RESEARCH SYNOPSIS.
Introduction
Salmonella typhi is a gram-negative bacterium that is responsible for typhoid fever. It is a rod-shaped, flagellated facultative anaerobe bacterium that belongs to the family Enterobacteriaceae, of which the only reservoir is the human body. Salmonella typhi is transmitted by ingestion of food and water that is contaminated with the excrements of those that carry the organism and must survive the gastric pH barrier in the stomach prior to adherence in the small intestine.
Genus Salmonella
Order Enterobacterales
Domain Bacteria
Phylum Proteobacteria
Class Gammaproteobacteria
These species which are Salmonella enterica and Salmonella bongori, are grouped into Salmonella of the S.Enterica, on the basis of its genomic relationship and biochemical properties, can be further categorized into six subspecies. Classifying Salmonella by serotype based on 3 important antigenic determinants somatic (O), capsular (K), and Flagellar (H) About two thousand and six hundred serotypes have been made via the use of the standard Kauffman-White system most of these serotypes have the potential to adapt across a number of animal hosts, including humans.
The human-specific gram-negative pathogenic bacteria, Salmonella typhi and Salmonela paratyphi, are main cause for typhoid fever and also for paratyphoid fever. Transferred from person to person via the fecal oral path by ingesting contaminated water or food or by contact with fecal matter from chronic infected individuals.
Contaminated water is the main source of dissemination. It is also present in poultry, eggs. The major transmission routes for pathogens is livestock and uncooked animal food products [1].
Almost all strains of Salmonella are pathogenic because they are free to invade, reproduce and live in human host cells, resulting in potentially fatal diseases [2]. Typhoid fever triggers the presence of S.typhi in infected water or food. Salmonella ingested in food survives its passage through the barrier of gastric acid and invades the small and large intestine mucosa and produces toxins.
The acute inflammatory reaction induces diarrhea and can contribute to mucosal ulceration and destruction. Bacterial infection spreads from intestine to other parts of body resulting in systematic diseases [3, 4].
S.typhi causes typhoid fever. After an inoculation period of 10 to 14 days, symptoms start. In the initial asymptomatic phase, patients with associated fever develop an influenza-like disease.
During in the phase of the illness, abdominal disease symptoms are frequently present and can include discomfort, nausea, vomiting, or constipation. In all patients, however, abdominal pain is experienced and can differ from diffuse in nature to pain that mimics appendicitis. Typically, hepatomegaly and splenomegaly are formed during the course of a patient's disease. Enteric fevers are serious infections that can be fatal if antibiotics are not given promptly [5].
Environmental risk elements for typhoids are living in the vicinity of open wells or sewers, residence at low elevation and climatic events such as the rainy seasons. The greatest risk of transmission is in heavily populated regions and environments which lack sanitation and access to safe drinking water [6].
A research group discovered the bacteria S.typhi species present in tap water in Tamale Metropolis.Tamale is the capital town of the Northern Region of Ghana. To show the prevalence of pathogenic bacteria found in tap water, this research was conducted to find out if the sources of drinking water in Tamale are polluted with species of Salmonella. The antibiotic resistance of Salmonella species isolated from water samples was also examined in the study [7].
Problem statements
The research study will focus on the presence of S.typhi in the tap water sources of district Peshawar. As it is reported in a study in 2008, Pakistan and India are one of the major countries having higher number of cases of enteric fever, while another study showed that almost 412.9(100,000) [8].
Peshawar is also one of effected city from enteric fever. As reported in one of the study almost 1,000 patient in tertiary care hospital of Peshawar receives blood culture for it.In this regard is a menace for the local populace. In light of such events to mitigate the effects of typhoid fever, a through research is need of the time in order to devise a plan. This research work in this regard will help the health authorities and water sanitation department to introduce such strategies to provide better health facilities and ensure availability of clean drinking water respectively [9].
Aims and Objectives
To isolate and identify S.typhi from drinking water sources in district Peshawar.
To determine the pattern of antibiotic susceptibility of bacterial isolates.
Material required
Sterile jars
Incubator
Nutrient agar plate
Inoculation loop
Disposable gloves
Xylose Lysine Deoxycholate agar
Brilliant Green Agar
Filter paper
Autoclave
Plan of work
This research will be carried out in the laboratory of Health sciences department of City University of science and information technology Peshawar in the following phases.
Phase: 1.Sample collection
Sample will be collected in the sterile jar. This method will trap S.Typhi [10]; the sterile jar will be carried to the laboratory and subjected to enrichment culture for S.Typhi [11].
Phase: 2.Enrichment Culturing and sub culturing of samples
Two selective media will be used in culturing these are:
1. xylose lysine deoxycholate (XLD) agar
2. brilliant green agar (BGA)
A 10 μL loop will be used to collect discrete colonies and the selective enrichment cultures to pre-warmed Xylose Lysine Deoxycholate agar and to pre-warmed Brilliant Green Agar media after incubation subculture is done. Incubation will be done for 24 ± 3 hours at 37 ± 1 ° C [12].
Phase: 3.Identification of colonies
Examination of selective agar plates for Salmonella colonies after 24± 3 hours to 48± 3 hours of extended incubation.
XLD
Xylose Lysine Deoxycholate agar is a selective medium of growth used in Salmonella typhi isolation. In 1000 ml of distilled water, 56.68 grams of XLD is dissolved.
Heat supplied. Until the medium boils with frequent agitation. Then switch immediately to 50° C at water bath. It will be poured into sterile Petri plates after cooling.
BGA
A selective growth medium used in the isolation of Salmonella typhi is Brilliant Green Agar.
In 1000 ml of purified/distilled water, 58.09 grams of media are dissolved .It was heated until it boiled to fully dissolve. Sterilized for 15 minutes at a pressure of 15 lbs (121 °C) by autoclaving. Cooled it in the temperature of 45-50°C.When mixed well and poured into sterile Petri plates.
Phase: 4.Confirmatory tests
A typical colony from one plate subjected to serological and biochemical confirmation.
Serological confirmation
We performed an oxidase test from Nutrient agar plates on S,typhi colonies. On NA colonies appear as colorless. Salmonella typhi are oxidase negative. Sub-culture of oxidase negative colonies from NA to slopes of NA are done then added a few drops of sterile water to the container prior to inoculation [13]. Inoculation of the colony into the water is done and streak up the slope. Incubate it at 37 ± 1°C for 5 ± 1 h, or overnight if insufficient growth is evident.
Biochemical confirmation
All suspected colonies of Salmonella typhi are selected from the agar plates and inoculated for confirmation in the following biochemical test tubes:
Triple sugar iron (TSI) test
The Triple Sugar Iron agar test is used to confirm that S.typhi has been identified.The top of a well-isolated colony will be touched with an inoculating loop, then the TSI agar inoculation will be done by first stabbing through the middle of the medium to the bottom of the tube and then streaking the agar slant's surface. The tube is incubated in the ambient air at 35 °-37 ° C for 18 to 24 hours.
Citrate test
The citrate test is used to confirm that S.typhi has been identified. 24.28 grams of laboratory-prepared medium is applied to thousand ml of distilled water in a beaker. In order to completely dissolve the medium the solution is then boil. The medium which is dissolved is then dispensed into tubes and sterilized at a pressure of 15 lbs (121 °C) in an autoclave for 15 minutes. The tubes are taken out and cooled to a temperature of about 40-45 ° C at a slanted position if the autoclaving process is complete.From a well-isolated colony, an 18-24 hour culture of S.typhi with a sterile inoculating needle is taken.By streaking the surface of the slope the tubes of citrate agar are inoculated. With the loop the slant runs back and forth. The cap of the test tubes was loosened to guarantee sufficient aeration. For up to 4 days, at 35-37 °C, the tubes are then incubated aerobically.
Urease test
The Urease test is used to verify S.typhi detection. S.typhi is urease-negative.The urease test ingredients are dissolved in hundred milliliter of distilled water and sterilized by the filter. The agar is suspended in nine hundred milliliter of distilled water and boiled until fully dissolved. Autoclave for 15 minutes at one hundred twenty one degrees. Cool the agar to fifty to fifty-five degrees hundred milliliter of filter sterilized urea, added to the agar which are cooled solution and mix thoroughly. Distribute four to five milliliter per sterile tube and slant the tubes during cooling until solidified. The urea agar slant surface was streaked with a part of isolated colony of S.typhi. Hold the cap loosely on and incubate the tube in ambient air at 35°-37°C for 48 hours to 7 days.
Indole test
The Indole test is used to confirm Salmonella typhi identification. A few drops of Indole Spot Reagent are placed on a piece of filter paper. With an inoculating loop, a portion of the 18-24-hour isolated colony was collected from the media and rubbed onto the filter paper's saturated reagent area. Check the filter paper immediately [14].
Phase: 5.Antibiotic susceptibility pattern
Antibiotic susceptibility is accomplished by the system of Kirby-Bauer disc diffusion. Selected antibiotic disc containing S.typhi cultured on MHA (Muleller hingon Agar) using standard procedures. In the current research, the antibiotics used include:
Name Class Generation
Amikacin (30 μg) Pencillins 1st generation antibiotic
Kanamycin (30 μg) Aminoglycosides 1st generation antibiotic
Gentamicin (10 μg) Aminoglycosides 1st generation antibiotic
Amikacin (30 μg) Aminoglycosides 1st generation antibiotic
Ciprofloxacin (5 μg) Fluoroquinolones 2nd generation antibiotic
Ampicillin (10 μg) Pencillins 2nd generation antibiotic
Cefmetazole (30 μg) Cephems(parental) 2nd generation antibiotic
Tetracycline (30 μg) Tetracyclines 2nd generation antibiotic
Cefoxitin (30 μg) Cephems(Parental) 2nd generation antibiotic
Ceftizoxime (30 μg) Cephems(Parental) 3rd generation antibiotic
Imipenem (10 μg) Carbapenems 3rd generation antibiotic
Meropenem (10 μg) Carbapenems 3rd generation antibiotic
Doxycline (30 μg Tetracyclines 3rd generation antibiotic
Cefdinir (5 μg Cephems(oral) 3rd generation antibiotic
Aztreonam (30 μg) Monobactams 3rd generation antibiotic
Ticarcillin (75 μg) Pencillins 4th generation antibiotic
Socioeconomic benefits
First step in solving
any problem is to identify its root cause. It has been reported in many studies
that typhoid fever is increasing day by day due to contaminated water, so this
is need of the day to device strategies for the better management of clean
drinking water.
This study will help
in better management of cleaning the water from S.typhi and low the risks for the typhoid fever. This study will
raise awareness among people about typhoid fever via this study health
authorities of Peshawar can know in which area water is contaminated with S.typi so they can take action against
it and can clean water and in which area people are suffering from typhoid
fever they can do treatment of them. After this research People of Peshawar
Pakistan can be prevented from S.typhi
and can save money spend on treatments on people which is very helpful
financial statement of Peshawar.
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