ISOLATION AND ANTIBIOTIC SENSITIVITY PROFILE OF Salmonella typhi FROM

STOOLS OF PATIENTS IN UMUAHIA

BY

IBENACHO, UGOCHI CONCILIA

MOUAU/11/17907

A PROJECT RESEARCH WORK SUBMITTED TO THE DEPARTMENT OF

MICROBIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE AWARD OF THE BACHELOR OF SCIENCE (B.SC) DEGREE IN
MICROBIOLOGY.

1
 CERTIFICATION

This is to certify that Ibenacho, Ugochi Concilia, a student of the Department of Microbiology

with MOUAU/11/17907 has satisfactorily completed the requirements for the award of the

Bachelor of Science (B.Sc) Degree in Microbiology. The project is original and has not been

submitted in part of in full for any other University.

……………………………. …….. ……………….

Dr. E. O. Ekundayo Date
Supervisor

……………………………. …….. ……………….

Prof. V. C. Eze Date
Head of Department

2
 DEDICATION

This work is dedicated to my beloved husband for his care and financial assistance throughout

my stay as an undergraduate and also family, friends and well wishers for the support and

encouragement

3
 ACKNOWLEDGMENTS

I acknowledge the Almighty God for his mercies and favour upon my life throughout my stay as

an undergraduates in the pursuit for this course. My sincere gratitude also goes to my project

supervisor Dr. E.O Ekundayo. Also my amiable Head of Department Prof. V.C Eze and the

Dean of the College of Natural Science (COLNAS) Prof. I.I Ijeh, also to the project co-ordinator

Mrs. Kelechi Edward for her advices and assistances and other lecturers for impacting greatly

into my life. My profound gratitude also goes to my parents Mr. and Mrs. Paschal Ibenacho for

his parental and financial supports and to my loving husband Engr. Emmanuel Chidi for his

caring throughout my academic persuit and my siblings, Engr. Ikenna, Uchechi, Chukwuebuka

for their encouragement.

Finally my sincere thanks goes to my worthy friends for their great support and contributions and

also to my colleagues worthy to mention for their support during my academic career. I say God

bless you all. Amen

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 TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgements iv

Table of Contents v

.List of Table viii

List of Figure ix

Abstract x

CHAPTER ONE

1.1 INTRODUCTION 1

1.2 Aims and objectives 4

CHAPTER TWO

2.0 LITERATURE REVIEW 5

2.1 Prevalence / incidence 5

2.2 Isolation and identification of Salmonella 7

2.3 Culture 8
2.4 Anti-microbial resistance 8

5
2.5 Widal agglutination test 11

CHAPTER THREE

3.0 MATERIALS AND METHODS 13

3.1 Study area 13

3.2 Sample collection 13

3.3 Media preparation 14

3.4 Isolation and identification of salmonella species 14

3.5 Culture 14

3.6 Widal tests 14

3.7 Gram Staning 15

3.8 Anti microbial Susceptiblity testing 15

3.9 Biochemical tests 16

CHAPTER FOUR

4.0 RESULTS 18

CHAPTER FIVE
5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION 27
5.1 Conclusion 29
5.2 Recommendation 30
References 31
Appendix 37

6
 LIST OF TABLES

Table Title Page

1: Comparison of culture and widal tests for diagnosis of typhoid

among the study population 21

2: Antibiotic sensitivity profile of the isolates 22

3: Diameters of zone inhibition of various antimicrobial agents against

the isolates (stated interpretative reference range in brackets) 23

7
 LIST OF FIGURE

Figure Title Page

1 Prevalence of Salmonella spp in stool sample in Umuahia 20

2 In vitro Antimicrobial Susceptibility profile of 33 Salmonella 26

isolate

8
 ABSTRACT

Fifty (50) stool samples and corresponding venous blood specimens were collected from patients
who presented for widal test diagnosis of typhoid fever in five Hospitals or Clinics in Umuahia.
The stool samples were cultured on Salmonella Shigella agar (SSA) and the sera from the blood
specimens were used to screen for antibodies to febrile antigens of Salmonella typhi and
Salmonella paratyphi A, B, and C. Out of the 50 stool samples cultured, 33(73.33%) produced
significant growth on SSA. The isolates were characterized by colonial morphology, Gram
staining reaction and Biochemical tests and were provisionally identified as Salmonella specie.
The antibiotics sensitivity profile of the 33 isolates was done using the Kirby-Bauer disc agar
diffusion technique. All the 33(100%) isolates were sensitive to Nitrofurontoin, 27(81.8%) were
sensitive to Gentamicin, 22(66.7%) to Ciprofloxacin and 19(57.5%) to Ofloxacin, while one
(3.30%) isolates was sensitive to Cefuroxime, all the isolates were resistant to Augmentin,
Ceftazidime and Ceftriaxone. Comparison of culture widal test results revealed that only
7(21.2%) of the culture positive cases had significant antibody titres (> 1:80) to Salmonella
febrile antigens. The result of this study show that there is a high level of antibiotic resistance
among Salmonella isolates and that widal test detected less than 50% of cultured positive cases.
The results of this work suggest there is a need for culture and sensitivity as the basis for
diagnosis and treatment of typhoid disease in the study area.

9
 CHAPTER ONE

1.1 INTRODUCTION

Salmonellae, a genus belonging to the family of Enterobacteriaceae. Comprises a large

and complex group of human pathogen that have been long associated with a wide

spectrum of infectious diseases, including typhoid fever and non typhoidal. Is one of

pathogen that causes death in infants and immune compromised and aged individual

through out the world. These organisms are transmitted through food, water,

environmental contacts, pet (animals) and from person to person, with morbidity rates in

developing countries 3 to 6 higher than in developed countries.

(Guerrant, 2001).

Among the enteric pathogens, Salmonella is a particular concern as the cause of enteric

fevers, food poisoning and Gastroenteritis. Although more prevalent in developing

countries, Salmonella, with its more than 2500 different serotypes is a leading cause of

food borne infections worldwide. Two hundred million to more than one billion cases of

diarrhea result worldwide due to Salmonella every year leading to 3 million deaths

(Goburn, et al., 2007), and this occurs mostly in developing countries especially in

children and young adult. (Kasper, et al., 2005; Niyogi, 2005; Khatun, et al., 2011).

Typhoid and paratyphoid fevers (enteric fevers) are acute systemic infections caused

mainly by the bacterium Salmonella enterica serotype typhi and other serotypes of

Salmonella paratyphi A, B. and C (Buckle et al., 2012). It continues to be a global health

problem, especially in the tropics and sub-tropical countries. Over 27 million person

suffer from this disease annually (Buckel et al., 2012). Salmonella

10
causes mild to severe forms of intestinal tract infections. It can cause self-limited and the

more severe form of systemic typhoid fever (Kasper et al., 2005; Goburn et al., 2007).

These infections are prevalent in developing countries where lack of clean water supply,

lack of proper sewage disposal system and flies aggravate the spread of the disease

(Kasper, et al., 2005). Salmonella infections can be divided into two major groups of

clinical importance; Typhoidal salmonellosis (Salmonella typhi and Salmonella

paratyphi) and non Typoidal salmonellosis (all Salmonella serovars) Abdullahi, 2010).

Human Salmonellosis is usually self-limiting, and antimicrobial treatment is seldom

required. Reports have indicated that antimicrobial treatment for uncomplicated

Gastroenteritis does not reduce the severity or duration of symptoms; in contrast, it may

prolong faecal excretion at convalescence and result in the emergence of resistant strains

(Abdullahi, 2010). Nevertheless, if spread beyond the intestine occurs, effective

antimicrobial treatment is essential, and the knowledge of the likelihood of resistance to

commonly available drugs could be of considerable value to the clinicians.

Humans spread Salmonella mainly through the stool. Food borne illness among the

people and the transmission can occur when food and water are contaminated with stool

or through direct faecal – oral route. Human stool acts as an important reservoir of

Salmonella serovars that are grouped based on their cell surface antigen. Species isolated

from human stool are Salmonella typhi, S. paratyphi A, S. typhimurium, S. wrothington,

and S. enteritidis (Kumar, et al., 2009). Salmonella is a worldwide issue in public health

sector. People most at risk for serious complications due to food poisoning include older

adults, pregnant women, infant and people who have compromised immune systems.

Salmonellosis is manifested clinically in all hosts by one of three major syndromes, acute

11
systemic infection, acute enteritis or chronic enteritis (Dworkin, et al., 2001). Symptoms

usually include headache, nausea, vomiting, fatigue, gastroenteritis, abdominal cramps,

bloody diarrhea with mucus and sometimes reactive arthritis (Reiters syndrome)

(Dworkin, et al., 2001). Following dehydration with renal insufficiency and death may

occur.

Drug resistance is the decreased sensitivity or the complete insensitivity of

microorganisms to drugs that cause cell death or inhibition of growth (Sharma, et al.,

2005). Microbial resistance of Salmonella is an emerging global challenge, especially in

developing countries where there is an increased misuse and abuse of antimicrobial

agents in humans and animals (Kasper, et al., 2005). In most developing countries,

laboratory investigations of Salmonella are diagnostic challenges due to the lack of

adequate facilities that enable culture and antimicrobial susceptibility testing. As a result,

there is limited awareness of the prevalence of infections and antimicrobial resistance

(Cook, et al., 2003; Sharma, et al., 2005). Also, the injudicious use of antibiotics by

patients and physicians like in many developing countries has led to increased antibiotic

resistance, and in turn reduced therapeutic efficacy in these countries (Okeke, et al.,

2007; Asrat, 2008). Antimicrobial resistance has complicated the selection of antibiotics

for the treatment of enteric bacterial pathogens, particularly to commonly used

antimicrobial agents such as Ampicillin, Tetracycline and Trimethoprim –

Sulfamethoxazole (ISenbarger, et al., 2002). Examining the antibiotic susceptibility

pattern of pathogens is important towards folioing treatment to the ever changing

resistance patterns and distribution of pathogenic bacteria. Hence, there is a great need

susceptibility pattern of different bacterial agents which cause enteric infections in order

12
 to introduce effective treatment for gastrointestinal illness.

1.2 Aims and objectives

1. To access the frequency of isolation of Salmonella species from stool samples of people

undergoing widal test

1.3. LITERATURE REVIEW

Salmonellae, a genus belonging to the family Enterobacteriaceae, comprises a large and

complex group of human pathogens that have long been associated with a wide spectrum

of infectious diseases, including typhoid fever and non-typhoidal Salmonellosis. They are

non lactose fermenters, motile and gas producer Gram negative rods (Arisa and Murray,

2009). Transmission of these organisms are from to person through faecally contaminated

food, water or through direct faecal oral route (Coburn, et al., 2006; Kumar et al., 2009).

They invade the mucosa of the small and large intestines and produce inflammation.

Invasion of epithelial cells induces an inflammatory reaction which causes diarrhea due

to Salmonella (Huruy et al., 2008). Salmonellosis is a disease condition caused by a

large group of bacteria of the genus Salmonella. Human Salmonellosis is usually self-

limiting and antimicrobial treatment is hardly required.

1.4 Prevalence / incidence

Salmonellosis caused by Salmonella species is a global infection with about 200, 000

deaths annually (Crump et al,, 2004; Nagshetly et al., 2010). The disease is a cause for

concern and major public health problem in developing countries (Asia and Africa)

especially in Nigeria due to poor sanitary conditions and lack of inadequate potable water

13
supply (WHO, 1996; Anita, et al., 2002; Doughari, 2005).The importance of

salmonellosis in public health sector is growing concern day by day through the world

and over the several decades, there have been significant shift in predominant Salmonella

serovars associated in human infection (Steven et al., 2011).

The World Heath Organization (WHO) estimated an annual infection rate of 21.6 million

with approximate death rate of 600 000 per year, with highest percentage of these rates

occurring in Africa and Asia. With the high incidence of Salmonellosis worldwide, the

highest incidence of infection is among the very young and elderly. Mortality higher in

children less than one year old. The increase of the susceptibility of this age group may

be due to the fact that the children less than two month old produce little hydrochloric

acid, a natural barrier of many microorganisms (Corburn, 2006).

Salmonella in the past has cause tremendous loss to the society in many countries around

the world . Two to four million cases have been reported annually and yet a significant

number of cases have been unreported world wide.

In Nigeria , student in the past have carried out a convoluted analysis to explore the

incidence of Salmonellosis among different sub-population. Reports have suggested a

high rate of prevalence up to 60% in Anyigba, Kogi State. Among patients suspected to

be suffering from Typhoid fever based on physical and cultural examination (Sule,et ,al,

2012).

Another study was undertaken to determine the prevalent Salmonella enteric serovars in

Jos, Abuja and Lagos to evaluate their Antimicrobial Suseptibities to routinely used

antibiotics with the ultimate aim of establishing which antibiotic can be used for empiric

14
 treating of Salmonella infection in this locality.

From this study, the prevalence rate of Salmonella was 1% and 2.3% and17.5%

respectively (Akinyemi et al 2007, Cejetan et al , 2013,Opajobi et al, 2014)

The wide spread occurrence and distribution of Salmonella infection in Ethiopia has

increase, this due to contamination of food, water, and poor sewage disposal system. The

very young, the elderly, and the immune compromised individuals are particularly more

susceptible to Salmonella infection at a lower infective dose than healthy adults

(Birhanseslassie and Williams, 2013).

Another study conducted in Gondar town health institution in Ethiopia among the

patients have diarrhea showed the isolation rate of Salmonella species to be lower than

the previous years.

This is attributed to the increase awareness of the people about personal and

environmental hygiene, made by the health institutions and other partner (Mache, 2001).

1.5 Isolation and identification of Salmonella

In this study, Salmonella species isolated were S. typhimurium and S. paratyphi. The

frequency of isolated of salmonella from diarrhea cases in developing countries is low

(Georges et al., 1984, WHO, 1995). The isolated Salmonella species is 2.7% of young

urban children (Suan et al., 1995), that Salmonella causes about 1.5% Gastroenteritis

cases in most developed countries. S. typhimurium belongs to the groups that has been

described as ubiquitous, members of there groups are known of causing classical food

poisoning and infantile and traveler’s diarrhea (Varnam and Evans 1991). Transmission

of infection may result from person to person or through contaminated water. The
15
 isolation of this bacteria from clinical and laboratory samples can also increase the

susceptibility of confirming typhoid fever. (Shrivastara et al., 2011 and Hohman EL,

2011). Theortically, it is possible to eliminate the Salmonella causing enteric fever since

they can survive in human host and spread by contaminated food and water. The high

prevalence of this disease in developing countries due to lack of adequate sewage

disposal and water treatment. (Gizachew, Andualem 2011) and World Health

Organization (2006).

1.6 Culture
Stool culture remains the Gold standard method for the detection of Salmonella infection,

because of its high level of specificity. However, it could be used to in resource limited

areas where laboratory capacity is limited with clinical investigation. The basis for

diagnosis of Salmonella infection (typhoid fever) is a positive result for widal test, stool

culture. Many developed countries, lack adequate infrastructure to perform culture and

may depend on cheaper serological tests Kariuki. S. (2008).

1.7 Anti-microbial resistance

The emergence of antibiotic resistant Salmonella strains is increasing, and multi-drug

resistant (MDR) serovars with substantial resistance to the traditional first –line

antibiotics have been reported (Fashae, 2010; Crump, et al., 2011; Wandili, et al., 2003).

Antimicrobial resistance of Salmonella is an emerging global challenge especially in

developing countries, where there is an increased misuse of Antimicrobial agents by

humans and animals (Kasper, et al., 2005). This has become a major problem associated

with the control of diarrhea. Various authors have reported high rates of resistance to

commonly used, inexpensive, oral antimicrobial agents such as Ampicllin, Tetracycline,

16
and Trimethoprim Sulphamethaxozole (Kesper, et al., 2005). Antibiotic therapy for

Salmonella Gastroenteritis has long been a debated matter because of the idea that

antibiotic administration prolongs Salmonella excretion needs antibiotic therapy (Sur, et

al. 2004). In recent years, an increase in occurrence of antimicrobial resistance. Among

Salmonella has been observed in many countries such as Asia, Africa and China (Xia et

al., 2009; Yan et al., 2010). This includes resistance to quinolones and third generation

cephalosporins.

The progressive increase in antibiotic resistance because of overuse and misuse of

antibiotics in the treatment of diarrhea in developing countries is becoming a critical area

of concern (Vu Nguyen et al., 2006; Ecker, et al., 2011; Reda, et al., 2011).

Most Ethopican studies conducted respectively , the prevalence of Salmonella (5.3% -

15.4%), was high with antibiotic resistance pattern ranged from 0% in case of

Ciprofloxacin and Nalidixic acid to 100% case of Ampicillin (Tiruneh, 2009; Bayene et

al., 2011; Reda, et al., 2011). With the increasing use of extended spectrum

Ceohalosporins and Fluoroquinolones, antimicrobial resistance in this part of the world

may have complicated the problem. Emerging drug resistance among circulating serotype

typhi strains in Vietnam and elsewhere has complicated the treatment of Salmonellosis

and heightened the need for rapid accurate diagnosis, and the appropriate and selective

use of antimicrobial agents to which the organism the organism has thus far remained

susceptible.

The have become increasingly resistant to most commonly used antimicrobials from time

to time, which results in some challenges in selecting the drug for therapeutic

17
management (Sharma, et al; 2005). The emergence of antibiotic resistant Salmonella is a

serious problem in antimicrobial therapy globally. The incidence varies with the area of

isolation of these strains. The progressive increase in antibiotics resistance among these

pathogens in developing countries is also becoming a critical area of concern (Huruy et

al., 2008). In developing countries like Ethiopia, resistance maybe acquired mostly by

selective pressure due to indiscriminate and misuse of antibiotics (Chesbrough, 2009;

Huruy et al., 2011). This leads to the emergence of resistant strains of is 2011). This leads

to the emergence of resistant strains of Salmonella, which may be difficult for treatment

and prevention (Asrat, 2008; Beyene, et al., 2008). In the 1940s, was treated successfully

with sulfa-drugs. In the 1950’s, it was treated with Tetracycline (Coburn et al., 2006). In

the 1970’s, Ampicilin was the drug of choice for the treatment of bacilliary dysentery.

After the pathogen began to develop resistance to Ampicilin, the new drug

Trimethoprim-sulphamethoxozde (TMP-SXT) was used. Until about 1960, nearly all

Salmonella were Sensitive to a wide range of antimicrobial agents, but since 1962,

emergence of resistance, frequently plasmid mediated have appeared in Salmonellae

worldwide.

The relative importance of antibiotic resistance, and the serotypes in which it occurs

differs from country to country (Coburn, et al., 2006). Antimicrobial resistance has really

become an unfortunate outcome which is simply an adaptive response in which

microorganisms begin to tolerate an amount of drug to which it was previously

susceptible. The development of these mechanisms for circumventing or inactivating

antibiotic drugs is largely due to the genetic versatility and adaptability of microbial

populations. Strains of Salmonella resistant to Chloramphenicol and other recommended

18
 antibiotics have been identified in several parts of Latin America, Asia and Africa. While

resistance to single antibiotics occurs, development of multi-drug resistance by this

bacterium has complicated the health problem. Salmonella is one of the most resistant

organisms with multi-drug phenotype (Keith and John, 2005). Infections caused by

resistant Salmonella and indeed other pathogens results in significant morbidity and

mortality and contribute to escalating health care cost worldwide.

A convoluted study in Egypt and the Egyptain ministry of health estimated infant

mortality as 72 per 1,000 (7.2%), of which 43 cases (4.3%) were due to acute enteric

infections. In a more recent report, mortality decreased in the early 1990s to 34 cases per

1000, of which 9 cases were due to diarrhea.

Descriptive characterization of bacterial pathogens involved, which induces antimicrobial

sensitivity remains poor as few such studies have been conducted in Egypt on the

causative bacterial agents and their antibiotic resistance profiles in the indigenous

population (Miller and Hirschhorn, 1995).

Knowing the prevalence and the local patterns of antimicrobial susceptibility of

Salmonella species is important for reducing the burden of the disease (Hurny et al.,

2008). There is therefore a great need to establish the identify and antibiotic susceptibility

patterns of Salmonella which is an important bacterial agent in gastroenteritis in order to

augment effective treatment of Gastroenteritis.

1.8 Widal agglutination test

Typhoid fever, caused by Salmonella typhi (S. typhi) is a global infection that occur

19
annually with the highest incidence (1000 cases per 100,000 people per year) in

Southeast Asia (Thong, et al., 1995). The incident rate of typhoid fever in Asia pacific

region is estimated at more than 100 cases per 100, 00 population per year. The highest

burden of disease has been observed in children. Widal, a serological diagnosis test for

enteric fever was founded in 1896 by Georges Fernand Isidore Widal (Sndhar Rao,

2009). It is an agglutination reaction demonstrating the presence of lipopolysaccharides

(LPS), somatic (0) and flagella (H) agglutinins to Salmonella typhi in the serum of a

patient using suspensions of H and antigens. Commercial kits are available for antigens

of Salmonella paratyphi A. B. and C. the recommended method of performing the widal

test is by the tube agglutination technique, where two-fold serial dilutions of the subject

is serum from 1:20 to 1: 280 are tested (Sndhar Roa, 2009). The widal test has been used

extensively in the serodiagnosis of typhoid fever and so remains the only practical test

available in most developing countries. Isolation of Salmonella typhi from the blood,

faeces, urine, bone marrow or other body fluids is an important disgnoistic tool. In

addition, unavailability of microbiological facilities and the long waiting time for culture

results have been identified as reasons for the preference for the widal tests

(Cheesbrough, 2009). One of the major challenges of the widal test is cross-reactivity due

to some bacteria for same genus often produce false positive results. Hence, the positive

results must correlate clinically before prescribing medicine. Typhidot is another rapid

test used to ascertain the diagnosis of typhoid fever, but not cost effective as widal. So,

widal test is the choice for typoid fever especially in rural areas.

20
 CHAPTER TWO

MATERIALS AND METHODS

2.1 Study area

This study was conducted in Umuahia, the Capital City of Abia State, located in the

South –Eastern parts of Nigeria. The state is surrounded by other states such as Imo,

Anambra and some South-South States like Akwa-Ibom and Cross River States.

Blood and stool specimens were collected from patients undergoing medical examination

at different hospitals and laboratories in Umuahia, including Michael Okpara University

Health Services and from National Root Crops Research Institute, Umudike. The patients

that participated in this study range from rural and urban dwellers of diverse socio-

economic and ethnic backgrounds. The consent of the patients were sought after they

were fully educated on the scope of the research, and its potential benefits. Consented

patients were enrolled in the study.

2.2 Sample collection

Clinical samples of blood and stool were collected from each patient and processed for

the investigation of Salmonella infection.

Fifty (50) stool samples were collected in clean, sterile, wide mouthed containers,

without disinfectant or detergent residue and tight-fitting leak-proof lids. Blood samples

21
 were collected using 5ml syringe and transferred into small, clean containers, without

anti-coagulant (EDTA).

2.3 Media preparation

The media used were Salamonella Shigella agar (SSA), nutrient agar and Muller Hinton

agar. The Salmonella Shigella agar was used to culture bacteria. The media was prepared

according to the manufacturers’ instructions. The prepared were then sterilized by

autoclaving for 15minutes at 1210C pressure, and allowed to cool for some minutes, and

depense into agar plate and allowed to cool for Gelification.

3.4 Isolation and identification of Salmonella species

Stool samples were streaked on Salmonella- Shigella agar (SSA) with the aid of a sterile

wire loop. The plates were then inverted and incubated at 37 oC for 18-24hr. Cultural and

morphological characteristics of the isolates were observed thereafter, and the isolates

duly identified following standard microbiological methods.

3.5 Culture

All the stool samples were brought to the laboratory within the space of one hour

30minutes of collection. A portion of the stool was inoculated into peptone water and

incubated at 370C over night. The broth culture was shaken after which a loopful was

inoculated into SSA and incubated at 370C for 18-24hr.

The colonial morphology of the isolates were observed. The isolates were sub-cultured

into the Nutrient agar using streak method, and incubated at 370C for 18-24hr.

22
3.6 Widal tests

Widal agglutination test was performed detect antibodies to Salmonellae typhi using O

and H antigens of Salmonellae typhi and Salmonella Paratyphi A,B,C antigens as

described by (House, et al., 2001). Spin react kit were used, before carrying out the test,

the serum samples were places into four places on clean tile, serum was dropped on the

tile and was performed according to the manufacturers’ instruction, and emulsified and

rocked about 5second to achieve agglutination which indicates the presence of typhoid.

3.7 Gram Staining

A smear was made by picking colony using a sterile wireloop. A drop of physiological

saline was drop on clean, grease free glass slide. This smear was air-dried, then heat fixed

by passing the slide over a busen flame four times. The slide was then placed on the

staining rack and flooded with crystal violet for 60 seconds. This was washed off with

distilled water, Lugols iodine solution was added left for 30 seconds, and it washed off

with distilled then it was decolorized with alcohol for 20seconds and washed

immediately. Few drops of safarnin was added, left for 30seconds and washed with

distilled water, and was viewed under the microscope using x 40 objectives and x 100

objectives for oil immersion.

3.8 Anti microbial Susceptibility testing

Organisms isolated were tested for antimicrobial susceptibility. Colonies of each

isolated was picked using sterile wire loop and used to make turbid suspension which

was checked and against the turbidity of a chemical standard 0.5 McFarland

suspension. Sterile swab stick were dipped into the standardized of bacterial isolates and

23
 swabbed on Muller Hinton agar and allowed to penetrate the medium for 8 minutes.

Antibiotics disc Gram negative disc were aseptically placed on inoculated plates with the

aid of sterile forceps. Plates were inverted after 20 minutes of application and incubated

aerobically at 370C for 18-24hours. After 18-24hr incubation period, transparent ruler was

used to measure the diameter of each of zone of inhibition in millimeter.

3.9 Biochemical tests

Biochemical tests were carried out on each isolate obtained from the stool culture, among

the biochemical test are citrate test, indole test, methyl red test and voges proseakeur.

a. Indole test

The test is important in the identification of Enterobacteria. The organisms breakdown

the amino acid, typtophan to release indole. The test organisms was inoculated into 2ml

of peptone water and was incubated at 37 0C for 24-48hours. 10ml of kovacs reagent was

added, it was allowed to stand for 5mins. The absence of red colour was indicative of

negative tests, while the appearance of red colour was indicative of positive test.

b. Citrate tests

This tests is carried out to differentiate Entrobacteriaceae from other bacteria. Citrate

tests utilizes simmons citrate media to determine if a bacteria can utilizing citrate at

source of carbon and energy source.

The simmons was weighed and sterilized by autoclaving, it was transferred into bijou

bottles and allows to cool, using a sterile wire loop. The medium was streaked with tests

organisms and stabbed. Incubated at 37 oC for 24-48hrs. The presence of blue color

24
 indicate a positive tests.

c Methyl red Tests

This test is used in identification of Enterobacteria. The methyl red tests were conducted

by inoculating a colony of the bacteria culture in 0.5ml sterile glucose phosphate peptone

broth and incubated at 370C for 120 hours (5 days), 5 drops of methyl red indicator was

added using pasture pipette and was mixed thoroughly. Bright red colouration was

indicative of positive result while yellow colouration indicated a negative result.

d. Voges Proskeauer tests

This tests is occasionally used to assists in differentiation of Enterobacteriacea. 2ml of

glucose phosphate peptone water was inoculated into the tests organisms was incubated

at 37oC aerobically for 24- 48hours. 1% of potassium hydroxide was added and left for

60 minutes. No pink colouration indicates negative tests while pinkish color indicates

positive tests.

25
 CHAPTER FOUR

4.0 RESULTS

A total of fifty (50) patients were diagnosed for typhoid fever for this study, out of this,

27 were female (54%) and 23 (45%) were male. Of the 50 samples 33(73.33%) shows

significant growth 17(34%) shows no significant growth. This is summarized in figure 1

the ages of this study participants ranged from 10-80, isolation rate from stool culture

was more than that of the serological examination (blood specimen). The number of

isolates for both sexes decreased with increase in age from 50. This is summarized in

table 1 comparison of culture and widal tests for diagnosis of typhoid among the study

population.

Antimicrobial susceptibility testing was determined by the observation of various

antimicrobial agents against the isolates (stated interpretative reference range in

brackets). This is summarized in table 3.

The highest number of samples was from the age groups of (10-20) and the least was

from the age group of 51-60 n=1, 61 – n = 1. Salmonella was isolated in all the age

groups, of has high incidence of 21(63.63%) in the age group 10-20yrs, 7(21.21%) in the

age group of 21-30years, 1(30.30%) in the age group of 31-40 2(60.60%) in the age

group of 5 1-60 and 1(30.30%) in the age group of > 61. 0ut of the 8 antibiotics used,

Salmonella typhi were only sensitive to five, they were Gentamicin, Ciprofloxacin

26
Nitfurontoin, Ofloxacin, Cefuroxime. Only the Nitrfurontoin was able to show (100%) to

the isolates. In all the age groups, the highest level of susceptibility was detected for

Nitrfurontoin 33(100%), followed by Gentamicin, Ciprofloaxcin, Cefuroxime.

Nitrfurontoin (100%), Gentamicin (81.8%) 3(9.09%) was intermediate, 3(9.09%) was

resistant, Ciprofloaxcin 22(66.66%) 3(9.09%) was intermediate, 8(24.24%) was resistant,

Ofloxacin 19(57.5%) 10(30.30%) was intermediate, 4(12.12%) was resistant

respectively. This is summarized in table 2.

In vitro antimicrobial susceptibility profile of 33 Salmonella isolates. This is summarized

in figure 2

The isolates were completely resistant to Augmentin, Ceftriaxone, and Ceftazidime

probably because of multi drug resistant or due to overuse of these drugs.

27
 50 Stool Samples

17(34%) No significant 33(73.33%)

growth Significant growth

21(53%) from 12(36%) from male

female patients patients

Antibiotic
sensitivity
test

NIT = 33(100%), R = (0%), I = (0%) Aug, Ceftri, and

Cefta did not
Gen = 27 (81.8%) R = 3(9.09%), I = 3(9.09%)
produce observable
CPR = 22(66.66%) R = 8(24.24%), I = zone of inhibition
3(9.09%)

OFL = 19(57.5%) R = 4(12.12%), I =

10(30.30%)

I = Intermediate

S = Sensitive

R = Resistance

NTI = Nitfurontion

GEN = Gentamicin

CPR = Ciprofloxacin

Ofl = Ofloxacin

28
Aug = Augmentin

Ceft = Ceftazidime

Ceftri = Ceftriaxome

Figures 1: Prevalence of Salmonella spp in stool samples in Umuahia

Table 1: Comparison of culture and widal tests for diagnosis of typhoid among the study
population.

Sex No. of Culture Widal test

samples
(No. with Samlonella spps (No. (%) with significant
isolates) antibody titre)

Female 27(54%) 21(63.63%) 5(74.42%)

Male 23(46%) 12(36.36%) 2(28.6%)

Total 50 33 7

29
Table 2: Antibiotic sensitivity profile of the isolates

Antibiotic No of sensitive No with intermediate No of resistant

isolates response isolates

Nitrofurontoin 33(100%) 0(0%) 0(0%)

Gentamicin 27(81.8%) 3(9.09%) 3(9.09%)

Ciprofloxacin 22(66.66%) 3(9.09%) 8(24.24%)

Ofloaxcin 19(57.5%) 10(30.30%) 4(12.12%)

Cefuroxime 1(3.030%) 0(0%) 0(0%)

30
Table 3: Diameters of zone inhibition of various antimicrobial agents against the isolates (stated
interpretative reference range in brackets)

No. of Nit Gen Cpr Ofl Cxm Aug Ceft Cefta

isolates (> 17) (> 15) (> 21) (> 21) (> 35)
1 20 18 21 22 - - - -
2 21 17 23 24 - - - -
3 18 20 22 19(I) - - - -
4 19 22 25 20 - - - -
5 22 12(R) 14(R) 18(I) - - - -
6 19 19 24 22 - - - -
7 18 17 15(R) 18 (I) - - - -
8 20 18 22 14(R) - - - -
9 23 18 22 18(I) - - - -
10 21 17 15(R) 22 - - - -
11 19 22 21 19(I) - - - -
12 24 17 24 23 - - - -
13 20 18 15(R) 18(I) - - - -
14 22 12(R) 23 24 - - - -
15 25 18 22 22 - - - -
16 19 19 15(R) 19(I) - - - -
17 18 17 23 21 - - - -
18 20 19 15(R) 22 - - - -
19 17 21 22 18(I) - - - -
20 20 18 21 19(I) - - - -
21 19 17 15(R) 22 - - - -
22 18 20 22 23 - - - -
23 23 19 21 25 - - - -
24 18 14(I) 16(I) 14(R) 35(S) - - -
25 20 18 23 24 - - - -
26 19 13(I) 22 12(I) - - - -
27 20 17 17(I) 23 - - - -
28 22 13(I) 22 12(R) - - - -
29 19 18 14(R) 25 - - - -
30 20 17 21 23 - - - -
31 18 23 16(I) 14(R) - - - -
32 23 12(R) 22 24 - - - -
33 19 17 24 21 - - - -

31
R = Resistant
I = Intermediate
S = Sensitive

Interpretative reference rage

Cipro = 21 Susceptible

= 16 -20 intermediate

= 15resistant

Gentamicin = 15 susceptible

= 13 – 14 intermediate

= 12 resistant

Nit = 17 Nitrofurontoin

= 15 -16 intermediate

= 14 resistant

Ofloxacin = 21 susceptible

= 12 resistant

= 18 – 19 intermediate

Cxm = 35 susceptible

Antibiotics

Nit = Nitrofurontoin

Gen = Gentamicin

Cpr = Ciprofloxacin

32
Ofl = Ofloxacin

Cxm = Cefuroxime

Aug = Augmentin

Ceft = Ceftazidime

Ceftri = Ceftriaxone

R = Resistant

I = Intermediate

33
Series1

34
 CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION

Hospital patients are generally assumed to reflect the relative importance and seriousness

of prevalent illness. Our investigation took advantage of random selection of patients

diagnosed for typhoid fever in hospital within Umuahia meteropolis (Abia State, Nigeria)

of the 50 samples of stools collected from patients in Umuahia, 33 produced growth,

while 17(34%) did not produce any significant growth. Salmonella typhi which attributes

to typhoid fever is a major public health associated with significant morbidity and

mortality in many counties. (Chang, et al., 1990). The prevalent rate of Salmonella in this

study is much higher than 1% obtained by (Opajobi et al., 2014). From Jos Nigeria and

higher than the 2-3% reported by (Cejetan et al., 2013) from Abuja, Nigeria.

The prevalence rate is also than the (60%) reported by (Sule, et al., 2012) in Ayiba Kogi

State.

The increase in prevalence rate could be dependent on the study centres as in the case of

Jos which is a referral centre where only complicated cases are treated, the increase is

also highly dependent on low sanitary condition of the town that characterizes most poor

resource countries, poor isolation of carriers which spread the disease the negligence of

the people to take antibiotics of ignorance of the spread and mode of transmission of

Salmonella or over the counter use of antibiotics with prescription. In our setting,

33(73.33%) of the isolates were confirmed to be Salmonella typhi, this confirms the high

prevalence of Salmonella typhi in Umuahia, due to lack of proper treatment and isolation

of carriers.

35
The highest number of isolates was recovered from 10-20 years. The high susceptibility

of young adult (Adolescent) to Salmonella infections has been documented. Gastric

hypoacidity or gut immaturity maybe contributing factor to the susceptibility, poor

hygiene and exposure to other diseases (Commonly found in young adult may suppress

their immune system, and high rate of exposure to quarantine (Vugia et al., 2004)

(Corburn, 2006). The number of isolates decreases with increase in age. Lowest number

of isolates was from male patients above the age of 70, this could be attributed to small

samples sizes finding from this study indicate that Salmonella isolation rates were higher

in females then in males, this have been observed by several researchers. Among the

reasons which have been given to account for this include behavioural factors propensity

of women to participate in food handling, preparation and consumption, most factors,

poor hygiene of cleaning the anus and washing of hands most seen in women (Opaejobi,

et al., 2014).

In this study, there was 100% resistant of the isolated strain to Augmentin Ceftazidime,

Ceftriaxone, this work showed that all the isolates were sensitive to Nitrofurontoin, due

to the fact Nitrofurontoin is not commonly used even in the primary care settings,

followed by Gentamicin 27 (81.8%) Ofloxacin 19(57.5%) Ciprofloxacin 22(66.66%) and

than Ceftriaxome, 1(30.30%).

In this study, Augmentin, Ceftazidime and Ceftrixome did not produce any inhibitory

zones which signify that, the isolates were completely resistant to these antibiotics. This

could be because these isolates produced enzymes that inactivated the antibiotics, or

probably because the Salmonella serotypes must have generated different types of hybrid

plasmid which must have consisted of the serotypes – specific virulence plasmids which

36
 carried a gene cassettes and most of gene cassette contains resistance gene responsible to

resistance of there conventional antibiotics.

Data reported in this study were generally similar to those of earlier (Tesfanye, et al.,

2014).

Studies in terms of distributions among various age groups and antimicrobial resistance

to the convention antibiotics. This maybe due to those fact that Salmonella typhi causing

human illness were colonial in origin (Su et al., 2001; Nastabi et al., 1994).

The antibiotic sensitivity profile of this work showed that susceptibility patterns of

bacteria isolates are constantly changing and as such the antibiograms of the bacteria

isolates should be known before the initiation of treatment by a physician.

The observation of this study is however, cause of concern and an indication that

presently antibiotics may not longer effective for that treatment of typhoid fever in

Umuahia. Factors which encourage antibiotic resistance development such as the over the

counter sale of potent antibiotics, under dose, overdose, practiced in Umuahia contributed

that problem of antibiotic prevalent.

5.1 Conclusion

Indiscriminate use of antibiotics has resulted to the emergence and survival of resistant

strains of bacteria isolates, thus antibiotic sensitivity is important to determining

antibiotic of choice. Nitrofurontoin and Gentamicin and Ciprofloxacin as revealed this

work were effective against most of the by bacterial isolates.

I suggest that more work should be done on there antibiotics to determine their toxicity

37
 and hence their potency for treatment of S. typhi infection. I conclude that indiscriminate

use of antibiotics both in primary care settings should be restricted and I suggest that

Salmonella isolates should be confirmed in a reference laboratory to avoid

misidentification, and that the susceptibility zones for the reference strain against various

antimicrobial agent be within the acceptable ranges hence, the results of then evaluation

should be properly recorded and appropriate measure taken to solve any problem that are

encountered.

5.3 Recommendation

i. A more cautious selection and use of antimicrobial agents along with a continuous

monitor of resistance are critical to combat the increasing antimicrobial resistance in

Salmonella.

ii. Culture methods should be used for the isolation or the organisms, so as to subject than to

susceptibility test before administering drugs.

iii. Control strategies such as improving person public hygiene, preventing judicious use of

existing enteric vaccines and antibiotics.

iv. Production development of newer, more broadly protective vaccines.

v. Society should be enlightened especially on hygiene, drug abuse, eating habit.

vi. Carrier should be isolated and treated.

vii. Pre-disposing ailments should be treatment to make the immune system active.

viii. Over the counter use of antibiotics should be discouraged.

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APPENDIX 1

Antibiotic sensitive profile of the isolates to all the antimicrobial agents

Antibiotics % Sensitive % Intermediate % Resistance

Nit 33(100%) (0%) 0(0%)

Gen 27(81.8%) 3(9.09%) 3(9.09%)

Cpr 22(66.66%) 3(9.09%) 8 (24.24%)

Ofl 19(57.5%) 10(30.30%) 4 (12.12%)

Cxm 1(3.030%) 0(%) 0(%)

Aug 0(%) 0(%) 0(%)

Ceftri 0(%) 0(%) 0(%)

Cefta 0(%) 0(%) 0(%)

45
 APPENDIX 2

Distribution of antibiotic sensitivity profile of the isolates according to age groups of patients.

Age No of Nit Gen Cpr Ofl Cxm Agu Ceft Cefta

group isolates

10-20 21 S I R S I R S I R S I R S T R

33 0 0 19 0 2 15 0 6 12 9 1

21- 30 7 7 0 0 4 3 0 5 2 0 5 0 2 1 0 0 - - - - - - - -

31 – 40 1 1 0 0 1 0 0 1 0 1 1 0 0 - - - - - - - - - - -

41 – 50 2 2 0 0 1 0 0 1 1 0 0 1 0 - - - - - - - - - - -

51 – 60 1 1 0 0 1 0 1 1 0 1 1 0 1 - - - - - - - - - - -

>61 1 1 0 0 1 0 0 1 0 0 0 0 0 - - - - - - - - - - -

46