Table of Contents

Certification

Acknowledgements

Table of Contents

Abstract

CHAPTER ONE

Introduction

1.0 Aim of the Study

1.1 Objectives of the Study

CHAPTER TWO

2.0 Literature Review

2.1 Definition of Terms

2.2 Description of the species of Organisms

2.3 The Role of Lactobacillus and enteropathens in the Gastrointestinal Tract

2.4 The Gastrointestinal Defence Ecosystem of the Host

2.5 The Resident Microbiota as a Partner of the Gastrointestinal Defence Ecosystem of

the Host

2.6 Antimicrobial Activity of Lactobacillus against Intestinal Microbial Pathogens

2.7 Mechanisms of the Antimicrobial Effects

CHAPTER THREE

3.0 Materials and Methods

3.1 Sources and Collection of Samples

3.2 Media Used

3.3 Microbiological Analysis

3.4 Statistical Analysis

CHAPTER FOUR

4.0 Result

4.1 Screening of Antimicrobial Activity and Expression of Inhibition Effect

4.2 Inhibition due to Acids

4.3 Inhibition due to Hydrogen Peroxide

4.4 Evaluation of pH Effect

4.5 Interpretation and Justification of Result

CHAPTER FIVE

5.0 Discussion

5.1 Conclusion

5.2 Recommendation

References

Appendix
 CHAPTER ONE

The gastrointestinal ecosystem constitutes a variety of resident microbiota which play

important role in the health and well-being status of humans and animals. However, certain

organisms are capable of initiating infection in the gastrointestinal tract which results in the

emergence of abnormalities or disorders requiring serious medical attention.

Variety of enteropathogens such as Shigella species, Helicobacter pylori, Salmonella

species, Vibrio cholerae, Candida albicans, Clostridium difficile, E. coli, and Campylobacter

are known to be agents of enteritis and food intoxication with principal symptoms of diarrhea

and typhoid (Yung et al., 2002). Diarrheal disease is one of the critical diseases caused by

enteropathogens and can be fatal when it occur in people with compromised and debilitated

immune system including infants and adults (Yungh et al., 2002). The control of these

bacteria necessitate the prevention of diarrheal diseases in humans and animals (Yungh et al.,

2002).

The gastrointestinal tract constitutes varieties of microorganisms whose change in

population are susceptible in response to various environmental factors such as medication,

antibiotics and diet. The enteropathogens induce diseases when the host’s normal microflora

has been distorted or suppressed by these factors (Jin-Seong et al., 2002). The resistance of

these microorganisms to first line drugs and common antibiotics such as Chloramphenicol,

Ciprofloxacin, Fluroquinolones and other antimicrobial agents has resulted in lack of control

of certain enteric infections such as typhoid and diarrhea (Amira et al., 2013). Hence, the

need for the management or treatment of enteric infections caused by enteropathogens.

In general, gastrointestinal microbiota plays important role in the health and well-

being of humans and animals. Saad et al., (2013) defined probiotics as the concept where

certain microorganisms, when supplied in sufficient amount confer direct benefits to the host.
This research focuses on the activities of Lactobacillus species as probiotic organisms well-

known as beneficial microorganisms that contribute to immune function as well as preventing

the emergence of pathogenic and/or opportunistic species in the gastrointestinal tract of

human and animals (Mancilla et al., 2015).

Different species of Lactobacillus are capable of functioning as microbial barriers

against gastrointestinal pathogen through competitive exclusion of pathogen binding,

modulation of the host’s immune system, and production of inhibitory compounds such as

organic acid (e.g. lactic acid and acetic acid), oxygen catabolites (e.g. hydrogen peroxide),

proteinaceous compounds (bacteriocins), fat and amino acid metabolites and other

compounds (e.g. reuterin).

According to Suskovic et al., (2010), the ability to inhibit pathogenic species and

undesirable microorganisms is the most important property of probiotic such as

Lactobacillus. in addition, probiotic organisms have been associated with a number of other

beneficial effects, which are noticed when regularly consumed in sufficient quantities (see

table 1 in chapter two).

According to O’Brien and Wright (2011), the main objective in the production of

antimicrobial compounds is establishing an adaptive advantage in highly competitive

environments. In such situations, according to Marroti et al., (2011), antagonism constitutes

an important means by which a particular species overthrow competing species, though this

ability comes at an energetic cost. Explaining further, antagonistic interaction is an

interaction between different populations of organisms in which one is able to either inhibits

or kills the other population for a number of reasons. But in this context, the mechanisms

underlying this antagonistic interaction between Lactobacillus species and enteropathogenic

organisms are evaluated in vitro.

 Aim of the Study

The aim of this study was to evaluate the antagonistic activities of Lactobacillus species

against some enteropathogens of clinical significance.

Objectives of the Study

 To isolate and identify Lactobacillus species capable of inhibiting the growth of

enteropathogens.

 To evaluate the activities of Lactobacillus species against enteropathogens.

 To detect the susceptibility of enteropathogens to Lactobacillus species

 To evaluate the factors responsible for antagonistic activities of Lactobacillus species.

 CHAPTER TWO

2.0 Literature Review

2.1 Definition of Terms

Antagonism: Microbial antagonism refers to the presence of normal microbiota that

protect the body by competing with pathogen in a variety of ways; and that prevent pathogens

from invading the body. According to Marrot et al., (2011), antagonism constitutes an

important means by which a particular species overthrow competing species. Antagonism is

commonly thought of as an association between different, although may occur between

members of the same species through competition.

Lactobacillus: It is a genus of Gram positive, facultative, anaerobic or

microaerophilic, rod-shaped, non-spore-forming bacteria from the family of lactobacillaceae.

Also, Lactobacillus is a genus of bacteria that sometimes contains coccobacilli that lack

catalase and cytochromes, produce lactic acid as principal fermentation product, and have

complex nutritional requirement. The genus either exhibits a homolactic fermentation using

the Embden-Meyerhof Pathway or heterolactic fermentation with the pentose phosphate

pathyway (Prescott et al., 2005).

Enteropathogens: These are microorganisms that are capable of causing or initiating

infection in the intestinal lumen or tract of human or animal. Examples of enteropathogens of

clinical significance include Salmonella species, Shigella, Hellicobacter pylori, Vibrio

cholera, Candida albicans, Clostridium difficile, Campylobacter etc.

2.2 Description of the Species of Organisms

Lactobacillus acidophilus: This species may also be referred to as Doderleins bacillus.

It is specie of gram positive bacteria in the genus Lactobacillus. L. acidophilus is a

homofermentative, microaerophilic species, fermenting sugars into lactic acid, and grows
readily at rather low pH value (below 5.0) and has an optimum growth temperature of around

37°C. L. acidophilus occurs naturally in the human and animal gastrointestinal tract and

mouth. Some strains may be considered to have probiotic characteristics. These strains are

commercially used in many dairy products, sometimes together with Streptococcus

thermophiles and Lactobacillus delbrueckii subspecies bulgaricus in the production of

acidophilus-type yoghurt.

Some strains may be able to survive gastrointestinal transit, being resistant to bile,

low pH, and digestive enzymes. They may then be able to adhere to human epithelial cell

lines and human intestinal mucus. L. acidophilus can decrease the incidence of pediatric

diarrhea and can lead to a significant decrease in levels of toxic amines in the blood of

dialysis patients with small bowel bacterial overgrowth. Sufficient intake of L. acidophilus

may facilitate lactose digestion in lactose-intolerant persons through the production of

enzymes (Honda et al., 2007). L. acidophilus is helpful in reducing serum cholesterol level

(Guo et al., 2011). The specie is capable of producing lactase, bacteriocins such as acidolin,

acidophilin and lactocidin; and vitamin k.

Lactobacillus plantarum

Lactobacillus plantarum is a Gram positive aerotolerant bacteria that grows at 15°C and

produces both isomers of lactic acid (D and L). It is commonly found in many fermented

food products as well as anaerobic plant matter. It is present in saliva (from which it was first

isolated) and has the capacity to liquefy gelatin. Lactobacillus plantarum has one of the

largest genomes known among the lactic acid bacteria and is very flexible and versatile

species. This species and related Lactobacilli are unusual in that they can respire oxygen but

possess no respiratory chain or cytochromes. The consumed oxygen ends up as H2O2 which is

presumed, acts as weapon to eliminate or overthrow competing bacteria from food source.
Instead of the present of superoxide dismutase as in other oxygen-tolerant cells, they

accumulate millimolar quantities of manganese polyphosphate. L. plantarum can be cultured

using MRS media. It is commonly found in Ogi, pickels, brined olives etc.

Lactobacillus fermentum

This is a Gram-positive species of bacterium in the genus Lactobacillus. It is associated with

active dental carried lesions. It is commonly found in fermenting animal and plant material. It

is also found in Sourdough and a few strains are often considered probiotic or friendly

bacteria. Testing of L. fermentum against different pH concentration solutions revealed that it

has a strong pH tolerance by its ability to grow and survive a few hours after being incubated

in a 3-pH level solution.

Lactobacillus casei

This particular species of Lactobacillus is documented to have a wide pH and temperature

range, and complements the growth of acidophilus, a producer of the enzyme amylase (a

carbohydrate digesting enzyme). The commonest application of L. casei is industrial,

specifically for dairy production. In a study published in 2003 in the “Canadian Journal of

Gastroenterology and Hepatology”, patient with chronic constipation saw significant

improvement when drinking a daily beverage containing the Lactobacillus casei strain L.

casei shirota. In a 2007 study, published in the journal “BMJ” hospital patients consuming a

drink containing the Lactobacillus casei, L. bulgaricus and S. thermophilus bacteria had

fewer cases of antibiotic- and infection-associated diarrhea.

2.3 The Role of Lactobacillus and Enteropathogens in the Gastrointestinal Tract

Lactobacillus plays an important role in the gastrointestinal tract of both humans and

animals. It is used for treating and preventing diarrhea, including infectious type such as
rotaviral diarrhea in children and traveler’s diarrhea. It is also used to prevent and treat

diarrhea associated with using antibiotics.

Lactobacillus plays role in digestion problems; irritable bowel syndrome; colic in

babies; crohn’s disease; inflammation of the colon; and a serious gut problem called

neurotizing enterocolitis (NEC) in babies born prematurely. The genus is also used for

infection caused by Helicobacter pylori, the type of bacteria that causes ulcers, and also for

other types of infections associated with urinary tract, vaginal yeast infections, to prevent

common cold and in adults, and to prevent respiratory infections in children attending

daycare centers. Also plays role in skin disorders such as fever blisters, canker sores, eczema

(allergic dematitis) and acne. It is also used for high cholesterol, lactose intolerance, lyme

disease, hives, and to boost the immune system (Liong, 2005; Guo et al., 2011).

However, table 1 explains further, the beneficial roles or properties associated with

this genus of probiotic microorganisms.

Beneficial property Reference

Adherence to intestinal mucosa and Uchida; Kunakasu, 2004; Lam et al., 2007

maintenance of its integrity

Attenuation of intestinal allergic reactions Yoshida et al., 2011

Production of vitamin; increased in Narva et al., 2004; Pompei et al., 2007

bioavailability of mineral

Alleviation in constipation cases as well Guerra et al., 2010; Riezzo et al., 2012

as increase intestinal motility

Accelerate healing of wound (topical use) Peral et al., 2009; Nasiabudi et al., 2011;

Huseini et al., 2012

Production of digestive enzymes Honda et al., 2007

Anticarcinogenic effect Kumar et al., 2012

Decrease cholesterol and triglyceride levels Liong; Sha, 2005; Guo et al., 2011

Immunomodulation Tsai et al., 2008b; Tsai et al., 2010a;

Tsai et al., 2010b

Inhibition of pathogen and modulation of

intestinal microbiota through bacteriocin production Cursino et al., 2006; Pingitere et al.,

2009; Toclorov et al., 2011

Reduction of obesity by inducing significant

reduction in adipcytesize Choi et al., 2007; Takemura et al.,

2009; Kadooka et al., 201

Table 1: Beneficial properties of Lactobacillus

Enteropathogens: Enteropathogens play a major role in the causation of intestinal disorders

or abnormalities. They are known to cause various kinds of infectious and deadly diseases

such as diarrhea and typhoid. Helicobacter pylori is known to cause an infection called ulcer,

which is a very serious disease that distort the functionality of the intestinal ecosystem.

Clostridium defficile and Salmonella cause diarrhea and typhoid fever infections respectively.

Other pathogens such as Shigella and Vibrio cholera are also involved in the causation of

intestinal infections or disorders.

2.4 The Gastrointestinal Defence Ecosystem of the Host

The host has protection from internal and external attack by potentially harmful or

unfriendly microbes by the physical and chemical barriers exerted by the intestinal

epithelium. The gastrointestinal epithelial cells and the resident microbiota synergistically

function to promote an effective and efficient host defensive system.

 The gastrointestinal cells that protects the host against the unwanted intrusion of

microorganisms into the gastrointestinal microbiota, and against the penetration of harmful or

unfriendly microorganisms which can hijack the cellular molecules and signaling pathways

of the host to become pathogenic (Cursino et al., 2006; Pinjitore et al., 2009; Todorov et al.,

2011).

The intestinal mucosa exhibit a surface coated with mucus that is secreted by

specialized gobut cells. The intestinal mucein-secreting polarized cells exhibit two secretory

pathways, which include, first the regular vesicular constitutive pathway of mucein

exocytosis, in which no storage occurs as the small vesicles transporting the mucins through

the constitutive pathway are guided directly to the cell surface by microtubules and undergo

rapid exocytosis of their contents. The second pathway for mucins exocytosis involves the

packaging and storage of mucins in large vesicles, from which mucin release is regulated by

specific stimuli consisting signaling molecules (Alain, 2004). The entrapment of bacteria

within he mucus which contains secretory immunoglobulin, coupled with peristalsis,

enhances rapid elimination of bacteria from the intestine.

In line with the innate defences of the host, the components of the intestinal immune

system of defence re-strengthen the barrier function of the gastrointestinal epithelium. In

effort to boast the defensive system, the intestinal epithelium and gut-assoicated immune

cells enhances sensing of the microbial environment by the host to promote effective defence

response by the release of signaling molecules such as cytokines and chemokines that attract

leukocytes and initiate attraction of immune cells. Also, the gut has the potential of

distinguishing its commensal microbiota from the enterovirulent organisms. The production

of antimicrobial peptides offers the first line of chemical defence in the gastrointestinal tract

of the host.
2.5 The Resident Microbiota as a Partner of the Gastrointestinal Defence Ecosystem

of the Host

One physiological function of the resident normal flora is its ability to function as a microbial

barrier against microbial pathogens. The intestinal lumen is a nutritionally rich environment

in which microbial concentrations of up to 10¹²CFU/ml can be found (Whitman et al., 1998;

Alain, 2004). Microbial interaction in the gut is known to be complex and dynamic; hence,

the constituent of the microbial community fluctuates regularly owing to certain factors.

Large or selective shifts in the gut microbiota composition promoted by changes in diet,

medications and environment may stimulate specific microbial interactions or host-

microbiota associated events, which can significantly influence host health (Sun and Chang,

2004).

In light of this, predominance of beneficial microorganisms contribute to immune

function and prevent the emergence of pathogen and/or opportunistic pathogens (Tsai et al.,

2010; Todorov et al., 2011). Lactobacillus as inhabitant of the gastrointestinal tract develops

antimicrobial activities that participate in the host’s gastrointestinal system of defence.

2.6 Antimicrobial Activity of Lactobacillus against Intestinal Microbial Pathogens

2.6.1 In Vitro Demonstration of Antimicrobial Activity

According to Lam et al., (2007) experiment has shown that Lactobacillus species

express adhesiveness properties that enable them to inhibit the adhesion of bacterial

pathogens to host cells. In the light of this demonstration, it has been assumed that

Lactobacillus may use similar mechanism to that of Bifidobacteria to fight gastrointestinal

microbial pathogens.
 In vitro studies using cultured human intestinal cell lines have investigated the

adhesiveness and the bacterial interference effect of Lactobacillus (Alain, 2004). Many of

the cell phenotypes that line the intestinal epithelium have been extensively used to study

specific human intestinal cell functions. For instance, T84 cells are known to exhibit crypt cell

morphology and functions. However, these cell models form functional complexes, and so

constitute a monolayer that mimics the intestinal epithelial barrier, that pathogenic

microorganisms have to cross before they can infect the systemic circulation of the host and

spread within the organism.

2.6.2 Adhesiveness Properties

The adhesiveness of intestinal Lactobacillus strains has been investigated using Caco-

2 and HT29-MTX cell lines (Alain, 2004). It was observed that not all strains of

Lactobacillus examined adhere onto enterocyte-like caco-2 cells, indicating that adhesiveness

is strain specific. Strains such as L. johnsonii La1, L. acidophilus HN017, L. acidophilus

BG2FO4 and L. casei shirota and others adhere to enterocyte-like caco-2 cells. Also,

adhesion to cultured human intestinal cells and isolated muceins is not sufficient to describe

the mucosal interactions of Lactobacilli. The author proposed that adhesiveness of

Lactobacilli were better examined using human intestinal tissue pieces. However, some

processes enhance adhesion of the bacteria.

The processes that facilitate adhesion of Lactobacilli include passive forces,

electrostatic interactions, hydrophobic steric forces, lipoteichoic acids, and specific

structures, such as lectin-covered external appendages. Lipoteichoic acid has been identified

as a factor responsible for the adhesion of the L. johnsonii La1 bacteria. This molecule has

been isolated from the bacterial cell wall and from the culture supernatant and is responsible

for the concentration-dependent inhibition of La1 adhesion to caco-2 cells. Lactobacillus

fermentum and L. animalis surfaces, and L. animalis has been shown to have ribitol teichoic

acids in its cell wall.

2.6.3 Bacterial Interference in vitro

Microbial pathogens develop several mechanisms of interaction to ensure that they

remain associated with the gut mucosa and withstand the flow of the intestinal chime.

Microbial pathogens, specifically bacteria form associations with the intestinal mucosa that

require specialized factors encoded by the bacteria, and if this attempt fails, they are rapidly

removed from the gut. According to Candela et al., (2010), this defeat is promoted by the

activities of the mucus layer and the gut microbiota.

Experiment has demonstrated that urovaginal Lactobacilli compete with

uropathogens. In like manner, Lactobacilli of intestinal origin have the ability to interfere

with the adhesion of pathogenic bacteria onto intestinal cells, resulting in the inability of the

intestinal pathogen to establish association. Lactobacillus strains including L. casei, L.

rhamnosus and L. plantarum, develops inhibitory activity against enterohemorrhagic E. coli

(EHEC) infecting the human colon ephithelial cell line. Strains of L. fermentum, L.

acidophilus and L. salivarius isolated from the gastrointestinal tract of piglets at the time of

weaning, were found to affect the attachment of enterotoxigenic E.coli to porcine enterocytes

by co-aggregating with E. coli. Also, examination of the antimicrobial activity against

Clostridium defficile of a high number of Lactobacilli strains isolated from healthy infants

shows that only 8 of the 109 strains showed activity against C. defficile and that 19 strains

were active against E. coli 015:H7 (Alain, 2004). Hence, some selected adhesive Lactobacilli

strains develop antagonistic activity against adhesion of gastrointestinal pathogens onto the

mucosal wall of the gut.

2.6.4 Demonstration of Antimicrobial Activity in Infectious Animal Models

The antimicrobial activity of Lactobacilli has been evaluated using two infected

mouse models. The first involves germ-free (probiotic) mice, in which the microbiota is

missing and the epithelium is not completely differentiated. The second model employ

conventional mice, in which the microbiota is present and the epithelium completely

differentiated. However, the antagonistic activity of Lactobacilli against some bacteria and

viruses involved in diarrhea in human cannot be evaluated using murine models, since these

microbial pathogens are highly specific for human tissues, principally as a result of

differences in structure of human and murine intestinal brush border-associated molecules

that act as microbial receptors.

2.6.5 Adhesiveness Properties and Persistence in the Gut

Scientists have maintained that in vitro adhesion to intestinal cells of Lactobacilli is

not sufficient to ensure the persistence of living bacteria in the digestive tract of mice. The

strength of many Lactobacillus species already demonstrated to adhere in vitro, to colonize

the gastrointestinal tract of mice in vivo has been investigated. This investigation shows that

some strains of Lactobacilli when given orally to certain protobiotic mice, established

themselves in all the segments of the gut while to other mice survived but were eliminated,

indicating that they do not establish themselves in the gut. Clinical studies have equally

shown that some strains of Lactobacillus bacteria survived passage through the human gut

when administered in humans. The rate of survival has been extrapolated to be 20-40% for

selected strains; the major hindrances to survival being gastric acidity and the activity of bile

salt making survival strain specific (Servin, 2004).

2.6.6 Activity of Lactobacillus against Helicobacter pylori

 An antagonistic effect of Lactobacilli against H. pylori has been demonstrated in a

murine model. H. pylori urease catalyzes the conversion of urea to carbon (iv) oxide and

ammonia; ammonia in turn forms ammonium hydroxide, which neutralizes the local acidity

in favour of H. pylori survival. In an H. pylori infected protobiotic murine model, L.

salivarius strain that produces a large amount of lactic acid and completely inhibits the

growth of H. pylori in a mixed culture, suppressed H. pylori, and reduced the H. pylori-

induced inflammatory response. In conventional mice, oral treatment with spent culture

supernatant of the human L. acidophilus LB, conferred protection against infection by H.

felis, reducing the urease activity of H. felis in the stomach, inhibiting colonization of the

stomach by H. felis, and abolishing the H. pylori-induced gastric histopathological lesions

(Seruin, 2004).

However, the anti H. pylori activity exerted by Lactobacilli bacteria has been

proposed to be due to organic acids produced by the bacteria. L. plantarum and L. gasseri

have the potential of being able to inhibit H. pylori adherence to gastric epithelial cells.

2.6.7 Activity against Salmonella

Spent culture supernatant of Lactobacillus rhamnosus GG has been reported to

exhibit antibacterial activity against Salmonella (Keersmacecker et al., 2006). Lactobacillus

rhamnosus GG produce a low-molecular weight, heat-stable, non-proteinaceous bactericidal

substance, and active at acidic pH against a wide spectrum of bacterial species.

Consequence upon various experimental approaches, it could be ascertained that

strong antimicrobial activity of L. rhamnosus GG against Salmonella is mediated by lactic

acid. However, the most common infectious model used to investigate the antibacterial

activity of Lactobacillus is that of protobiotic or convential mice infected by Salmonella

(Alain, 2004). In one of such investigations, it was observed that L. johnsonii La1 and GG
strains, which colonize the gut of protobiotic mice, developed antibacterial activity when the

mice were orally infected by S. enterica serovar Typhimurium c5, increasing the survival of

mice. Similarly, L. acidophilus has been found active against experimental infections with S.

flexneri and S. enteritidis serovar Typhimurium in protobiotic mice.

2.6.8 Activity against Rotavirus

The investigation of Neha Pant et al., (2007) is used in this study to explain the

activity of Lactobacillus against rotavirus pathogen. In the investigation, six different

Lactobacillus were tested for protection against rotavirus induced diarrhea. Mouse pups

received two prophylactic doses of bacteria before challenge with rotavirus, followed by

daily therapeutic administration of respective bacteria and monitoring for diarrhea symptoms.

A strong anti-rotavirus activity of L. rhamnosus strain GG, corroborating results obtained

previously by other researchers in clinical trials was noted. However, the same bacterium,

when heat-killed was unable to impact protection against rotavirus challenge, indicating that

the inhibitory effect against rotavirus is either dependent on viability or is a heat labile factor.

Explaining further, a clinical investigation of treatment with live or heat inactivated L.

rhamnosus GG against rotavirus diarrhea found that the inactivated bacteria did not

effectively stimulate local IgA production, hence raising the chances of reinfection.

2.6.9 Activity against Listeria monocytogenes

Clinical trials revealed that administration of L. casei species to mice injected with L.

monocytogenes indicate that the growth of L. monocytogenes in the liver of these mice was

suppressed (Alain, 2004). Similarly, ingesting viable L. casei shirota significantly reduced

the numbers of L. monocytogenes in the tissue of orally infected host resistance of L.

monocytogenes infection induced by L. casei may be mediated by macrophages migrating

from the blood stream to the reticuloendothelial system in response to L. casei intake before

or after infection with Listeria monocytogenes.

2.7 Mechanisms of the antimicrobial effects

The main mechanisms of action include enhancement of the epithelial barrier,

increased adhesion to intestinal mucosa and concomitant inhibition of pathogen adhesion,

competitive exclusion of pathogenic microorganisms, production of anti-microorganism

substances and modulation of the immune system.

2.7.1 Enhancement of the Epithelial Barrier

The intestinal epithelium is in permanent contact with huminal contents and the

variable, dynamic enteric flora. The intestinal barrier is a major defence mechanism used to

maintain epithelial integrity and to prevent the organism from the environment. Defences of

the intestinal barrier consist of the mucous layer, antimicrobial peptides, secretory IgA and

the epithelial junction adhesion complex (Carolina et al., 2012). Once this barrier function is

disrupted, bacterial and food antigens can reach the submucosa and can induce inflammatory

responses, which may result in intestinal disorders such as inflammatory bowel disease.

However, consumption of non-pathogenic bacteria can contribute to intestinal barrier

function.

2.7.2 Increased Adhesion to Intestinal Mucosa

Adhesion to intestinal mucosa is regarded as a prerequisite for colonization and is

important for the interaction between Lactobacillus strains and the tract. The adhesion is also

important for modulation of the immune system and antagonism against pathogens.

Lactobacillus displays various surface determinants that are involved in their

interaction with intestinal epithelial cells and mucus. The intestinal epithelial cells secrete
mucin, which is a complex glycoprotein mixture that is the main component of mucous,

thereby preventing the adhesion of pathogenic bacteria. In addition, lipids, free proteins,

immunoglobulin and salt are present in mucous gel, to further facilitate the activity.

Lactobacillus also causes qualitative alteration in intestinal mucins that prevent

pathogen binding. The bacterial component involved in the adhesion of the LB and BG2FO4

L. acidophilus strains is protease resistant and is associated with the bacterial surface. This

bacterial component is also degraded into an antimicrobial peptide, which lends anti-

pathogenic properties to the host and provides an illustration of how large surface proteins

may exhibit evolutionarily beneficial pleiossstrophic effects.

2.7.3 Competitive Exclusion of Pathogenic Microorganisms

In a report published in 1969 by Greenberg, addressing the total exclusion of

Salmonella typhimurium from maggots of blow-flies, ‘competitive exclusion’ term was used

for the first time to denote a situation in which one species of bacteria more vigorously

competes for receptor sites in the intestinal tract than another species (Carolina et al., 2012).

The mechanisms used by one species of bacteria to exclude or reduce the growth of another

species are varied, including the following mechanisms: creation of a hostile microecology,

elimination of available bacterial receptor sites, production and secretion of antimicrobial

substances and selective metabolites, and competitive depletion of essential nutrients.

Specific adhesiveness properties was due to the interaction between surface protein

and mucins may inhibit the colonization of pathogenic bacteria and are a consequence of

antagonistic activity by some strains of Lactobacillus against adhesion of enteropathogens.

Lactobacilli have been known to inhibit a broad spectrum of pathogens, including

Salmonella, E. coli, Listeria monocytogenes, Helicobacter pylori and Rotavirus. Exclusion is

seen as a consequence of different pathogen adhesion, including the production of substances

and the stimulation of intestinal epithelial cells. Competitive exclusion by intestinal bacteria

is based on a bacterium-to-bacterium interaction mediated by competition for available

nutrients and for mucosal adhesion sites. Bacteria gain competitive advantage through

modification of their environment to make it less suitable for their competitors. The

production of antimicrobial substances such as lactic and acetic acids is an example of this

type of environmental modification performed by the intestinal bacteria.

2.7.4 Production of Antimicrobial Substances

One beneficial activity of Lactobacillus is the fact that it is involved in the formation

of low molecular weight (LMW) compounds (less than 1, 000Da), such as organic acids, and

the production of antimicrobial substances termed bacreriocin (greater than 1000Da).

Organic acids, in particular acetic and lactic acids, have a strong inhibitory effect

against Gram-negative bacteria, and have been considered the principal antimicrobial

compounds responsible for the inhibitory activity of Lactobacillus against enteropathogens

(Ogara et al., 2001; Tsai et al., 2005; Tsai et al., 2008; Zhang et al., 2011). However, the

production of de-conjugated bile acids which is a derivative of bile salt is another beneficial

antimicrobial activity of Lactobacilli. De-conjugated bile acids show a stronger antimicrobial

activity compared to that of the bile salts synthesized by the host organism (Carolina et al.,

2012).

Explaining further, some strain of Lactobacilli produce metabolites that inhibit the

growth of fungi and other species of bacteria. However, some researchers have reported that

Lactobacillus can produce antifungal substances, such as benzoic acid, methyl-hydantoin,

mevalonolactone and short-chain fatty acids.

 In the light of the foregoing facts, Lactobacilli exhibit various antimicrobial

mechanisms as well as antagonistic activities against enteropathogenic organisms, as could be

detected by using appropriate materials and methods.

 CHAPTER THREE

3.0 Materials and Methods

Materials Used

The materials used for this study were purchased from Core Biomedical Enterprise

Umoren Lane, Uyo and Effective Medical Enterprise Ikpa Road, Uyo respectively. The

materials were stored, prepared and used according to the manufacturer´s instructions.

The materials are as listed: test tubes, forceps, glass slide, spirit lamp, petri dishes,

inoculating wire loop, conical flask, syringe and needle, reagents, nutrient agar, MRS agar,

test tube rack, Salmonella Shigella Agar (SSA), measuring cylinder, beaker, stock bottles,

MacConkey agar, EMB agar etc.

Staining reagent used in this study include Gram´s staining reagents such as basic

crystal violet, Lugol´s iodine solution, alcohol and safranin. Other reagents employed were

malachite green, catalase reagent, normal saline, oxidase reagent etc.

Methods Used

All the methods used in this work are approved standard microbiological methods.

3.1 Sources and Collection of Samples

Infant and adult faecal samples from 10 patients already confirmed with typhoid and

diarrheal infections were collected at the University of Uyo Medical Centre. Also, in addition

to raw milk bought at Itakinyang along Itu-Calabar Road in Uyo, faecal sample was collected

from infants aged 0-6 months old for isolation of Lactobacillus species. The samples were

immediately transported to the University of Uyo Microbiology Laboratory for analysis.

 The different media employed in the study were Salmonella Shigella Agar (SSA),

MacConkey Agar, EMB Agar, Nutrient Agar, MRS Agar, Mueller Hinton Agar, Urea Agar,

peptone water and Simon citrate Agar respectively.

3.3 Isolation and Characterization of the Test Isolates

The test isolates were isolated from different sources which include faecal samples of

typhoid and diarrheal patients and cultured on Salmonella Shigella Agar, MacConkey Agar

and EMB Agar respectively.

In addition, another category of the test isolates used in this study was isolated from

raw cow milk (nunu) and faecal sample from infants aged 0-6months old respectively. These

samples were cultured on MRS agar for 24-72 hours at 37°C; growth was observed as clear

colonies with some rod-shape. Salmonella on SSA showed colonies with black spot after

24hours of incubation at 37°C while E. coli on MacConkey agar demonstrated strong lactose

fermentation indicated by bright pink halo, bile precipitant around the colonies and formed

green metallic sheen on EMB agar after 24hours of incubation at 37°C. also, Shigella on the

SSA was observed to be colourless.

3.4 Biochemical Tests

These tests were performed for a conclusive identification and characterization of the

test isolates. The biochemical test conducted included Gram staining, catalase test, oxidase

test, spore staining, urease test, indole test, MR test, Voges Proskauer test, sugar fermentation

test, citrate test and motility test.

3.4.1 Gram Staining Test

This test was performed to differentiate the test isolates into either Gram positive or

Gram negative organisms based on their permeability of the cell wall to staining reaction.
 Procedure

A smear of the organisms were prepared on different clean glass slides and were allowed to

air-dry. The slides were passed gently over a flame 3 times to heat fix the smear. The heat

fixed smear was placed on a staining rack and was flooded with crystal violet stain for 60

seconds which after was rinsed off with slow running distilled water.

The slides were then flooded with iodine solution for 60 seconds and then rinsed off

with slow running distilled water. A decolourizing agent (alcohol) was then applied for 20

seconds and rinsed off with slow running water. The secondary stain (safranin) was applied

to counter stain for 30 seconds and thereafter rinsed with gentle running sterile water and left

to air-dry. The slides were observed under x100 oil immersion objective lens of a

microscope. Organisms with purple appearance under the microscope were regarded as Gram

positive while those with pink or red appearance were regarded as Gram negative.

3.4.2 Catalase Test

Catalase test is performed based on the fact that certain organisms possesses the

ability to produce the enzyme, catalase which has the potential of breaking down hydrogen

peroxide. The presence of catalase enzyme in the test isolates was detected using hydrogen

peroxide (H2O2).

Procedure

A drop of freshly prepared 3% hydrogen peroxide was placed on a clean grease free slide. A

sterile wire loop was used to aseptically transfer a loopful of the isolates on the slide. The

isolates were emulsified with the H 2O2 and for 10seconds were observed for production of

bubbles which indicate a positive result.

3.4.3 Oxidase Test

This test is used to identify organisms that produce cytochrome c oxidase, an enzyme

of the bacterial electron transport chain. It differentiates oxidase producers from non-oxidase

producers. All bacteria that are oxidase positive are aerobic, though not strict and can use

oxygen as a terminal electron acceptor in respiration.

Procedure

A clean filter paper was placed on a sterile petri dish, 2 to 3 drops of oxidase reagent

was placed on the filter paper. A wire loop was used to make a smear on the filter paper and

observation was made for 10seconds. A change in colour of the reagent to deep purple

indicates that the test isolate is oxidase positive and vice versa.

3.4.4 Sugar Fermentation Test

The sugar (carbohydrate) fermentation test is used to determine whether or not a

bacterium can utilize a certain carbohydrate as a carbon source. Common sugars employed in

this test were glucose, lactose, sucrose and mannitol. 1g of each of the sugars was added to

1.5gram of peptone powder and dissolved in 100ml of distilled water and was filtered. Phenol

red indicator was added and was aseptically dispensed into test tubes. Sterile Durham´s tubes

were inserted invertedly and each tube was inoculated with the test organism following

aseptic technique. The test tubes were incubated at 37°C overnight. Each tube was examined

for gas and acid production. Acid production was indicated by colour change while gas

production was indicated by air space in the Durham´s tubes.

3.4.5 Motility Test

Motility test was carried out to detect motile and non-motile microorganisms based on

their ability to move.

Procedure

Nutrient agar was prepared and poured into test tubes and allowed to set. A sterile

inoculating needle was used to pick the test organism and inoculated into the test tube by

making a single stab down the centre of the tube to about half depth of the media and it was

incubated at 35°C for 18hours to 24hours. Non-motile bacteria grow only on the stab line and

the surrounding media is transparent while motile bacteria show a diffused spread of growth

beyond the stab line and the surrounding media is turbid.

3.4.6 Citrate Utilization Test

This test is based on the ability of an organism to use citrate as its only source of

carbon. The test was carried out to assist in the identification of enteropathogens.

Procedure

According to the manufacturer´s instruction, six grams of Simmon´s citrate agar was

dissolved in 250ml of distilled water and sterilized by autoclaving at 121°C for 15minutes.

The prepared medium was allowed to cool and poured into sterile test tubes and left to set.

The test organism was inoculated into the test tube and incubated at 37°C for 24hours.

After 24hours of incubation, observation of a bright blue colour in the test tube shows a

positive citrate test. No change in colour indicates a negative citrate test.

3.4.7 Urease Test

Testing for urease is also necessary in differentiating enterobacteria. The test is

performed to detect the ability of an organism to break down ammonia and carbon dioxide.
 Procedure

5.2g of urea agar was weighed and suspended into 250ml of distilled water in a

conical flask and sterilized by autoclaving for 15minutes, allowed to cool and poured into

sterile test tube. The prepared media in the tube was left to set and the test organism was

inoculated. The tubes were incubated at 37°C for 5-7 days. A colour change to pink indicates

a positive urease test. No change in colour indicates a negative urease test.

3.4.8 Indole Test

This is usually performed to detect the production of trptophanase enzyme which

decomposes amino acid called typtophan to indole, which accumulates in the medium to give

a red ring colouration on addition of Kovac´s reagent (p-dimethylaminobenzaldehyde).

Procedure

Three grams of typtone soy broth was weighed and dissolved in 100ml of water and

sterilized by autoclaving at 121°C for 15minutes.

The media was dispensed into sterile test tubes; the test organism was inoculated and

incubated for 48hours. After incubation, 0.5ml of Kovac´s reagent was added into the test

tubes and observed. Red surface layer in the test tube is a positive indole test. No red surface

layer is a negative test.

3.4.9 Methyl Red Test

Microbes produce acid by fermentation of glucose which changes the pH of the

medium.

Procedure
 Glucose phosphate broth was prepared and poured into test tubes. The test organism

was inoculated into the test tubes and incubated at 37°C for 1-5 days. After incubation, 5

drops of 0.04% solution of alcoholic methyl red solution was added into the tubes and the

result read immediately. A bright red colour indicates a positive test result while a yellow

colour indicates a negative test.

3.4.10 Voges Proskauer Test

Bacteria ferment carbohydrate resulting in the production of acetone. In the presence

of alkali and atmospheric oxygen, acetone is oxidized to diacetyl which reacts with peptone

present in the broth to give a red colour.

Procedure

Glucose phosphate broth was prepared and poured into test tubes. The test organism

was inoculated and incubated at 37°C for 48hours. After incubation, 1ml of 40% potassium

hydroxide containing 0.3% creatine and 3ml of 5% solution of α-naphthol in absolute alcohol

was added into the test tubes respectively. It was observed for results after 2-5minutes. Pink

colour indicates positive reaction whereas no colour change indicates negative reaction.

3.4.11 Spore Stain Test

This test was performed to detect whether the test isolates were spore formers or non-

spore formers.

Procedure

A smear of the organisms were prepared on different clean glass slides and were

allowed to air-dry. The slides were flooded with malachite green and heated to produce

bubbles for 3 times. The slides were then washed off by gentle running water and flooded
again with safranin for 30-60seconds and washed off with slow running water. The slides

were left to air-dry and then observed under x100 oil immersion objective. Organisms that

form spores appear green under the microscope while the non-spore formers appear pink to

red in colour.

3.5 Test for Antimicrobial Activity

Antimicrobial activity of Lactobacillus species was assessed using procedure of

Agar-Well diffusion on solid medium.

The detailed procedure of agar diffusion test was as follows: the test organisms were

subcultured in peptone water in and incubated for 24hours at 37°C. With the aid of couvet

centrifuge, the culture of Lactobacillus species was centrifuged at 4000rpm for 5minutes.

0.5ml of organism from the broth culture of enteropathogens were inoculated into Mueller

Hinton Agar plate and spread evenly to cover the entire surface of the plate, which was then

left to dry. Using a cork borer, a well was made on the plate and the supernatant from the

broth culture of Lactobacillus was inoculated into the well and incubated for 24hours at

37°C. However, commercially prepared antibiotics were used as control to check the

effectiveness of Lactobacillus against enteropathogens.

3.6 Statistical Analysis

The statistical analysis employed in this study is a one-way analysis of variance

(ANOVA 1).
 CHAPTER FOUR

4.0 RESULT

This chapter gives explanation of the result obtained in this study and their analysis.

4.1 Morphological and Biochemical Analysis

Different biochemical test were carried out to identify the enteropathogens and the

Lactobacillus species. The morphological and biochemical characterization of the bacterial

isolate used in this work are presented in table 1 and 2

Table 1: Morphological and Biochemical Characterization of Test Isolates
Urease

Glucsoe
ReactionGram

Shape

Oxidase

Indole

Lactose
Sucrose

Mannitol
ProskauerVoges
Catalase

MR test

Citrate
Motility
Isolates

Probable Organism

OA - Rod + - + - + + - - AG AG AG AG E. coli spp

OB - Rod + - - - - + - - AG - - AG Shigella spp

OC - Rod + - + - + + - - AG AG AG AG E. coli spp

OD - Rod + - - - + + + - AG - - AG Salmonella spp

OE - Rod + - - - - + - - AG - - AG Shigella spp

OF - Rod + - - - + + + - AG - - AG Salmonella spp

OG - Rod + - - - + + - - AG AG AG AG E. coli spp

OH - Rod + - - - + + + - AG - - AG Salmonella spp

OI - Rod + - + - + + - - AG AG AG AG E. coli spp

OJ - Curved - + + - + - - + - - AG AG Vibrio cholerae

Rod
OK - Rod + - - - + + - - AG AG AG AG E. coli spp

OL - Curved - + + - + - - + - - AG AG Vibrio cholerae

Rod

Key: A = acid, G = Gas, + = Positive, - = negative

 Oxidase

Glucose

Sucrose
Shape

Indole

Lactose

Mannitol

Spore test
Catalase

Citrate
Motility
Isolates

Probable Organism
ReactionGram

1 + Rod - - - - - AG AG AG AG - Lactobacillus spp

2 + Rod - - - - - AG AG AG AG - Lactobacillus spp

3 + Rod - - - - - AG AG AG - - Lactobacillus spp

4 + Rod - - - - - - AG AG - - Lactobacillus spp

5 + Rod - - - - - - AG AG AG - Lactobacillus spp

6 + Rod - - - - - AG AG AG AG - Lactobacillus spp

7 + Rod - - - - - AG AG AG AG - Lactobacillus spp

8 + Rod - - - - - - AG AG - - Lactobacillus spp

Key: A = acid, G = Gas, + = Positive, - = negative

S/N Probable Organism Frequency of Occurrence Percentage of occurrence (%)

1 E. coli spp 5 41.67

2 Salmonella spp 3 25

3 Shigella spp 2 16.67

Table
4 2: Morphological and Biochemical
Vibrio cholera 2 Characterization of Test 16.67
Isolates of Lactobacillus Species

Table 3: Frequency of occurrence of bacterial isolates

4.2 Antimicrobial Assay

The antimicrobial activity of Lactobacillus species against Salmonella spp, E.coli, Shigella

spp and Vibrio cholera was assessed. A clear free zone of bacterial growth was observed

indicating the antimicrobial activity of Lactobacillus species against the enteropathogens.

The result of this assay is presented in figures 1, 2, 3 and 4 in this chapter. However, the

antimicrobial activity of ciprofloxacin against the various enteropathogens was evaluated.

This was done to assess the effectiveness of the Lactobacillus against the pathogens using the

ciprofloxacin as the ‘gold standard’.

30

25
Zone of Inhibtion (mm)

20

15
Salmonella
10 Ciprofloxacin

0
1 2 6 7

Salmonella spp
Figure 1: Antimicrobial activity of Isolated Lactobacillus against Salmonella spp
 30

25

Zone of Inhibition (mm)

20

15
E. coli
10 Ciprofloxacin

0
1 2 6 7

E. coli
Figure 2: Antimicrobial activity of Isolated Lactobacillus against E. coli

25
Zone of Inhibition (mm)

20

15

10 Shigella spp
Ciprofloxacin
5

0
1 2 6 7
Shigella spp
Figure 3: Antimicrobial activity of Isolated Lactobacillus against Shigella spp

25
Zone of Inhibition (mm)

20

15

10 Vibrio cholerae
Ciprofloxacin
5

0
1 2 6 7
Vibrio cholerae

Figure 4: Antimicrobial activity of Isolated Lactobacillus against Shigella spp

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