Prevalence and Antibiotic Resistance Patterns of Salmonella Isolates in
Patients Visiting Worabe Health Center: Worabe, Southern Ethiopia.
M. Sc Thesis
By
Abdulmejid Muteba
May 2012
Haramaya University
Prevalence and Antibiotic Resistance Patterns of Salmonella Isolates in
Patients Visiting Worabe Health Center: Worabe, Southern Ethiopia.
A Thesis Submitted to the College of Natural and Computational Sciences,
Department of Biology, School of Graduate Studies
HARAMAYA UNIVERSITY
In Partial Fulfillment of the Requirement for the Degree of
MASTER OF SCIENCE IN MICROBIOLOGY
By
Abdulmejid Muteba
May 2012
Haramaya University
ii
APPROVAL SHEET OF THESIS
SCHOOL GRADUATE STUDIES
HARAMAYA UNIVERSITY
As Thesis Research advisor, I hereby certify that I have read and evaluated this thesis
prepared, under my guidance, by Abdulmejid Muteba, entitled ‘‘Prevalence and Antibiotic
Resistance Patterns of Salmonella Isolates in Patients Visiting Worabe Health Center,
Worabe, Southern Ethiopia’’. I recommend that it can be submitted as fulfilling of the
thesis requirement.
Ameha Kebede (PhD)
Major Advisor
Sissay Menkir (PhD)
Co-advisor
_________________
Signature
_______________
Date
_________________
_______________
Signature
Date
As member of the Board of Examiners of the Masters of Science Thesis Open Defense
Examination, we certify that we have read, evaluated the thesis prepared by Abdulmejid
Muteba and examined the candidate. We recommended that the thesis be accepted as
fulfilling the Thesis requirement for the Degree of Master of Science in Microbiology.
______________________
Chairperson
______________________
Internal Examiner
______________________
External Examiner
_________________
Signature
_________________
Signature
_________________
Signature
iii
_______________
Date
_______________
Date
_______________
Date
DEDICATION
I dedicate this thesis to my family for their love, care, unforgettable and valuable contribution
and encouragements in my education career and for their dedicated partnership while I was
performing this study and in the success of my life.
iv
STATEMENT OF THE AUTHOR
First, I declare that this thesis is my bona fide work and that all sources of materials used for
this thesis have been dully acknowledged. This thesis has been submitted in partial fulfillment
of the requirement for Masters of Science Degree at Haramaya University and is deposited at
the University Library to be made available to borrow under rules of the Library. I solemnly
declared that this thesis is not submitted to any other institutions anywhere for the award of
any academic degree, diploma, or certificate.
Brief quotations from this thesis are allowed without special permission provided that
accurate acknowledgement of source is made. Requests for permission for extended quotation
from or reproduction of this manuscript in whole or in part may be granted by the Dean of
School of Graduate Studies or the Head of Biology Department when in his or her judgment
the proposal use of the material is in the interest of scholarship. In all other instances,
however, permission must be obtained from the author.
Name of the author: Abdulmejid Muteba
Signature……………
Place: Haramaya University, Haramaya.
Date of Submission: April 2012
v
LIST OF SYMBOLS, ACRONYMS AND ABBREVIATIONS
AOR
Adjusted Odds Ratio
aw
Water Activity
CDC
Center for Disease Control
CI
Confidence Interval
COR
Crude Odds Ratio
EFSA
European Food Safety Agency
EHNRI
Ethiopian Health and Nutrition Research Institute
GPW
Glucose Peptone Water
LIA
Lysine Iron Agar
m.a.s.l.
Meters above Sea Level
MPW
Mannitol Peptone Water
NCCLS
National Committee for Clinical Laboratory Standards
PI
Pathogenicity Islands
SCV
Salmonella Containing Vacuole
SPI
Salmonella Pathogenicity Island
SSA
Salmonella Shigella Agar
T3SS
Type III Secretion System
TSI
Triple Sugar Iron
WHC
Worabe Health Center
WHO
World Health Organization
WTA
Worabe Town Administration
XLD
Xylose Lysine Deoxycholate
vi
BIOGRAPHICAL SKETCH OF THE AUTHOR
The author was born on August 7, 1984 in Awro village located near by a small town called
Mugo, Southern Nations, Nationalities and Peoples’ Regional State, which is about 240 km
far from Addis Ababa. He attended his elementary education at Chenchen Elementary School
and Quante Primary and Secondary School and secondary education at Mugo Secondary High
School and Dalocha Secondary High School. He completed his high school education at
Dalocha Secondary High School in 2005.
Upon successful completion of his high school studies, he joined Bahir Dar University in
2005 and graduated in July, 2007 with B.Ed. degree in Biology.
Soon after completion of his undergraduate studies, he became employed in Samara
University as a Graduate Assistant. He stayed and served in this University for one and half
years until he joined the School of Graduate Studies of Haramaya University to pursue his
M.Sc. degree in Microbiology in January 2009.
vii
ACKNOWLEDGEMENTS
First and foremost, it is my pleasure to express my heartfelt appreciation and special gratitude
to my major thesis advisor Dr. Ameha Kebede, for his advice, unreserved guidance, and
constructive suggestions, follow up and critical comments from the beginning to the end of
this study. In addition, my heart-felt thanks and special gratitude go to my thesis co-advisor
Dr. Sissay Menkir for his advice, unreserved guidance, and constructive suggestions and
follow up throughout this study.
I would like to thank all patient participants of Worabe Health Center, who gave me the
required information, as well as the health center laboratory technicians and out patient
department physicians for their cooperation in providing valuable in this study.
I would like to express my great gratitude to School of Graduate Studies of Haramaya
University for funding my research and Samara University for sponsoring my Masters of
Science education as well as the members of Institutional Research Ethics Review Committee
in the Harar Campus, Haramaya University for the genuine comments they made to produce
the ethical clearance.
I am especially grateful to the Ethiopian Health and Nutrition Research Institute, Addis
Ababa, particularly Mr. Endris Mohammed, the head of bacteriology section, as well as the
bacteriology section staff for providing me laboratory facilities to accomplish this work.
Last but not least, my special and heartfelt thanks are due to my colleagues and all those
people who have assisted me in various ways while doing this thesis.
viii
TABLE OF CONTENTS
DEDICATION
iv
STATEMENT OF THE AUTHOR
v
LIST OF SYMBOLS, ACRONYMS AND ABBREVIATIONS
vi
BIOGRAPHICAL SKETCH OF THE AUTHOR
vii
ACKNOWLEDGEMENTS
viii
TABLE OF CONTENTS
ix
LIST OF TABLES
xii
LIST OF FIGURES
xiii
ABSTRACT
xiv
1. INTRODUCTION
1
2. LITERATURE REVIEW
5
2.1. General Characteristics of Salmonella
5
2.2. Taxonomy of Salmonella
6
2.3. Salmonellosis
6
2.4. Epidemiology and Transmission of Salmonellosis
7
2.5. Signs and Symptoms of Salmonellosis
8
2.6. Pathogenesis
9
2.7. Global Prevalence and Incidence of Salmonellosis
10
2.8. Prevalence of Salmonellosis in Ethiopia
12
2.9. Control and Prevention of Salmonellosis
12
2.10. Treatment of Salmonellosis
13
2.11. Antibiotic Resistance
14
2.11.1. Effects of antibiotic resistance
15
2.11.2. Antibiotic resistance in Salmonella
15
3. MATERIALS AND METHODS
17
3.1. Description of the Study Area
17
3.2. Study Design
17
ix
TABLE OF CONTENTS Continued
3.3. Study Population
17
3. 4. Sample Size Determination and Sampling Procedure
17
3.6. Methods of Data Collection and Quality Control
18
3.7. Isolation and Identification of Salmonella
19
3.7.1. Bacteriological culture
19
3.7.2. Isolation of Salmonella Species
19
3.7.3. Biochemical identification
20
3.8. Antibiotic Resistance Test
21
3.8.1. Inoculum Preparation for Antibiotic Sensitivity Test
21
3.9. Data Analysis
22
3.10. Ethical Considerations
22
4. RESULTS AND DISCUSSION
23
4.1. Biochemical tests
23
4.2. General Prevalence of Salmonella Isolates
26
4.3. Prevalence of Salmonella Isolates from Different Age Groups
27
4.4. Distribution of Salmonella Isolate positive patients by sex
28
4.5. Stool Culture Results of Salmonella in EHNRI Laboratory Vs Laboratory Results of
the Patients in WHC
29
4.6. Antibiotic susceptibility of Salmonella Isolates
30
4.7. The Association between the Prevalence of Salmonella infection and the Identified
Risk Factors in the Study Area
33
5. Summary, Conclusions and Recommendations
38
5.1. Summary
38
5.2. Conclusions
39
5.3. Recommendations
39
6. REFERENCES
41
x
TABLE OF CONTENTS Continued
7. APPENDICES
53
7.1. Appendix I: Participant Information Sheet (English Version)
53
7.2. Appendix II: Written and Signed Consent Form (English Version)
54
7.3. Appendix III: Questionnaires (English Version)
55
7.4. Appendix IV: Quetionarreis, Amharic Verssion
56
7.5. Appendix V : Quetinarries, Siltigna Verssion
57
7.6. Appendix VI: Zone diameter of antibiotics and sensitivity of Salmonella isolates
58
7.7. Appendix VII: Standard Zone Diameter of the Antibiotics and their Sensitivity
59
xi
LIST OF TABLES
List ofTables
Pages
1. Biochemical tests results of Salmonella isolates …………………………………..…25
2. Distribution of Salmonella positive patients by age group …………………………..…28
3. Distribution of Salmonella positive patients by sex…………………………………………..29
4. Antibiotics tested against Salmonella isolates on Muller Hinton agar and the
proportion of their sensitivity…………………………………………………………30
5. Risk factors associated with Salmonella infection among patients in WHC from July
2011 to January 2012………………………………………………………...……….34
6. Zone Diameter of the antibiotics tested for salmonella isolates and their sensitivity on Muller
Hinton Agar……………………………………………………………………………….......58
7. Standard Zone Diameter of the Antibiotics and their Sensitivity…………………………….62
xii
LIST OF FIGURES
Figures
Pages
1. Fig-2: The agar plate with antibiotics susceptibility test for Salmonella isolates …………..32
xiii
Prevalence and Antibiotic Resistance Patterns of Salmonella Isolates in
Patients Visiting Worabe Health Center: Worabe, Southern Ethiopia.
ABSTRACT
Salmonellosis, a disease caused by Salmonella, remains an important public health problem
worldwide, particularly in the developing countries. A cross sectional hospital-based survey
was designed to investigate the prevalence and antibiotic resistance patterns of Salmonella
isolates among patients visiting Worabe Health Center(WHC) from July 2011 to January
2012. A total of three hundred eighty four stool samples were collected from WHC. The
samples were transported to Ethiopian Health and Nutrition Research Institute laboratory
within 48 hours using Cary-Blair transport media kept in an ice box. Out of the 384 stool
specimens analyzed, 19 (4.9%) were proved to be positive for Salmonella isolates. In this study, the
high ratio of Salmonella infection predominated children whose age were from six to fourteen
year. Antibiotic sensitivity test was performed for these Salmonella isolates against 9
currently recommended antibiotics following the methods of the National Committee for
Clinical Laboratory Standard by using Kirby-Bauer disk diffusion technique on Muller
Hinton agar plate. The study indicated that all Salmonella isolates were sensitive to
ciprofloxacin (5µg), amikacine (30µg) and norfloxacine (10µg). In addition to this,
4/19(21%) of the isolates were found to be sensitive for all applied antibiotics, however, least
sensitivity was observed in amoxicillin. Likewise, 5.3%, 15.8%, 10.5%, 10.5% and 15.8%
antibiotic resistant Salmonella were observed for ceftriaxone (30µg), Chloramphenicole
(30µg), Cotrimoxazole (25µg), Amoxicillin (30µg) and Tetracycline (30µg). Thus, wise use of
antimicrobials should be practiced in order to prevent further spread and large scale
emergence of Salmonella against new antibiotics. Moreover, the study indicated that
Salmonella infection was associated with drinking unprotected (river) water and consumption
of raw products of animals such as meat, eggs and milk.
Key words: - Diarrhea, Fever, Headache, Salmonellosis
xiv
1. INTRODUCTION
Salmonella are a genus of Gram-negative, facultative anaerobes, rod-shaped and mostly
motile bacteria of the family Enterobacteriaceae that cause a wide range of human diseases
such as enteric fever, gastroenteritis and bacteremia. Gastroenteritis associated with foodborne disease outbreaks is probably the most common clinical manifestation of the infection
(Bennasar et al., 2000). There are 2 species of Salmonella: Salmonella enterica and
Salmonella bongori and more than 2,300 Salmonella serovars that may cause human and
animal diseases (Brenner et al., 2000).
S. Enteriditis and S. Typhimurium are ubiquitous serovars which affect humans and animals
causing gastroenteritis that is less severe than enteric fever (Velge et al., 2005). These two
serovars are the predominant serotypes associated with human disease in most countries
(EFSA, 2005). They have been reported as having the potential to cause epidemics and
become the dominant serovars in many countries in the foreseeable future (WHO, 2005). The
spread of S. Typhimurium in sub-Saharan Africa is a public concern too. As a result of HIV in
Sub-Saharan Africa, it is believed that there is a high prevalence of non-typhiodal Salmonella,
mainly S. Typhimurium and S. Enteriditis serotypes that cause bacteremia in these areas.
Some S. Typhimurium strains are particularly important because of their multidrug resistance
genes and worldwide dissemination (Ruiz et al., 2008).
Salmonellosis, the disease caused by Salmonella is one of the most frequently occurring foodborne diseases worldwide (Puthucheary et al., 2004). As a result it continues to be a major
health burden worldwide. Even in developed countries fore instance USA, there was an
estimate of 1.4 million non-typhoid Salmonella infection, resulting in 168,000 visits to
physicians, 15,000 hospitalizations and 580 deaths annually (WHO, 2005). As Reported by
Mikhail et al., (1990), there was a prevalence of 2.9% Salmonella in human diarrheal causes in
Djibouti. In Ethiopia, Reda et al. (2011) reported 11.5% Salmonella in Harar among patients
who were admitted to hospital. Ashenafi and Gedebou (1985) reported a prevalence of 4.5%
Salmonella in Adult Diarrhea in Addis Ababa. Likewise, Beyene et al. (2011) reported 5.3%
Salmonella infection in children of Addis Ababa and Jimma in Tikur Anbessa Addis Ababa
University Hospital, Addis Ababa and Jimma University Hospital, southern Ethiopia.
The primary route of Salmonella infection in humans and other animal species is the fecal-oral
transmission of the organism (Vought and Tatini, 1998). Food handlers during contact with
food or materials used for preparing food and poor food handling techniques are the main
causes of food-borne diseases (Mohan et al., 2006). Typically, Salmonella infections result
due to consumption of food products of animal origin (Angulo et al., 2000) such as poultry,
beef, pork, eggs, milk and seafood. In addition to this, direct contact with infected animals
(Tauxe, 1991) and water contaminated with one or the other Salmonella strains (Puthucheary
et al., 2004) also serve as a source for Salmonella infections. Other routes of infection besides
fecal-oral transmission are the respiratory system and tonsils, especially in cattle and swine
(Fedorka-Cray et al., 1995).
Salmonellosis is characterized by acute onset of fever, abdominal pain, diarrhea, nausea and
sometimes vomiting. In a small percentage of cases, septicemia and invasive infections of
organs and tissues can occur, leading to diseases such as osteomyelitis, pneumonia, and
meningitis (CDC, 2001). These diseases can generally become serious problems in an area in
the presence of other diseases and factors that weaken the immune system as well as with the
development of antibiotic resistance in Salmonella species.
In human hosts, well adapted Salmonella strains are known to produce systemic diseases such
as typhoid and paratyphoid fever (Rotger and Casadesus, 1999). As typhoid continues to be a
global problem, even in non endemic areas, e.g. in developed countries, imported cases
continue to cause problems (Bokkenheuser, 1983). The global incidence of typhoid is
estimated to be about 21 million with 700,000 deaths each year primarily in South East Asia,
Africa and Latin America attributed to rapid population growth, inadequate and improper
waste disposal, and lack of safe water supply (Shanahan et al., 1998). That is why, in the late
1970’s, typhoid fever was the major health problem in Addis Ababa (Beyene et al., 2008).
2
According to personal observation of the investigator, in the southern part of Ethiopia,
particularly amongst the Siltie and Guragae people, eating raw meat is a more common
practice. The most common raw meat consumed in these areas is the traditional spiced and
butter soaked minced meat (ktfo). In addition to ktfo, raw sliced meat (kurt) as well as mildly
fried meat (tibs) are frequently eaten as part of their meals. Giving raw milk to children,
patients and elderly as well as other people is also a common habit in this area in such a way
that such habits of their feeding may create great chance to food-borne diseases such as
salmonellosis.
There are also several unprotected water sources such as ponds, streams, and rivers which are
directly or indirectly exposed to contamination with fecal material originating from human
and animals. These water sources, which are undoubtedly laden with a variety of microorganisms, are used as sources of drinking water by the nearby resident people especially
rural people and those who do not have pipe water. Thus, according to Puthucheary et al.,
(2004), it is these contaminated waters and foods that are responsible for initiating
salmonellosis.
Furthermore, poor food handling is the other major problem in the study area especially in the
rural people which contribute to the development of the disease. The rural people live together
with their domestic animals in the same shelter. Pet animals such as cats feed on dead
animals and offal’s of animals such as cattle and poultry. Since, according to Tauxe (1991),
direct contact with infected animals may be a source of Salmonella infection; these pet
animals can transmit the disease if they are infected by the organism.
At present, several researchers have reported the prevalence of antimicrobial resistant
Salmonella isolates from Iran (from the year 1996 – 2005), China, Morocco, Thailand,
France, England and Wales, Spain Netherland, Southern Asia, Taiwan and Africa
(Mohammed et al., 2009; Hengli et al., 2008; Abdellah et al., 2009; Angkititrakul et al.,
2005; Cailhol et al., 2006; Threlfall et al., 2000; Valdezate et al., 2007; Duijkeren et al.,
2003; Hakanen et al., 2001; Hsueh et al., 2002; and Kariuki et al., 2006). Likewise, in
Ethiopia studies suggested an increase in the antibiotic resistance of Salmonella to commonly
3
used antimicrobials (Reda et al., 2011; Beyene et al., 2011 and 2008; Endrias, 2004 and
Molla et al., 2003).
Salmonella infection in animals and humans is the cause for the emergence of antimicrobial
resistance and the risk of transfer to animal and human population as either resistant
Salmonella or resistant genes into communal flora or pathogens affecting man (McEwen and
Fedorka-Cray, 2002).
Informal communication made with health workers has also revealed that antibiotic sensitivity
test is not practiced in Worabe Health Center. No published data exists at the moment on the
antibiotic resistance patterns of Salmonella as well as other bacterial pathogens in this area.
Knowledge of antibiotic resistance patterns of Salmonella is very important for effective
treatment of salmonellosis and limiting the resistance to antimicrobials to a low level by using
the correct antibiotics.
The information obtained in this regard will primarily benefit Worabe and the nearby health
centers that do not have adequate laboratory facilities and qualified trained laboratory
technicians as well as the entire population living in Worabe. Thus, this study was initiated
with the aim of investigating the prevalence and antibiotic resistance profiles of Salmonella
isolates among patients visiting Worabe Health Center (WHC).
General objective:
 To evaluate the prevalence and antibiotic resistance patterns of Salmonella isolates
patients visiting WHC.
The specific objectives were:
 To determine the prevalence of Salmonella isolates in patients visiting WHC.
 To determine the antibiotic resistance patterns of Salmonella isolated from patients
visiting WHC.
 To asses the impact of known risk factors of salmonellosis in the study area.
4
2. LITERATURE REVIEW
2.1. General Characteristics of Salmonella
The genus Salmonella had been named after the American microbiologist, D.E. Salmon, who
identified the organism. It is a non-sporing, non-acid fast, non-capsulated bacillus (Arora and
Arora, 2008), which consists of Gram-negative, facultative anaerobe, straight, rod-shaped
bacteria of the family Enterobacteriaceae (Bennasar et al., 2000). Its members are closely
related to Escherichia and Shigella. Members of the family Enterobacteriaceae contain a
single, circular chromosome of DNA that usually falls between 4.3 and 5Mb in length.
Salmonella colonizes the host organism in the intestinal lumen and transmitted to other hosts
through the environment (Baker and Dougan, 2007). It can invade non-phagocytic cells
through its T3SS which induces a trigger entry process. It seems to be the first bacterium
found to be able to induce both zipper and trigger mechanisms to invade host cells (Rosselin
et al., 2010). It has long been recognized as an important food-borne pathogen which can
cause symptoms in humans ranging from self-limiting enteric infections to enteric fever
(EFSA, 2007). They cause a wide range of human diseases such as enteric fever,
gastroenteritis and bacteremia. Gastroenteritis associated with food-borne outbreaks is
probably the most common clinical manifestation of the infection (Bennasar et al., 2000).
Salmonella usually produce gas from glucose, lysine and ornithine decarboxylase, but not
urease or tryptophanase and can use citrate as their only carbon source (Romo and Rodriguez,
2004). The ability of Salmonella to survive in multiple environment stresses including
extreme pH, nutrient deficiency, O2 stress, osmotic shock and heat is responsible for the
persistence of the bacteria to survive in vivo and vitro (Foster and Spector, 1995). Salmonella
can resist dehydration for a very long time (aw ≥ 0.93), both in feces and in foods for human
and animal consumption. In addition, it can survive for several months in brine with 20%
salinity, particularly in products with a high protein or fat content, such as salted sausages and
resists smoking. It can survive for a long period of time in soil and water (WHO, 1988).
5
Salmonella grows in 2-47oC with rapid growth between 25 and 43oC. Heat resistance of
Salmonella increases at low water activity (Romo and Rodriguez, 2004). Foods with pH < 4.5
do not normally support the growth of Salmonella, but some serovars grow at pH 4.0 (e.g.
Salmonella Infantis). Pasteurization temperature readily inactivates Salmonella serovars;
however, Salmonella Senftenberg 775w is known to have exceptional resistance to heat
(Yousef and Carlstrom, 2003).
2.2. Taxonomy of Salmonella
Salmonellae belong to genus Salmonella and family Enterobacteriaceae (Bennasar et al.,
2000). Of all Enterobacteriacea, the genus Salmonella is the most complex (Arora and Arora,
2008). There are a number of Salmonella serotypes that are capable of causing human
diseases. Historically, Salmonella was divided into separate species based on the results of
serotyping. For each serovar, there was a separate species. According to Arora and Arora,
(2008), there are more than 2,300 identified Salmonella serotypes. However, because they
share a high degree of genetic similarity, they are broadly divided into two species namely,
Salmonella enterica and Salmonella bongori. Over 99% of the serotypes are grouped into the
species S. enterica, which contains all of the major serovars that are pathogenic to humans
(Brenner et al., 2000). There are six sub species of Salmonella enterica, the most important of
which is Salmonella enterica subspecies enterica (subspecies I) which includes the typhoid
and paratyphoid bacilli and most other serotypes responsible for causing diseases in mammals
(Arora and Arora, 2008).
2.3. Salmonellosis
Salmonellosis is one of the most important public health problems, affecting more people and
animals than any other single disease. The incidence of human cases of salmonellosis is
thought to be many times greater than the number of reported and confirmed cases, even in
countries with well-organized surveillance activities (Davd et al., 1997). During the
progression of the disease there may be more severe manifestations such as bacteremia.
6
Antimicrobial therapy is often administered to treat the infection (Mead et al., 1999, Foley
and Lynne, 2008).
The most common Salmonella infection is gastroenteritis, with bacterial multiplication in
intestinal sub-mucosa and diarrhea, caused by the inflammatory response and perhaps also by
toxins. In specific hosts, adapted Salmonella produce systemic diseases such as typhoid and
paratyphoid fevers in humans. If host defenses are impaired, as in elderly or AIDS patients,
Salmonella can enter the bloodstream and cause septicemia, which is often fatal (Rotger and
Casadesús, 1999).
Salmonella infections in animals have serious world-wide implications for public health. They
result in the emergence of antimicrobial resistance and the risk of transfer to human
population either as resistant Salmonella or resistance genes into communal flora or
pathogens affecting man (McEwen and Fedorka-Cray, 2002). As antimicrobials are frequently
misused and overused in many developing countries, resistance to antimicrobials has led to an
increase in morbidity, mortality and cost of health care. To maintain the useful activity of
antimicrobial drugs in developing countries there is a need to improve access to diagnostic
laboratories, improved surveillance of the emergence of resistance and better regulation of the
use of antibiotics (Sharma et al., 2005).
2.4. Epidemiology and Transmission of Salmonellosis
The epidemiology of food-borne problems like salmonellosis is complex and expected to
vary with change in the pathogens themselves, industrialization, urbanization and change of
lifestyles, knowledge, belief and practices of food handlers and consumers, demographic
changes (increased susceptible population), international travel and migration, international
trade in food, animal feed and in animals, and poverty and lack of safe food preparation
facilities (WHO, 1988).
The evolution of specific Salmonella serotypes in intensive animal husbandry and
subsequently in humans has been observed over the past three decades. S. Enteritidis caused
7
the most recent epidemic, which peaked in humans in 1992 in many European countries
(WHO, 2005).
Salmonella serotypes are important zoonotic pathogens in humans and animals. The most
common animal reservoirs are chickens, turkeys, pigs and cows; dozens of other domestic and
wild animals also harbor these organisms (Carli et al., 2001). Humans as well as livestock
and poultry share most of the serovars indicating the potential hazard of interspecies sharing
of these organisms. It has been reported that livestock and their products can contribute as
much as 96 % of the total Salmonella infection in humans (Aggarwal et al., 1983).
Human salmonellosis is initiated by the ingestion of food or water contaminated with one or
the other Salmonella strain (Puthucheary et al., 2004). Food products of animal origin for
Salmonella infection includes: poultry, beef, pork, eggs, milk and seafood. Direct contact with
infected animals may also serve as a source for Salmonella infections (Tauxe, 1991).
Salmonella is also among the commonly isolated pathogens associated with fresh fruits and
vegetables. Outbreaks of salmonellosis have linked to a wide variety of fresh produce
including alfalfa sprouts, lettuce, fennel, cilantro, cantaloupes, unpasteurized orange juice,
tomatoes, melons, mango, celery and parsley (Lapidot et al., 2006). An increasing number of
uncommon but characteristic serotypes associated with exotic pets are being observed in
association with cases of salmonellosis in humans (David et al., 1997).
2.5. Signs and Symptoms of Salmonellosis
Human salmonellosis is usually characterized by acute onset of fever, abdominal pain,
diarrhea, nausea and sometimes vomiting (WHO, 2005). Typically, symptoms of
gastroenteritis develop within 6 to 72 hour after ingestion of the bacteria. The symptoms are
usually self-limiting and typically resolve within 2 to 7 days. In a small percentage of cases,
septicemia and invasive infections of organs and tissues can occur, leading to diseases such as
osteomyelitis, pneumonia, and meningitis (CDC, 2001). In some cases, particularly in the
very young and in the elderly, the associated dehydration can become severe and lifethreatening. In such cases, as well as in cases where Salmonella causes bloodstream infection,
8
effective antimicrobials are essential drugs for treatment. Serious complications occur in a
small proportion of cases (WHO, 2005). Although most cases are self-limiting, the degree to
which a person becomes sick depends on his or her health status and the number and
virulence of Salmonella species ingested. In general, the poorer the individuals health and the
more Salmonella ingested, the greater the probability for serious illness and death (Mead et
al., 1999).
2.6. Pathogenesis
The primary route of Salmonella infection in humans and other animal species is the fecal-oral
transmission of the organism. The estimates of the number of organisms required to cause
disease are quite variable, ranging from about 30 to more than 109 infectious organisms. The
infectious dose appears to be lower if the contaminated food that is consumed has a high fat
content, such as cheese or ice cream (Vought and Tatini, 1998). In order to reach their sites of
colonization, Salmonella must be able to survive the antimicrobial properties of the stomach,
including the low pH and the presence of many organic acids; subsequently, Salmonella have
evolved mechanisms that allow for survival at low pH value (Foster, 1991).
Interestingly, there have been reports of other routes of infection besides fecal-oral that appear
to lead to colonization of the gastrointestinal tract. In cattle and swine, the respiratory system
and tonsils are potential sites of invasion by Salmonella. If the lungs are the initial site of
colonization, Salmonella may be able to more easily enter the bloodstream due to the
proximity of the circulatory system and lead to the development of septicemia (Fedorka-Cray
et al., 1995).
Salmonella entering via the fecal-oral route that survive the low pH environment of the
stomach are able to colonize multiple sites including the small intestine, colon, and cecum.
Intestinal adhesion is mediated by fimbriae or pili present on the bacterial cell surface (Darwin
and Miller, 1999).
9
Salmonella have evolved intricate measures to invade host cells following epithelial
attachment. After interaction with host cells, Salmonella can express a type III secretion
system (T3SS), which facilitates endothelial uptake and invasion (Lostroh and Lee, 2001).
The genes that encode the T3SS machinery are associated with Salmonella pathogenicity
island 1 (SPI-1). Pathogenicity islands (PI) are genetic elements that carry genes encoding
virulence factors, such as adhesion, invasion, and toxin genes. The PI can be located on the
chromosome or on a plasmid (Hacker et al., 1990).
The plasmids that are known to carry virulence gene clusters are called virulence plasmids.
Strains from many serovars lack virulence plasmids; however, some of the most important
serovars for human health, including Typhimurium, Enteritidis, and Choleraesuis are known
to harbor virulence plasmids (Foley and Lynne 2008).
2.7. Global Prevalence and Incidence of Salmonellosis
Food-borne diseases continue to be a major public health problem in the developed and
developing worlds. Current statistics for food-borne illness in various industrialized countries
show that up to 60% of cases may be caused by poor food handling techniques, and by
contaminated foods served in food service establishments. No valid data are available for
most developing countries (Mohan et al., 2006).
In developing countries, a rapidly growing industry of intensive animal production is
accompanying the process of urbanization with all its environmental and behavioral changes
favorable for Salmonella to prevail (WHO, 1988).
Infections by Salmonella enterica are a significant public health concern around the world. On
a global scale, an estimated 1.3 billion cases of acute nontyphoidal gastroenteritis occur
annually, resulting in 3 million deaths. In the United States alone, it is estimated that there are
approximately 1.4 million cases of Salmonella infections resulting in 17,000 hospitalizations
and 585 deaths each year, which is 30.6% of the total number of yearly deaths caused by
10
known food-borne pathogens. Salmonella is responsible for an estimated 26% of all infections
caused by food-borne pathogens in the United States, with 95% of human salmonellosis cases
associated with the consumption of contaminated food products (Mead et al., 1999 ). In the
European Union, serovars Salmonella Enteritidis and S.Typhimurium are the most frequent
causes of gastroenteritis in humans. In 2006, more than 160,000 cases of salmonellosis were
reported in the EU resulting in an annual incidence of 34.6 cases per 100,000 populations
(EFSA, 2007).
Salmonellosis was reported in Switzerland and Italy by Essers et al. (2000), and Caprioli et al.
(1996), respectively, in Trinidad by Mohammed (2005), in Iran by Mohammed et al. (2009),
in Nigeria by Ogonsanya et al. (1994), in India, by Udgaonkar et al. (1995), and in Israel, by
Yagupsky et al. (2002). In addition, in England and Wales human Salmonellosis was a major
public health problem and the disease had important economic and social consequences
(Humphrey et al., 1988). In Canada there were 216 human endemic salmonellosis cases with
incidence rate of 14.7 cases/100,000 person/years (Andre et al., 2010). In Yucatan, Mexico,
reported salmonellosis was caused by mainly Salmonella Typhimurium, followed by
Salmonella Agona, nevertheless, asymptomatic children were also found to carry many
Salmonella isolates (Mussaret et al, 2012). In Ghana, Salmonella bloodstream infections
especially due to non-typhoidal strains, was a potential health problem for Ghanaian children
(Wilkens et al., 1997).
Non-typhoidal Salmonella infection is estimated to cost nations billions of dollars annually
thereby draining funds that could have been used for development (WHO, 1988). Nontyphoidal Salmonella are especially problematic in a wide variety of immune-compromised
individuals (Levine et al., 1991). Salmonella bacteremia is one manifestation of immunesuppression in patients with human immunodeficiency virus infection. Salmonella is more
likely to cause severe invasive disease in persons with acquired immunodeficiency syndrome
than in immune-competent persons (Tocalli et al., 1991).
Typhoid continues to be a global problem. Even in non endemic /developed areas, imported
cases continue to cause problems. In developed countries, the incidence and fatality rate is
11
very low. In the year 1980, there was about 12.5 million cases of typhoid in the world
(excluding China), an incidence of 365 cases per 10,000 populations. 3-5% of patients with
typhoid fever become life-long carriers (Bokkenheuser, 1983). However, after 19th century the
global incidence of typhoid is estimated to be 21 million with 700,000 deaths each year
primarily in South East Asia, Latin America and Africa attributed to rapid population growth
and unplanned urbanization, inadequate and improper waste disposal, lack of potable water
supply (Shanahan et al., 1998). The causative organism, Salmonella Typhi has rapidly gained
resistance to antibiotics like ampicilline, chloramphenicole and cotrimoxazole and previously
efficacious drugs like ciprofloxacin (Jesudason and John 1992).
2.8. Prevalence of Salmonellosis in Ethiopia
Even though there is no sufficient published data on the prevalence of salmonellosis in all
parts of Ethiopia; salmonellosis in Ethiopia was reported in Tikur Anbessa University
Hospital, Addis Ababa, and Jimma University Hospital, South West Ethiopia by Beyene et al.
(2011), from Addis Ababa by Ashenafi and Gedebou (1985), and (Beyene et al., 2011), in
Harar, Eastern Ethiopia by Reda et al. (2011) and by Beyene et al. (2008), in their review
article conducted on Salmonellosis in Ethiopia.
In Ethiopia, as in other developing countries, it is difficult to evaluate the burden of
salmonellosis because of the limited scope of studies and lack of coordinated epidemiological
surveillance systems. In addition, under-reporting of cases and the presence of other diseases
considered to be of high priority may have overshadowed the problem of salmonellosis.
Salmonellosis is particularly common in children of developing countries such as Ethiopia
(Beyene et al., 2008).
2.9. Control and Prevention of Salmonellosis
Salmonella are difficult to eradicate from the environment. However, as the major reservoir
for human infection is poultry and livestock, to significantly reduce human exposure would
mean a reduction in the number of Salmonella harbored in live stock (Wegener et al., 2003).
12
Measures to reduce disease in animals include specific measures such as vaccination and the
prevention of spread through biosecurity and general operational hygiene management
measures. Improved hygiene at all steps of the food chain, including primary production, is
effective in reducing the number of food-borne pathogens in food. This will also reduce the
numbers of food-borne pathogens that are resistant to antimicrobials. The use of program
aimed at the prevention and control of Salmonella and other zoonotic bacteria in primary
animal production, can lead to a reduction in the level of contamination of related food
products at retail, and thereby also reduce the risk of human exposure to antimicrobial
resistant Salmonella from those food products. The occurrence of Salmonella and
antimicrobial resistant Salmonella in other food commodities is also likely to be reduced as
the risk of cross-contamination is reduced (EFSA, 2008).
There is no effective immunization against infection by Salmonella, except against typhoid
fever. This is because of large number of Salmonella serotypes that would have to be included
in vaccines. Typhoid fever, however, is caused by only one serotype and two vaccines are
available. One consists of killed cells of Salmonella Typhi and is administered by injection.
The other vaccine consists of live, attenuated strain of Salmonella Typhi. It is administered
orally, in the form of capsules that can be swallowed (Pelczar et al., 1993).
2.10. Treatment of Salmonellosis
Antimicrobial agents are not essential for the treatment of Salmonella infections which are
manifested as uncomplicated gastroenteritis because such infections usually are self limiting,
and may result in the emergence of resistant Salmonella in the treated person. However,
effective antimicrobial agents are essential for the treatment of patients with bacteremia,
meningitis, or other extra intestinal Salmonella infection (Wilcox and Spencer, 1992). The
antimicrobials most widely regarded as optimal for the treatment of salmonellosis in adults is
the group of fluoroquinolones. They are relatively inexpensive, well tolerated, have good oral
absorption and are more rapidly and reliably effective than earlier drugs. Third-generation
cephalosporins (which need to be given by injection) are widely used in children with serious
infections, as quinolones are not generally recommended for this age group. The earlier drugs
13
chloramphenicole, ampicillin and amoxicillin are occasionally used as alternatives (WHO,
2005).
Outbreaks of drug resistant Salmonella can result in multiple hospitalizations and death
among individuals with the most severe infections. The multidrug-resistant nature of these
organisms makes treatment failure more likely (Angulo et al., 2000). Previously;
sulfamethoxazole-trimethoprim was the drug of choice for the treatment of diarrhea.
However, Salmonella isolated from salmonellosis patients has recently been found to have
increasing resistance to this type of antimicrobial therapy. The resistance caused failure of
regular antimicrobial therapy and has increased the severity of infection. Patients infected
with antimicrobial resistant strains were more likely to be hospitalized (Angkititrakul et al.,
2005).
2.11. Antibiotic Resistance
Resistance is a natural biological response of microbes to antimicrobials and is currently
irritating situation affecting many parts of the world. Apart from intrinsic resistance, gene
transfer and mutation are among the underlying mechanisms involved in the development of
antimicrobial resistance by microbes. Several factors contribute to resistance by pathogens
causing gastroenteritis in the setting of a developing country like Ethiopia. These include
frequent overuse, misuse and factors related to the potency and quality of antimicrobials and
the distribution of resistant strains (Sharma et al., 2005). Many bacterial species have the
ability to produce antimicrobial compounds. This ability is needed to give the bacteria an edge
in micro-organism rich environments. Antibiotic-resistance likely emerged as bacteria began
producing compounds in order to survive in their environment and competing species found
ways to counteract these compounds (Matthew et al., 2007).
Antibacterial drugs are readily available over the counter in many countries. Such drugs can
be easily obtained by the community and used improperly, which contributes to the selection
of resistant strains and multiple drug-resistant strains, especially when a broad-spectrum
antibiotic, such as tetracycline, is used (Spika, et al., 1987). Antimicrobial agents are currently
14
used for three main reasons: to treat infections in humans, animals, and plants;
prophylactically in humans, animals, and plants; and sub- therapeutically in food animals as
growth promoters and for feed conversion (Angulo et al., 2000).
When antibiotic use became the norm in both human and animal medicine, selection pressure
increased the bacterial advantage of maintaining and developing new resistance genes that
could be shared among bacterial populations (Matthew et al., 2007).
The development of resistance in Salmonella toward antimicrobial agents is attributable to
one of multiple mechanisms, including production of enzymes that inactivate antimicrobial
agents through degradation or structural modification, reduction of bacterial cell permeability
to antibiotics, activation of antimicrobial efflux pumps, and modification of the cellular target
for drug (Sefton, 2002). In addition to inactivation of the drug itself, other resistance is
associated with the modification of the drug binding target within the cell (Heisig, 1993).
2.11.1. Effects of antibiotic resistance
If the frequency of drug resistance increases, the choice of antibiotics for treatment of
systemic salmonellosis in humans will become more limited (Angulo et al., 2000, White et
al., 2001). Outbreaks of drug resistant Salmonella can result in multiple hospitalizations and
death among individuals with the most severe infections. The multidrug-resistant nature of
these organisms makes treatment failure more likely (Angulo et al., 2000). Recent appearance
of isolates with multidrug resistance (e.g. S.Typhimurium DT104) is a potential threat to the
safety of consumers and raises a great health concern worldwide (Yousef and Carlstrom,
2003).
2.11.2. Antibiotic resistance in Salmonella
The production of antibacterial peptides is a host defense strategy used by various species,
including mammals, amphibians, and insects. Successful pathogens, such as the facultative
15
intracellular bacterium Salmonella Typhimunum, have evolved resistance mechanisms to this
ubiquitous type of host defense (Eduardo et al., 1992).
Strains of Salmonella have emerged that show antimicrobial resistance which can lead to
treatment failure in both humans and animals (Rabsch et al., 2001). The increasing prevalence
of multidrug resistance among Salmonella and resistance to clinically important antimicrobial
agents such as fluoroquinolones and third generation cephalosporines has also been an
emerging problem (Fey et al., 2000). The frequency of multidrug resistant serotypes such as
Typhymurium and Newport is reportedly increasing. One major concern to public health has
been the emergence of Definitive Type 104 which was recognized in the UK in 1984
(Molback et al., 1999). This phage type commonly exhibits resistance to five antimicrobial
agents: ampcillin, chloramphenicole, streptomicine, sulfamethoxazole and tetracycline (Fey et
al., 2000). Known resistance genes of tetracycline, ampicillin, chloramphenicole and
gentamicin were also reported from Salmonella by Türkylmaz, 2009. Thus, these resistance
genes are also a health concern due to the potential of the organism to transfer resistant genes
by means of conjugal transfer to other Salmonella or organisms related to it (Zhao et al.,
2003).
Antimicrobial-resistant Salmonella strains are increasing due to the use of antimicrobial
agents in food animals, which are subsequently transmitted to humans usually through the
food supply (Angulo et al., 2000 and White et al., 2001). Plasmid-mediated spread of
antibiotic resistance genes is likely an important means for Salmonella to acquire resistance.
Experimental conjugal transfer of antibiotic resistance among Salmonella and related
organisms was observed (Zhao et al., 2003). In India, twenty eight multi-drug resistant
Salmonella were obtained from patients (Udgaonkar et al., 1995). In Ethiopia, there was
antibiotic resistant Salmonella to commonly used antimicrobials (Molla et al., 2003, Endrias,
2004, Beyene, 2008 and 2011, Reda et al, 2011).
16
3. MATERIALS AND METHODS
3.1. Description of the Study Area
The study was conducted in Worabe Heath Center (WHC) at Worabe town of Silte Zone,
which is found 172 km south of Addis Ababa. According to the official report of Zonal
Administration of Silte Zone Health Department, the Zone has a total area of 3047.83 sq km.
The geographical location of Silte is between 7o 43′ – 8 o 10′ North latitude and 37 o 86′ – 38 o
53′ East longitude. The Zonal administration is bounded in the North by Guragae Zone, in the
West Hadia Zone, in the South East by Alaba Special Woreda and in the East by Oromia
Regional State. Out of the total land size, 3.42%, 73.5% and 23.01% are climatically
categorized as ‘Kola’, ‘Weynadega’ and ‘Dega’, respectively. The annual mean temperature
is between 10.10C – 22.50C and the annual rain fall ranges from 650 – 1818 mm. The altitude
ranges from 1501 to 2500 m.a.s.l. The Zone has about 789,187 populations. In Worabe town,
there are one governmental health center, two private clinics and two private pharmacies.
There is also one hospital, which is currently under construction.
3.2. Study Design
The study involved a hospital-based cross-sectional survey design in determining the
prevalence, antibiotic resistance patterns of Salmonella isolates, and risk factors associated
with Salmonella infection.
3.3. Study Population
The study population constituted those patients who had fever and headache that were
examined for widal test as well as patients with diarrheal cases visiting Worabe Health Center
for medical treatment.
3. 4. Sample Size Determination and Sampling Procedure
17
Based on the 95% confidence limits and 5% sampling error, the sample size was calculated
using the following formula provided for single population proportion (Bland, 1998). As
indicated below, in the formula, the prevalence was taken as 50% since there was no previous
study made on the prevalence and antibiotic resistance of Salmonella isolates in the selected
study area.
n = (Zα/2)2 P (1-P)/d2
Where:
n = Required sample size.
P = Prevalence of Salmonella isolates. (0.5).
d = Marginal error between the samples and populations (0.05).
Zα/2 = Standard score corresponding 95% confidence level, i.e. 1.96,
Therefore, the calculated sample size for this study was 384.
Patients that came to WHC with diarrheal cases and those who were examined for widal test
between July 2011 and January 2012 were selected and used as source of stool samples and
information. The selection of patients was done using systematic serial sampling technique. In
short sample patients with diarrhea case and those who were examined for widal test were
serially selected until the desired sample size was reached.
3.6. Methods of Data Collection and Quality Control
Fresh stool samples along with important information in the prepared questionnaires (See,
Appendix III) were collected from 384 selected patients with the help of experienced
laboratory technicians of WHC. The stool samples were collected using sterile stool cups and
immediately kept at WHC in a refrigerator for 48 hours until transported to Ethiopian Health
and Nutrition Research Institute (EHNRI) bacteriology laboratory, Addis Ababa. All samples
were transported to EHNRI in an ice box using Cary-Blair transport media within 48 hours
after collection. The prevalence and antibiotic resistance patterns of Salmonella isolated from
stool samples of patients were determined at EHNRI using standard methods. The
bacteriological analyses were done by the principal investigator with the assistance of
18
professional laboratory technicians of EHNRI. The strain Escherichia coli ATCC 25922 was
used for determining the quality of media used in the experiment and controlling antibiotic
susceptibility tests (NCCLS, 2006).
3.7. Isolation and Identification of Salmonella
Isolation and identification of Salmonella isolates were done by appropriate bacteriological
culture techniques and biochemical tests (Yousef and Carlstrom, 2003 and ISO 6579, 1998).
3.7.1. Bacteriological culture
Stool samples collected from patients were placed in containers containing Cary-Blair
transport medium and transported to EHNRI. They were then streak plated on MacConkey,
XLD and Salmonella-Shigella agar (selective media used for isolation of Salmonella and
Shigella) (ISO 6579, 1998). The high bile salt concentration and sodium citrate in the later
two media inhibits all gram positive bacteria, coli-forms and other gram negative bacteria
(Arora and Arora, 2008).
Differentiation of Salmonella and non-Salmonella strains is accomplished by inclusion of
suitable carbohydrate and pH indicator combinations. Lactose, sucrose and salicin are not
typically fermented by Salmonella, and thus production of acid from these carbohydrates
indicates non-Salmonella isolates. If lysine is present in the medium, Salmonella
decarboxylates the amino acid, producing alkaline products that change the color of the pH
indicator in the agar surrounding the colony (Yousef and Carlstrom, 2003). Colonies of
Salmonella grown on XLD agar were characterized with a black centre and a lightly
transparent zone of reddish color due to the color change of the indicator.
3.7.2. Isolation of Salmonella Species
Stool samples obtained from WHC were directly streaked on to MacConkey, XLD and SSA,
followed by plating overnight enriched samples at 37°C in selenite F broth (indirect method),
19
which enriches the number of Salmonella in stool samples, onto Salmonella-Shigella agar
(SSA). The plates were incubated for 24 hrs at 37°C and examined for presence of colonies
which were morphologically similar to Salmonella. In order to get pure colonies, the isolates
were screened on a fresh medium. Finally, a series of biochemical tests were made and used
as identification of Salmonella isolates (Wallace et al., 2011).
3.7.3. Biochemical identification
Decarboxylation of lysine in LIA, fermentation of glucose anaerobically in TSI agar,
production of H2S in TSI agar and LIA media, and utilization of citrate are important
biochemical properties for identification of Salmonella. Inabilities of Salmonella to hydrolyze
urea, produce indole from tryptophan, and grow in potassium cyanide broth are also useful
biochemical tests for the characterization of the micro-organism. Biochemical identification
of Salmonella, therefore, requires testing presumptive isolates in TSI agar and LIA as well as
running additional biochemical tests (Yousef and Carlstrom, 2003).
The Centers of isolated colonies were lightly touched to be picked with sterile inoculating
needle and a suspension of the sample was made in a sterile test tube containing nutrient
broth. Three drops of this suspension was added to TSI agar, LIA, citrate, urea agar, nutrient
agar, MPW and GPW and were incubated at 37°C for 24 ± 2 h. The butt of TSI, LIA and
nutrient agar were stabbed. MPW and GPW tubes were tightly capped to check production of
gas during the reaction. TSI and LIA tubes were loosely capped to maintain aerobic
conditions while incubating slants and to prevent excessive H2S production. Salmonella in
TSI culture typically produces alkaline (red) slant and acid (yellow) butt, with or without
production of H2S (blackening of agar) in TSI. In LIA, Salmonella typically produces alkaline
(purple) reaction in butt of tube. Most Salmonella cultures produce H2S in LIA (Wallace et
al., 2011). The isolates were identified based on the standard guidelines of ISO 6579 (1998).
20
3.8. Antibiotic Resistance Test
The antibiotic resistance patterns of Salmonella isolates were determined for the commonly
used antibiotics (Oxoid Ltd., Basingstoke, England) such as amikacine (30µg), amoxicillin
(30µg), ciprofloxacin (5µg), tetracycline (30µg), chloramphenicole (30µg), ceftriaxone
(30µg), cefepime (30µg), norfloxacine (10µg) and cotrimoxazole (25µg) by using the KirbyBauer disk diffusion technique on Muller Hinton agar plate. The diameter of the zone of
inhibition was measured and results were interpreted using a standard table that relates the
zone diameter along with the degree of microbial resistance. Escherichia coli (ATTC25922)
was used as a control strain for interpretation of their resistance based on the guidelines of the
National Committee for Clinical Laboratory Standards (NCCLS, 2006).
3.8.1. Inoculum Preparation for Antibiotic Sensitivity Test
There are two major methods of inoculum preparation for antibiotic susceptibility test. These
are the direct colony suspension and the growth method. The later method is preferable when
colony growth is difficult to suspend directly and a smooth suspension cannot be made. This
method is also used for non-fastidious organisms when fresh 24h old colonies, as required for
the direct colony suspension method, are not available. Thus, in this study the growth method
was used to prepare inocula for all antibiotic susceptibility tests.
In order to prepare inoculum at least 2-3 well isolated pure colonies of the same morphologic
type were selected from nutrient agar plate. The top of each colony was touched with a loop
and the growth was transferred into a tub containing 4-5 ml of tryptic soy (TSY) broth. The
broth cultures were adjusted to the turbidity of the 0.5 McFarland standard.
After preparation of the inoculum and adjusting the turbidity of the inoculum suspension, a
sterile cotton swab was dipped into the adjusted suspension. The swab was rotated several
times and pressed firmly on the side wall of the tube above the fluid level in order to remove
excess inoculums from the swab. The dried surface of MHA plate was inoculated by streaking
the swab over the entire sterile agar surface. The procedure was repeated by striking three
21
more times, rotating the plate approximately 60 degree each time to ensure an even
distribution of the inoculum. Finally, the rim of the agar was swabbed and the antibiotic disks
were placed on the surface of Muller Hinton agar medium by using sterile forceps at equal
distance from each other. The disks were gently pressed onto the agar surface to ensure firm
contact. The plates were then incubated at 370C for 24h. A standardized reference strain of E.
coli ATCC25922, sensitive to all the antimicrobial drugs being tested, was used as a positive
control. The diameter of the zone of inhibition around the disk on the incubated plates was
measured to the nearest whole number mm using a metal caliper to differentiate the sensitive,
intermediate, and resistant isolates and compared to standard table (NCCLS, 2006).
3.9. Data Analysis
The whole data obtained from the participants in WHC as well as the experiments done in
EHNRI bacteriology laboratory was carefully recorded. Descriptive statistics were used for
the analysis of recorded data by using SPSS version 16.0 computer software. The antibiotic
resistance profiles obtained from the isolates were compared with those of the control strain,
Escherichia coli (ATTC25922), to determine their resistance to the antimicrobial agents.
3.10. Ethical Considerations
Ethical approval was obtained from the ethical reviewer committee of the College of Health
Sciences, Haramaya University. Institutional permission was also obtained through
communicating Worabe Health Department before starting the study. Through these ethical
certificates, the medical director of WHC was first briefed about the study before meeting
with patients. The patients and parents/caretakers/ of children were informed about the
objectives of the study. The stool samples obtained from the patients were used only for the
purpose of this study and the information obtained from the study was kept confidential.
22
4. RESULTS AND DISCUSSION
This section presents the data obtained from laboratory examination of stool samples and the
responses obtained in the questionnaires from respondents using appropriate tables and
figures. Attempts are also made to explain the results and relate them with similar research
findings from available literature.
4.1. Biochemical tests
Three hundred eighty four stool samples collected from patients who were attending WHC for
treatment were examined for Salmonella isolates by culturing the organism on different
media. The colonies appeared transparent and colorless in SS and MacConkey agar, and pink
in XLD. Colonies with black centers were also observed in some of SS and XLD agar. Pure
colonies which were morphologically similar to Salmonella were selected followed by
screening the isolates on nutrient agar (Wallace et al., 2011, WHO and CDC, 2010). Finally, a
series of biochemical test was made for the screened isolates and their results are presented in
Table 1.
23
Table 1: Biochemical test results of Salmonella
isolates
TSI
Biochemical test results
LIA
Citrate
Color change
H2S g
S
B
Urea
Motility
GPW
MPW
Indole
+
-
+
+
+
-
-
-
-
+
+
+
-
-
-
-
-
+
+
+
-
NCC
NCC
+
-
-
-
+
+
+
+
+
+
-
-
Yellow
+
+
+
-
+
+
+
-
-
-
NCC
+
-
+
-
+
+
+
-
+
+
-
NCC
+
-
+
-
+
+
+
-
Yellow
+
+
-
Yellow
+
-
+
-
+
+
+
-
Red
Yellow
-
-
-
NCC
-
-
+
-
+
+
+
-
291
Red
Yellow
+
-
-
NCC
+
-
+
-
+
+
+
-
294
Red
Yellow
-
-
-
NCC
-
-
+
-
+
+
+
-
299
Red
Yellow
-
-
-
NCC
-
-
+
-
+
+
+
-
314
Red
Yellow
+
+
-
NCC
+
-
+
-
+
+
+
-
320
Red
Yellow
+
+
-
Yellow
+
-
+
-
+
+
+
-
355
Red
Yellow
-
-
-
NCC
-
-
-
-
+
+
+
-
365
Red
Yellow
-
-
-
NCC
-
-
-
-
+
+
+
-
369
Red
Yellow
+
+
-
NCC
+
-
+
-
+
+
+
-
385
Red
Yellow
+
+
-
NCC
+
+
+
-
+
+
+
-
Isolates
Color change
S
B
H2S
g
24
Red
Yellow
+
+
-
NCC
+
-
71
Red
Yellow
+
+
-
NCC
+
73
Red
Yellow
-
-
-
NCC
96
140
Red
Red
Yellow
Yellow
+
+
-
185
Red
Yellow
+
+
259
Red
Yellow
+
272
Red
Yellow
286
Red
288
GPW= Glucose Peptone Water, MPW= Mannitol Peptone Water, S= Slant, B= Butt, g= gas production, H2S= hydrogen sulfide production,
Red= alkaline, Yellow= acidic, NCC = No Color Change.
25
During the experiment, majority of the organisms utilized urea liberating ammonia that
resulted in color change (urea test positive). Since Salmonella species do not utilize urea, urea
test negative isolates, were kept for further identification of the organism. Alkaline reaction in
LIA test tubes and blackening of it due to production of H2S was formed in the test tubes,
which is the main feature of Salmonella. All isolates showed red /alkaline/ slants and
yellow/acid/ butt/, with or without gas formation and hydrogen sulfide formation in TSI agar
test tubes. This is also the main distinguishing features of Salmonella. Citrate negative, which
is the characteristics of few Salmonella isolates and bacterial growth accompanied by color
change from green to blue (citrate positive) that is the characteristics of most Salmonella was
observed. Colorless to pink color change in both MPW and GPW test tubes, and obvious
production of gas was observed from some of GPW test tubes which indicated utilization of
mannitol and glucose by Salmonella. Motility was confirmed by stabbing a test tube having
nutrient agar which showed the movement of the organism. Motile isolates started to diffuse
from the stabbed centre to the edge of the test tubes; in contrast to this, non-motile organisms
did not show any movement as a result they grown on the stabbed part of the test tubes only.
Indole test negative that is the characteristics of the organism, were formed in the surface of
test tube after addition of 0.2-0.3 ml of kovacs’ reagent to 24 hrs cultured inoculum within a
test tube. Finally, on the basis of these characteristics, the isolates were identified as
Salmonella according to ISO- 6579 (1998) and WHO and CDC (2010) and Wallace et al.
(2011).
4.2. General Prevalence of Salmonella Isolates
Out of 384 stool samples collected from patients attending WHC for treatment 19 were found
to be Salmonella test positive with over all prevalence of 4.94%. The prevalence of
Salmonella isolates in stool samples of this study is closer to the findings of previous studies
made in china by Hengli et al. (2008) who reported a prevalence of 5.8% and 4.8%
Salmonella in the years 2006 and 2007, respectively. On the other hand lower prevalence was
reported by Mikhail et al. (1990) from Djibouti who reported a prevalence rate of 2.9%.
However, the prevalence found in this study was found to be lower than the 11.5%
Salmonella prevalence reported by Reda et al. (2011) who conducted a study on Antibiotic
susceptibility patterns of Salmonella and Shigella isolates in Harar, Eastern Ethiopia and
much lower than the 16.7%, 17%, 19% and 20.5% prevalence of Salmonella reported by
Mussaret et al. (2012), Kabir et al. (2007), Caprioli et al. (1996) and Murugkar et al. (2005)
in Mexico, Nigeria, Italy and northeastern India respectively.
4.3.
Prevalence of Salmonella Isolates from Different Age Groups
The patients were divided into 5 groups based on their age (http:// www.Whoindia.org). As
shown in Table 2 below, the prevalence of Salmonella varied among the different age groups
of the population. Twelve isolate were obtained from 6-14 years old. Only one isolate was
recorded from the age group 15 to 24. Three isolates of Salmonella were also positive from
both age groups of late 25-49 and above 49 years old, however, Salmonella isolates were not
obtained from children under five years old with the overall 0-5 to 6 -14 to 15-24 to 25-49 to
above 49 year ratio of 0:12:1:3:3 respectively. In this study the majority of the isolates were
obtained from age group 6-14 years old. The prevalence of Salmonella in pediatrics (0-5 and
6-14) was 12.4%. The prevalence obtained from the pediatrics, is close to 12% and 15.3%
prevalence in Switzerland and South West Ethiopia reported by Essers et al. (2000) and
Abebe, 2002. However, this finding in the pediatrics is higher than 5.3% prevalence rate
reported by Beyene et al. (2011) in Ethiopia, Ogonsanya et al. (1994) from Nigeria, and
Mohammed et al. (2005) from Trinidad who reported prevalence rates of 5.3%, 3.3% and
1.7%, respectively.
Likewise, several researchers reported higher prevalence of Salmonella from children. For
instance, Salmonella infection was a common phenomenon in children of developing
countries such as Ethiopia (Beyene et al., 2008). Salmonella bloodstream infection was also a
potential health problem for Ghanaian children (Wilkens et al., 1997). In Israel, the incidence
of children’s bacteraemia, caused by Salmonella infection, has experienced a significant
increase (Yagupsky et al., 2002). Salmonella isolates were also found mainly from the Indian
pediatric. Two cases of meningitis caused by Salmonella were also found to be health
problem in Indian children (Udgaonkar et al., 1995).
27
Table: 2 Distribution of Salmonella positive patients by age group
Age group
Test for presence of Salmonella species
(years)
Positive
Negative
Total
N
%
N
%
N
%
0–5
0
0
20
100
20
100
6 – 14
12
13.8
75
86.2
87
100
15 – 24
1
1
101
99
102
100
25 – 49
3
2
150
98
153
100
Above 49
3
9
30
90.9
33
100
Total
19
4.9
95.1
385
100
366
N = Frequency
4.4. Distribution of Salmonella Isolate positive patients by sex
Out of the nineteen Salmonella isolates 12/180 (5.9%) were obtained from females and 7/205
(3.9%) were obtained from males with the male to female ratio of 1:1.5. The greater
prevalence value observed in females may be due to the activity of the females’. According to
the investigator, females prepare all types of food, fetch river water for house use and wash
their family clothes in river. Furthermore, they are also responsible for milking cows, remove
and distribute feces of farm animals into the farm, which lead them to have greater contact to
organisms that may present in unsafe food, river water and the feces of animals. If they do not
wash their hand properly, contamination may occur during eating. A major risk of food
contamination also lies with the food handlers. Dangerous organisms present in or on the food
handler’s body can multiply to an infective dose when they come into contact with food or
surfaces used to prepare food (Mohan et al., 2006).
28
Table 3: Distribution of Salmonella positive patients by sex
Sex
Male
Female
Total
Test for Salmonella
N
%
N
%
N
%
Positive
7
3.9%
12
5.9%
19
4.9%
Negative
173
96.1%
193
94.1%
366
95.1%
Total
180
100%
205
100%
385
100%
N = Frequency
4.5.
Stool Culture Results of Salmonella in EHNRI Laboratory Vs Laboratory Results
of the Patients in WHC
During 6 months study, there were a total of 263(68.3%) diarrheal causes that were either
diagnosed for stool examination only to detect parasitic infection, or both Widal test to
confirm Salmonella Typhi and stool examination in order to detect parasitic infection. Many
parasitic infections (88 P. falciparum and vivax, 52 G. lamblica, 32 E. histolitica, 3
Hookworm and 2 H. nana) were detected from the patients in WHC. Similar cases of this
result was also reported in Tikur Anbessa Addis Ababa University Hospital and Jimma
University Hospital, Ethiopia by Beyene et al., 2011, who reported parasites in 337,
Salmonella in 65, and Shigella in 61 cases on the study conducted on Multidrug Resistant
Salmonella Concord in Children.
Similarly, 234(60.8%) patients had headache and fever with or without diarrhea; as a result
Widal test and blood film was made for the confirmation of Salmonella Typhi and malaria.
The majority of the patients result was malaria test positive and Widal test positive with only
one strongly reactive and the other being weakly to moderately reactive to Widal test. Despite
this, the majority of Salmonella stool culture gave negative result. This contradictory result
may be due to the reason that malaria and typhoid fever often present with mimicking
symptoms especially in the early stages of typhoid. Thus, it is very common to see patients
undergoing both typhoid and malaria treatments even if their diagnosis has not been
confirmed and a cross reaction between malaria parasites and Salmonella antigens may cause
29
false positive test (Mohan, et al., 2006). Furthermore, rheumatoid arthritis, chronic liver
disease, nephritic syndrome and ulcerative colitis may give false positive Widal test. This
problem can be solved if rapid identification tests such as IgM dipstick are used, which
detects IgM antibodies against whole cell serotype S. Typhi. It is also more sensitive than
Widal test (Samuel et. al., 2004).
4.6. Antibiotic susceptibility of Salmonella Isolates
The antibiotic resistance patterns of 19 Salmonella isolates were performed for nine currently
used antibiotics (Oxoid, England with expiration date of 2014) on Muller Hinton agar, based
on the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS,
2006). Antibiotic resistant Salmonella were observed in five antibiotics and the results are
presented in Table 4 below:
30
Table 4: Antibiotics tested against Salmonella isolates on Muller Hinton agar and the
proportion of their sensitivity.
Antibiotics
Concentration
Patterns of antibiotic susceptibility
(µg)
Susceptible
Intermediate
Resistant
N
%
N
%
N
%
Ceftriaxone
30
17
(89.5%)
1
(5.3%)
1
(5.3%)
Norfloxacine
10
19
(100%)
0
0
0
0
Chloramphenicole
30
15
(79%)
1
(5.3%)
3
(15.8%)
Amoxicillin
30
7
(36.8%)
10
(52.6)
2
(10.5%)
Cotrimoxazole
25
17
(89.5%)
0
0
2
(10.5%)
Cefepime
30
18
(94.7%)
1
(5.3%)
0
0
Ciprofloxacin
5
19
(100%)
0
0
0
0
Tetracycline
30
12
(68.4%)
3
(15.8%)0
3
(15.8%)
Amikacine
30
19
(100%)
0
0
0
N = Frequency µ= micro g= gram
As shown above in the table, most of Salmonella isolates were found to be sensitive to the
antibiotics tested on Muller Hinton agar plates. All were sensitive to ciprofloxacin (5µg),
amikacine (30µg) and norfloxacine (10µg). In addition to this, four isolates were sensitive for
all tested antibiotics (See, Appendix IV). The same finding in ciprofloxacin and amikacine
sensitivity of Salmonella was also reported from the previous study conducted on antibioticresistant Salmonella species from human and non-human sources in Oman by Al-Bahry et al.
(2007) who reported Salmonella isolates that were sensitive to ciprofloxacin and amikacine.
Furthermore, Mussaret et al. (2012) reported 100% susceptible for ciprofloxacin. In the study
conducted by Mohammed et al. (2009) in Iran increased resistance in amikacine was reported
from 2.3% to 9.2% in the year 1996 to 2005 respectively. In contrast to this, no increased
resistance to ciprofloxacin was found among Shigella and Salmonella isolates in Iran at the
31
same year. In Eastern Ethiopia, a study conducted on antibiotic susceptibility patterns of
Salmonella and Shigella isolates, reported by Reda et al. (2011) showed that 89.3% of isolates
were sensitive to Norfloxacin with only 7.1% resistance, which is closer to 100% sensitivity
found in this finding.
Nevertheless, 1(5.3%), 3(15.8%), 2(10.5%), 2(10.5%) and 3(15.8%) resistant isolates were
observed for ceftriaxone (30µg), Chloramphenicole (30µg), Cotrimoxazole (25µg),
Amoxicillin (30µg) and Tetracycline (30µg) respectively. However, Beyene et al. (2011)
reported that 70% of Salmonella isolates were resistant to ceftriaxone, which is a drug of
choice in children. Tetracycline 30μg 71.4% and 72.1% resistance was reported by Reda et
al. (2011) and Mussaret et al. (2012) respectively. These results are much higher than these
findings. Increase in antibiotic resistance of Salmonella to tetracycline from 1 to 42%,
chloramphenicole from 1.7 to 26% throughout their 7-years study was reported from enteropathogenic bacteria by Prats et al. (2000). Later on, the study made in Iran from 1996 to 2005,
chloramphenicole resistance among Salmonella had increased from 17.2% to 27.9% in 1996
to 2005, respectively (Mohammed et al., 2009). In addition to this, 22% and later 62.3%
chloramphenicole resistance was reported by Al-Bahry et al. (2007) and Reda et al. (2011)
respectivelly. Likewise, Beyene et al. (2011) reported that, 70% of Salmonella isolates from
Addis Ababa were resistant to chloramphenicole. Al-Bahry et al. (2007) also reported 42%
resistance in cotrimoxazole.
On the other hand, three isolates revealed intermediate value of 5.3% for ceftriaxone (30µg),
Chloramphenicole (30µg) and Cefepime (30µg). Moreover, 15.8% and 52.6% intermediate
value of the isolates were also observed in Tetracycline (30µg) and Amoxicillin (30µg)
respectively (See, Appendix IV). As shown above in table 4, only 36.8% susceptibility was
reported for amoxicillin in this study. Salmonella isolates were reported with 100% resistance
to amoxicillin (30μg) by Reda et al. (2011) in Eastern Ethiopia. It is also a common antibiotic
in the study area. This much lower sensitivity deviation of amoxicillin from the other
antibiotics observed in both this and the previous studies, could be due to the fact that,
amoxicillin have been used for a long period of time, their easy availability and potential for
misuse and the frequent use of this antibiotic by the people for different pain they had. The
32
emergence of antibiotic resistant Salmonella serotypes was most likely due to Self-medication
due to easy access to antibiotics in the nearby pharmacies without prescription and the
indiscriminate use of antimicrobials (WHO, 1988). The agar plate with antibiotics
susceptibility test for Salmonella isolates on MHA after 24 hour incubation are shown in
Figure-2.
Fig-2: The agar plate with antibiotics susceptibility test for Salmonella isolates
4.7. The Association between the Prevalence of Salmonella infection and the Identified
Risk Factors in the Study Area
Primary data was obtained from the participants in WHC from the prepared questionnaires to
asses the availability of risk factors for the transmission of Salmonella (See, Appendix III).
Bivariate and multivariate statistical analyses were done to determine the association between
infection of Salmonella and the selected risk factors. Subsequently, the following variables
were found to be associated with infection of Salmonella using logistic regression analysis: (I)
consumption of raw or mildly fried meat (AOR = 3.8, CI = 1.260-12.041, P < 0.002) (II)
consumption of raw or partially cooked eggs (AOR = 2.282, CI = 1.057-4.924, P = 0.014)
(III) raw milk or raw milk product consumption (AOR = 4.537, CI = 2.054-9.453, P < 0.013)
33
and (IV) drinking unprotected (river) water (AOR = 0.564, CI = 0.245-0. 983, P = 0.021). As
a result, these four factors were important variables that affected the prevalence of Salmonella
infection in the study area. However, sex (AOR 0.501, CI 0.182-1.382 P = 0.182), daycare to
children (AOR 0.456 CI 0.057-3.657 P = 0.460), absence or presence of toilet (AOR 1.164 CI
0.345-3.928 P = 0.806) and exposure to farm or pet animals (AOR 0.968, CI .221-19.322)
were not found to have significant association with infection of Salmonella in both crude odds
ratio and adjusted odds ratio. Thus, according to this statistical analysis, patients with or
without each of these factors were found to be at equal risk for salmonellosis. The detailed
associations between Salmonella infections and risk factors are presented in Table 5.
34
Table 5 Risk factors associated with Salmonella infection among patients in WHC from July
2011 to January 2012.
Salmonella infection
Risk factors
Positive
Negative
COR (95% CI)
AOR (95%CI)
Used
17(5.3)
303(94.7)
2.806(1.438-1.482) *
3.846(1.260–12.041) *
Not used
2 (3)
63(97)
1
1
Used
15(5.9)
239(94.1)
0.389(0.050-3.022)
2.282(1.057-4.924) *
Not used
4(3.0)
126(96.9)
1
1
Exposed
7(5.7)
116(94.3)
0.889(0.098-8.079)
0.968(.221-19.322)
Not exposed
12(4.6)
250(95.4)
1
1
Present
14 (4.9)
267 (95.0)
1.038 (0.364-2.957)
1.164 (0.345-3.928)
Absent
5 (4.8)
99(95.2)
1
1
Owned
0
2(100)
0.394(0.051-3.022)
0.456(0.057-3.657)
Not owned
0
17(100)
1
1
Used
17(6.0)
266 (94.0)
2.392(1.689-4.572) *
4.537 (2.054-9.453) *
Not used
2 (2.0)
100 (98.0)
1
1
Pipe
5 (1.9)
260(98.1)
0.561(0.219-1.143)
0.564 (0.245-0. 983) *
River
14 (11.7)
106(88.3)
1
1
Male
7 (3.9)
173 (96.1)
0.651(0.251-1.690)
(0.182-1.382)
Female
12 (5.9)
193 (94.1)
1
1
Raw or mildly fried meat
Raw or partially cooked
eggs
Exposure to farm or pet
animals contact
Toilet
Daycare
Raw milk or raw milk
product
Drinking water
Sex
COR = Crude Odds Ratio, AOR = Adjusted Odds Ratio, CI. = Confidence Interval, *significant at p <0.05.
35
Fresh milk drawn from a healthy cow normally contains a low microbial load (less than 1000
ml-1), but the loads may increase up to 100 fold or more once it is stored for some time at
normal temperatures (Richter et al., 1992). In this study, 283(73.5%) of the patients used raw
milk or raw milk product which was stored at room temperature. Since milk serves as a good
medium for growth of many micro-organisms, including pathogenic bacteria such as
Salmonella (Adesiyun et al., 1995), the consumption of raw milk and raw milk products from
a dairy, may result in infection by Salmonella and many other pathogenic bacteria (CDC,
2007). This finding also revealed significant (AOR = 4.537, P= 0.013) association between
consumption of raw milk or raw milk products and Salmonella infection. The odds ratio of
patients indicated that, people who consumed raw or raw milk products were 4.5 folds at risk
of having Salmonella infection than those who do not consumed it.
Farm animals were found to carry Salmonella, affecting meat, dairy products and eggs
(Moreira and Lima, 2001). Salmonella Enteritidis, which is one of the most common types of
Salmonella causing human illness, was found to be associated with consumption of eggcontaining products (Voetsch et al., 2009). Poultry and cattle are a reservoir for Salmonella,
consuming unsafe meat or their products lead to disease caused by Salmonella (Carli et al.,
2001). Thus, human salmonellosis was initiated by the consumption of contaminated products
such as meat and eggs (Foley, 2008 and Mead et al., 1999). In this study, significant (AOR =
3.846, P = 0.002) association between consumption of raw or mildly fried meat and
Salmonella infection was observed. Those patients who consumed raw or mildly fried meat
were 3.8 times at risk of having salmonellosis compared to those who did not consume it.
Furthermore, significant association was observed for Salmonella infection and consumption
of raw or partially cooked eggs in the adjusted odds ratio. Accordingly, patients those who
consumed raw or partially cooked eggs were two folds at risk of having Salmonella infection
than that do not consumed it (AOR = 2.282; CI = 0.181-0.972 P = 0.014). However, the
association was not significant or did not appear in the crude odds ratio of logistic regression
for eggs.
Likewise, significant association in water was not obtained from crude ratio of logistic
regression. In contrast to this, significant association was appeared in the adjusted odds ratio
36
of logistic regression (AOR = 0.564, CI = 0.245-0. 983) and people who used to drink river
water were found to be 50% fold at risk of salmonellosis than that used protected water. This
result is closer to the finding of Guane et al., (2000) who reported the transmission of
Salmonella Typhi infections to human which was associated with ingestion of contaminated
water.
According to the researcher as well as personal observation of local residents, in the study
area, sometimes it is common to leave dead bodies of animals to hyena on the field near to
rivers. There was dead body of cow above the river through which flooding to the river takes
place, in such a way that, its bodies are taken into the river during flooding. There were also
many children swimming, taking bath and washing their cloth in this river. This activity of the
children may be one factor to the high ratio of Salmonella infection in this finding from 6-14
years old.
Out of 384 patients, 111 (28.8%) used river and 274(71.2%) protected water for drinking.
Among these, WTA(Worabe Town Administration) patients used protected water with the
highest percentage 183(97.3%) and unprotected water with the least percentage 5(2.7%)
followed by Dalocha Woreda patients that use 91.7% protected and 8.3% unprotected water.
In contrast to this, Wulbarag Woreda patients were found with the highest percentage of
drinking river water 79(92%) and lowest percentage of protected water 7(8%) especially
amongst the rural people. Furthermore, out of 384 patients, 281(73.0%) used toilet, however,
104 (27.0%) do not had a toilet. As a result, they defecate in the forest, valley or anywhere on
the field. Thus, infected humans and animals shed micro-organisms like Salmonella into the
environment via faeces. Since, Salmonella can survive for a long period of time in soil and
water and resist dehydration for a very long time (aw ≥ 0.93), both in feces and in foods for
human and animal consumption (WHO, 1988) re-infection takes place by ingestion of water
which is contaminated by these shaded micro-organisms by flood. So, Wulbarag Woreda
people are more likely to be under the victim of this problem particularly the rural people
(Clyde, et al., 1997). As reported by Moreira and Lima, (2001) exposure to contaminated
water is known to be associated with diarrhea caused by ingestion of microorganisms.
37
5. Summary, Conclusions and Recommendations
This chapter summarizes findings of the study and the conclusions drawn based on the
findings. At the end, recommendations that were thought to be addressing the problems were
forwarded.
5.1. Summary
Salmonellosis remains an important public health problem worldwide. Transmission of the
organism is through the consumption of contaminated products such as meat, poultry, eggs,
milk, seafood, and water.
Routine cultures are not available in many parts of Ethiopia, furthermore, the prevalence and
antimicrobial drug resistance of Salmonella isolates in the study area was not known. As a
result, a cross sectional hospital based study was designed to investigate the prevalence and
antimicrobial resistance patterns of Salmonella isolates among patients visiting WHC.
A total of three hundred eighty four stool samples were collected among patients attending
WHC for treatment. The samples were transferred to EHNRI laboratory within 48 hours in an
ice box containing Cary Blair transport medium within a test tube. The samples were
inoculated on SSA, XLD and MacConkey media followed by further inoculation of 24hrs
incubated samples within selenite F broth on to SSA. After 24hrs incubation at 37oC typical
colonies of Salmonella were further screened on a fresh media and a series of biochemical test
had been made to identify the organism.
Finally 19 (4.9%) Salmonella isolates were
identified from a total of 384 patients stool sample.
Antibiotic sensitivity test was performed for the identified Salmonella isolates against 9
currently recommended antibiotics (Oxoid Ltd., Basingstoke, England) based on the National
Committee for Clinical Laboratory Standards, (2006) by using Kirby-Bauer disk diffusion
technique in Muller Hinton agar plate. Control strain of Escherichia coli (ATTC 25922) was
used to control the quality of the result.
38
In this finding, all Salmonella isolates were sensitive to ciprofloxacin (5µg), amikacine
(30µg) and norfloxacine (10µg). In addition to this four isolates were sensitive to all tested
antibiotics. However, least sensitivity was observed in amoxicillin and multiple antibiotic
resistances were observed in three isolates of Salmonella. 5.3%, 15.8%, 10.5%, 10.5% and
15.8% resistance were observed to ceftriaxone (30µg), Chloramphenicole (30µg),
Cotrimoxazole (25µg), Amoxicillin (30µg) and Tetracycline (30µg) respectively.
Consumption of raw meat, eggs, milk and milk products were the major common
phenomenon, which were associated with infection of Salmonella.
5.2. Conclusions
This finding indicated that Salmonella isolates are prevalent in the study area, except under
five years old children. In recent years antibiotic resistant Salmonella were reported in this
study as well as in different parts of the world from the previous study. All of Salmonella
isolates were sensitive to Ciprofloxacin (5µg), norfloxacine (10µg) and amikacine (30µg). In
contrast to this, amoxicillin was the least sensitive antibiotics. Similarly, higher resistance was
observed in chloramphenicole and tetracycline in this finding as well as in the previous study
made so far. Special care should be given to those people that drink river water because
water-borne diseases caused by ingestion of contaminated water killed many people in the
world (WHO 2005). Consumption of unsafe meat, eggs, milk and milk products were
associated with Salmonella infections in the study area.
5.3. Recommendations
Based on this finding, the following major recommendations seem to have a great
significance value in order to combat the transmission and antibiotic resistance development
of Salmonella.
1. Protected water supply should be given to population of the study area to control the
transmission of Salmonella through drinking river water.
39
2. Raw eggs, meat, and milk should not be consumed in order to limit the spread of
salmonellosis.
3. Animals that die without known reason and the remains of slaughtered animals should
be buried to limit microbial contamination of the river, which is used by the nearby
residents and the environment as a whole.
4. The emergence of Salmonella strains that are resistant to commonly used
antimicrobials should be particularly noted by clinicians, microbiologists and those
responsible for the control of communicable diseases.
5. Ciprofloxacin, amikacine and norfloxacin should be drugs of choice for treating
salmonellosis in the study area.
6. Amoxicillin should not be used and chloramphenicole and tetracycline should not be
used without antibiotic sensitivity test.
7. Updated bacterial susceptibility data are crucial to physicians and infection control
practitioners in the study area.
8. Surveillance and research on the prevalence and antibiotic susceptibility pattern
should be undertaken with the collaboration of public health institutions, veterinary
and food hygiene, particularly in areas with limited knowledge on drug resistance and
inadequate health facilities.
40
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52
7. APPENDICES
7.1.
Appendix I: Participant Information Sheet (English Version)
My name is ---------------------------------------------------------------Title of the study: Prevalence and Antibiotic Resistance Patterns of Salmonella Isolates in
Patients Visiting Worabe Health Center: Worabe, Southern Ethiopia.
Procedure and Duration of the study: stool samples will be collected from the participants
from October 2010 to February 2011.
Purpose of the study: for the partial fulfillment of Masters of Degree in Microbiology
Benefit and compensation: Fee free treatment will be given at their home to those
individuals who are antibiotic resistant salmonella isolates positive for the prescribed
antibiotics by WHC physicians along with the nearby health extension professionals.
Risks: the study will not have any risks on the patients.
Rights of the participant: they have the right to not participate or refuse their /their child/
participation in the study.
Confidentiality of information: the information obtained from the participants will be
treated confidentially and not used for other purpose.
If you have question or complaint you can ask the contact person by the following address:
Abdulmejid muteba
Tell: 0913159256
Email: abdulmejidm@gmail.com
Institutional research Ethics Review Committee (IRERC)
Tell 0256661899
P. O Box 235
Harar
Will you participate in the study?
A) Yes, I want to participate
B) No, I don’t want to participate
53
7.2.
Appendix II: Written and Signed Consent Form (English Version)
I undersigned have been informed and understand the purpose of this particular research
project. I have also been informed that the information I give will be treated confidentially.
Furthermore, I have been told that I can refuse my /my child/ participation in the study.
Hence, with this understanding, I hereby agree to my /my child/ participation in this particular
research voluntarily.
Signature of the patient/parent (caretaker) ------------------Date -------------------
54
7.3.
Appendix III: Questionnaires (English Version)
Prevalence and Antibiotic Resistance Patterns of Salmonella Isolates in Patients
Visiting Worabe Health Center: Worabe, Southern Ethiopia
Questionnaires: to be filled by the participants.
A. Demographic Information
1. Name ----------------------------------------------------------------age----------sex-------Woreda-----------------------------kebele-----------------------sample code------------2. Did you have a toilet?
A. Yes
B. No
3. Does the child have a day care/ care taker? If the patient is a child.
A. Yes
B. No
4. Where is the source of your drinking water?
A. Pipe
B. Underground
C. River
5. Did you consume any fruit? If your answer for the above question is ‘‘yes’’,
A. Washed
B. Unwashed
6. Are there any animals in your house?
7.
If your answer for the above question is yes, A. Pet B. Farm
8. Did you have exposure for the above animals? If ‘‘yes’’,
A. Pet
B. Farm
9. Did you consume any meat? If ‘‘yes’’,
A. Raw
B. Partially cooked
C. Cooked
10. Did you eat eggs? If yes,
A. Raw
B. Partially cooked
C. Cooked
11. Did you consume milk? If ‘‘yes’’
A. Raw
B. Boiled
C. Yogurt
B. Sign and Symptoms
12. Which one of the following did you experience?
A. Abdominal pain. C. Diarrhea D. Fever
B. Bloody diarrhea E. Head ache F. Vomiting
55
D. cheese
7.4.
Appendix IV: Quetionarreis, Amharic Verssion
የየየየ የየየ: የየየየየየ የየየ የየየየየ የየየየየ የየየየየ የየየ የየየየ የየ የየየ
የየየየየ የየየየ የየየየየ
የየየየየየየ የየየየ የየ የየየየ የየየየየ የየየየ
የ. የየ የየየ የየየ
1. የየ ------------------------------------------------------የየየ------------------የየ-------የየየ-----------------------------የየየ-----------------------የየየየ የየየ---------------2. የየየ የየ የየየ? የ. የየየ የ. የየየየ
3. የየየየ የየየየየየ የየ የየየየ? የየየየ የየየ የየየ የ. የየየ የ. የየየየ
4. የየየየ የየ የየየየየ የየየ የየ?
የ. የየየየ የ. የየየየየ የየየየ የየየ የየ የ. የየየየ የየ
5. የየየየ የየየየየ የየ? የየየየየ የ. የየየየ የ. የየየየየ
6. የየየየየየየ የየ የየየ የየየየየየ የየ የየ? የየየ የየየየየ የየየየየ የ የየየየ
የየየየየ
7. የየየየ የየየየየየ የየ የየየ የየየ የየ?የየየ የ የየየየየ የየየየየ የ የየየየ
የየየየየ
8. የየ የየየየየየ የየ? የየየየየየ የ. የየ የ. የየየየየ የ. የየየየየ
9. የየየየየ የየየየየየ የየ? የየየየየየ የ.የየ የ. የየየየየ የ. የየየየየ
10.
የየየ የየየየየየ የየ? የየየየየየ የ. የየ የ. የየየ የ. የየየ የ. የየየ
የ. የየየየ የየየየየ
11.
የየየየ የየየየ የየየየ የየየየየየየ? የ. የየየየየየ የ. የየ የየየ
የየየየ
የ. የየየየ የ. የየየየ የ. የየየየየ የ. የየየየ
56
7.5.
Appendix V : Quetinarries, Siltigna Verssion
የየየየየ የየየየየ: የየየየየየ የየየ የየየየየየ የየ የየየየየ የየየየ የ የየየየ የየ
የየየ የየየየ የየየየ የየየየ
የየየየ የየየየ የየየ የየየየየየ የየ
የየየየየ የየ
የ. የየየየየ የየየየየ
1. የየ ------------------------------------------------የየየ-----------------የየየ/የየየየየ--------የየየ-----------------------የየየ-----------------የየየየየ የየየ------------------------------2. የየየ የየ የየየ የየ? የ. የየየ የ. የየየ
3. የየየየየየ የየ የየ የየየ: የየየየ የየየየየ የየ የየየ የየ? የ.
የየ
የ. የየየ
4. የየየየየየ የየ የየየ የየየየየ?
የ. የየየየ
የ. የየየየየ የየየየየየ የየ
የ. የየየ የየ
5. የየየየ የየየየ የየ? የየየየ የ. የየየየ የ. የየየየ
6. የየየየየየየየየ የየ የየየ የየየ የየየየ የየ? የየ የ የየየየየ የየየየ የ
የየየ የየየየ
7. የየየ የየየየ የየየየየየየየ የየየ የየየየየየየየየ? የየየየየየየየ የየየ
የየ የ የየየየየየ የየየየ የ የየየየየ የየየ
8. የየየ የየየየየየ? የየየየ የ. የየየ የ. የየየየየየ የ. የየየየ
9. የየ የየየየ የየ? የየየየ የ.የየየ የ. የየየየየየ የ. የየየየ
10.
የየየ የየየየየየ? የየየየ የ. የየ የየየ የ. የየየየ የ. የየየ
የ. የየ
የ. የየየየየ የየየየየየ
11.
የየየየ የየየየ የየየየ የየየየየየየ?
የ. የየየ የየየየ የ. የየ የየየ የየ የየየየየየየ
የ. የየየየ የ. የየየ የ. የየየየ
57
የ. የየ የየየየየየየ
7.6.
Appendix VI: Zone diameter of antibiotics and sensitivity of Salmonella isolates
Table 8: Zone Diameter of the antibiotics tested for salmonella isolates and their sensitivity on Muller
Hinton Agar
Zone Diameter (mm) of the antibiotics with the degree of resistance
Sample
CRo
Nor
C
AMc
TS
FEP
Cip
T
Ak
(30µg)
(10µg)
(30µg)
(30µg)
(25µg)
(30µg)
(5µg)
(30µg)
(30µg)
24
28S
31S
24S
17I
28S
31S
35S
21S
23S
71
27S
30S
23S
17I
26S
32S
32S
20S
24S
73
28S
32S
18I
27S
20S
33S
33S
9R
24S
96
36S
30S
28S
12R
20S
35S
35S
25S
24S
140
32S
33S
10R
25S
9R
39S
30S
10R
25S
185
30S
30S
22S
17I
27S
32S
31S
18S
32S
259
27S
35S
20S
24S
19S
37S
34S
15I
25S
272
38S
26S
25S
15I
26S
32S
25S
20S
24S
286
37S
30S
24S
25S
28S
35S
29S
23S
25S
288
32S
36S
20S
19S
24S
40S
36S
18S
26S
291
27S
30S
<6R
15I
<6R
30S
32S
<6R
24S
294
36S
30S
26S
25S
28S
33S
29S
25S
25S
299
39S
27S
26S
14I
26S
34S
30S
25S
23S
314
29S
33S
26S
25S
25S
35S
38S
20S
26S
320
30S
30S
20S
17I
25S
30S
32S
20S
22S
355
28S
33S
25S
9R
20S
33S
35S
14I
23S
365
22I
32S
20S
13I
21S
33S
35S
15I
25S
369
8R
30S
10R
16I
39S
16I
35S
33S
21S
385
30S
32S
22S
13I
23S
36S
35S
18S
21S
code
R – Resistant, I - Intermediate and S – Sensitive. µg- microgram CRo- ceftriaxone (19-23)
Nor- Norfloxacine (12-17) C- Chloramphenicole (12-18) AMc- Amoxacyline (13-18) TSCotrimoxazole (10-16) FEP- Cefepime (14-18) Cip- Ciprofloxacin (15-21) T- Tetracycline
(11-15) Ak- Amikacine (14-17).
58
7.7.
Appendix VII: Standard Zone Diameter of the Antibiotics and their Sensitivity
Table 7: Standard Zone Diameter of the Antibiotics and their Sensitivity
Antibiotics
Concentration
Standard zone diameter for susceptibility
(µg)
Susceptible
Intermediate
Resistant
Ceftriaxone
30
>23
19-23
<19
Norfloxacine
10
>17
12-17
<12
Chloramphenicole
30
>18
12-18
<12
Amoxicillin
30
>18
13-18
<13
Cotrimoxazole
25
>16
10-16
<10
Cefepime
30
>18
14-18
<14
Ciprofloxacin
5
>21
15-21
<15
Tetracycline
30
>15
11-15
<11
Amikacine
30
>17
14-17
<14
59