 HOURS

(mm)

72 HOURS

(mm)

1-1 52 55 59

1-2 31 38 38

1-3 20 61 74

2-1 37 50 55

2-2 22 48 51

2-3 20 48 53

3-1 25 62 65

3-2 25 35 43

3-3 25 50 54



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Appendix I

Zone of Inhibition data of E.coli

Number of

trials and its

corresponding

replicates

24 HOURS

(mm)

48 HOURS

(mm)

72 HOURS

(mm)

1-1 25 32 38

1-2 20 26 33

1-3 27 35 46

2-1 20 22 35

2-2 19 24 36

2-3 26 31 41

3-1 21 24 29

3-2 18 42 42

3-3 24 37 30



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Appendix J

Descriptive Statistics for Staphylococcus aureus

Zone of Inhibition: High Land



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Zone of Inhibition: Intermediate



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Zone of Inhibition: lowland



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Appendix K

Descriptive Statistics for Escherichia coli

Zone of Inhibition: High Land



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Zone of Inhibition: Intermediate



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Zone of Inhibition: lowland



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Appendix L

Picture of Zone of inhibition



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Appendix M

Letter to UPLB Biotech

April 8, 2014

DR. REYNALDO V. EBORA

BIOTECH Director

PNCM-BIOTECH, UPLB

Los Banos, Laguna 4031

Dr. Ebora:

Good day Sir!

I would like to request for the purchase of activated cultures

of Staphylococcus aureus and Escherichia coli that will be used

by my undergraduate advisees, Ma. Sophia Estella C. Tajolosa and

Kristine Joy R. Estrella for their antibacterial research study.

Aside from my supervision, our Biology department has also a

registered microbiologist to guide and assist them in handling

these bacteria. Our microbiology lab is also provided with the

necessary equipment and gadgets in order to qualify and fit for

a Biosafety level 2 organisms manipulation.

Attached herewith are the accomplished forms needed for our

request and for your perusal. I look forward for your favorable

acceptance of my request for the success of my students’

undergraduate research study as part of their chosen career

path. Thank you and more power.

Very yours truly,

REY G. TANTIADO

Biological Science Instructor and Research Adviser

Bio. Sci., CAS, West Visayas State University



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Appendix N

Letter of Permission

April 29, 2014

MR. AUDIE A. SULADAY

Head

Central Laboratory Section

West Visayas State University

Dear Mr. Suladay:

Greetings!

We, Ma. Sophia Estella C. Tajolosa and Kristine Joy R. Estrella,

incoming fourth year students of BS Biology will be conducting a research

experiment as a partial fulfillment of the requirement in our course subject,

Thesis Writing in Biological Sciences (BIO 230A).

In line with this, we would like to request the following chemicals and

laboratory apparatus for our thesis entitled “Antibacterial Screening of Soil

Bacterial Isolates from Sinapsapan, Jordan Guimaras against Escherichia coli

and Staphyloccocus aureus” this May 2014 at RH 104.

Attached hereof is the list of the following apparatus and chemicals

that we are to use in the said study.

We are looking forward for the granting of our request.

Thank you very much.

Respectfully Yours,

______________________________

MA. SOPHIA ESTELLA C. TAJOLOSA

Researcher

_____________________________

KRISTINE JOY R. ESTRELLA

Researcher

Noted by:

______________________

PROF. REY G. TANTIADO

Thesis Adviser

_____________________

DR. NANCY S. SURMIEDA

Dean, College of Arts and Sciences

_______________________

MR. AUDIE A. SULADAY

In Charge, Central Science Laboratory



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April 29, 2014

MR. RUBEN C. ITABAG

Chief Administrative Officer

West Visayas State University

Sir:

Greetings!

We, Ma. Sophia Estella C. Tajolosa and Kristine Joy R. Estrella,

incoming fourth year students of BS Biology will be conducting a research

experiment entitled “Antibacterial Screening of Soil Bacterial Isolates from

Sinapsapan, Jordan Guimaras against Escherichia coli and Staphyloccocus

aureus”.

In view of this, we are asking permission to use the facilities of the

the Rizal Hall Room 104 from May 2014 – October 2014 from 7:30 am to 5:00 pm.

We are hoping for your kind approval regarding our request.

Thank you very much.

Respectfully Yours,

______________________________

MA. SOPHIA ESTELLA C. TAJOLOSA

Researcher

_____________________________

KRISTINE JOY R. ESTRELLA

Researcher

Noted by:

_______________________

PROF. REY G. TANTIADO

Thesis Adviser

Endorsed By:

_______________________

DR. NANCY S. SURMIEDA

Dean, College of Arts and Sciences

_______________________

Romeo Y. Solano

GSO Supervisor

Approved by:

____________________

MR. RUBEN C. ITABAG

Chief Administrative Officer



Iloilo City

Apparatus and Materials:

(10) 20 ml Test tube

(1) Test tube rack

(2) 500 ml Erlenmeyer flask

(2) 250 ml Erlenmeyer flask

(15) Petri dish

(3) Spatula

(5) Stirring rod

(1) Electric Weighing balance

(1) Autoclave Machine

(1) Incubator

(3) Alcohol Lamp

(3) 100 ml Beaker

(3) 250 ml Beaker

(1) Colony Counter

(1) 100 ml Graduated Cylinder

(1) 25 ml Graduated Cylinder

(15) Pipette

Chemicals:

Nutrient agar

Nutrient broth

Mc.Farland 0.5

Normal Saline Solution

Mineral oil

Safranin

Gram’s iodine

70% Alcohol



Iloilo City

Appendix O: Soil Sample Collected

Appendix P: Pure Culture of Escherichia coli Biotech 1634



Iloilo City

Appendix Q: Pure Culture of Staphylococcus aureus Biotech 1582

Appendix R: Prepared Nutrient Agar and NSS



Iloilo City

Appendix S: Preparation of Nutrient Agar Plates

Appendix T: Labelling of Agar Plates



Iloilo City

Appendix U: Agar Slant Preparation

Appendix V: Serial Dilution of Soil Isolates



Iloilo City

Appendix W: Soil Bacterial Isolates in Preparation for

Incubation

Appendix X: Incubation



Iloilo City

Appendix Y: Recorded Characteristics of Soil Bacterial Isolates

Appendix Z: Stains used in Gram’s Staining



Iloilo City

Appendix A1: Gram staining of Bacterial Isolates with

Antibacterial Properties

Appendix A2: Stained Slides for Microscopic Observation



Iloilo City

RESEARCH CERTIFICATION

This is to certify that KRISTINE JOY R. ESTRELLA and MA.

SOPHIA ESTELLA C. TAJOLOSA have submitted to me their manuscript

entitiled “ANTIBACTERIAL SCREENING OF SOIL BACTERIAL ISOLATES

FROM SINAPSAPAN, JORDAN, GUIMARAS AGAINST Escherichia coli

AND Staphylococcus aureus.”

I have examined the same and found it in order.

REY G. TANTIADO Ph. D. ________________

Research Adviser Date

JEANNEMAR GENEVIEVE YAP-FIGUERAS M. Sci. ________________

Department Editor Date

PRENCY C. YERRO M. A. ________________

Student Research Coordinator Date

GERARD L. PENECILLA Ed. D. ________________

Department Chair Date

NANCY S. SURMIEDA Ph. D. ________________

Dean Date

ANTIBACTERIAL SCREENING OF SOIL BACTERIAL ISOLATES FROM SINAPSAPAN, JORDAN, GUIMARAS AGAINST Escherichia coli AND Staphylococcus aureus

Documents

study soil

soil bacteria

study chapter

species of pathogenic

soil particles

notable pathogenic bacteria

billions of bacteria

different bacteria species