Salmonella enterica IN BLOOD SAMPLES OF PATIENTS CLINICALLY SUSPECTED OF ENTERIC FEVER IN SHANKARAPUR HOSPITAL

Transcription

1 Salmonella enterica IN BLOOD SAMPLES OF PATIENTS CLINICALLY SUSPECTED OF ENTERIC FEVER IN SHANKARAPUR HOSPITAL A Dissertation Submitted to the Department of Microbiology St. Xavier s College (Affiliated to Tribhuvan University) In the partial fulfillment of the requirements for the degree of Master of Science in Microbiology (Medical) BY SUDESH ADHIKARI DEPARTMENT OF MICROBIOLOGY ST. XAVIER S COLLEGE MAITIGHAR, KATHMANDU, NEPAL 2011 i

2 Certificate of Recommendation This is to certify that this dissertation entitled Salmonella enterica in Blood Samples of Patients Clinically Suspected of Enteric Fever in Shankarapur Hospital is the genuine work by Mr. Sudesh Adhikari under our direct supervision and guidance. His observations have been checked by us from time to time. The study submitted as thesis for Master of Science in Microbiology is in accordance with the rules and regulations of the Tribhuvan University. Principal Supervisor: Dr. Ramesh Dhakhwa, MD Department of Pathology Shankarapur Hospital Kathmandu, Nepal Date: Supervisors: Mr. Pradeep Kumar Shah MSc Visiting Faculty Department of Microbiology St. Xavier s College Ms. Anima Shrestha, MSc Department of Microbiology St. Xavier s College Tribhuvan University Date: Date: ii

3 Certificate of Approval On the recommendation of the Principal Supervisor Dr. Ramesh Dhakhwa, and Supervisors Senior Lecturer Mr. Pradeep Kumar Shah and Lecturer Ms. Anima Shrestha, the dissertation work of Mr. Sudesh Adhikari entitled Salmonella enterica in Blood Samples of Patients Clinically Suspected of Enteric Fever in Shankarapur Hospital has been approved for the examination. This research work submitted as thesis for Master of Science in Microbiology is in accordance with the rules and regulations of the Tribhuvan University Mr. Sudhakar Pant Head of the Department St. Xavier s College Maitighar, Kathmandu Date: iii

4 Board of Examiners Recommended by: Principal Supervisor (Dr. Ramesh Dhakhwa) Supervisor (Mr. Pradeep Kumar Shah) Supervisor (Ms. Anima Shrestha) Approved by: Mr. Sudhakar Pant Head of the Department St. Xavier s College Examined by: Internal Examiner (Ms. Pallavi Gurung) Lecturer, St. Xavier s College Date: 23/09/ External Examiner (Mr. Binod Lekhak) Central Department of Microbiology Tribhuvan University iv

5 Acknowledgements Firstly, I would like to thank the Almighty God and to my parents for giving me the courage and strength to complete this study, without them this study would not have been possible. I would also like to give my sincere gratitude to my principal supervisor Dr. Ramesh Dhakhwa, and supervisors Mr. Pradeep Kumar Shah and Ms. Anima Shrestha for their guidance, invaluable suggestions, verification and constant encouragement to complete this assignment. I am very grateful to Dr. Ramesh Dhakal, Director, Shankarapur Hospital for giving me a space in his hospital to carry out my research. Many thanks to the Laboratory team of the Department of Pathology, Shankarapur Hospital providing me the precious encouragement and generous technical support to complete this research work. I must sincerely thank Ms. Maunata Khadka and Ms. Samjhana Acharya Lab. Assistants who have constantly helped me in sample collection and processing, and Ms Ambika Shrestha and Ms Sunita Brahmasakha for helping me in media and reagents preparations, cleaning and sterilization works. I also express my gratitude to Rev Dr A Antonysamy SJ, Principal of my college for giving me space to study in this college. I am also thankful to HOD Mr. Sudhakar Pant, Former HOD Dr. Hari Prasad Thapaliya and all faculty members and laboratory team of my college for help and coordination. I would also acknowledge my friends Mr. Raju Patel, Mr. Shailendra Chaudhary, Mr. Milan KC, Mr. Bharat Pangeni, Mr. Dev Raj Awasthi, Mr. Ramesh Pakka, Dr. Chandra Bhushan Yadav, Dr. Bikash Shah, Dr. Mamata Shakya and everyone who delivered me valuable suggestions and for helping me in collecting the materials for the literature review. v

6 I am equally thankful to the patients who provided me the blood samples without which the research would not be possible to carry out. I am also grateful to the physicians and consultants who provided me the clinical history that served as a great tool in completing my research. Last but not the least, I express my warm thanks to my brother Mr. Himal Adhikari, my sister Ms. Shreesti Adhikari and my beloved wife Ms. Susma Koirala Adhikari who rendered me their valuable time and untiring effort in the editing of the thesis work.. Sudesh Adhikari Date: vi

7 Abstract Enteric fever (Typhoid and Paratyphoid fever) continues to be major public health problem in under-developed countries including Nepal. Since the antimicrobial susceptibility profile of the organism may be changing over the time, current knowledge on antimicrobial susceptibility profile is essential for appropriate therapy. ESBL producing bacteria may not be detected by routine disk diffusion susceptibility test leading to inappropriate use of antibiotics and treatment failure. The aim of this study was to isolate Salmonella enterica in blood of the suspected enteric fever patients visiting Shankarapur Hospital as well as identification of MDR, ESBL producing and NAR strains in the population. A cross-sectional study was carried out from February 2010 to October A total 512 samples were processed for culture and sensitivity as well as leukocyte counting and hemoglobin percentage. Out of the total 512 blood cultures samples, only 49 (9.57%) showed bacterial growth. The growth of Salmonella enterica (n=45, 8.78%) was significantly higher (p < 0.05) among the total bacterial isolates. Non- Salmonella included Streptococcus pneumoniae (n=3) and Staphylococcus aureus (n=1). The isolation of the Salmonella in the age group less than 30 years was significantly higher (p<0.05). The isolation rate of Salmonella from the male patients (6.83%) was not found significantly different (p>0.05) from the female patients (11.11%), however, the growth rate was found significantly higher (p<0.05) in out-patient sample (9.09%) than in in-patient sample (7.44%). Four (12.1%) out of thirty three Salmonella enterica (all serovar Typhi) were MDR where as three (24.2%) out of thirty three Salmonella enterica (all serovar Typhi) were NAR. Interestingly, all NAR strains appeared susceptible to Ciprofloxacin and Ofloxacin in in-vitro susceptibility test. Three (9.1%) S. enterica serovar Typhi were found to be ESBL producer while all were susceptible to Imipenem and Meropenem. This study revealed that there is an increase in number of NAR Salmonella. Also there is increasing resistance to many antibiotics. Key Words: Enteric fever, MDR, ESBL, NAR, Antimicrobial Susceptibility Profile vii

8 Table of Contents Title Page i Certificate of Recommendation ii Certificate of Approval iii Board of Examiners iv Acknowledgements v Abstract vii Table of Contents viii List of Appendices x List of tables xi List of figures xii List of Photograph xiii Abbreviations xiii Chapter I: Introduction 1 Chapter II: Objectives of the study 2.1 General Objective Specific Objectives 4 Chapter III: Literature Review 3.1 Salmonella Antibiotic Susceptibility Testing Therapeutic Failure Relapse of Enteric Fever Typhoid carrier Prevention and Control of Enteric Fever Quality Control 41 Chapter IV: Methodology 4.1 Materials Methods Validity and reliability Quality assurance 46 viii

9 4.5 Limitation of the study Data management and analysis 47 Chapter V: Results 48 Chapter VI: Discussion and Conclusion 64 Chapter VIII: Summary and Recommendations 74 References 76 ix

10 List of Appendices Appendix I Questionnaires I Appendix II List of equipments and materials used during the study III Appendix III I. Composition and preparation of different culture media IV II. Composition and preparation of different biochemical tests media VII III. Composition and preparation of different staining and tests reagent X Appendix IV Gram staining procedure XV Appendix V Methodology of biochemical tests used for identification of bacteria XVI Appendix VI Method of collection of blood for Culture XIII Appendix VII Method of performing total leukocyte counting XXIV Appendix VII I Method of determining hemoglobin percentage XXV Appendix IX Serological identification XXVI Appendix X I. Distinguishing biochemical reactions of the isolated Gram negative bacteria XXVII II. Kauffman and White scheme of serological classification of Salmonella XXVIII Appendix XI Zone size interpretative chart XXXI Appendix XII Calculation using χ 2 test. XXXII x

11 List of Tables Table 1: Distribution of positive isolates 49 Table 2: Age wise distribution of Salmonella enterica 50 Table 3: Gender wise distribution of Salmonella enterica among total population 51 Table 4: Symptom wise distributions of Patients with Salmonella enterica 52 Table 5: Hemoglobin Level in Patients with Salmonella enterica 53 Table 6: Antimicrobial susceptibility pattern of S. enterica 54 Table 7: Antimicrobial susceptibility pattern of S. enterica serovar Typhi 56 Table 8: Antimicrobial susceptibility pattern of S. enterica serovar Paratyphi A 58 Table 9: Antimicrobial susceptibility pattern of S. enterica serovar Typhimurium 60 Table 10: Prevalence of NAR and MDR strains in Salmonella enterica 61 Table 11: Prevalence of ESBL in Salmonella enterica serovar Typhi 61 Table 12: Antibiotic sensitivity patterns of ESBL producing Salmonella enterica serovar Typhi 62 Table 13: Comparative susceptibility to antibiotics between ESBL positive and ESBL negative S.enterica serovar Typhi xi

12 List of Figures Figure 1 Distribution of different bacterial species 48 Figure 2 Chart showing Positive and Negative Growth of Salmonella enterica in culture 49 Figure 3 Distribution of Salmonellae among Out and In-Patients 50 Figure 4 Ratio of male: female 51 Figure 5 WBC count in Enteric Fever Patient 53 xii

13 List of Photographs Photograph 1 Photograph 2 Photograph 3 Photograph 4 Photograph 5 Photograph 6 Photograph 7 Colonies of S. enterica on agar plates S. enterica serotype Typhi showing different biochemical reactions Blood Culture bottles (Left: BHI broth, Right 5ml Blood + 45 ml BHI broth) Antibiotic susceptibility profile of S. enterica Salmonella enterica serotyping ESBL producing S. enterica serovar Typhi Researcher performing work on Salmonella enterica xiii

14 ABBREVIATIONS A/A ALK/A AMC ATCC BA CA CE CI CLSI DDST DNA e.g. EDTA ESBL Fig. H2S Hrs LF MA MBC MDR MHA MIC μg μl μm ml mm N/No Acid/Acid Alkaline/Acid Amoxycillin-Clavulanic acid American Type Culture Collection Blood Agar Chocolate Agar Cefotaxime Ceftriaxone Clinical and Laboratory Standards Institute Double Disk Synergy Test Deoxyribonucleic Acid For example Ethylenediaminetetraacetic acid Extended Spectrum Beta Lactamases Figure Hydrogen Sulfide Hours Lactose Fermenter MacConKey Agar Minimum Bactericidal Concentration Multi-Drug Resistant Mueller Hinton Agar Minimum Inhibitory Concentration Microgram Microlitre Micrometer Milliliter Millimeter Number xiv

15 NA NAR NLF O/F OPD PCR SIM TSI TUTH VP WBC WHO ZOI Nutrient Agar Nalidixic acid resistant Non-lactose Fermenter Oxidative/Fermentative Out-patient Department Polymerase chain reaction Sulfide Indole Motility Triple Sugar Iron Tribhuvan University Teaching Hospital Voges Proskauer White blood cell World Health Organization Zone of Inhibition xv

16 CHAPTER I INTRODUCTION The enteric fevers are severe systemic forms of Salmonellosis. The best studied enteric fever is typhoid fever. The causative organism of typhoid is Salmonella enterica serovar Typhi, and Salmonella enterica serovar Paratyphi A, B or C of the paratyphoid fevers. Salmonella organisms continue to be responsible for significant number of gastrointestinal infections (Emmeluth, 2004). In the late 1990s, a new naming scheme for the Salmonella was proposed. This new nomenclature is based on DNA relatedness. This new system would recognize only two species: Salmonella bongori and Salmonella enterica. All human pathogens are regarded as serovars within subspecies I of Salmonella enterica. The proposed nomenclature would change Salmonella typhi to Salmonella enterica serovar Typhi, abbreviated S. Typhi. Some official agencies have adopted the new nomenclature including this dissertation. However, most articles and books still continue to use the name of Salmonella typhi. The term serovar is now used in place of the term serotype. It refers to a serological variety of a species characterized by its ability to induce antibody formation (Emmeluth, 2004; Pegues and Miller, 2010). Infection with organisms of the Salmonella spp. is an important public health problem throughout the world. Non-typhoidal Salmonellosis is a common cause of food-borne infection, and typhoid continues to exact a considerable death toll in developing countries (Jenkins and Gillespie, 2006). Enteric fever still remains as one of the endemic problem in Nepal. S. enterica serotype Typhi and S. enterica serotype Paratyphi A have been reported as the most common isolates from patients in major hospitals (Murdoch et al., 2004, Biswas et al., 2004). In Nepal typhoid fever is also known as "bisham joauro" meaning fever with poison. The fever is prevalent in mountains, valleys and southern belts of Nepal as an endemic disease with its peak incidence in May to August (Lewis et al., 2002, Rauniar et al., 2000). Typhoid outbreak was responsible for the deaths of many Nepalese from the time it was known as one of the causes of the fever. However, typhoid fever was considered responsible for deaths of many patients admitted in the hospitals in Kathmandu, the 1

17 capital of Nepal, in late 1960s when the National Public Health Laboratory (NPHL) came into being (Sharma et al., 2003, Pokharel et al., 2006). There have been reports of seasonal typhoid outbreaks with recent one in 2002 in Bharatpur, a town in central Nepal. The multi-drug resistant typhoid epidemic in Bharatpur affected more than 6,000 patients in a 4- to 5- week period and was from a single source the municipality water supply (Lewis et al., 2002, Gulati et al., 1992). The isolation of NAR strains has become the most interesting subject of research as it has resulted into therapeutic dilemma for physicians. The prolonged defervescence or treatment failure in enteric fever associated with fluoroquinolones have been reported from many countries (Threlfall and Ward, 2001; Ackers et al., 2000). Patients infected with NAR strains were found to have high treatment resistance rates (up to 25%) (Threlfall and Ward, 2001). Since Salmonella infects both humans and animals, bacteria are generally transmitted to humans through the consumption of contaminated food of animal origin, mainly meat, poultry, eggs and milk (WHO, 2003). Salmonella is a widespread illness-causing agent and the intestinal infections like salmonellosis are common in people of all age. Salmonellosis is usually caused by Salmonella enterica serovar Enteritidis and symptoms typically include fever, diarrhea and abdominal cramps. The other prevalent Salmonella infection is typhoid fever, caused by Salmonella enterica serovar Typhi, which is highly endemic in Indian subcontinent and in other developing countries. S. Typhi can invade into the bloodstream and cause life-threatening infections, like bacteremia. These severe generalized infections need to be treated with antimicrobials (Pegues and Miller 2010; CDC, 2009). The extensive uses of antimicrobial agents have invariably resulted in the development of antibiotic resistance, which in recent years has become a major problem worldwide. The emergence of complete resistance to ciprofloxacin in S. enterica serovars with high- level resistance to fluoroquinolones are therefore particularly worrying (Adachi, 2005). The targets of fluoroquinolones are the two enzymes, dna gyrase and topoisomerase IV, whose subunits are encoded respectively by gyra and gyrb and parc and pare genes. The alteration caused by single point mutations within the quinolone resistancedetermining region (QRDR) of the DNA gyrase subunits gyr A leads to quinolone resistance (ie, decreased susceptibility to ciprofloxacin). In Salmonellae, the most 2

18 common residues associated with mutation leading to quinolone resistance have been Ser-83 and Asp-87 in the gyra gene, either alone or together (Wain et al., 1997). ESBL producers do not respond to even third generation cephalosporins in vivo although they are susceptible in vitro tests as well as they are associated with multi drug resistance. Enteric fever is endemic in most developing regions, specially the Indian Subcontinent, South and Central America and Asia, including Nepal (Pegues and Miller, 2010). Nowadays in medical practice, newer antibiotics have been used extensively without studying antibiotic susceptibility pattern which has resulted in emergence of MDR bacterial strain. The organism s intrinsic mutation in the gene is the main cause for emergence of resistant strains. Beside this, the underuse, overuse, misuse of antibiotics, inadequate prescription by the physician and the patients self-prescribing habit could have been contributing in the emergence of MDR Salmonella enterica. There are scanty studies done regarding the MDR, ESBL producing and NAR Salmonella enterica in Nepal which are only a pilot study. Therefore, this dissertation was intended to address the issues regarding the MDR, ESBL-producing and NAR strains and their sensitivity towards broad spectrum antibiotics. This study can be proved important for clinicians in order to facilitate the empirical treatment of patients and management of patients with symptoms of enteric fever. Moreover, the data would also help relevant authorities to formulate antibiotics prescription policies. 3

19 CHAPTER II OBJECTIVES OF THE STUDY 2.1 GENERAL OBJECTIVE To determine the prevalence of Salmonella enterica among suspected patients visiting Shankarapur Hospital SPECIFIC OBJECTIVES To isolate and identify Salmonella enterica. To study the antibiotic sensitivity pattern of isolated Salmonella enterica. To determine the multi drug resistance among isolated Salmonella enterica. To determine the Prevalence of ESBL producing Salmonella enterica. To determine total Leukocyte counting during enteric fever. To determine hemoglobin percentage during enteric fever. 4

20 CHAPTER III 3.1 Salmonella Historical Consideration LITERATURE REVIEW The history of enteric fever goes as long as before 2,500 years. Around BC, a devastating plague, which some believe to have been typhoid fever, killed one third of the population of Athens. Ancient historian Thucydides ( BC) also contracted the disease, but he survived to write about the plague. Alexander the Great died mysteriously in 323 BC and Salmonella was the cause of death, based on a description of Alexander s symptoms written by the Greek author Arrian of Nicomedia. Prince Albert, the consort of Queen Victoria, died of a Salmonella infection in During the Victorian era, an estimated 50,000 cases per year occurred in England. Typhoid outbreak was responsible for deaths of over 6,000 settlers between 1607 and 1624 in Virginia. During Spanish- American War (1898) 20,738 soldiers acquired Typhoid and caused 1,590 deaths. Typhoid outbreak in British camps during the South African War ( ) caused 13,000 deaths. The most notorious carrier was Mary Mallon known as Typhoid Mary. She worked as a cook in domestic service in and around New York at the beginning of the 20 th Century. She may have been the source of infection for several hundred people. Fifty cases and five deaths were confirmed as being associated with her (Ray, 2002; Emmeluth, 2004). In the early 19 th century a group of Pathologists in France reported the association of human intestinal ulceration with a contagious agent which was later identified as typhoid fever. Before that typhus and typhoid fever were confused. In 1829 A. Louis separated typhoid from other fevers on the basis of intestinal lymph node and spleen pathology. In 1849 William Jenner explained typhoid was a different case than typhus and relapsing fever (Jenner, 1850). In 1869, Budd pointed out that the disease was transmitted through the excreta of the patient and the same year the term enteric fever was proposed for typhoid fever by Wilson on the basis of anatomic site of infection. Eberth (1880) described the typhoid bacillus and Gaffky (1884) isolated it in pure culture. Its causative 5

21 role was confirmed by Metchnikoff and Besredka (1900) by infecting apes experimentally (Doherty, 2000). About two decades after during 1920s a great step was forwarded for serological detection of somatic and flagellar antigens within Salmonella groups. An antigenic scheme for classification of Salmonellae was first proposed by White (1926) and in 1941by Kauffmann (Kauffmann, 1950) and till now more than 2,500 serovars are included in the Kauffmann-White scheme (Popoff et al., 2006) Distribution Habitats The principal habitat of the salmonellae is the intestinal tract of humans and animals. Salmonella serovars can be found predominantly in one particular host, can be ubiquitous, or can have an unknown habitat. Typhi and Paratyphi A are strictly human serovars that may cause grave diseases often associated with invasion of the bloodstream. Typhimurium and related ubiquitous Salmonella are mostly responsible for food borne toxic infections. Salmonellosis in these cases is transmitted through fecal contamination of water or food. Gallinarum, Abortusovis, and Typhisuis are respectively avian, ovine, and porcine Salmonella serovars. Such host-adapted serovars cannot grow on minimal medium without growth factors Salmonella in the Natural Environment Salmonellae are disseminated in the natural environment (water, soil, sometimes plants used as food) through human or animal excretion. Humans and animals (either wild or domesticated) can excrete Salmonella either when clinically diseased or after having had salmonellosis, if they remain carriers. Salmonella organisms do not seem to multiply significantly in the natural environment (out of digestive tracts), but they can survive several weeks in water and several years in soil if conditions of temperature, humidity, and ph are favorable (Todar, 2011) Classification and Taxonomy Salmonella is a genus of the family enterobacteriaceae. Before 1983, multiple Salmonella species were taxonomically accepted based on immune reactions with two surface structures O and H. O antigen is a carbohydrate antigen and is the outermost component of lipopolysaccharide and H antigen is a protein antigen called flagellin which is present 6

22 in the flagella. Currently, as a result of experiments indicating a high degree of DNA similarity, the genus Salmonella is divided into two species: Salmonella enterica, which contains six subspecies (I, II, IIIa, IIIb, IV, and VI), and Salmonella bongori subspecies V (Brenner et al., 2000). On March 18, 2005, a new species, Salmonella subterranean was validly approved by the Judicial Commission which was isolated by Shelobolina et al., (2004). S. enterica subspecies I contains almost all the serotypes pathogenic for humans, except for rare human infections with subspecies IIIa and IIIb that were formerly designated by the genus Arizonae. Members of the seven Salmonella subspecies can be serotyped into one of more than 2,500 serotypes (serovars) according to antigenically diverse surface structures. The names of subspecies usually refer to the location where the Salmonella serotype was first isolated. Salmonella species, subspecies, serovars, and their usual habitats, Kauffmann-White scheme a Salmonella species and No. of serotypes subspecies Within subspecies Usual habitat S. enterica subsp. enterica (I) 1,504 Warm-blooded animals S. enterica subsp. salamae (II) 502 Cold-blooded animals and the environment b S. enterica subsp. arizonae (IIIa) 95 Cold-blooded animals and the environment S. enterica subsp. diarizonae (IIIb) 333 Cold-blooded animals and the environment S. enterica subsp. houtenae (IV) 72 Cold-blooded animals and the environment S. enterica subsp. indica (VI) 13 Cold-blooded animals and the environment S. bongori (V) 22 Cold-blooded animals and the environment Total 2,541 a The Kauffmann-White scheme has been described elsewhere (Popoff et al., 2004). b Isolates of all species and subspecies have occurred in humans. 7

23 According to the present Salmonella nomenclature system in use at CDC and recommended by WHO the serotype name is not italicized and the first letter is capitalized (Popoff et al., 2004). The terms serotype and serovar are both frequently used, but according to the Rules of the Bacteriological Code (1990) established by the Judicial Commission of the International Committee on the Systematics of Prokaryotes, the term serovar is preferred to the term serotype. Thus serovar is used in the Kauffmann-White scheme (for example, Salmonella enterica serotype or serovar Typhimurium). Subsequently, the name may be written with the genus followed directly by the serovar name (for example, Salmonella Typhimurium or S. Typhimurium). Both versions of the serotype name are listed as key words in manuscripts to facilitate the search and retrieval of information on Salmonella serotypes from electronic databases (Popoff et al., 1997). Examples of Salmonella nomenclature currently seen in the table: Complete name CDC designation Other designations S. enterica a subsp. enterica ser. Salmonella ser. Typhi Salmonella typhi Typhi S. enterica a subsp. enterica ser. Typhimurium S. ser. Typhimurium Salmonella typhimurium S. enterica a subsp. arizonae ser. 18: S. IIIa 18:z4,z23:- Arizona z4,z23:- hinshawii ser. 7a,7b:1,2,5:- S. enterica a subsp. diarizonae ser. 60:k:z S. IIIb 60:k:z A. hinshawii ser. 24:29:31 S. enterica a subsp. houtenae ser. Marina S. ser. Marina S. IV 48:g,z51:-, S. marina S. enterica a subsp. indica ser. S. ser. Srinagar S. VI 11:b:e,n,x, S. Srinagar srinagar a S. choleraesuis and S. enteritidis are also used (Brenner et al., 2000). 8

24 3.1.4 The Genome The generation of complete genome sequences provides a blueprint that facilitates the genetic characterization of pathogens and their hosts. A comparison of the genomes of several sequenced S. enterica highlights some important common traits. All have a single chromosome, normally Mb in size (Parkhill et al., 2001, McClelland et al., 2004). Different strains may also harbor extra chromosomal DNA in the form of plasmids. Plasmids often carry genes associated with virulence or antibiotic resistance and can be considered to be a rapidly evolving gene pool. Comparison of the chromosomes of different S. enterica identifies a common set of so called core genes that are, in general, shared among enteric species (Anjum et al., 2005). The genome of Salmonella enterica serovar Typhi (S. Typhi) harbors about 5 million base pairs encoding some 4000 genes, of which 1200 are functionally inactive. Comparison of S. Typhi isolates from around the world indicates that they are highly related (clonal) and that they emerged from a single point of origin about 30,000 50,000 years ago. Evidence suggests that, as well as undergoing gene degradation, S. Typhi has also recently acquired genes, such as those encoding the Vi antigen, by horizontal transfer events. The genome sequences of 15 complete salmonellae serotypes, including two S. Typhi strains, two S. Paratyphi A strains, and one S. Paratyphi B strain, six non-typhoidal strains, and numerous other species-specific serotypes including Arizonae, Choleraesuis, Dublin, and Gallinarum, are available in Genbank. Twenty-five other partially completed genomes using next generation sequencing technology also are available, including 15 S. Typhi sequences (Holt et al., 2008). The salmonellae genomes contain approximately 4.8 to 4.9 million base pairs with approximately 4400 to 5600 coding sequences. A characteristic phenomenon of host restriction such as that found for S. Typhi is gene loss. In S. Typhi strain CT18 there are 204 inactivated pseudo genes, which may explain its host restriction to humans, though a recent publication comparing Salmonella Gallinarum, which is host restricted to poultry, to Salmonella Enteritidis phage type 4, which infects poultry and is broad host range, found significant overlap between the defined pseudo genes in both S. Typhi and S. Gallinarum (Pegues and Miller, 2010). 9

25 3.1.5 Microbiology Salmonellae are gram-negative, non-spore-forming and are non-encapsulated, facultatively anaerobic bacilli that measure 2 to 3 by 0.4 to 0.6 µm in size. Like other Enterobacteriaceae, they produce acid on glucose fermentation, reduce nitrates, and do not produce cytochrome oxidase (Pegues and Miller, 2010). They grow optimally at 37 o C on ordinary culture media, with developed small colonies of 2 to 4 mm in diameter, smooth, shiny and homogenous. Salmonella growth may still occur in a wide ph range (4.5 to 9.5) depending on the surrounding conditions. The temperature range at which Salmonella has been growing is 2 o C to 54 o C (S. Typhimurium). Regarding available moisture, growth inhibition has been reported for water activity (aw) values below 0.93 (Doyle et al., 2001). A salt content of 3-4% generally inhibits the growth of Salmonellae, but increasing temperature is increase salt tolerance. However, a salt content above 8% is bactericidal for salmonellae (Jay et al., 2005). Except the rare non-motile Salmonella serovars such as S. Gallinarum and S. Pullorum, the vast majority of Salmonella is motile as a result of peritrichous flagella. The motile Salmonella may lose their ability to develop flagella under the effect of sublethal stress, caused by external physicochemical influence such as refrigeration or high temperatures (Doyle et al., 2001). Most species do not ferment lactose. However, approximately 1% of organisms is able to ferment lactose and therefore may not be detected if only MacConkey agar or other semi selective media are used to identify Salmonella based on colorimetric assay for fermentation of lactose. The differential metabolism of sugars can be used to distinguish many Salmonella serotypes; serotype Typhi is the only organism that does not produce gas on sugar fermentation (Pegues and Miller, 2010). Salmonellae catabolize D-glucose and other carbohydrates with the production of acid and gas. They are oxidase negative and catalase positive, grow on citrate as a sole carbon source, generally produce hydrogen sulfide, decarboxylate lysine and ornithine, and do not hydrolyze urea. Many of these traits have formed the basis for the presumptive biochemical identification on Salmonella isolates, as listed in table in next page. 10

26 Biochemical characteristic of Salmonella (Maza et al., 1997) Biochemical characteristic Reaction Indole - Methyl Red + Voges-proskauer - Citrate +/- Oxidase - Catalase + Urease - Phenylalanine deaminase - Hydrogen sulphide + (except S. Paratyphi A) Lysine decarboxylate + Ornithine decarboxylate + Motility ( 36ºC) + Acid produced from lactose - Acid produced from glucose Epidemiology The etiological agents of enteric fever, Salmonella enterica serovar Typhi, S. enterica serovar Paratyphi A, B and C have no known hosts other than humans. Most commonly, food borne or waterborne transmission occurs as a result of fecal contamination by infected or asymptomatic chronic carriers. Usually, waterborne transmission involves the ingestion of fewer microorganisms and therefore has a longer incubation period and low attack rate compared with food borne transmission. Although direct person to person transmission is uncommon, S. Typhi can be transmitted sexually, including by anal and oral sex (Reller et al., 2003). Health care workers can acquire the disease from infected patients as a result of poor hand hygiene or handling laboratory specimens (Weikel and Guerrant, 1985). 11

27 Magnitude of the problem Enteric fever still remains as a global health problem, with an estimated about 22 million cases caused by S. Typhi and about 5.5 million cases caused by S. Paratyphi A, B, or C annually and an incidence ranging from 25 to 1000 cases per 100,000 population in endemic regions (Crump, 2004; Ochiai, 2008). Reports show about 200,000 to 600,000 deaths annually in endemic regions (Crump, 2004). High incidence of enteric fever (>100/100,000 cases /year) include south-central Asia and Southeast Asia. Regions of medium incidence (10-100/100,000 cases/year) include the rest of Asia, Africa, Latin America and the Caribbean, and the Oceania. The low incidence region (<10/100,000cases/year) includes USA, New Zealand, Australia and Rest Europe (Crump, 2004). The incidence of enteric fever is directly related with poor sanitation and lack of access to clean drinking water. In endemic regions cases are reported more in urban than rural areas and among young children and adolescents particularly between age groups 1 to 15 years. Reported risk factors include contaminated water and ice, flooding, food and drinks purchased from street vendors, raw fruits and vegetables grown in fields fertilized with sewage, ill contacts in household, lack of hand washing and toilet, and evidence of prior Helicobacter pylori infection (Pegues and Miller, 2010). Outbreaks of enteric fever in developing and underdeveloped countries leads to high morbidity and mortality, especially among children less than 5 years of age and when caused by antibiotic resistant strains (Bhutta, 1996). In between many Salmonella enterica developed plasmid-mediated multidrug resistance to common first line antibiotics Chloramphenicol, Ampicillin, and Trimethoprim in many regions of the world, especially in Indian subcontinent and south Asia (Rowe, 1997). In 1990s, the increase use of fluoroquinolones for the treatment of enteric fever resulted in the chromosomal and plasmid encoded resistance towards Ciprofloxacin in S. Typhi and S. Paratyphi isolates from Indian subcontinent and South Asia (Wain, 1997). In 2003, in Kathmandu, Nepal 73.3% of S. Typhi and 94.9% of S. Paratyphi A strains acquired resistance to Nalidixic acid, and had decreased susceptibility to fluroquinolones increasing the failure of fluoroquinolones (Shirakawa et al., 2006). 12

28 Though enteric fever has been burden to developing and undeveloped countries, in developed nations it has become rare occurring infection with the marked improvements in food handling and water/sewage treatment. A data of 2006 shows only 353 cases of enteric fever in USA compared with 35,994 cases in 1920 (Pegues and Miler, 2010). However in Africa and South Asia the burden is still same. In 2009, cases per 100,000 persons were reported in Mediterranean North African countries (Ghenghesh et al., 2009) while in India case incidence ranged from per 100,000 population (Chowta and Chowta, 2005). In our country Nepal, different studies conducted from time to time show more than 100 cases per 100,000 populations (Rai et al., 2005; Shirakawa et al., 2006) Nontyphoidal Salmonellae Besides enteric fever, the incidence rate of non-typhoidal Salmonella infection has markedly increased in the last two decades, with an estimated 1.4 million cases and more than 600 deaths occurring annually (Mead, 1999; Voetsch, 2004). Non typhoid Salmonella (NTS) are zoonotic agents and a wide variety of animals have been identified as a reservoir (Mead et al., 1999). Individuals infected with NTS exhibit mild gastrointestinal illness involving diarrhea, chills, abdominal cramps, fever, head and body aches, nausea and vomiting (Kariuki et al., 2006). A variety of foods have been implicated as vehicles transmitting salmonellosis to humans, including poultry, beef, pork, eggs, milk, cheese, fish, shellfish, fresh fruit and juice and vegetables (Varma et al., 2005). The increasing centralization and industrialization of food supply have enhanced the distribution of these hardy organisms (Hohmann, 2001). NTS may sometimes lead to mycotic aneurysm of the aortic arch which is a rare but potentially fatal condition (Wong et al., 2007). Nontyphoid Salmonella (NTS) is also a common cause of gastroenteritis, which is usually a self-limited illness in healthy children that does not require antimicrobial treatment (Hohmann, 2001) however, children with Salmonella gastroenteritis below 6 months of age who are febrile and dehydrated should be treated empirically with antibiotics until the result of blood culture is available (Araque, 2009). 13

29 Identification of non-typhoidal Salmonella species in the bacteriology laboratory is not difficult (Bopp et al., 2003). The best recovery of such species from fecal samples can be achieved by the use of direct plating and inoculation of standard enrichment broths. Rectal swabs are inferior to fecal specimens and are not recommended. Many selective agar plates are available for NTS. Most laboratories use one medium with low selectivity, such as MacConkey agar, and one with higher selectivity, such as Hektoen enteric agar or XLD agar; a medium with higher selectivity is also useful for the identification of Shigella species. Newer, more-selective chromogenic agars may be useful, but have not yet made their way into routine clinical use. Less than 1% of nontyphoidal Salmonella are lactose positive but this is rarely a problem in the laboratory if one of the aforementioned plates that look for hydrogen sulfide production by Salmonella is also used. Salmonellae are facultative anaerobes that grow well both in bottles of standard, paired automated systems for culture of blood samples and on culture media routinely used for urine, tissue, and respiratory cultures (Hohmann, 2001) Pathogenesis Enteric fever begins with the ingestion of Salmonella enterica in contaminated food and water. In a healthy person the median dose required to produce disease is approximately 10 6 S. enterica serovars however dose may be even less depending upon volunteer to be affected (Blaser and Neuman, 1982). The primary barrier is gastric juice that affects Salmonella colonization and those bacilli that tolerate acid and survive in stomach pass to the small intestine that leads to infection (Selsted et al., 1992). The pathogenesis begins with the interaction of Salmonella enterica with intestinal epithelium and induction of enteritis. Salmonellae invade intestinal epithelial cells through a process termed as bacteria-mediated endocytosis (Francis et al., 1992). Salmonellae encode a type ΙΙΙ secretion system (T3SS) within Salmonella pathogenecity island 1 (the SP Ι-1 T3SS) which is required for bacteria mediated endocytosis and intestinal epithelial invasion (Pegues and Miller, 2010). The second phase follows interactions of bacilli with macrophages which lead to systemic infection. After crossing the epithelial barrier, salmonellae encounter and enter macrophages present in submucosal space and invade through Payer s patches. Pathology in the Payer s patches 14

30 assumes four phases. These phases correspond approximately to the weeks of disease if treatment has not been given. Phase 1: Hyperplasia of lymphoid follicles. Phase 2: Necrosis of lymphoid follicles during the second week involving both mucosa and submucosa. Phase 3: Ulceration in the long axis of the bowel with the possibility of perforation and hemorrhage. Phase 4: Healing takes place from the fourth week onward, and unlike tuberculosis of the bowel with its encircling ulcers, does not produce strictures (Singh, 2001). (Source: Pathophysiology of Enteric Fever 15

31 Bacteria are then transported to intestinal lymph nodes, where they multiply within mononuclear cells. Monocytes, unable to destroy the bacilli early in the disease process, carry these organisms into the mesenteric lymph nodes. Organisms then reach blood stream through the thoracic duct, causing a transient bacteremia. The number of bacteria that initially reach the blood is not very large, not enough to cause noticeable symptoms. They are rapidly filtered by the fixed macrophages of the reticuloendothelial system, especially in the liver, spleen, and bone marrow. They continue to multiply within these cells (Pegues and Miller, 2010). When the intracellular population reaches a critical level, the organisms emerge and invade the blood-stream. This marks the end of the incubation period and the start of the clinical illness, with the beginning of a sustained period of high fever. These events take time, which is why the incubation period of typhoid is relatively long, 1-2 weeks from ingestion of the organisms to onset of symptoms. Secondary bacteremia leads to the localization of the now abundant organisms at many of the body, accounting in part for the numerous clinical manifestations of enteric fever. Many of these manifestations can be better understood if one thinks of typhoid fever as a disease of the reticuloendothelial system (RES) and a disorder of the areas of the body where there are localized collections of cells of this system, especially macrophages (e.g. spleen, bone marrow, liver, payer s patches of the gut). Aggregate of the infected mononuclear cells forms the basic pathological lesion, the typhoid nodule. Typhoid bacilli eventually reach the gallbladder, where they grow actively in the bile. This localization is somewhat unusual among bacterial pathogens and is due in part to the particular resistance of the organisms to the bile salts. Note that what appears to be a specific tropism for the gallbladder is due to the combination of properties not shared with many other bacteria, i.e., the ability to multiply within Kupffer cells and to resist the damaging effects of bile. It is fortunate that few other bacteria have evolved these characteristics. The usual course of typhoid fever is 4 to 6 weeks in uncomplicated untreated cases. Circulating organisms reach the reticuloendothelial cells in the liver, spleen and bone marrow and may seed other organs. After proliferation into the reticuloendothelial system, bacteremia recurs. The gallbladder is particularly susceptible to being infected. Local multiplication in the walls of the gallbladder produces large numbers of salmonellae, which reach the intestine through the bile (Pegues and Miller, 2010; Jenkins 16

32 and Gillespie, 2006). Several virulence factors play important role in the pathogenesis. The surface Vi capsular antigen found in S. enterica interferes with the phagocytosis by preventing the binding of C3 to the surface of the bacterium. Circulating endotoxin, a lipopolysaccharide component of the bacterial cell wall, is thought to cause the prolonged fever and toxic symptoms of enteric fever, although its level in symptomatic patients is low. Alternatively, endotoxin induced cytokine production by human macrophages may cause the systemic symptoms. Cell mediated immunity is important in protecting the human host against enteric fever. Decreased number of T lymphocytes is found in patients who are critically ill with typhoid fever. Carriers show impaired cellular reactivity to S. enterica antigens in the leukocyte inhibition test. In carriers, a large number of virulent bacilli pass into intestine daily and are excreted in the stool, without entering the epithelium of the host (Cleary, 2003). Fig: Salmonella membrane ruffles. These extensions of the plasma membrane are stimulated by the Salmonella (arrow) and are related to internalizing the bacteria (Source: Ryan, 2004). 17

33 3.1.9 Clinical Manifestations of Salmonellosis The clinical patterns of salmonellosis can be divided into gastroenteritis, bacteremia with and without focal extra intestinal infection, enteric fever, and the asymptomatic carrier state. Any Salmonella serotype can probably cause any of these clinical manifestations under appropriate conditions, but in practice the S. enterica serovars are associated primarily with gastroenteritis. Typhi and a few related serovars (Paratyphi) cause enteric fever. The incubation period is usually 7-14 days, but it may range from 3-30 days, depending mainly on the size of the ingested inoculums (Clearly, 2003) Gastroenteritis Typically, the episode begins 24 to 48 hours after ingestion, with nausea and vomiting followed by, or concomitant with, abdominal cramps and diarrhea. Diarrhea persists as the predominant symptom for 3 to 4 days and usually resolves spontaneously within 7 days. Fever (39 C) is present in about 50% of the patients. The spectrum of disease ranges from a few loose stools to a severe dysentery-like syndrome Bacteremia and Metastatic Infection The acute gastroenteritis caused by S. enterica can be associated with transient or persistent bacteremia. Frank sepsis is uncommon, except in those with a compromised cell mediated immune system. Salmonella infection in patients with acquired immunodeficiency syndrome (AIDS) is common and often severe. Bacteremia occurs in 70% of these patients and can cause septic shock and death. Despite adequate antimicrobial coverage, relapses are frequent (Gordon, 2008). Patients with lymphoproliferative disease, perhaps owing to T-cell defects similar to those in patients with AIDS, are also highly susceptible to disseminated salmonellosis. Metastatic spread by salmonellae is a significant risk when bacteremia occurs. These organisms have a unique ability to colonize sites of preexisting structural abnormality including atherosclerotic plaques, sites of malignancy, and the meninges (especially in infants). Salmonella infection of the bone typically involves the long bones; in particular, sites of trauma, sickle cell injury, and skeletal prosthesis are at risk. 18

34 Enteric Fever The disease was initially called typhoid fever because of its clinical similarity to typhus. However, in the early 1800, typhoid fever was clearly defined pathologically as unique illness on the basis of its association with enlarged peyer s patches and mesenteric lymph node. In 1869, given the anatomical site of infection, the term enteric fever was proposed as an alternative designation to distinguish typhoid fever from typhus. S. enterica serovar Typhi is not primarily an opportunistic organism: Typhoid fever readily affects healthy non-immune persons, not just debilitated people. This attests to the intrinsic invasiveness of the organism and to its ability to overcome constitutive host defences. It consists of a series of definable steps, which are not always so sharply delineated. Enteric fever is a multi-organ system Salmonella infection characterized by prolonged fever, sustained bacteremia, and profound involvement of the RES, particularly the mesenteric lymph nodes, liver, and spleen. The manifestations of typhoid have been well documented in human volunteer studies conducted during vaccine trials. The mean incubation period is 13 days, and the first sign of disease is fever associated with a headache. The fever rises in a stepwise fashion over the next 72 hours. A relatively slow pulse is characteristic and out of phase with the elevated temperature. In untreated patients, the onset of illness is insidious and non-specific, with intermittent fever, headache and abdominal pain. Physical findings in the early stages include abdominal tenderness, hepatosplenomegaly, lymphadenopathy and a scanty maculopapular rash ( rose spots ). Few in number, these spots are readily overlooked, especially in darkskinned individuals. Many patients are constipated, although perhaps one third of patients have a mild diarrhea. As the untreated disease progresses, an increasing number of patients complain of diarrhea. Without treatment (and occasionally even after treatment) serious complications can arise, usually in the third week of illness. These include meningitis, lobar pneumonia, osteomyelitis, intestinal perforation and intestinal hemorrhage. The fourth week of the illness is characterized by gradual improvement, but in developing countries up to 30% of those infected will die, and 10% of untreated survivors will relapse (Finch et al., 2009). 19

35 Obviously, chronic infection of the bloodstream is a serious disease, and the effects of endotoxin can lead to myocarditis, encephalopathy, or intravascular coagulation. Moreover, the persistent bacteremia can lead to infection at other sites. Of particular importance is the biliary tree, with reinfection of the intestinal tract and diarrhea late in the disease. UTI and metastatic lesions in bone, joint, liver, and meninges may also occur. However, the most important complication of typhoid fever is hemorrhage from perforations through the wall of the terminal ileum at the site of necrotic Peyer s patches or in the proximal colon. These occur in patients whose disease has been progressing for 2 weeks or more. The classical sign of typhoid is fever. Fever is continuous and increases as the disease progresses. A few patients may show abdominal discomfort associated with diarrhea. Non specific symptoms: Chills, sweating, headache, loss of appetite, weakness, sore throat, and dry cough, constipation, and muscle pains are often present. Patients may lose interest in their surroundings. In case of enteric fever in children, the onset of the symptoms is insidious. Headache, malaise, generalized aches in the muscle joints and abdominal pain are fluently observed. Vomiting may be present with loss of appetite. However step ladder pattern of fever as observed in adults and rose spots as in enteric fever may not be seen in small children but ronchi and scattered rales may be heard on asculation of the chest. Case definition (i) Confirmed case of typhoid fever: A patient with fever (38 C and above) that has lasted for at least three days, with a laboratory-confirmed positive culture (blood, bone marrow, bowel fluid) of S. enterica serovar Typhi. (ii) Probable case of typhoid fever: A patient with fever (38 C and above) that has lasted for at least three days, with a positive serodiagnosis or antigen detection test but without S. Typhi isolation. (iii) Chronic carrier: Excretion of S. Typhi in stools or urine (or repeated positive bile or duodenal string cultures) for longer than one year after the onset of acute typhoid fever. Short-term carriers also exist but their epidemiological role is 20

36 not as important as that of chronic carriers. Some patients excreting S. Typhi have no history of typhoid fever Laboratory Diagnosis The laboratory diagnosis of enteric fever is very important mainly because in postantibiotic era most of the patients are treated empirically by the local medical practitioners and when the fever does not subside, these cases are labeled as pyrexia of unknown origin (PUO) and investigated for various causes of PUO including enteric fever. At this stage the typical signs and symptoms as described above are hardly observed (Singh, 2001). Laboratory diagnosis of enteric fever depends mainly on the isolation and identification of the causative organism from a specimen of blood, stool, urine, bile and bone marrow cultures. Whenever the cultures fail to isolate the organism, serological tests available for the diagnosis of enteric fever are applied to check the rise in the antibody titer (Sherwal et al., 2004). Culture of Salmonella from the blood or feces is the primary diagnostic method. Early in the course of enteric fever, blood is far more likely to give a positive culture result than culture from any other site. The media used for stool culture are the same as those used for Shigella. Failure to ferment lactose and the production of hydrogen sulfides from sulfur containing amino acids are characteristic features used to identify suspect colonies on the selective isolation media. Characteristics of biochemical tests are used to identify the genus, and O serogroup antisera are available in larger laboratories for confirmation. Typhi has a pattern of biochemical reactions which are sufficient to characterize it without reference to its serogroup (D). All isolates should be referred to public health laboratories for confirmation and epidemiologic tracing. Serologic tests are no longer used for diagnosis (Forbes et al., 2007) Blood Culture: Blood cultures are easy to perform and do not require special technique-only one kind of bacteria is likely to be present in the sample. Enteric fever cannot be diagnosed clinically or microbiologically during the asymptomatic incubation period when blood cultures are negative. The organisms are usually isolated from the blood during the acute phase of the 21

37 disease i.e. within the first 7-10 days of infection and febrile period. The percentage blood culture isolation of Salmonella falls after first week of infection (Gasem et al., 1995). The volume of blood cultured is one of the most important factors in the isolation of S. enterica from suspected patients ml should be taken from school children and adults in order to achieve optimal isolation rates; 2-4 ml is required from toddlers and pre-school children (Wain et al., 1998). This is because children have higher levels of bacteremia than adults. In some regions it may be impossible to collect such large volumes of blood and so alternative diagnostic methods may be necessary for cases in which blood cultures are negative. Because reducing the blood volume reduces the sensitivity of the blood culture, however, an effort should be made to draw sufficient blood if at all possible. Blood should be drawn by means of a sterile technique of venous puncture and should be inoculated immediately into a blood culture bottle with the syringe that has been used for collection. Several reports of pseudobacteremia have been associated with the reinoculation of blood culture bottles after the collection of blood in contaminated vessels. The practice of inoculating blood culture bottles from specimens taken for biochemical or hematological analysis should therefore be avoided. The optimum ratio of the volume of blood to traditional culture broth should be 1 to 10 or more (e.g. 1:12). In general, if 5 ml of blood are drawn they should be inoculated into 45 ml or more of broth. If ml of blood is drawn the specimen can be divided into equal aliquots and inoculated into two or more blood culture bottles. This allows the use of standard blood culture bottles of 50 ml. For small children the volume of blood drawn can be reduced but should still be inoculated into 45 ml of culture broth. In order to assist the interpretation of negative results the volume of blood collected should be carefully recorded. The blood culture bottle should then be transported to the main laboratory at ambient temperature (15 C to 40 C) as indicated above. Blood cultures should not be stored or transported at low temperatures. If the ambient temperature is below 15 C it is advisable to transport blood cultures in an incubator. In the laboratory, blood culture bottles should be incubated at 37 C and checked for turbidity, gas formation and other evidence of growth after 1, 2, 3 and 7 days. For days 1, 2 and 3, only bottles showing 22

38 signs of positive growth are cultured on agar plates. On day 7 all bottles should be subcultured before being discarded as negative (Forbes et al., 2007; Gasem et al., 1995). Subculturing is performed on days 1, 2, 3 and 7 on non-selective agar. The best agar is blood agar (horse or sheep blood) as this allows the growth of most bacterial pathogens (WHO, 2003). Blood Culture has great advantage over stool and urine cultures, as they are positive even in the asymptomatic carrier (Gilmen, 1975) Stool or rectal swab Culture: This involves inoculating 1 g of stool into 10 ml of selenite F broth and incubating at 37 C for hours. Because selenite broth is very sensitive to heat the manufacturer s instructions should be carefully followed during preparation and overheating of the broth during sterilization should be avoided. Once a batch is prepared it should be stored at 4 C. Selenite broth inhibits the motility of E. coli found in stools but does not kill this bacterium. A subculture of selenite broth on a selective agar is therefore made from the surface of the broth without disturbing the sediment. The choice of agar media includes MacConkey agar, deoxycholate citrate agar, xylose-lysine deoxycholate agar, and hektoen enteric agar or SS (Salmonella-Shigella). The plate is incubated at 37 C for 24 hours Urine Culture: Urine samples become positive later in infection, after when secondary bacteremia has seeded bacteria in the kidneys Colony characteristics Blood agar On blood agar, S. Typhi and S. Paratyphi usually produce non-hemolytic smooth white colonies. MacConkey agar On MacConkey agar, salmonellae produce lactose non-fermenting smooth colonies. 23

39 SS agar On SS agar, salmonellae usually produce lactose non-fermenting colonies with black centers (except S. Paratyphi A, whose colonies do not have black centers). Deoxycholate agar (DCA) On DCA, salmonellae produce lactose non-fermenting colonies with black centers (except S. Paratyphi A, whose colonies do not have black centers). Xylose-lysine-deoxycholate (XLD) agar On xylose-deoxycholate agar, salmonellae produce transparent red colonies with black centres (except S. Paratyphi A, whose colonies do not have black center). Hektoen enteric agar On hektoen enteric agar, salmonellae produce transparent green colonies with black centers (except S. Paratyphi A, whose colonies do not have black centers). Bismuth sulfite agar On this medium, salmonellae produce black colonies Biochemical identification Suspected Salmonella enterica colonies obtained on the above media are screened by means of the following media/tests: Salmonella TSI Agar Motility Indole Citrate Urea enterica serovar Slant Butt H2S Gas Typhi Pink Yellow weak Paratyphi A Pink Yellow Other salmonellae Pink Yellow +/- +/ / Serological Procedures (i). Serological identification Salmonellae can be characterized by their somatic (O) and flagellar (H) antigens, the latter existing in some serotypes in phases 1 and 2. Some salmonellae also have an envelope antigen called Vi (virulence). Agglutination with polyvalent O and H antiserum Salmonella species should agglutinate with Polyvalent O antiserum. Some serotypes eg 24

40 Salmonella Typhi may produce a Vi antigen, which can prevent agglutination with Polyvalent O antiserum. Not all O serotypes are included in Polyvalent O antisera. H antigens may not be well developed on some solid agar and should again be subcultured to semi-solid agar if necessary. The following limited ranges of antisera are available for routine use: Polyvalent O Single factor O (2, 4, 6, 7, 8, 9, 3, 10) Polyvalent H Rapid H sera (RSD 1, 2, 3) Polyvalent H phase 2 (1-7) Single factor H (a, b, c, d, E, G, i, r) (ii) Felix-Widal Test for Antibody detection This test measures agglutinating antibody levels against O and H, AH and BH antigens. The levels are measured by using doubling dilutions of sera in large test tubes. Usually, O antibodies appear on days 6-8 and others on days after the onset of the disease. The test is usually performed on an acute serum (at first contact with the patient). A convalescent serum should preferably also be collected so that paired titrations can be performed. In practice, however, this is often difficult. At least 1 ml of blood should be collected each time in order to have a sufficient amount of serum. In exceptional circumstances the test can be performed on plasma without any adverse effect on the result. The test has only moderate sensitivity and specificity. It can be negative in up to 30% of culture-proven cases of enteric fever. This may be because of prior antibiotic therapy that has blunted the antibody response. On the other hand, S.Typhi shares O and H antigens with other Salmonella serotypes and has cross-reacting epitopes with other Enterobacteriacae, and this can lead to false-positive results. Such results may also occur in other clinical conditions, e.g. malaria, typhus, bacteremia caused by other organisms, and cirrhosis. In areas of endemicity there is often a low background level of antibodies in the normal population. Determining an appropriate cut-off for a positive result can be difficult since it varies between areas and between times in given areas (WHO, 2003). 25

41 It is therefore important to establish the antibody level in the normal population in a particular locality in order to determine a threshold above which the antibody titer is considered significant. This is particularly important if, as usually happens, a single acute sample is available for testing. If paired sera are available a fourfold rise in the antibody titer between convalescent and acute sera is diagnostic. Quality control of the test is achieved by running a standard serum with a known antibody titer in parallel in each batch of assays. The variations in the standard serum should not exceed one tube, i.e. double dilution New diagnostic tests: current status and usefulness There is a need for a quick and reliable diagnostic test for typhoid fever as an alternative to the Widal test. Recent advances include the IDL Tubex test marketed by a Swedish company, which reportedly can detect IgM O9 antibodies from patients within a few minutes. Another rapid serological test, Typhidot, takes three hours to perform. The dipstick test, developed in the Netherlands, is based on the binding of S. Typhi-specific IgM antibodies in samples to S. Typhi lipopolysaccharide (LPS) antigen and the staining of bound antibodies by an anti-human IgM antibody conjugated to colloidal dye particles (WHO., 2003). Recently a 60 minutes dot enzyme immunoassay for the rapid detection of Salmonella typhi specific IgM and IgG antibodies has been introduced. The test is reported to be 95% sensitive (Ismail et al, 1992) Salmonella Gastroenteritis ( Salmonella Food Poisoning ) Many Salmonellae other than those that cause typhoid fever are involved in human disease. The most common Salmonella disease by far is an acute gastroenteritis. It is often referred to as food poisoning, but this is a misnomer because the condition is not caused by a bacterial toxin present in the blood but is a true infection. Salmonella is the most common cause of gastroenteritis with about two million documented cases a year. Staphylococci and other bacteria cause food poisoning by producing toxins in the food. Salmonella gastroenteritis is possibly the most common zoonosis or animal derived disease, with Campylobacter jejuni closing in on the leader. Chicken, turkeys, and cattle 26

42 may acquire the organisms from contaminated feed and either become asymptomatic carriers or acquire infection ( shipping fever ) during transport to market. The symptoms of Salmonella food poisoning are noticed 24 to 36 hours after eating contaminated food, much later than the time after ingesting food containing, say, preformed Staphylococcal enterotoxin. This relatively long incubation time is due to the need for the organisms to multiply to a sufficient concentration in the small intestine. The main symptom is a watery diarrhea, sometimes bloody, and accompanied by low-grade fever. These symptoms may be due to the production of a cholera-like toxin (though this is still a controversial matter) and the initiation of an inflammatory reaction in the intestinal mucosa (Pegues and Miller, 2010). 3.2 Antibiotic Susceptibility Testing Introduction to Antibiotics Since therapy of infection normally begins, quite properly, before laboratory results are available, antibiotic sensitivity testing primarily plays a supplementary role in confirming that the organism is susceptible to the agent that is being used. Sometimes it may enable the clinician to change from a toxic to a less toxic agent or from an expensive to a cheaper one. Usually the laboratory report will influence treatment only if the patient is failing to respond. By this time, the laboratory should have succeeded in establishing the sensitivity pattern of the offending organism (if it is bacterial) sufficiently for the clinician to be able to make an informed decision as to how treatment might be modified. Sensitivity testing of non-bacterial pathogens is not usually possible, although limited antifungal testing is carried out in some centers, and methods for testing antiviral agents are being developed (Chambers, 2007) Classification of Antibiotics Traditionally, antimicrobial agents have been classified based on their mechanism of action, chemical structure, or spectrum of activity. The primary mode of action is the inhibition of vital steps in the growth or function of the microorganism. These steps include inhibiting bacterial or fungal cell wall synthesis, inhibiting protein synthesis, 27

43 inhibiting nucleic acid synthesis, or disrupting cell membrane function (Calderón and Sabundayo, 2007). Antibiotic Mechanisms of Action and Description of Activity a. Inhibition of Bacterial Cell Wall Synthesis Penicillins (cidal) a Cephalosporins (cidal) Carbapenems (cidal) Monobactams (cidal) Glycopeptides (cidal) b. Inhibition of Nucleic Acid Synthesis Fluoroquinolones (cidal) Rifamycins (cidal) Sulfonamides (static) Trimethoprim (static) Cyclic lipopeptides (cidal) c. Inhibition of Protein Synthesis Aminoglycosides (cidal) Tetracyclines (static) Glycylcyclines Chloramphenicol (static) Clindamycin (static) Macrolides (static or cidal) Ketolides Oxazolidinones Streptogramins (cidal) d. Inhibition of Cell Membrane Function Amphotericin B Imidazoles (fungistatic) Triazoles (fungistatic) Echinocandins a cidal = bactericidal; static = bacteriostatic. By the spectrum of their biological action antibiotics are also commonly classified as: Broad spectrum: Antibiotics of broad spectrum activity are active against both gram-positive and gramnegative bacteria. The broad spectrum antibiotics are Tetracyclines, Chloramphenicol. 28

44 Narrow spectrum: Antibiotics of narrow spectrum are active against gram-positive or gram-negative bacteria e.g. Penicillin, Bacitracin, Cloxacillin against gram-positive bacteria Antimicrobial susceptibility test for enteric fever organisms Antimicrobial susceptibility testing is crucial for the guidance of clinical management. Isolates from many parts of the world are now multidrug-resistant (MDR) (Rowe et al., 1997; Bhutta., 1996). Isolates are usually resistant to Ampicillin, Chloramphenicol, Sulfonamide, Trimethoprim, Streptomycin and Tetracycline. Alternative drugs that are used for treatment include: fluoroquinolones (e.g. Ciprofloxacin), third-generation cephalosporins (e.g. Ceftriaxone, Cefotaxime), a monobactum beta-lactam (Aztreonam) and a macrolide (Azithromycin). Even though resistance to the first two has been noted they nevertheless remain useful (Saha et al., 1999). Reduced susceptibility to fluoroquinolones is indicated by in vitro resistance to Nalidixic acid. In vitro susceptibility testing usually involves disc diffusion. The choice of antimicrobial agents for the test is dictated by the agents that are currently being used for treatment and the desire to determine the prevalence of MDR strains (WHO, 2003). It is therefore recommended that susceptibility tests be performed against the following antimicrobial agents: a fluoroquinolone, a third-generation cephalosporin and any other drug currently used for treatment, Nalidixic acid (for determining reduced susceptibility to fluoroquinolones because of the possibility of false in vitro susceptibility against the fluoroquinolone used for treatment), and the previous first-line antimicrobials to which the strains could be resistant. Azithromycin disc test results should be interpreted with caution. The appropriate break-point recommendations for Azithromycin against S. enterica are still not clear. Patients may respond satisfactorily to Azithromycin even if isolates are intermediate according to current guidelines (WHO, 2003) Antibiotic Resistance Antibiotic resistance is defined as the microbe which is sensitive to certain antibiotic starts gaining resistance against it. The MDR strain is defined as the strain that shows 29

45 resistance to two or more antibiotics among the six commonly prescribe drugs (Tuladhar et al., 2003) Antibiotic resistance mechanisms (Smith, 2004) An antibiotic is said to be resistant when that antibiotic in prescribed amount and concentration is unable to kill/ suppress the growth of the pathogens. Environmentally mediated antimicrobial resistance Environmentally mediated resistance is defined as resistance that directly results from physical or chemical characteristics of the environment that either directly alter the antimicrobial agent or alter the microorganism's normal physiologic response to the drug. Examples of environmental factors include ph, anaerobic atmosphere, cation concentration and thymine-thymidine content. Antibacterial activities of Erythromycin and Aminoglycosides diminish with decresing ph while the activity of tetracycline decreases with increasing ph. Aminoglycoside activity requires intracellular uptake across the cell membrane, much of which is driven by oxidative processes so that in the absence of oxygen, uptake and hence activity is substantially diminished. Microorganism mediated antimicrobial resistance Microorganism mediated resistance refers to antimicrobial resistance that is due to genetically encoded traits of the microorganism and is the type of resistance that in-vitro susceptibility testing methods are targeted to detect. Organism-based resistance can be divided into two subcategories: intrinsic or inherent resistance and acquired resistance. A. Intrinsic resistance Antimicrobial resistance resulting from the normal genetic, structural or physiologic state of a microorganism is referred to as intrinsic resistance. For example, Aminoglycosides are ineffective against Enterococci due to lack of sufficient oxidative metabolism to drive uptake of Aminoglycosides. Similarly sulphonamides, Trimethoprim, Tetracycline or Chloramphenicol are futile against P.aeruginosa due to lack of uptake resulting from 30

46 inability of antibiotics to achieve effective intracellular concentrations (Forbes et al., 2007). B. Acquired resistance Antibiotic resistance that results from altered cellular physiology and structure caused by change in a microorganism's usual genetic makeup is known as acquired resistance. The genetic basis of antimicrobial resistance The genetic change from drug sensitivity to resistance may come about in bacteria by following modes: Spontaneous mutation The appearance of resistant cells can be explained by the relatively infrequent occurrence (approximately 1 per 10 7 cells per cell division) of spontaneous gene mutations which confer drug resistance. Transfer of genetic information i. Conjugation In conjugation, there is physical contact between two genetically different bacterial cells of the same or closely related species. There is no exchange of genetic material during conjugation, only unilateral transfer. Genetic material that mediates resistance is most often transferred as plasmids or transposons. Resistance may, therefore, pass between species, including from commensals to pathogens, and vice versa (Hawkey, 1998). ii. Transduction Transduction is the transfer of genetic information between bacteria by bacteriophages. In the clinical setting, transduction may be more important in spreading resistance among gram-positive bacteria than gram-negative bacteria. iii. Transformation It is a process in which a free DNA molecule is transferred from a donor to a recipient bacterium. The DNA released from the donor cell upon cell lysis may be absorbed by competent cells and integrated into their genomes. 31

47 Biochemical Mechanisms of antimicrobial resistance (Rice et al., 2003) The following are the important possible mechanisms by which cells might resist the toxic effects of growth-inhibiting drug. 1. Modification of the Antibiotic Many antibiotic-modifying enzymes have been described, including the β-lactamases, the aminoglycoside-modifying enzymes, and Chloramphenicol acetyltransferases. These enzymes in general confer high level resistance to the antibiotic against which they have activity. 2. Modification to the Target Molecule Since antibiotic interaction with target molecule is generally quite specific, minor alterations of the target molecule can have important effects on antibiotic binding. Modifications can affect the affinities of these molecules for antibiotics. Generally modifications are very common in cell wall, nucleic acid, ribosomes and bacterial enzymes. 3. Restricted Access to the Target Antibiotics need to cross many barriers before they reach its target. Reductions in the quantities of known or presumed porins (cellular permeability) and oxygen dependent factors play vital role in restriction against antibiotics. 4. Efflux Pumps Several classes of pumps have been described in bacteria that are present in cytoplasmic membrane. The combination of more than one pumps result in higher level of resistance. Also sometimes the down-regulation of efflux pumps result in resitance against antibiotics. Resistance Mechanisms for Different Antimicrobial Classes (Rice et al., 2003; Chambers, 2007) A. Resistance to penicillins and other -lactams This involves one of four general mechanisms: (1) inactivation of antibiotic by - lactamase, (2) modification of target PBPs, (3) impaired penetration of drug to target PBPs, and (4) efflux. -Lactamase production is the most common mechanism of resistance. Many hundreds of different -lactamases have been identified. Some, such as 32

48 those produced by Staphylococcus aureus, Haemophilus sp, and Escherichia coli, are relatively narrow in substrate specificity, preferring penicillins to cephalosporins. Other -lactamases, eg, AmpC -lactamase produced by Pseudomonas aeruginosa and Enterobacter sp, and extended-spectrum -lactamases (ESBLs), hydrolyze both cephalosporins and penicillins. Carbapenems are highly resistant to hydrolysis by penicillinases and cephalosporinases but they are hydrolyzed by metallo- -lactamase and carbapenemases. Altered target PBPs are the basis of methicillin resistance in staphylococci and of penicillin resistance in pneumococci and enterococci. These resistant organisms produce PBPs that have low affinity for binding -lactam antibiotics, and consequently they are not inhibited except at relatively high, often clinically unachievable, drug concentrations. Resistance due to impaired penetration of antibiotic to target PBPs occurs only in gramnegative species because of their impermeable outer cell wall membrane, which is absent in gram-positive bacteria. -Lactam antibiotics cross the outer membrane and enter gramnegative organisms via outer membrane protein channels (porins). Absence of the proper channel or down-regulation of its production can greatly impair drug entry into the cell. Poor penetration alone is usually not sufficient to confer resistance, because enough antibiotic eventually enters the cell to inhibit growth. However, this barrier can become important in the presence of a -lactamase, even a relatively inactive one, as long as it can hydrolyze drug faster than it enters the cell. Gram-negative organisms also may produce an efflux pump, which consists of cytoplasmic and periplasmic protein components that efficiently transport some -lactam antibiotics from the periplasm back across the outer membrane. B. Resistance to Aminoglycoside Three principal mechanisms have been established: (1) production of a transferase enzyme or enzymes inactivates the aminoglycoside by adenylylation, acetylation, or phosphorylation. This is the principal type of resistance encountered clinically. (2) There is impaired entry of aminoglycoside into the cell. This may be genotypic, ie, resulting from mutation or deletion of a porin protein or proteins involved in transport and maintenance of the electrochemical gradient; or phenotypic, eg, resulting from growth conditions under which the oxygen-dependent transport process described above is not 33

49 functional. (3) The receptor protein on the 30S ribosomal subunit may be deleted or altered as a result of a mutation. C. Resistance to Tetracycline Three mechanisms of resistance to tetracycline analogs have been described: (1) impaired influx or increased efflux by an active transport protein pump; (2) ribosome protection due to production of proteins that interfere with tetracycline binding to the ribosome; and (3) enzymatic inactivation. The most important of these are production of an efflux pump and ribosomal protection. Tet(AE) efflux pump-expressing gram-negative species are resistant to the older tetracyclines, doxycycline, and minocycline. They are susceptible, however, to tigecycline, which is not a substrate of these pumps. Similarly, the Tet(K) efflux pump of staphylococci confers resistance to tetracyclines, but not to doxycycline, minocycline, or tigecycline, none of which are pump substrates. The Tet(M) ribosomal protection protein expressed by gram-positives produces resistance to the tetracyclines, doxycycline, and minocycline, but not to tigecycline, which because of its bulky t- butylglycylamido substituent has a steric hindrance effect on Tet(M) binding to the ribosome. Tigecycline is a substrate of the chromosomally encoded multidrug efflux pumps of Proteus sp, and Pseudomonas aeruginosa, accounting for their intrinsic resistance to all tetracyclines including tigecycline. D. Resistance to Macrolides Resistance to erythromycin is usually plasmid-encoded. Three mechanisms have been identified: (1) reduced permeability of the cell membrane or active efflux; (2) production (by Enterobacteriaceae) of esterases that hydrolyze macrolides; and (3) modification of the ribosomal binding site (so-called ribosomal protection) by chromosomal mutation or by a macrolide-inducible or constitutive methylase. Efflux and methylase production are by far the most important resistance mechanisms in gram-positive organisms. Crossresistance is complete between erythromycin and the other macrolides. Constitutive methylase production also confers resistance to structurally unrelated but mechanistically similar compounds such as clindamycin and streptogramin B (so-called macrolidelincosamide-streptogramin, or MLS-type B, resistance), which share the same ribosomal binding site. Because nonmacrolides are poor inducers of the methylase, strains expressing an inducible methylase will appear susceptible in vitro. However, constitutive 34

50 mutants that are resistant can be selected out and emerge during therapy with clindamycin. E. Resistance to Clindamycin Resistance to clindamycin, which generally confers cross-resistance to macrolides, is due to (1) mutation of the ribosomal receptor site; (2) modification of the receptor by a constitutively expressed methylase and (3) enzymatic inactivation of clindamycin. Gramnegative aerobic species are intrinsically resistant because of poor permeability of the outer membrane. F. Resistance to Chloramphenicol Low-level resistance to Chloramphenicol may emerge from large populations of chloramphenicol-susceptible cells by selection of mutants that are less permeable to the drug. Clinically significant resistance is due to production of chloramphenicol acetyltransferase, a plasmid-encoded enzyme that inactivates the drug. ESBL Not long after Cefotaxime came into clinical use in Europe, strains of Klebsiella pneumonae were discovered in Germany with transferable resistance to the oxyiminocephalosporins (e.g. Cefotaxime, Ceftazidime, and Ceftriaxone). The enzyme responsible was related to SHV and was named SHV-2. TEM-related ESBLs were discovered in France in 1984 and in the United States in The ESBL family is heterogeneous. SHV and TEM-type ESBLs arose by amino acid substitutions that allowed narrower spectrum enzymes to attack the new oxyimino betalactams. Others, notably members of the CTX-M family, represent plasmid acquisition of broad spectrum beta-lactamases originally determined by chromosomal genes. ESBLs vary in activity against different oxyimino beta-lactam substrates but cannot attack the cephamycins (Cefoxitin, Cefotetan and Cefmetazole) and the carbapenems (Imipenem, Meropenem and Ertapenem). They are also generally susceptible to betalactamase inhibitors, such as clavulanate, sulbactam, and tazobactam, which consequently can be combined with a beta-lactam substrate to test for the presence of this resistance mechanism (Bradford, 2001). 35

51 ESBLs have been found exclusively in Gram-negetive organisms, primarily in Klebsiella pnuemoniae, Klebsiella oxytoca, and Escherichia coli but also in Acinetobacter, Burkholderia, Citrobacter, Enterobacter, Morganella, Proteus, Pseudomonas, Salmonella, Serratia, and Shigella spp (Kliebe, 1985). Although ESBLs have been described in a range of Enterobacteriaceae and Pseudomonadaceae from different parts of the world, they are most often identified in Klebsiella pneumoniae and Escherichia coli. These enzymes belongs to the Ambler class A and D beta-lactamses. ESBL varieties: TEM beta-lactamases, SHV beta-lactamases, CTX-M beta-lactamases, OXA-type ESBLs, for example, are poorly inhibited by clavulanate. Some ESBLs are best detected with ceftazidime and others with cefotaxime (such as most CTX-M enzymes). Consequently, susceptibility to several oxyimino-lactams must be tested; criteria for ESBL detection have changed over time; and clinical laboratories vary in their success in diagnosis (Stevenson et al., 2003). Treatment option The only current proven therapeutic option for severe infections caused by ESBL producing organisms is the carbapenem family (Imipenem, Meropenem and Ertapenem). ESBL-producing isolates typically show greater than average resistance to other agents including aminoglycosides and fluoroquinolones (Paterson et al., 2004). Treatment with Imipenem or Meropenem has produced the best outcomes in terms survival and bacteriologic clearance. Ertapenem has good in-vitro activity (Jacoby et al., 1997). Clinical and Laboratory Standards Institute (CLSI) recommends that such ESBLproducing organisms should be reported as resistant (CLSI, 2006). Plasmids responsible for ESBL production may also carry genes encoding resistance to other drug classes, for example, aminoglycosides, trimethoprim, and fluoroquinolones. Therefore, antibacterial drug options in the treatment of patients with ESBL producing organisms may be very limited. ESBLs are an increasingly important cause of multi-drug resistant in infections throughout the world (Turner, 2005). 36

52 3.2.3 Methods of Testing (Forbes et al., 2007; Miles and Amyes, 2006; Singh, 2001) The antibiotic sensitivity of bacteria can be assessed in a variety of ways according to individual preference, the constraints of cost, the nature of the bacterium, the number of strains requiring investigation and the degree of accuracy required. Traditional methods fall into one of three main categories: (i) Agar diffusion tests, in which the antibiotic is allowed to diffuse from a point source, commonly in the form of an impregnated filter paper disc, into an agar medium that has been seeded with the test organism. (ii) Broth dilution tests, in which serial (usually twofold) dilutions of antibiotic in a suitable fluid medium are inoculated with the test organism. The highest dilution of the antibiotic to prevent the development of visible growth after overnight incubation is the minimum inhibitory concentration (MIC). (iii) Agar incorporation tests, which are essentially similar to broth dilution tests except that the antibiotic dilutions are incorporated in an agar medium in a series of Petri dishes. These are spot-inoculated with a number of test organisms, usually by means of a semiautomatic inoculating device. A specialized version of the agar diffusion test that has been introduced commercially is the â Etestâ. A plastic strip with a linearly decreasing concentration of antibiotic is applied to the surface of an inoculated agar plate. An elliptical zone of inhibition is formed after incubation, and the point at which this intersects with the strip corresponds to the MIC of the antibiotic, which can then be read off from graduations on the strip. The test is suitable for many types of organisms and provides a simple, if expensive, means of assessing the MIC if this is required Measurement of Antibiotic Sensitivity Antimicrobial susceptibility testing methods are divided into types based on the principle applied in each system. They include: Diffusion Dilution Diffusion&Dilution Stokes method Minimum Inhibitory Concentration E-Test method Kirby-Bauer method (i) Broth dilution (ii) Agar dilution 37

53 Using an appropriate standard test organism and a known sample of drug for comparison, these methods can be employed to estimate either the potency of antibiotic in the sample or the susceptibility of the microorganism. There are various types of disc diffusion sensitivity tests which vary in their methods of standardization, reading and control. Modified Kirby-Bauer disc diffusion method The Kirby-Bauer method (Bauer et al., 1966) and its modifications recognize three categories of susceptibility: susceptible, intermediate and resistant. Susceptible: An organism is called "susceptible" to drug when the infection caused by it is likely to respond to treatment with this drug, at the recommended dosage. Intermediate: It covers two situations. It is applicable to strains that are moderately susceptible to an antibiotic that can be used for treatment at a higher dosage because of its low toxicity or because the antibiotic is concentrated in the focus of infection (e.g. urine). The classification also applies to strains that show intermediate susceptibility to a more toxic antibiotic that cannot be used at a higher dosage. Resistant: This term implies that the organism is expected not to respond to a given drug, irrespective of the dosage and on the location of the infection. For clinical and surveillance purpose and to promote reproducibility and comparability or results between laboratories, WHO recommends the CLSI recommended modified Kirby- Bauer disc diffusion technique (CLSI, 2006). 3.3 Therapeutic Failure If the patient with the documented Salmonella enterica infection doesnot show response to the antibiotic used as per culture sensitivity report within a period of 10 days is considered as therapeutic failure. 3.4 Relapse of Enteric Fever It is due to persistence of the organisms in certain tissues, which is inaccessible to antibiotics to certain reticuloendothelial cells, or due to early withdrawal of the antibiotics. Relapse usually occurs within 14 days of defervescence of fever. 38

54 3.5 Typhoid Carrier The patients who are treated for the enteric fever usually excrete the organisms in the stool and sometimes even in urine during recovery and convalescent period of enteric fever. Carriers may be temporary or incubatory or convalescent or chronic. Chronic carriers pose a potent danger to others. Convalescent carriers excrete the organisms even after one year after the recovery or sub-clinical illness. If they are not responding to antibiotics, cholecystectomy may be considered in them. 3.6 Prevention and Control of Enteric Fever Prevention The major routes of transmission of typhoid fever are through drinking water or eating food contaminated with Salmonella enterica. Prevention is based on ensuring access to safe water and by promoting safe food handling practices. Health education is paramount to raise public awareness and induce behavior change (WHO, 2003) Safe water Enteric fever is a waterborne disease and the main preventive measure is to ensure access to safe water. The water needs to be of good quality and must be sufficient to supply all the community with enough drinking water as well as for all other domestic purposes such as cooking and washing. During outbreaks the following control measures are of particular interest: a. In urban areas, control and treatment of the water supply systems must be strengthened from catchment to consumer. Safe drinking water should be made available to the population trough a piped system or from tanker trucks. b. In rural areas, wells must be checked for pathogens and treated if necessary. c. At home, a particular attention must be paid to the disinfection and the storage of the water however safe its source. Drinking-water can be made safe by boiling it for one minute or by adding a chlorine-releasing chemical. Narrow-mouthed pots with covers for storing water are helpful in reducing secondary transmission of typhoid fever. Chlorine is ineffective when water is stored in metallic containers. 39

55 d. In some situations, such as poor rural areas in developing countries or refugee camps, fuel for boiling water and storage containers may have to be supplied Food safety Contaminated food is another important vehicle for typhoid fever transmission. Appropriate food handling and processing is paramount and the following basic hygiene measures must be implemented or reinforced during epidemics: washing hands with soap before preparing or eating food; avoiding raw food, shellfish, ice; eating only cooked and still hot food or re-heating it. During outbreaks, food safety inspections must be reinforced in restaurants and for street food vendors activities. Typhoid can be transmitted by chronic carriers who do not apply satisfactory food-related hygiene practices. These carriers should be excluded from any activities involving food preparation and serving. They should not resume their duties until they have had three negative stool cultures at least one month apart Sanitation Proper sanitation contributes to reducing the risk of transmission of all diarrhoeal pathogens including Salmonella enterica. Appropriate facilities for human waste disposal must be available for all the community. In an emergency, pit latrines can be quickly built. Collection and treatment of sewage, especially during the rainy season, must be implemented In areas where typhoid fever is known to be present, the use of human excreta as fertilizers must be discouraged Health education Health education is paramount to raise public awareness on all the above mentioned prevention measures. Health education messages for the vulnerable communities need to be adapted to local conditions and translated into local languages. In order to reach 40

56 communities, all possible means of communication (e.g. media, schools, women s groups, religious groups) must be applied. Community involvement is the cornerstone of behavior change with regard to hygiene and for setting up and maintenance of the needed infrastructures. In health facilities, all staff must be repeatedly educated about the need for: excellent personal hygiene at work; isolation measures for the patient; disinfection measure Typhoid Vaccination There are two vaccines to prevent typhoid. One is an inactivated (killed) vaccine gotten as a shot, and the other is live, attenuated (weakened) vaccine which is taken orally (by mouth) Inactivated Typhoid Vaccine (Shot) This vaccine should not be given to children younger than 2 years of age. One dose provides protection and should be given at least 2 weeks before travel to allow the vaccine time to work. A booster dose is needed every 2 years for people who remain at risk Live Typhoid Vaccine (Oral) This vaccine should not be given to children younger than 6 years of age. It has four doses, given 2 days apart, are needed for protection. The last dose should be given at least 1 week before travel to allow the vaccine time to work. A booster dose is needed every 5 years for people who remain at risk. Either vaccine may be given at the same time as other vaccines (CDC, 2006). 3.7 Quality control The steps involved in the accurate laboratory diagnosis of typhoid fever include specimen collection and transport, the performance of laboratory procedures, and reporting. It is important that the correct specimen is collected in the correct volume, that it is transported to the laboratory in the right condition, that correct laboratory procedures are followed and that reporting is accurate. These steps should therefore be monitored at all 41

57 levels and correction should take place if unacceptable performance is identified. Quality assurance is vital to the success of such investigations. Quality control programs ensure that the information generated by laboratories is accurate, reliable and reproducible. This is accomplished by assessing the quality of specimens and monitoring the performance of test procedures, reagents, media, instruments, and personnel. Laboratories should have internal quality control programs. A panel of reference isolates consisting of typhoid and non-typhoid salmonellae and other Enterobacteriaceae should be maintained. At periodic intervals, e.g. monthly, the laboratory supervisor should submit a random selection of the reference isolates under code to laboratory technologists for evaluation. Quality control of the disc susceptibility test should take place. E. coli ATCC (Source: TUTH, Kathmandu) should be run in parallel with the test strains. The susceptibility zones for the reference strain against various antimicrobials should be within the acceptable ranges. The results of these evaluations should be entered in a quality control monitoring book. Appropriate measures should be taken to solve any problems that are encountered. It is important to confirm Salmonella isolates in a reference laboratory because of the possibility of their misidentification (WHO 2003). 42

58 CHAPTER IV MATERIALS AND METHODS 4.1 Materials Materials used in this study are listed out in the Appendix II. 4.2 Methods Study design This research was carried out as hospital based cross-sectional study Study site The research was carried out at Pathology Department of Shankarapur Hospital, Narayantar, Jorpati, Kathmandu Sample population This research included blood samples received for culture of suspected enteric fever patients attending Shankarapur Hospital, Narayantar, Jorpati, Kathmandu Duration The study period for this research work was from February 2010 to October Inclusion criteria This research has included all blood samples submitted for culture and sensitivity Sample size A total 512 blood culture samples were studied during the study period. During this research work, blood culture sample from the suspected enteric fever patients collected aseptically in the Pathology laboratory or in the hospital wards were processed at Microbiology laboratory. The age of patients included neonates to elderly. The history of all the patients including age, gender, symptoms were recorded in the data collection form from the requisition form obtained along with the sample Sampling procedure Purposive sampling was taken prospectively from patients of the suspected enteric fever. All blood culture samples suspected for enteric fever received during the study period was used for the study. The methods for the collection, isolation and identification were followed as described by American Society for Microbiology (ASM) and World Health 43

59 Organization (WHO). The antibiotic sensitivity tests (AST) of the pathogens was determined by Kirby-Bauer method of disk diffusion technique as recommended by CLSI and the phenotypic confirmation of ESBL producing strains was done by using combination disk method. We considered Salmonella as MDR when it was resistant to at least two or more different groups of antibiotics Sample collection and processing Blood samples were collected aseptically and 0.5 ml blood was collected in EDTA bottle for leukocyte counting and hemoglobin percentage while five ml was poured in 45 ml Brain heart infusion (BHI) broth (Hi-Media, India). The culture bottles were incubated at 37 o C for overnight. Subculture was done on Blood agar (BA), Chocolate agar (CA), MacConkey agar (MA) and Salmonella-Shigella (SS) agar (Hi-Media, India) in day 2 and day 3. The BA, CA, MA and SS agar plates were incubated at 37 o C for 24 hour. The culture bottles were examined daily for any visual evidence of microbial growth, such as turbidity, hemolysis, gas production and clot formation of discrete colony. On the day 7, a blind sub-culture was done on SS agar before discarding these culture bottles. Identification of bacteria from positive culture plates was done with the use of standard Microbiological techniques which included colony morphology, Grams reaction and Biochemical reactions that includes Catalase, Oxidase, Triple Sugar Iron (TSI), Sulphide Indole Motility (SIM), Citrate utilization and Urea hydrolysis test. The organisms were fully identified using biochemical and serological profiles. Serotyping was done by using polyvalent O-antisera A-G and individual O and H-antisera (Denka Seiken, Japan) to confirm Salmonella enterica serovar Typhi and serovar Paratyphi A. S. aureus ATCC 25923, E. coli ATCC and Ps. aeruginosa ATCC (Source: TUTH, Kathmandu) were used parallel as a part of quality control of test system. Both positive and negative controls were included during tests Purity plate This technique was done to determine whether the biochemical tests were done in aseptic condition or not. Specimen was cultured in respective media before and after the biochemical tests performed and pure growth in both the plate indicated the aseptic condition. 44

60 Antibiotic susceptibility test The antibiotic sensitivity tests of the pathogens isolated from the blood against different antibiotics was determined by Kirby-Bauer method of disk diffusion technique as recommended by CLSI using Mueller Hinton agar (MHA). At least three to five wellisolated colonies of the same morphological types was selected from MHA plate. The base of each of the colony was touched with a inoculating wire and the growth was transferred into a tube containing 5 ml of nutrient broth and was incubated at 37 o C (usually 2 to 6 hours and observed at an interval of 30 min) until it achieved the turbidity equivalent to McFarland tube number 0.5. In case of overgrowth, the broth was diluted with sterile physiological saline to match with McFarland tube number 0.5. A sterile cotton swab was dipped into the broth and the swab was rotated several times and pressed firmly on the inner side wall of the tube above the fluid level to remove excess inoculum from the swab. Then, dried MHA plate was inoculated by streaking the swab over the entire agar surface three times, turning the plate 60 o C between streaking. Finally, the inoculum was left to dry for few minutes at room temperature with the lid closed. The predetermined antimicrobial disks were placed on the surface of the prior inoculated agar plate such that there will be 25 mm distance from disk to disk and 15 mm from the side. The disks were pressed down to ensure complete contact with the agar surface. The plates were left at room temperature for about 15 minutes after applying the disks to allow antimicrobial agents to diffuse from the disks. These plates were then incubated overnight aerobically at 37 0 C. After overnight incubation, the diameter of zone of inhibition (ZOI) of each disk was measured (including diameter of the disk) and recorded in millimeter. It was then compared with standard chart developed by Kirby-Bauer to determine bacterial susceptibility towards different antimicrobial agents in terms of Sensitive, Resistant and Intermediate (moderately sensitive). The measurement was made with a ruler on the under surface of the plate without opening the lid. S. aureus ATCC 25923, E. coli ATCC and Ps. aeruginosa ATCC were also tested in every set of experiment, in parallel, as a part of quality control (CLSI, 2006). In this study if the isolates were resistant to at least two or more different groups antimicrobial agents, it was regarded as MDR. 45

61 Tests for ESBL production The initial screening test for the production of ESBL was performed by using both Ceftazidime (CAZ, 30 µg) and Cefotaxime (CE, 30 µg) disks. If the zone of inhibition was < 22 mm for CAZ and /or <27 mm for CE, the isolate was considered a potential ESBL producer as recommended by CLSI. Combination disk method was used for the confirmation of ESBL producing strains in which CE and CAZ (30µg), alone and in combination with Clavulanic acid (CA, 10µg) was used. An increase ZOI of > 5mm for either antimicrobial agent tested in combination with CA versus its zone when tested alone was confirmed ESBL. E. coli ATCC and K. pneumoniae ATCC (Source: TUTH, Kathmandu) were used as negative and positive controls respectively Test for NAR screening A Nalidixic acid disk (30 µg) was used along with other antibiotics during antibiotic susceptibility testing. If the strain was resistant to Nalidixic acid, then it was considered as NAR strain (Hakanen et al., 1999). 4.3 Validity and Reliability For validity and reliability of this research, research proposal preparation, questionnaire development, check and verification of set of questionnaire was made under the close guidance of Research supervisor and concerned teachers. Quality/Reference procedures were followed with appropriate controls/ standards. 4.4 Quality assurance It is essential to maintain quality control to obtain reliable microbiological result. During this study, quality control was applied in various areas. During sample collection, aseptic technique was followed for collecting blood culture samples in order to avoid contamination. During sample processing, all the tests were carried out appropriately in aseptic conditions (CLSI, 2006; Isenberg, 2004). While using readymade dehydrated media, the manufacturer's instructions for preparation, sterilization and storage were followed to prevent the alteration of the nutritional, selective, inhibitory and biochemical properties of media. 46

62 The performances of newly prepared media were tested using control species of bacteria (i.e. known organisms giving positive and negative reactions). For stains and reagents, whenever new batches of them were prepared, control smear was stained to ensure correct staining reaction. Control strains of E. coli (ATCC 25922) and S. aureus (ATCC 25923) were used for the standardization of the Kirby-Bauer test and also for correct interpretation of zone of diameter. For ESBL test standardization, E. coli ATCC and K. pneumoniae ATCC were used as negative and positive controls respectively (Source: TUTH, Maharajgunj, Kathmandu). 4.5 Limitation of the study This research was limited only to the patients attending Shankarapur Hospital with suspected enteric fever and time framework for the study was 8 months. 4.6 Data management and analysis Editing, Coding and Classification of the collected data were done thoroughly. Main focus was on frequency and percentages. Chi-square (χ 2 ) test was done wherever applicable with a p value <0.05 regarded as significant (Feinstein, 2002). 47

63 CHAPTER V RESULTS A total of 512 blood culture samples of the suspected enteric fever patients (out patients and in patients) submitted to the bacteriology laboratory were processed for culture and sensitivity from February 2010 to October The results obtained were analyzed which are shown in the following tables and figures. 5.1 Distribution of different bacterial isolates Distribution of Bacterial Species 35 Number of Organisms S. enterica serovar Typhi S. enterica serovar Parayphi A S. enterica serovar Typhimurium S. pneumoniae S. aureus Frequency Organisms Fig 1: Distribution of different bacterial species Of the total 512 blood culture samples, only 49 (9.57%) were positive for culture which included 45 (8.78%), Salmonella enterica and 4 (0.78%) non-salmonellae. Among Salmonella enterica, 33 (73.33%) were Salmonella enterica serotype Typhi, 11 (24.44%) were Salmonella enterica serotype Paratyphi A and 1 (2.22%) was Salmonella enterica serovar Typhimurium. Non-salmonellae included 3 (0.58%) Streptococcus pneumoniae and 1 (0.2%) Staphylococcus aureus. 48

64 Table 1: Distribution of positive isolates Among 49 (9.57%) positive isolates, 33 (67.34 %) were Salmonellae enterica serovar Typhi, 11 were (22.44%) Salmonella enterica serovar Paratyphi A, 3 (6.12%) were Streptococcus pneumoniae, 1 (2.04%) was Salmonella enterica serovar Typhimurium and 1 (2.04%) was Staphylococcus aureus. The growth of Salmonella enterica was found significantly high (p<0.05) than non-salmonellae. Isolates Frequency % p-value Salmonella enterica serovar Typhi Salmonella enterica serovar Paratyphi A Salmonella enterica serovar Typhimurium Streptococcus pneumoniae p<0.05 Staphylococcus aureus Positive vs negative growth in culture Out of 512 (100%) samples, 49 (9.57%) were positive for culture while 463 (90.43%) were negative. Growth Positive 49, 9.57% Negative 463, 90.43% Fig 2: Chart showing Positive vs. Negative Growth of Salmonella enterica in culture 49

65 5.3 In-Patients vs Out-Patients growth in culture Out of the total 512 blood culture samples, 94 (18.35%) were from in-patients and 418 (81.64%) were from outpatient department. Thirty eight (9.09%) out of 418 out-patients were Salmonella positive while seven (7.44%) out of 94 in patients were Salmonella positive. The growth of Salmonella was significantly higher (p<0.05) in out- patients than in-patients In Patients 94, 7 Out Patients 418, 38 Fig 3: Distribution of Salmonellae among Out and In Patients Table 2: Age wise distribution of Salmonella enterica The highest percentage of isolation was among years (44.5%). Children less than 10 years and adults more than 70 years showed similar percentage of distribution (2.0%). Age (Years) Frequency % p-value Less than p< and above Total

66 Table 3: Gender wise distribution of Salmonella enterica among total population Out of 45 Salmonella enterica, 19 (42.2%) were isolated from male patients while 26 (57.8%) were isolated from female patients. The isolation rate of Salmonella enterica was found statistically insignificant (p>0.05) in both sex. Sex Frequency Growth positive Positive % p-value Male 278 (54.3%) Female 234 (45.7%) p>0.05 Total 512 (100%) Ratio of male: female Male were found comparatively higher in ratio than females (1.2: 1). female, 1 male, 1.2 Fig 4: Ratio of male: female 51

67 Table: 4 Symptom wise distributions of Patients with Salmonella enterica Fever was found in all 45 (100%) cases followed by headache 19 (42.2%), chill 12 (26.6%), abdominal pain 7 (15.5%), cough 3 (6.7%) and diarrhea 1 (2.2%). No cases with constipation and vomiting were reported in positive cases during the study. Presenting symptoms Number Percentage (%) Fever Headache Chill Abdominal pain Cough Diarrhea Constipation 0 0 Vomiting

68 WBC count in Enteric Fever Patient Normal leukocyte count was reported in 41 (91.1%) cases. Only 4 (8.9%) cases were reported with total count exceeding normal range. Fig: 5 WBC count in Enteric Fever Patient Table: 5 Hemoglobin Level in Patients with Salmonella enterica The hemoglobin level in 32 (71.1%) cases was in normal range. A reduced hemoglobin level in the range of was found in 13 (28.9%) cases. Hemoglobin level (in %) Number % >

69 Table 6: Antimicrobial susceptibility pattern of S. enterica (n=45) Sensitive Intermediate Resistant Antibiotics Ampicillin/Amoxicilli n No. % No. % No. % Amikacin Amoxiclav Azithromycin Cefixime Cefotaxime Ceftriaxone Ceftazidime Cefepime Chloramphenicol Ciprofloxacin Cotrimoxazole Gentamicin Imipenem (n=8) Meropenem (n=8) Nalidixic Acid Norfloxacin Ofloxacin Tetracycline All strains of Salmonella enterica were sensitive to Imipenem and Meropenem (100%). The Sensitivity was then highest to Chloramphenicol (97.8%) and lowest to Ampicillin 54

70 and Amoxicillin (33.3%). Amikacin (95.5%) and Azithromycin (93.3%) still had good sensitivity. Among cephalosporin, the fourth-generation antibiotic, Cefepime had better sensitivity (93.3%) compared to other generation cephalosporins; Cefotaxime (86.6%), Ceftriaxone (82.2%), Ceftazidime (82.2%) and Cefixime (77.8%). Cotrimoxazole (84.4%), Gentamicin (77.8%) and Tetracycline (73.3%) also had better antibacterial effect. The Fluroquinolones, Ciprofloxacin (75.6%), Ofloxacin (75.6%) and Norfloxacin (73.3%) still showed good activity towards Salmonellae while Nalidixic acid was found less effective (66.6%). The Amoxiclav had comparatively better activity (62.2%) than Ampicillin and Amoxicillin (33.3%). 55

71 Table 7: Antimicrobial susceptibility pattern of S. enterica serovar Typhi (n=33) Sensitive Intermediate Resistant Antibiotics Ampicillin/Amoxicilli n No. % No. % No. % Amikacin Amoxiclav Azithromycin Cefixime Cefotaxime Ceftriaxone Ceftazidime Cefepime Chloramphenicol Ciprofloxacin Cotrimoxazole Gentamicin Imipenem (n=8) Meropenem (n=8) Nalidixic Acid Norfloxacin Ofloxacin Tetracycline All strains of Salmonella enterica serovar Typhi were sensitive to Imipenem and 56

72 Meropenem. Chloramphenicol (97.0%), Amikacin (94.0%) and Azithromycin (91.0%) showed much susceptibility compared to other antibiotics. Among cephalosporins, the fourth-generation Cefepime had better sensitivity (91.0%) compared to other generation cephalosporins; Cefotaxime (82.0%), Ceftriaxone (75.8%), Ceftazidime (75.8%) and Cefixime (72.7%). Cotrimoxazole (84.9) and Gentamicin (75.8%) were more sensitive than Tetracycline (69.7%). The Fluroquinolones, Ciprofloxacin (72.7%), Ofloxacin (69.7%) and Norfloxacin (69.7%) show medium sensitivity towards S. Typhi. The sensitivity of Nalidixic acid was however found to be much reduced (60.6%). The Amoxiclav (57.5%), Ampicillin and Amoxicillin (33.3%) had reduced antibacterial effect. Four (12.1%) MDR and Three (9.09%) ESBL producing S. enterica serovar Typhi strains were isolated during the study. 57

73 Table 8: Antimicrobial susceptibility pattern of S. enterica serovar Paratyphi A (n=11) Sensitive Intermediate Resistant Antibiotics Ampicillin/Amoxicilli n No. % No. % No. % Amikacin Amoxiclav Azithromycin Cefixime Cefotaxime Ceftriaxone Ceftazidime Cefepime Chloramphenicol Ciprofloxacin Cotrimoxazole Gentamicin Nalidixic Acid Norfloxacin Ofloxacin Tetracycline All strains of Salmonella enterica serovar Paratyphi A were sensitive (100%) to Amikacin, Azithromycin, Chloramphenicol. Among cephalosporins except Cefixime (91.0%) rest all (Ceftriaxone, Cefotaxime, Ceftazidime, and Cefepime) were 100% sensitive. 58

74 Fluroquinolones (Ciprofloxacin, Ofloxacin, and Norfloxacin), Cotrimoxazole and Tetracycline were equally sensitive (82.0%). Nalidixic acid showed good activity (82.0%) to S. Paratyphi A strains compared to S. Typhi (60.6%). Ampicillin and Amoxicillin were less sensitive (45.5%) however Amoxyclav had still a better sensitivity (73.0%). None of the strains were multi-drug resistant and neither ESBL producers. Imipenem and Meropenem were not used against S. Paratyphi A strains. 59

75 Table 9: Antimicrobial susceptibility pattern of S. enterica serovar Typhimurium (n=1) Sensitive Intermediate Resistant Antibiotics Ampicillin/Amoxicilli n No. % No. % No. % Amikacin Azithromycin Cefixime Cefotaxime Ceftriaxone Ceftazidime Cefepime Chloramphenicol Ciprofloxacin Cotrimoxazole Gentamicin Imipenem Meropenem Nalidixic Acid Norfloxacin Ofloxacin Tetracycline The single strain of Salmonella enterica serovar Typhimurium was 100% sensitive to all tested antibiotics. Imipenem and Meropenem were not used against S. Typhimurium. 60

76 Table 10: Prevalence of NAR and MDR strains in Salmonella enterica Salmonella enterica serovar Number MDR NAR No. % No. % Typhi Paratyphi A Typhimurium Out of the total 33 Salmonella enterica serovar Typhi, Eight (24.2%) were Nalidixic acid resistant (NAR) while Four (12.1%) were multidrug-resistant. However, out of the total 11 Salmonella enterica serovar Paratyphi A and 1 Salmonella enterica serovar Typhimurium neither were Nalidixic acid resistant (NAR) nor multidrug-resistant (MDR). The occurrence of MDR and NAR strains in both serovars (Typhi and Paratyphi A) was not statistically significant (p>0.05). Table 11: Prevalence of ESBL in Salmonella enterica serovar Typhi Salmonella enterica serovar Number ESBL No. % Typhi Out of 33 Salmonella enterica serovar Typhi, only 3 (9.1%) strains were ESBL producers. The ESBL producing Salmonella enterica was found statistically insignificant (p> 0.05). 61

77 Table 12: Antibiotic sensitivity patterns of ESBL producing Salmonella enterica serovar Typhi (n= 3) Antibiotics Sensitive Intermediate Resistance n % n % n % Amikacin Ampicillin/Amoxic illin Amoxyclav Azithromycin Cefixime Cefotaxime Ceftriaxone Ceftazidime Cefepime Chloramphenicol Ciprofloxacin Cotrimoxazole Gentamicin Imipenem Meropenem Nalidixic Acid Norfloxacin Ofloxacin Tetracycline In the present study all three ESBL producers S. enterica serovar Typhi showed resistance to Ampicillin, Amoxiclav, Cefixime, Ceftazidime, Cefotaxime, Ciprofloxacin, 62

78 Norfloxacin and Cotrimoxazole. All the isolates were sensitive to Imipenem and Meropenem. They showed equal resistance pattern to Amikacin (33.3%), Azithromycin (33.3%) and Chloramphenicol (33.3%). The susceptibility S. Typhi towards Imipenem and Meropenem is significantly higher (p<0.05) than Chloramphenicol, Amikacin, Azithromycin and rest all. 63

79 CHAPTER VI Discussion DISCUSSION AND CONCLUSION A study of 512 cases of suspected enteric fever was conducted at Shankarapur Hospital, Narayantar, Jorpati. In this study, blood samples from different age group from both indoor and outdoor patients were included. The study shows the prevalence of MDR, NAR strain and ESBL-producing Salmonella enterica isolated from blood culture of suspected enteric fever patients visiting Shankarapur Hospital. Out of the total 512 blood culture samples, 94 (18.35%) were from patients admitted to the hospital (indoor patients) and 418 (81.64%) were from outpatient department (outdoor patients). Thirty eight (9.09%) out of 418 out-patients were Salmonella positive while seven (7.44%) out of 94 in patients were Salmonella positive. The growth of Salmonella was significantly higher (p<0.05) in out- patients than in-patients. Similarly, 278 (54.3%) samples were from male patients and 234 (45.7%) were from female patients. The ratio of male: female patient was found to be 1.2:1 approximately. Garg and Krashak (1993) reported the distribution of male to female was 1.5:1 and similar distribution was also reported by Buch et al., (1994). A study in Dhulikhel hospital, Nepal by Sharma et al., (2003) showed still higher ratio about 3:1. The exact reason for this distribution is not known, but outside eating habit might be one of the reasons for unequal distribution. Similar type of ratio was also reported by Adeleke et al., (2006) and Okonko et al., (2006). This difference in ratio may also be due to preference of male than female in society. In this study, only 49 (9.57%) samples were positive for culture. Among culture positive cases, forty five (8.78%) were Salmonella enterica and four (0.78%) were nonsalmonellae which included three Streptococcus pneumoniae (0.58%) and one Staphylococcus aureus (0.2%). The growth of Salmonella enterica is significantly higher (p<0.05) than non-salmonella. In addition, twelve (2.34%) Salmonella enterica were grown out of 94 inpatient blood samples and thirty three (6.44%) were out of 418 outpatient blood samples. The growth of Salmonella enterica in outpatient is significantly 64

80 higher (p<0.05) than in inpatient. The higher incidence of Salmonella enterica serovars were also reported by Dahal (2009). Out of the total 278 blood samples from male patients, nineteen (42.22%) showed growth of Salmonella enterica and out of 234 blood samples from female patients, twenty six (57.78%) showed its growth. The growth rate in male is not significantly higher (p >0.05) than in female. This study also showed significantly higher number (p<0.05) of Salmonella enterica isolated from age group 11 to less than 30 years. Out of the total 48 isolates, majority (n=158, 76%) were from this age group. This may be because eating outside and street foods are more familiar among age group 11 to less than 30 years. Similar findings in such age groups were also reported by Dahal (2010), Garg and Karshak (1993), Sharma et al., (2003). In this study fever was present in all 45 cases of Salmonella enterica followed by headache 19 (42.2%), chill 12 (26.6%), abdominal pain 7 (15.5%), cough 3 (6.7%) and diarrhea 1 (2.2%). No cases with constipation and vomiting were reported in positive cases during the study. Similar findings were also reported by Quimpo (1991), Sharma et al., (2003), and Gupta et al., (2009). But their findings also reported for a higher rate of diarrhea, abdominal pain, chills, constipation and vomiting which were not reported in this study. This may be due to difference in study area and sample population that was included in this study. Other clinical findings such as coated tongue, toxic look, relative bradycardia, rose spots, hepatomegaly, splenomegaly and similar symptoms that are clinician based were excluded in this study. The hematological investigation included total white blood cell counting (leukocyte counting) and hemoglobin percentage. In majority of cases in enteric fever the total leukocyte counting shows normal range (Dangana et al., 2010). In this study also the majority of enteric fever cases (41, 91.1%) showed a normal leukocyte counting. A raised level was reported in 5 (8.9%) cases. Leucopenia (low WBC count) was not found in this study. Similar pattern of leukocyte counting have been found by Dangana et al., (2010), Sarkinfada and Abubakar (2001), Sharma et al., (2003). Sood and Taneja (1995) noted increased total leukocyte count in 22.1% cases and leucopenia in 24.2% cases. Hence only leukocyte counting cannot be basis for detection of enteric fever. 65

81 Majority of cases (32, 71.1%) showed hemoglobin level in normal range. A reduced hemoglobin level in the range of was found in 13 cases (28.9%) which were considered as cases of mild anemia. Thus the incidence of anemia during enteric fever in this study was very low. This study shows that the hematological investigations alone cannot be reliable for detection of enteric fever. A study by Buzgan et al., (2007) in 18 year female patient revealed a case of leucopenia showing WBC count as low as 2,500/mm 3 (with differential: 47% lymphocytes, 43% polymorphonuclear leukocytes, and 10% monocytes) and hemoglobin 9.9 gm%. The gold standard for the study of enteric fever is done by isolation of Salmonella enterica from the blood culture. The yield of blood culture falls after first week and hence stool and urine culture are much fruitful in the isolation of organism after first week of infection. In this study only 8.78% of total culture was positive for Salmonella enterica. This low rate of isolation of Salmonella enterica in blood culture may be due to different reasons. The primary reason was because majority of patients had undergone some kind of therapeutic agents before admission to hospital. Another reason for lower isolation rate may be insufficient amount of blood (5 ml) withdrawn for culture. Many studies show that 10 ml blood is recommended for routing blood culture (Isenberg, 2004). In a study of adults it has been found that ml increased the yield by 19% while ml increased the yield by another 10% (Li et al., 1994). When whole blood is to be cultured, it is essential to prevent bactericidal serum effects either by adequate dilution of the sample in an adequate medium volume or by inhibition of serum bactericidal factors such as sodium polyanetholsulfate (SPS) in broth (Watson, 1978). Poor cultural results in this study may be due to this reason too. Some other reasons may be due to late timing for culture (after a week), only suspected enteric fever patient not the real one, patients with pyrexia of unknown origin and similar others. Literature reviews show the isolation of Salmonella enterica in blood culture vary from 6-80 percent. A higher rate (80-95%) is obtained when bone marrow is used for culture (Parry et al., 2002, Vallenas et al., 1985). In Nepal, the isolation rate varies from 6 to about 60% in blood culture. Dahal (2009) isolated 6.5% positive culture while Sharma et al., (2003) reported for 55% of positive blood culture. This study is also in the range of positive culture isolation rate in Nepal. 66

82 However, the study could even give a higher isolation rate if amount of blood for culture was taken 10 ml (or even more Wain et al., 2008). Nepal is one of the Typhoid endemic countries in Asia and is a hot bed of Enteric fever. Typhoid fever is most common in pre-school and school age children. In a study in Dhulikhel Hospital Kathmandu 71.4% were of less than 30 years of age (Sharma et al., 2003). This study shows still higher percentage (73.4%). Therefore early diagnosis and management of typhoid cases is economically beneficial as it affects the younger age group. Enteric fever is a major public health problem in Kathmandu and also in other parts of Nepal. Salmonella enterica serotype Typhi and Paratyphi A are isolated throughout the year in Kathmandu and its surroundings (Sharma et al., 2003). This reflects that water supply system of Kathmandu is constantly polluted with fecal material. Thus, a large number of carriers are present in the community. Isolation rates of Salmonella spp. have increased in recent years, particularly in the summer months due to lack of proper hygiene and sanitation and public health education. A large outbreak of multidrugresistant S. enterica serovar Typhi from a contaminated drinking water supply in a small Nepali town has recently been described (Pokharel et al., 2006). Although this study found a greater prevalence of serovar Typhi than serovar Paratyphi A, it is not statically significant (p<0.05). However, in some studies Paratyphi A has been reported in higher amount by other researcher in Nepal (Pokharel et al., 2006, Dahal, 2009). According to CLSI, all Salmonella should be routinely tested for susceptibility to Ampicillin, a quinolone and trimethoprim-sulfamethoxazole, and the extra intestinal isolates should be tested, in addition, for susceptibility to Chloramphenicol and an expanded-spectrum cephalosporin (CLSI, 2010). In this context, most laboratories evidently used one of the fluoroquinolones, since Nalidixic acid is not administered for the treatment of Salmonella infections. Until the mid-1980s, Ampicillin, Chloramphenicol or trimethoprim-sulfamethoxazole was the standard treatment for typhoid fever (Asna and Haque, 2000). Chloramphenicol has been the mainstay of treatment for enteric fever, while Ampicillin/ trimethoprimsulfamethoxazole are other cost-effective and well tried primary drugs of choice. Drug 67

83 resistance to Chloramphenicol in Salmonella enterica serovar Typhi first emerged in the United Kingdom (UK) in the 1550s and subsequently in Greece and Israel followed by the epidemics of MDR Salmonella in Mexico, neighboring country India and other regions (Gautam et al., 2002). The first report of multidrug resistant Salmonella enterica serovar Typhi in Nepal was published in 1991 (Watson and Pettibone, 1991). In this study Ampicillin, Amoxicillin and Amoxiclav (β-the lactam group) showed reduced sensitivity (Ampicillin/Amoxicillin 33.3% and Amoxiclav 62.2%) and hence are not recommended for using as first line therapy against enteric fever. Other first line antibiotics showed better sensitivity (Cotrimoxazole 84.4%, Chloramphenicol 97.8%). Among second line antibiotics the fluoroquinolones (Ciprofloxacin and Ofloxacin 75.6%, Norfloxacin 73.3%) had far better activity than Quinolone (Nalidixic acid 66.6%). The fourth generation cephalosporin (Cefepime 93.3%) had better activity than third generation cephalosporins (Cefotaxime 86.6%, Ceftriaxone and Ceftazidime 82.2%). The oral cephalosporin Cefixime was however found to be a less sensitive (76.6%) compared to intravenous cephalosporins (82-93%). Amikacin (95.5%) and Azithromycin (93.4%) had better sensitivity compared to Tetracycline (73.3%) and Gentamicin (77.8%). Such pattern of studies was also found by Sharma et al., 2003, Dahal 2009, Pokharel et al., Different from our study Mathura et al., (2005) found Ceftriaxone (100%), Ofloxacin (95.7%) Ciprofloxacin (95.7%) highly sensitive in compared to Gentamicin (17.4%), Chloramphenicol (37%) and Cotrimoxazole (15.2%). However, the sensitivity pattern of Amoxicillin was similar (37%). In a study by Onyango et al., (2008) 90% of S. enterica serovar Typhi isolates were resistant to Ampicillin and Streptomycin. Resistance to Chloramphenicol was 78.8% followed by Cotrimoxazole (66.7%). Emergence of bacterial resistance to well known and trusted antibiotics is widely recognized as one of the greatest challenges that physicians face in the management of adult and pediatric infections (Cohen, 1992). It has negatively affected the population by increasing morbidity and mortality rates, reducing desired treatment outcomes, and increasing the need for hospital admission while increasing the cost of patient care. Contributory factors may be drug over-use, misuse and inappropriate prescribing practices by physicians along with intrinsic microbiological plasmid and or chromosomal mediated factors. There is, therefore, need for more community-based antimicrobial 68

84 susceptibility surveys (Onyango et al., 2008). In the present study, it was observed that S. enterica serovar Typhi was more multidrug resistance as compared to S.Paratyphi A and S.Typhimurium. The study also showed four (8.8%) Salmonella enterica were multi-drug resistance (MDR). All the four strains were of S. Typhi (12.1%). In this study none of Paratyphi A strains or Typhimurium strain was MDR. This study was very much similar with that of Pokharel et al., (2006) in which 5% Salmonella as MDR were isolated. A higher ratio of MDR Salmonella enterica serovar Typhi were found by Achla et al., (2005) (60%) and Raghu Raman et al., (1993) (55.5%) in India. Prevalence of MDR Salmonella varies from 0 to 61% in the different parts of the world (Gupta et al., 1993). Similarly Pato- Masola and Donaldo (1997) reported 3 % MDR Salmonellae in Cebu city and smaller than 5 % in Manila in Philippines. The prevalence of low percent MDR Salmonella was also reported from various parts of Nepal (Malla et al., 2005), India (Madhulika et al., 2004) and Bangladesh (Rahman et al., 2002). However, the prevalence of MDR S. Typhi was found to be 26.5% by Khanal et al., (2007). Gupta et al., (1993) reported 22 % Salmonella enterica as MDR. Other few reports from different parts of world also reported high prevalence of MDR Salmonella. This difference in prevalence of MDR could be due to geographical variation and also the difference in antibiotic practice in that area. The epidemiological typing like phage typing or RFLP would help whether those strains belong to same or different clones. The fluoroquinolones and other second-line antibiotics, such as third-generation cephalosporins (eg Ceftriaxone and Cefixime) and Azithromycin (a macrolide antibiotic), are currently regarded as the antibiotics of choice for treating MDR strains (Thaver et al., 2008). These are highly effective drugs and reduce the duration of treatment from the traditional 14 days that is necessary with Chloramphenicol. This study also they show better sensitivity and the sensitivity of Azithromycin is more than 90%. Fluoroquinolones have good in-vitro and clinical activity against isolates of Salmonella spp. and are often the treatment of choice in case of life-threatening salmonellosis due to multi drug-resistant strains (Thaver et al., 2008). Fluoroquinolones, available since 1980 s have good in-vitro susceptibility and good in-vitro efficacy against Salmonella, including S. Typhi. In our study also sensitivity to fluoroquinolones is higher (73-75% 69

85 overall). Ciprofloxacin and Ofloxacin have similar sensitivity (75.6%) than Norfloxacin (73.3%). However in case of S. Typhi Ciprofloxacin had a bit higher sensitivity (72.7%) than Ofloxacin (69.7%) and in case of Paratyphi A; Ofloxacin sensitivity was fond higher (91%) than Ciprofloxacin (82%). This may be due to use of Ofloxacin higher than Ciprofloxacin in enteric fever. But to know the exact variation a large number of numples may be required. Some recent studies have also shown that fluroquinolones may also cause adverse effect in patients (Cross, 2001; Fish, 2001). The most common adverse effects associated with fluoroquinolones are gastrointestinal, such as nausea and diarrhea, and central nervous system effects, such as headache, dizziness, and drowsiness (Fish, 2001). Severe central nervous system events, such as psychosis and seizures are rare (Cross, 2001). Other adverse effects include dermatologic reactions, hepatic enzyme elevation, hypersensitivity, nephrotoxicity, hematological reactions, tendonitis, and tendon rupture. Tendon rupture can occur with short-term use and small doses (Cross, 2001). A potentially serious adverse effect is the prolongation of the QTc interval (Congeni and Thompson, 2002), which can lead to cardiac arrhythmias. In recent years, several treatment failures with fluoroquinolones have been reported (Asna et al., 2003). Since 1993, S. enterica serotype Typhi with decreased susceptibility to ciprofloxacin has been isolated in different parts of world (Thaber et al., 2008). The prolonged defervescence or treatment failure in typhoid failure associated with ciprofloxacin and other fluoroquinolone therapy have been reported from many centres (Ackers et al., 2000, Asna et al., 2003, Pokhrel et al., 2006). Nalidixic acid, a non-fluorinated quinolone is now seldom used due to the emergence of the resistant serotype of Salmonella. Patients infected with Nalidixic acid-resistant strains were found to have high treatment failure rates (up to 36%) and prolonged fecal carriage when treated with Ofloxacin (Chinh et al., 2000). Single point mutation in quinolone resistance determining region of the topoisomerase gene gyra in Salmonella usually leads to simultaneous resistance against Nalidixic acid and decreased susceptibility to fluoroquinolones (Parry et al., 2002). There is a region called QRDR (Quinolone resistance determining region) in both DNA gyrase (gyra, gyrb) and topoisomerase IV (parc, pare) where mutations usually occur. 70

86 This study found a higher prevalence of Nalidixic acid-resistant strains (24.2%) which included serotype Typhi. However serotype Paratyphi A was not NAR. Present study is close to that of Dahal (2009). In some few studies the prevalence of NAR strains are as much higher as 60% (Khanal et al., 2007). However, the occurrence of Nalidixic acid resistance in serotype Typhi is not significantly different (p>0.05) from serotype Paratyphi A. Interestingly, all Nalidixic acid-resistant strains were sensitive to Ciprofloxacin and Ofloxacin in disk diffusion test missing the detection of low level fluoroquinolone resistance. Compared to Ampicillin, Chloramphenicol and Cotrimoxazole were still found to be useful antibiotics against Salmonella. However, after the emergence of multidrugresistance, their uses have been narrowed. In this study, these antibiotics are almost equally sensitive to both serotypes i.e. 33.3%, 97.0% and 84.9% respectively to serotype Typhi and 45.5%, 100% and 82.0% respectively to serotype Paratyphi A. Chloramphenicol was found highly sensitive (97-100%) in this study. Many reviews show it is still effective for treatment of enteric fever (Manchanda et al., 2006; Pokharel et al., 2009) however in some studies a higher resistance has been reported (Agrawal et al., 1991). This may be due to low use of Chloramphenicol for the treatment of enteric fever due to its side effects and toxigenicity. The third generation Cephalosporins (Ceftriaxone, Cefotaxime, Ceftazidime, Cefexime and fourth generation cephalosporin, Cefepime had good activity against S. enterica in this study. A few resistances towards cephalosporins were shown by ESBL producing strains. Similar pattern of resistance were also reported by Khanal et al., (2007). Azithromycin has shown a good efficacy in the treatment of patients suffering from enteric fever (Chinh et al., 2000; Frenck et al., 2004; Threlfall et al., 2008). Azithromycin, a member of the macrolide group of antibiotics, has been used as an alternative drug for treating enteric fever. It achieves low intravascular levels, has high intracellular tissue penetration, and a long elimination half life of 72 hours. These properties make for once-daily administration and reduction in the duration of therapy. The drug is rapidly absorbed from the gut and is well-tolerated when used orally (Carbon 1998; Chambers 2007). In this study Azithromycin had good clinical response (93.4% 71

87 overall sensitive, 91.0% in S. Typhi and 100% in S. Paratyphi A strains). Similar pattern of sensitiveness were also reported by Butler et al., (1999). The emergence of ESBL producing micro-organism in the world is a great challenge to the treatment of the patients infected by these organisms. It was first reported in Germany in The prevalence of ESBLs among clinical isolates in Gram negative organisms varies from country to country and from institution to institution. A variety of Ambler class A and class C beta-lactamases have been described in different serotypes of Salmonella spp (Parry, 2003). Its prevalence in Salmonella has been reported to be very low so far. Infections with the ESBL producer organisms may result in avoidable treatment failure and increased cost in patients who have received inappropriate antibiotic treatment. Even if Ceftazidime and/or Cefotaxime appear to be sensitive in- vitro by routine disk diffusion method, it could be ESBL producer so that adequate attention should be given while measuring ZOI of those drugs. Although the prevalence of ESBLs is not known, it is clearly increasing. The study revealed three ESBL producer S. enterica serovar Typhi. No ESBL producing Paratyphi A and Typhimurium strains were reported. However, Pokharel et al., (2006) reported 0.3% ESBL producing S. enterica serotype Paratyphi A. A most recent study in India shows 19.04% of total salmonellae isolated were ESBL producers (Uma et al., 2010). However, the ratio was still low (about 2%) in Iran (Irajian et al., 2009). The low occurrence of ESBL production in Salmonella and limited number of sample could possibly explain its intermittent presence within the institution. Three cases of S. pneumoniae and one case of Staphylococcus aureus was reported in this study. Pneumonia is most common in streptococcal bacteremia and has been more common in children than adults (Raz et al., 1997; Musher, 2010). Similar findings were reported by Murphy and Fine (1984), and Metlay et al., (1997). Staphylococcus aureus is a leading cause of both community-acquired and hospital-acquired bacteremia. Patients with S. aureus bacteremia (SAB) can develop a broad array complication which may be difficult to recognize and which can lead to disability or death. Complications of SAB are common; frequencies range from 11 to 53 percent (Lowy, 1998; Fowler et al., 2005). A study by Sharma et al., (2006) showed similar prevalence of Streptococcus pneumoniae (11.5%) and Staphylococcus aureus (4.9%). 72

88 Conclusion Salmonella enterica serovar Typhi were found predominant (67.35%) among total isolates that included both Salmonella and non-salmonella. This study showed the prevalence of multi-drug resistance and ESBL production among Salmonella enterica was not so high. Only four (8.9%) out of fourty five Salmonellae were multidrug resistance and three (6.7%) among them were producers of ESBL. However, a high prevalence of NAR (24.2%) Salmonella enterica was found. The susceptibility of third generation cephalosporin was found significantly higher (p<0.05) than Ampicillin and Cotrimoxazole against serovar Typhi only. Imipenem, Meropenem, Amikacin, Azithromycin and Chloramphenicol showed good in vitro activity against ESBLproducing strains, so these can be regarded as the drug of choice in the treatment of infections caused by Salmonella enterica. Antibiotics showed however be prescribed only after culture and sensitivity report. Leukocyte counting and hemoglobin percentage were not found to be only reliable source for prediction of enteric fever as 91.1% cases showed normal leukocyte counting and 71.1% cases showed hemoglobin in normal range. Hence blood culture together with hematological investigations can give better results in studying enteric fever. 73

89 CHAPTER VIII Summary SUMMARY AND RECOMNENDATIONS The blood culture was found effective in detection of enteric fever. A total of 512 blood samples were processed during the study period. Of the total samples, 49 )9.57%) showed positive growth. Among the isolates, 49 different blood pathogens were isolated. In which 92% belongs to Gram negative and 8% were from Gram positive. Salmonella enterica serovar Typhi (73.3%) was found most predominant in Gram negative isolates followed by Salmonella enterica serovar Paratyphi A (24.5%), Salmonella enterica serovar Typhimurium (2.2%). Among gram positive isolates Streptococcus pneumoniae were highly predominant (75%) compared to S. aureus (25%). Of all the isolates, 8.9% were multi-drug resistant. The MDR isolates were Salmonella enterica serovar Typhi (100%) only. The most effective antibiotics for enteric fever were Imipenem and Meropenem (100%) followed by Chloramphenicol (97.8%), Amikacin (95.5%) and Azithromycin (93.3%). Among cephalosporin, the fourth-generation antibiotic, Cefepime had better sensitivity (93.3%). ESBL detection was carried out by Cephalosporins/Clavulanate Combination disks test. Among 512 blood samples 45 Salmonella enterica were isolated, among them 4 (8.9%) were ESBLs producers. All the isolates were sensitive to Imipenem and Meropenem but showed wide resistance to many groups of antibiotics. Amikacin, Chloramphenicol and Azithromycin were most among non β-lactam antibiotics. 74

90 Recommendations It is recommended to take 10 ml blood sample whenever requested for culture. As there is presence of MDR and NAR Salmonella in the community, it is strongly recommended for blood culture and sensitivity test before prescribing antibiotics for the treatment of enteric fever. Antibiotics should be strictly used only on the basis of Microbiology Laboratory results (Antibiotics susceptibility test). It would be better to carry out genotypic characterization of MDR and ESBL strains as an extension to this type of study in order to establish the location of drug resistance genes and to characterize the mechanism of drug resistance. It would be more fruitful if studies are carried out to know the prevalence of AmpC beta-lactamase and Metallo beta-lactamase producing strains. It is also recommended to launch effective health promotion programs at community to increase the awareness of people in eating behaviors. 75

91 REFERENCES Achla P, Grover SS, Bhatia R and Khare S (2005) Sensitivity index of antimicrobial agents as a simple solution for multidrug resistance in Salmonella Typhi. Indian Med Res 121: Ackers ML, Puhr ND, Tauxe RV and Mintz ED (2000) Laboratory based surveillance of Salmonella serotype Typhi infections in United States-antimicrobial resistance on the rise. JAMA 283: Adachi T, Sagara H, Hirose K and Watanabe H (2005). Floroquinolone-resistant Salmonella paratyphi A. Emerg Infect 11: Adeleke OE, Adepoju TJ and Ojo DA (2006) Prevalence of typhoid fever and antibiotic susceptibility pattern of its causative agent Salmonella typhi. Nig J Microbiol., 20(3): Agrawal S, Madhu SV, Guleria JS and Talwar V (1991) The problem of emerging chloramphenicol resistance in typhoid fever--a preliminary report. J Asso Phy Ind 39(6): Anjum MF, Marooney C, Fookes M, Baker S, Dougan G, Ivens A and Woodward MJ (2005) Identification of core and variable components of the Salmonella enterica subspecies I genome by microarray. Infect Immun 73: Araque M (2009) Nontyphoid Salmonella gastroenteritis in pediatric patients from urban areas in the city of Mérida, Venezuela. J Infect Developing Countries 3(1): Asna SM and Haque JA (2000) Decrease of antibiotic resistance in Salmonella Typhi isolated from patients attending hospital of Dhaka City over a 3 year period. Int J Antimicrob Agents 16: Asna SM, Haque JA and Rahman MM (2003) Nalidixic acid-resistant Salmonella enterica serovar Typhi and for non Typhi with decreased susceptibility to ciprofloxacin caused treatment failure: a report from Bangladesh. Jpn J Infect Dis 56:32-3. Antimicrobial susceptibility testing protocols, CRC Press, Boca Raton. Bauer AW, Kirby WM, Sherris JC and Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45(4):

92 Bhutta ZA (1996) Impact of age and drug resistance on mortality in typhoid fever. Arch Dis Child 75: Biswas R, Dhakal B, Das RN and Shetty KJ (2004) Resolving diagnostic uncertainty in initially poorly localizable fevers: a prospective study. Int J Clin Pract 58: Blaser MJ and Neuman LS (1982) A review of human salmonellosis: Infective dose. Rev Infect Dis 4:1096. Bopp CA, Brenner FW, Fields PI, Wells JG and Strockbine NA (2003) Escherichia, Shigella, and Salmonella.In: Murray P, Baron EJ, Pfaller MA, Jorgensen JH, Yolken RH (eds) Manual of clinical microbiology ASM Press, Washington DC 41: Bradford PA (2001) Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology and detection of this important resistance threat. Clin Microbiol Rev 14: 933. Brenner FW, Villar RG, Angulo FJ, Tauxe R and Swaminathan B (2000) Salmonella Nomenclature. J Clin Microbiol, 38(7): Brown JC, Thompson CJ and Amyes SG (1996) Mutation of the gyra gene of clinical isolates of Salmonella Typhimurium and three other Salmonella species leading to decreased susceptibilities to 4-quiniline drugs. J Ant Chemother 37: Buch NA, Hassan MU and Kakroo DK (1994) Enteric fever-a changing sensitivity pattern clinical profile and outcome. Ind Pdtr 31: Butler T, Sridhar CB, Daga MK, Pathak K, Pandit RB, Khakhria R, Potkar CN, Zelasky MT and Johnsonet RB (1999) Treatment of typhoid fever with Azithromycin versus chloramphenicol in a randomized multicentre trial in India. J Ant Chemother 44(2): Buzğan T, Evirgen Ö, Irmak H, Karsen H and Akdeniz H (2007) A case of typhoid fever presenting with multiple complications. Eur J Gen Med 4(2): Calderón CB and Sabundayo BP (2007) Antimicrobial Classifications: Drugs for Bugs. In Schwalbe R, Steele-Moore L, Avery C (eds) CRC Press, Boca Raton. Carbon C (1998) Pharmacodynamics of macrolides, azalides, and streptogramins: effect on extracellular pathogens. Clin Infect Dis 27(1):

93 Centers for Disease Control and Prevention (2006) Department of Health and Human Services, USA Centers for Disease Control and Prevention (2009) "Salmonellosis." Chambers HF (2007) Introduction to antimicrobial agents. In Bertram G. Katzung (ed) Basic & Clinical Pharmacology 10 th ed McGraw-Hill, New York. Chau T, Campbell JI, Galindo CM, et al (2007) Antimicrobial drug resistance of Salmonella enterica serovar Typhi in Asia and molecular mechanism of reduced susceptibility to the fluoroquinolones. Antimicrob Chemother 51: Cheryl BA, Brenner FW, et al (2003) Escherichia, Shigella, and Salmonella. In Manual of Clinical Microbiology, Murray PR editor-in-chief, 8 th edition, vol. 1, Washington, D.C., ASM Press. Chinh NT, Parry CM, Ly NT, Ha HD, Thong MX, Diep TS, Wain J, White NJ and Farrar JJ (2000) A randomized controlled comparison of azithromycin and ofloxacin for treatment of multidrug-resistant or nalidixic acid-resistant enteric fever. Antimicrob Agents Chemother 44: Chowta MN and Chowta NK (2005) Study of Clinical Profile and Antibiotic Response in Typhoid Fever. Ind J Med Microbiol 23: Cleary TG (2003) Salmonella. In Nelson testbook of Pediatrics, 17 th ed, Philadelphia, WB Saunder s company. Clinical and Laboratory Standards Institute (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard M7-A7, 7 th ed., CLSI, Wayne, PA. Clinical and Laboratory Standards Institute (2006) Performance standards for antimicrobial disk susceptibility tests, approved standard M2-A9, 9 th ed., CLSI, Wayne, PA. Clinical and Laboratory Standards Institute (2010) Performance standards for antimicrobial susceptibility testing, twentieth informational supplement. CLSI, Wayne, PA Cohen ML (1992) Epidemiology of drug resistance: implications for a post-antimicrobial era. Science 257:

94 Congeni BL and Thomson RB Jr (2002) Fluoroqinolones: consideration for future use. Ped Inft Dis J 21(4): Crosa JH, Brenner DJ, Ewing WH and Falkow S (1973) Molecular relationships among the Salmonelleae. J Bacteriol 115(1): Cross JT Jr (2001) New anti-infectives: Fluoroquinolones. Seminars in Pdtr Infect Dis 12: (211 23). Crump JA, Luby SP and Mintz ED (2004) The global burden of typhoid fever. Bull World Health Organ 82: Dahal, R (2009) Restriction Fragment Length Polymorphism (RFLP) Pattern of Salmonella enterica isolated from Blood of Suspected enteric fever Patients Visiting Tribhuvan University Teaching Hospital (TUTH), Kathmandu, Nepal. An academic dissertation (M.Sc. MLT) submitted to the Institute of Medicine, Tribhuvan University Kathmandu, Nepal. Dangana A, Ajobiewe J and Nuhu A (2010) Haematological changes associated with Salmonella typhi and Salmonella paratyphi in humans. Int J Biomed Hlth Sci 6(4): Doherty CP, Saha SK and Cutting WA (2000) Typhoid fever, ciprofloxacin and growth in young children. Ann Trop Paediatr 20: Doyle MP, Beuchat LR and Montville TJ (ed.) (2001) Food Microbiology, 2 nd ed. ASM Press, Washington, DC pp Emmeluth D (2004) Typhoid fever. Infobase Publishing, New York. Feinstein AR (2002) Principles of medical statistics. CRC Press, Boca Raton Finch RG, Irving WL, Moss P and Anderson J (2009) Infectious diseases, tropical medicine and sexually transmitted infection. In Kumar P, Clark M (eds) Kumar & Clark s Clinical Medicine 7 th ed Saunders Elsevier, Spain. Fish DN (2001) Fluoroquinolone adverse effects and drug interactions. Pharmacotherapy 21(10,2): Francis CL, Starnbach MN and Falkow S (1992) Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella Typhimurium grown under low oxygen conditions. Mol Microbiol 6:

95 Frenck RW Jr, Mansour A, Nakhla I, Putnam S, Wierzba T, Morsy M and Knirsch C (2004) Short-course azithromycin for the treatment of uncomplicated typhoid fever in children and adolescents. Clin Infect Dis 38(7): Forbes BA, Sahm DF and Weissfeld AS (2007) Bailey and Scott s Diagnostic Microbiology. Elsevier, New York. Fowler VG Jr, Justice A, Moore C, Benjamin DK Jr, Woods CW, et al., (2005) Risk factors for hematogenous complications of intravascular catheter-associated Staphylococcus aureus bacteremia. Clin Infect Dis 40:695. Garg RA and Krashak R (1993) Typhoid fever before two years of age. Ind Pdr 30: Gasem MH, Dolmans WM, Isbandrio BB, Wahyono H, Keuter M and Djokomoeljanto R (1995) Culture of Salmonella typhi and Salmonella paratyphi from blood and bone marrow in suspected typhoid fever. Trop Geo Med 47: Gautam V, Gupta NK, Chaudhari U and Arora DR (2002) Sensitivity pattern of Salmonella serotypes in Northen India. Braz J Infect Dis 6: Ghenghesh KS, Franka E, Tawil K, Wasfy MO, Ahmed SF, Rubino S, Klena JD (2009) Enteric Fever in Mediterranean North Africa. J Infect Dev Ctries 3(10): Gilmen RH (1975) Relative efficacy of blood, urine, rectal swab,bone marrow and rose spot culture for recovery of Salmonella Typhi in typhoid fever. Lancet 1: Gordon M (2008) Salmonella infections in immunocompromised adults. J of Infect 56: Gulati S, Marwaha RK, Prakash D, Ayyagari A, Singhi S, Kumar L, Singhi P, Walia BN (1992) Multi drug-resistant Salmonella typhi-a need for therapeutic reappraisal. Ann Trop Paediatr 12: Gupta B, Kumar R and Khurana S (1993) Multi drug-resistant Salmonella typhi in Ludiana (Punjab). Ind J Pathol Microbiol 36: 5-7. Gupta S, Handa A, Chadha DS, Ganjoo RK and Panda RC (2009) Profile of Culture Positive Enteric Fever from Bangalore. MJAFI 65: Hakanen A, Kotilainan P, Jalava J, Siitonen A and Huovinem (1999) Detection of decreased fluoroquinolone susceptibility in Salmonella and validation of nalidixic acid screening test. J Clin Microbiol 37:

96 Hawkey PM (1998) The origins and molecular basis of antibiotic resistance. BMJ 317: Hohmann EL (2001) Nontyphoidal salmonellosis. Clin Infect Dis 32: Holt KE, Parkhill J, Mazzoni CJ, Roumagnac P, Weill FX, Goodhead I, Rance R, Baker S, Maskell DJ, Wain J, Dolecek C, Achtman M and Dougan G (2008) High throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi. Nat Genet. 40: Hooper DC (2001) Mechanism of fluoroquinolone resistance. Emerg infect Dis 7: Irajian G, Ranjbar R and Moghada AJ (2009) Detection of Extended Spectrum Beta Lactamase Producing Salmonella spp and Multidrug Resistance Pattern. Iran J Pathol (3), Isenberg HD (2004) Clinical Microbiology Procedures Handbook 2 nd ed ASM press Washington,D.C. Ismail A, Abdul Kader Z and Ong KH (1992) Dot enzyme immunosorbent assay for the serodiagnosis of typhoid fever. S A J Trop Med Pub Health 22: Jacoby G, Han P and Tran J (1997) Comparative in vitro activities of carbapenem L- 749,345 and other antimicrobials against multiresistant gram-negative clinical pathogens. Antimicrob Agents Chemother 41:1830. Jay MJ, Loessner MJ and Golden DA (2005) Modern food microbiology, 7 th ed. Springer Science& Business media, Inc. United States pp Jenkins C and Gillespie SH (2006) Salmonella spp. In Gillespie SH, Hawkey MJ, Peter M (eds) Principles and Practice of Clinical Bacteriology 2 nd ed John Wiley & Sons Ltd, Philadelphia. Jenner W (1850) On the identity or non-identity of the specific cause of typhoid, typhus and relapsing fever. C and J Adrald, London. Kariuki S, Revathi G, Kariuki N, Kiiru J, Mwituria J, Muyodi J, Githinji JW, Kagendo D, Munyalo A and Hart CA (2006) Invasive multidrug resistant non-typhoidal Salmonella infections in Africa, zoonotic or anthroponotic transmission? J Med Microbiol 55: Kauffmann, F (1950) The Diagnosis of Salmonella Types. Blackwell Scientific Publications, Springfield. 81

97 KC M, Chaudhary D, Simkhada R, Pradhan M, Shrestha P and Gurubacharya DL (2005) Study of clinical profile and antibiotic sensitivity pattern in culture positive Typhoid fever cases. KU Med J 3(4, 12): Khanal B, Sharma SK, Bhattacharya SK, Bhattarai NR, Deb M and Kanungo R (2007) Antimicrobial susceptibility patterns of Salmonella enterica serotype Typhi in Eastern Nepal. J Health Popul Nutr 25: Kliebe C, Nies BA, Meyer JF, Tolxdorff-Neutzling RM and Wiedemann B (1985) Evolution of plasmid-coded resistance to broad-spectrum cephalosporins. Antimicrob Agents Chemother 28:302. Lewis M, Mason C. Pitarangsi C, Serichantalergs O, Chuanak N, Regmi LR, Pandey P, Laskar R, Shrestha CD, Malla S (2002) AFRIMS determines epidemics in Nepal is Typhoid. Global Emerg Infect Syst. Li J, Plorde JJ and Carlson LG (1994) Effects of Volume and Periodicity on Blood Cultures. J Clin Microbiol 32(11): Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339: 520. Madhulika U, Harish BN and Parija SC (2004) Current pattern in antimicrobial susceptibility of Salmonella Typhi isolates in Pondicherry. Ind J Med Res 120: Malla S, Kansakar P, Serichantalergs O, Rahman M and Basnet S (2005) Epidemiology of typhoid and paratyphoid fevers in Kathmandu: two years study and trends of antimicrobial resistance. J Nep Med Asso 44: Manchanda V, Bhalla P, Sethi M and Sharma VK (2006) Treatment of enteric fever in children on the basis of current trends of antimicrobial suseptibility of Salmonella enterica serovar Typhi and Paratyphi A. Ind J Med Microbio 24(2): Mathura KC, Chaudhary D, Simkhada R, Pradhan M, Shrestha P and Gurubacharya DL (2005) Study of clinical profile and antibiotic sensitivity pattern in culture positive typhoid fever cases. KUMJ 3(4): Maza LM de la, Pezzlo MT and Baron EJ (1997) Enterobacteriaceae. In Color Atlas of Diagnostic Microbiology St. Louis pp McClelland M, Sanderson KE, Clifton SW, Latreille P, Porwollik S, Meyer R, Bieri T, Ozersky P, McLellan M, Harkins CR (2004) Comparison of genome degradation 82

98 in paratyphi A and typhi, human-restricted serovars of Salmonella enterica that cause typhoid. Nat Genet 36: Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV (1999) Food-related illness and death in the United States. Emergency Infectious Disease 5: Metlay JP, Schulz R, Li YH, Singer DE, Marrie TJ, Coley CM, Hough LJ, Obrosky DS, Kapoor WN, Fine MJ. (1997) Influence of age on symptoms at presentation in patients with community-acquired pneumonia. Arch Intern Med 157: Miles RS and Amyes SGB (2006) Laboratory control of antimicrobial therapy. In Collee JG, Marmion BP, Fraser AG, Simmons A Mackie and McCartney Practical Medical Microbiology 14 th ed. Churchill Livingstone, New Delhi 8: Murdoch DA, Banatvaia NA, Bone A, Shoismatulloev BI, Ward LR and Threlfall EJ. (1998) Epidemic ciprofloxacin-resistant Salmonella Typhi in Tajikistan. Lancet 351:39. Murdoch DR, Woods CW, Zimmerman MD, Dull PM, Belbase RH, Keenam AJ, Scott RM, Basnyat B, Archibald LK and Reller LB (2004) The etiology of febrile illness in adults presenting to Patan hospital in Kathmandu, Nepal. Am J Trop Med Hyg 70: Murphy TF and Fine BC (1984) Bacteremic pneumococcal pneumonia in the elderly. Am J Med Sc 288: Musher DM (2010) Streptococcus pneumoniae. In Mandell GL, Bennett JE, Dolin R (eds) Principles and practice of infectious diseases, 7 th ed, Churchill Livingstone Elsevier, Philadelphia 200: Ochiai RL, Acosta CJ, Danovaro-Holliday MC, Baiqing D, Bhattacharya SK, Agtini MD, Bhutta ZA, Canh do G, Ali M, Shin S and Wain J (2008) A study of typhoid fever in five asian countries: disease burden and implications for controls. Bull World Health Organ 86: Okonko OI, Soleye A, Eyarefe OD, Amusan TA, Abubakar MJ, Adeyi AO, Ojezele MO, Fadeyi A (2006) Prevalence of Salmonella typhi among Patients in Abeokuta. Br J Pharm Toxicol 1(1):

99 Old DC (2006) Salmonella. In Collee JG, Marmion BP, Fraser AG, Simmons A Mackie and McCartney Practical Medical Microbiology 14 th ed. Churchill Livingstone, New Delhi Onyango D, Machoni F, Kakai R and Waindi EN (2008) Multidrug resistance of Salmonella enterica serovars Typhi and Typhimurium isolated from clinical samples at two rural hospitals in Western Kenya. J Infect Dev Coun 2(2): Pang T (1989) False positive Widal test in nontyphoid Salmonella infection. Southeast Asian J Trop Med Pub Hlth 20: Parkhill J, Dougan G, James KD, Thomson NR, Pickard et al., (2001) The complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18 provides insight into the evolution of host restriction and antibiotic resistance. Nature 413: Parry CM (2003) Antimicrobial drug resistance in Salmonella enterica. Curr Opin Infect Dis 16: Parry CM, Hien TT, Dougan G, White NJ and Farrar JJ (2002) Typhoid fever. NEJM 347: Paterson DL, Ko WC, Von Gottberg A, Casellas JM, Mulazimoglu L, Klugman KP, Bonomo RA, Rice LB, McCormack JG and Yu VL (2001) Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum beta-lactamases: implications for the clinical microbiology laboratory. J Clin Microbiol 39:2206. Paterson DL, Ko WC, Von Gottberg A, et al (2004) Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum betalactamases. Clin Infect Dis 39:31 Pato-Mesola VV and Donaldo S ME (1997) Antimicrobial susceptibility of S. typhi isolates from Government and privates hospitals in Cibu city. J Microbiol Infect Dis 26: 5-8. Pegues DA and Miller SI (2010) Salmonella species, including Salmonella Typhi. In Mandell GL, Bennett JE, Dolin R (eds) Principles and practice of infectious diseases, 7 th ed, Churchill Livingstone Elsevier, Philadelphia 223:

100 Pokharel BM, Koirala J, Dahal RK, Mishra SK, Khadga PK and Tuladhar NR (2006) Multidrug-resistant and extended spectrum beta-lactamase (ESBL) - producing Salmonella enterica (serotype Typhi and Paratyphi A) from blood isolates in Nepal: surveillance of resistance and a search for newer alternatives. Int J of Inf Dis 10: Pokharel P, Rai SK, Karki G, Katuwal A, Vitrakoti R and Shrestha SK (2009) Study of enteric fever and antibiogram of Salmonella isolates at a Teaching Hospital in Kathmandu Valley. Nep Med Coll J 11(3): Popoff MY, Bockemuhl J and Gheesling LL (2004) Supplement 2002 (no. 46) to the Kauffmann-White scheme. Res Microbiol 155: Popoff MY and Minor LLe (1997) Antigenic formulas of the Salmonella serovars 7th revision. World Health Organization Collaborating Centre for Reference and Research on Salmonella, Pasteur Institute, Paris. Quimpo VS (1991) Typhoid Fever in a Tertiary Metro Manila Hospital. Phil J Microbiol Infect Dis 20(2): Raghu Raman TS, Krishnamurthy L, Menon PK, Singh D and Jayaprakash DG (1993) Clinical Profile and Therapy in Enteric Fever Ind Pediatr 31: Rahman M, Ahmad A and Shoma S (2002) Decline in epidemic of multidrug-resistant Salmonella Typhi is not associated with increased incidence of antibiotic susceptible strain in Bangladesh. Epidemiol Infect 129: Rai DR, Kshetry NT, Tamang MD and Pokhrel BM (2005) Study of enteric fever and malaria incidence in southern part of Nepal. J Inst Med 27(3): Rao RS, Sundarraj R, Subramanian S, Shankar V, Morti SA and Kapor SC (1997) A study of drug resistance among Salmonella typhi and Salmonella paratyphi A in an endemic area Rauniar GP, Das BP, Baral DD and Naga Rani MA (2000) Treatment pattern of typhoid fever at a tertiary care teaching hospital in eastern Nepal. J Nepal Med Asso 39: Ray K (2002) Typhoid fever. The Rosen Publishing group, New York. 85

101 Raz R, Elhanan G, Shimoni Z, et al (1997) Pneumococcal bacteremia in hospitalized Israeli adults : Epidemiology and Resistance to Penicillin. Clin Infect Dis 24: Reller ME, Olsen SJ, Kressel AB, Moon TD, Kubota KA, Adcock MP, Nowicki SF and Mintz ED (2003) Sexual transmission of typhoid fever: a multistate outbreak among men who have sex with men. Clin Infect Dis 37: Rice LB, Sahm D and Bonomo RA (2003) Mechanisms of Resistance to Antibacterial Agents. In Murray P, Baron EJ, Pfaller MA, Jorgensen JH, Yolken RH (eds) Manual of clinical microbiology ASM Press, Washington DC 68: Rowe B, Ward LR and Threlfall EJ (1997) Multidrug resistance Salmonella Typhi: a worldwide epidemic. Clin Infect Dis 24(Suppll):S Ryan KJ (2004) Enterobacteriaceae. In Ryan KJ, Ray CG (eds) Sherris Medical Microbiology, 4 th ed, The McGraw-Hill Companies, Washington DC, pp Saha SK, Talukder SY, Islam M and Saha S (1999) A highly ceftriaxone resistant Salmonella typhi in Bangladesh. Pdtr Infect Dis 18(3): Sarkinfada F and Abubakar AG (2001) Leukocyte counts of typhoid fever patients in Kano, Nigeria. Nig Soc Exp Biol 1(1): Selander R, Li J and Nelson K. Evolutionary Genetics of Salmonella enterica (1996) In: Neidhardt F (ed) Escherichia coli and Salmonella. 2 nd ed. Washington D.C.: ASM Press Selsted ME, Miller SI, Henschen AH and Ouellettell AJ (1992) Enteric Defensins: Antibiotic Peptide Components of Intestinal Host Defense. J Cell Bio 118(4): Sharma N, Koju R, Karmacharya B, Tamang MD, Makaju R, Nepali N, Shrestha P and Adhikari D (2003) Typhoid fever in Dhulikhel hospital, Nepal. KU Med J 2(3) Sharma NP, Peacock SJ, Phumratanaprapin W, Day N, WhiteN and Pukrittayakamee S (2006) A hospital based study of bloodstream infections in febrile patients in Dhulikhel Hospital Kathmandu University Teaching Hospital Nepal. Southeast Asian J Trop Med Pub Hlth 37(2):

102 Shelobolina ES, Sullivan SA, O'neill KR, Nevin KP and Lovley DR (2004) Isolation, characterization, and U(VI)-reducing potential of a facultatively anaerobic, acidresistant bacterium from low-ph, nitrate- and U(VI)-contaminated subsurface sediment and description of Salmonella subterranea sp. nov. Appl Environ Microbiol 70 (5): Sherwal BL, Dhamija RK, Randhawa VS, Jais M, Kaintura A and Kumar M (2004) A Comparative Study of Typhidot and Widal Test in Patients of Typhoid Fever. Ind Acad Clin Med 5(3): Shirakawa T, Acharya B, Kinoshita S, Kumagaib S, Gotoha A and Kawabataa M (2006) Decreased susceptibility to fluoroquinolones and gyr A gene mutation in the Salmonella enterica serovar Typhi and Paratyphi A isolated in Kathmandu, Nepal, in Diagn Microbiol Infect Dis 54: Singh S (2001) Typhoid Fever: Pathogenesis and Laboratory Diagnosis. J Ind Aca Clin Med 2: Smith A (2004) Bacterial resistance to antibiotics. In Denyer SP, Hodges NA, Gorman (eds) Pharmaceutical Microbiology 7 th ed Blackwell science, Massachusetts. Sood SC and Taneja PN (1995) Typhoid fever clinical features and diagnosis. Ind J Ch Hlth 6: 123. Stevenson KB, Samore M, Barbera J, Moore JW, Hannah E, Houck P, Tenover FC and Gerberding JL (2003) Detection of antimicrobial resistance by small rural hospital microbiology laboratories: comparison of survey responses with current NCCLS laboratory standards. Diagn Microbiol Infect Dis 47:303. Thaver D, Zaidi AKM and Critchley JA (2008) Fluoroquinolones for treating typhoid and paratyphoid fever (enteric fever) (Review). The Cochrane Collaboration, JohnWiley & Sons, Ltd. Threlfall EJ, de Pinna E, Day M, Lawrence J and Jones J (2008) Alternatives to ciprofloxacin use for enteric Fever, United kingdom. Em Infect Dis 14(5): Todar K (2011) Salmonella and Salmonellosis. In Todar's Online Textbook of Bacteriology 87

103 Towner KJ (2000) Mechanisms of acquired resistance. In Greenwood D, Emmerson A, Finch R (eds) Antimicrobial Chemotherapy, 4 th ed, Oxford University Press, London. Tuladhar NR, Banjade N, Pokhrel BM, et al (2003). Antimicrobial resistant bacterial strains from inpatients of Tribhuvan University Teaching Hospital Kathmandu. J Inst Med 25: Turner PJ (2005) Extended-spectrum β-lactamases. Clin Infect Dis 41 (suppl 4): S Uma B, Prabhakar K, Rajendran S and Sarayu YL (2010) Prevalence of extended spectrum beta lactamases in Salmonella species isolated from patients with acute gastroenteritis. Ind J Gastroenterol 29: Vallenas C, Hernandez H, Kay B, Black R and Gotuzzo E (1985) Efficacy of bone marrow, blood, stool and duodenal contents cultures for bacteriologic confirmation of typhoid fever in children. Pediatr Infect Dis 4: Varma JK, Greene KD, Ovitt J, Barrett TJ, Medalla F and Angulo FJ (2005) Hospitalization and antimicrobial resistance in Salmonella outbreaks Emerg Infect Dis 11: Voetsch AC, Gilder TJV and Angulo FJ (2004) FoodNet Estimate of the Burden of Illness Caused by Nontyphoidal Salmonella Infections in the United States. Clin Infect Dis 38 (Suppl 3): S Wain J, Diep TS, Ho VA, Walsh AM, Hoa TTN, Parry CM and White NJ (1998) Quantitation of bacteria in blood of typhoid fever patients and relationship between counts and clinical features, transmissibility, and antibiotic resistance. J Clin Microbiol 36: Wain J, Hoa NT, Chinh NT, Vinh H, Everett MJ, Diep TS, Day NP, Solomon T, White NJ, Piddock LJ and Parry CM. (1997) Quinolone-resistant Salmonella Typhi in Vietnam: molecular basis of resistance and clinical response to treatment. Clin Infect Dis 25: Wain J, Song DT, Van Be B, Walsh AL, Vinh H, Nguyen MD (2008) Specimens and culture media for the laboratory diagnosis of typhoid fever. J Infect Dev Cntr 2(6):

104 Watson JP and Pettibone EC (1991) Chloramphenicol and ampicillin resistant Salmonella typhi in Nepal. J Nep Med Assoc 29: Watson KC (1978) Laboratory and Clinical Investigation of Recovery of Salmonella typhi from Blood. J Clin Microbiol 7(2) Weikel CS and Guerrant RL (1985) Nosocomial salmonellosis. Infect Contrl 6: WHO (2003) The diagnosis, treatment and prevention of typhoid fever. World Health Organization, Geneva. Wong SPY, Lai TKK, Ng WL and Luk WK (2007) Non-typhoid Salmonella mycotic aneurysm of the aortic arch. Hong Kong Med J 13:

105 Photograph 1: Colonies of S. enterica on agar plates (top left BA, top right CA, bottom left MA & bottom right SS agar).

106 Photograph 2: S. enterica showing different biochemical reactions (Starting from left: TSI, Urease, Citrate, NA slant, SIM) Photograph 3: Blood Culture bottles (left: BHI broth, right: 5ml Blood + 45 ml BHI broth)

107 Photograph 4: Antibiotic susceptibility profile of S. enterica Photograph 5: Salmonella enterica serotyping (Left: No agglutination non salmonellae; Right: Agglutination Salmonella enterica serovar Typhi)

108 Photograph 6: ESBL producing S. enterica serovar Typhi Photograph 7: Researcher performing work on Salmonella enterica