Antibiotic Resistance Pattern among Typhoid Patients and Reoccurrence in

Vaccinated Population in District Nowshera

SUBMITTED BY: Muhammad Tahseen

ROLL NO: 4854
STUDY LEVEL: M.PHIL
RESEARCH SUPERVISOR: DR. AYUB JADOON
Assistant Professor

CO SUPERVISOR:

DEPARTMENT OF MICROBIOLOGY

ABBOTTABAD UNIVERSITY OF SCIENCE AND

TECHNOLOGY

1
 Contents

SUMMARY................................................................................................................... ...1

1. INTRODUCTION.......................................................................................................1

2. GAP ANALYSIS/ BACKGROUND OR PROBLEM STATEMENT........................2

3. OBJECTIVES…...........................................................................................................3

4. RESEARCH DESIGN AND METHODS…..........................................................4 - 8

5. FLOW CHART……………………….………………………………………….......9

6. EXPECTED OUTCOMES….…………………………………………………..…..10

7. REFERENCES ....................................................................................................11 - 12

2
1. SUMMARY:

Typhoid is systemic infection classically caused by Salmonella Typhi. Typhoid fever can

vary fom a slight infection to clinical or radiologic pneumonia or severe infection with a kind of
the classical typhoidal condition which can be lethal. Annually, S. Typhi cause typhoid fever in
more than 20 million people and also responsible for 200,000 deaths. The rate of multidrug
resistant enteric fever is increasing day by day. Moreover, reoccurrence of typhoid in vaccinated
patients is alarming. Therefore, study was performed in order to check the pattern of antibiotic
resistance among the patient and also reoccurrence of typhoid in vaccinated population. 150
patients were selected based on available database. Patients were admitted in the hospital with
the history of fever more than one week duration and had positive blood culture for S. Typhi.
The overall number of patients presented was 507 out of which 150 were positive for S. Typhi.
Positive cultures processed for isolation of pathogens. Data was collected from the survey
questionnaire. The questionnaires were formed on the basis of references to existing literature
and consultancies with field professionals. Data related to gender, age, clinical features, blood
culture and anti-microbial sensitivity was collected. Kirby- Bauer disc diffusion method was
used to test the susceptibility. Mueller Hinton Agar plates were used for this purpose. 12
different antibiotic discs were used. Ampicillin, co-trimoxazole, chloramphenicol, ertapenem,
ciprofloxacin, ceftriaxone, ofloxacin, enoxacin, meropenem, imipenem and azithromycin were
used for susceptibility test.

3
1. INTRODUCTION:

Salmonella Typhi (S. Typhi), S. paratyphi A, and, in some cases, S. paratyphi B and C
produce typhoid fever, which is a systemic illness caused by Salmonella Typhi (S. Typhi), S.
paratyphi A, and, in rare cases, S. paratyphi B and C. Kidgell and colleagues (1). A rise in
body temperature, headache, anorexia, lethargy, constipation, and diarrhoea are the most
typical typhoid symptoms. Pathogens infect people of all ages and have developed their own
method of surviving in their hosts (2, 3). Typhoid fever can range in severity from a small
infection to clinical or radiologic pneumonia, or a severe illness with a form of typical
typhoidal disease that is lethal (4). Each year, S. Typhi causes typhoid fever in about 20
million individuals, resulting in 200,000 deaths (5). Enteric fever (TF) is a kind of typhoid
fever. Salmonella enteric server typhi is a gram-negative bacterium that causes a unique acute
multisystemic febrile illness. Humans are the only ones who get typhoid fever. 3-4 weeks of
persistent fever, relative bradycardia, substantial constitutional symptoms, and lymphoid
tissue involvement identify it (6). Humans are the only natural hosts and reservoirs of
infection for Salmonella typhi, and the disease is typically related with poor socioeconomic
level and excessive littering (7). With about 21.7 million cases reported each year and an
estimated 217000 deaths, it is considered as a major source of global morbidity (8). Nearly 78
percent of all deaths and cases occur in Asia. The attack rate in third-world countries has been
estimated to be as high as 1000 instances per 100,000 people (9). In developing nations, it is
still a major public health concern. Despite the fact that typhoid fever is regarded to be low
risk in such young children, young children under the age of five face a disproportionate
burden of S. Typhi infection in endemic areas such as India and Bangladesh (10)..In actuality,
the immune response and clinical features of S. Typhi bacteria-infected young infants must be
poorly defined. Infection with multi-drug resistance (MDR: resistant to trimethoprim,
ampicillin, chloramphenicol, and sulfamethoxazole) bacteria has also been suggested. Despite
the lack of precise data on MDR S. Typhi infection in young children, S. Typhi infection can
cause serious clinical problems and consequences (11). Typhoid vaccination was
recommended by the Advisory Committee on Immune Diseases in 1994..People who have
had close contact with a carrier, microbiologists who work with S.Typhi on a regular basis
and travellers were all advised immunizations. When visiting countries where possibly

4
 contaminated drinks and foods have been eaten for an extended period of time, a high risk of
exposure is usually recommended. Between 1994 and 1999, there were three typhoid vaccines
available in the United States: an oral (1)live-attenuated vaccine (Ty21a; Vivotif Berna); a (2)
parental heat-inactivated vaccine (Typhoid vaccine; Wyeth-Lederle); and, after December
1994, a (3) parental capsular polysaccharide vaccine (Typhim Vi; Aventis Pasteur SA) (12).
Typhoid vaccine is approved for children aged six months and up; Typhim Vi is approved for
children aged two to six; and oral Ty21a is approved for children aged six to six.

2. PROBLEM STATEMENT:
OBJECTIVES OF RESEARRCH:

The main objective of the study is mentioned below:

 To Isolation and identification of Salmonella typhi.

 To determine the efficacy of different detergents against the isolated microbial strains.
 To check the antibiotic susceptibility of isolated strains.
3. RESEARCH DESIGN AND METHODS:

3.1 Study area

The goal of this study was to see how antibiotic resistance differed between typhoid-
vaccinated and non-vaccinated patients in the Nowshera district.
The goal of this typhoid study is to determine the incidence and pattern of antibiotic
resistance in salmonella typhi and salmonella p-typhi isolated from adults and children at
medical centres in Nowshera district.

3.2 Sample population:

About 60 samples of different categories (garments, bed covers, footwear etc.) will be taken
from each flea market, to check the variety of microbial strains in different stuff.

3.3 Study Design:

The goal of this study was to see how antibiotic resistance differed between typhoid-
vaccinated and non-vaccinated patients in the Nowshera district.

5
The goal of this typhoid study is to determine the incidence and pattern of antibiotic resistance
in salmonella typhi and salmonella p-typhi isolated from adults and children at medical
centres in Nowshera district.
3.4 Study Area:
The present research will take place in the Nowshera district of Pakistan's Khyber
Pakhtunkhwa province. Blood samples will be taken from several locations throughout the
Nowshera district, although the primary lab facility will be located at Shifa International
Hospital Islamabad in Islamabad.
The goal of this study was to see how antibiotic resistance differed between typhoid-
vaccinated and non-vaccinated patients in the Nowshera district.
3.5 Study Setting and Sample Population:
In Tertiary Care Hospitals, District Head Quarter Hospitals, Qazi Hussain Ahmad
Medical Complex, Private Hospitals, and Nowshera, Khyber Pakhtunkhwa (KPK) Pakistan,
this study will be undertaken. A total of 200 blood/urine samples will be taken from patients,
outpatients, from the admitted in intensive care unit, and wards at the hospital. Shifa Labs'
facility will also be utilized.
3.6 Inclusion criteria:
Adult male and female patients with typhoid symptoms and a history of the disease will be
included in this study.
3.7 Data Collecting Of Typhoid Patients:
From confirmed typhoid fever patients, a complete history will be taken, including bio
demographical information, immunization history, previous typhoid fever and treatment
history, duration of fever, and so on.
3.8 Collection of Samples:
Sterile culture villas will be provided for the suspected samples (aerobic and anaerobic).
Antibiotics will not be utilized, and the patient will be informed of this 72 hours before the
sample collection.This research study will employ a total of 200 blood samples to culture
Salmonella typhi species. The following is the sample collection procedure:

3.9 Blood Specimen Collection:

6
The award should inform the patient that antibiotics should not be used for 72 hours after the
sample is taken. The region around the vein puncture will be cleansed with 70% alcohol and
allowed to dry naturally. The needle was destroyed after collecting the requisite amount of
blood in an aseptic manner.
a. Blood will be delivered into the lab via blood culture bottles when the seals on culture
bottles are removed.
b. BD BACTEC aerobic and anaerobic culture vials should be used for culture and
sensitivity.
c. The culture bottles shall all be correctly labeled.
d. The needle and syringe will be disposed of in a sharp container correctly.
e. Finally, the samples will be sent to the Microbiology lab for additional examination.
f. Blood samples will be sent as quickly as possible to the microbiology department and
deposited in an incubator for 24 hours at 37 degrees Celsius. Samples shall be inoculated
in BHI broth and incubated at 37 °C for 24 to 48 hours after incubation.

4. Materials and Methods:

4.1 Material:
This project used a variety of substrates for growing salmonella species, as listed below:
4.2 Cultures of the Media:
Five distinct types of culture media were employed to isolate and cultivate particular
Salmonella typhi bacteria. The agars are arranged as follows: Mueller Hinton agar, Brain
Heart Fusion agar, MacConkey agar, Salmonella Shigella agar All of the media were prepared
according to the manufacturer's directions; here are some additional details:
4.3 Broth for Brain and Heart Infusion:
BHI broth is a non-selective, enhanced media used to cultivate a wide range of fastidious
bacteria with complex nutritional needs. According to the company's criteria, the media was
generated.
4.4. MacConkey Agar:
It's a selective and differential media for gram-negative bacteria. On MacConkey agar
plates, lactose fermenting colonies appear pink, while non-lactose fermenting colonies look
yellow or straw colored. Because of its composition, this choice was made.
4.5 Salmonella Shigella Agar:

7
 Salmonella Shigella (SS) Agar is a medium for isolating, growing, and differentiating
Salmonella spp. and some Shigella spp. strains that is moderately selective and differential.
Desoxycholate Citrate Agar is a variation of SS Agar.
5.5 Mueller-Hinton Agar:
For sensitivity testing, Muller Hinton agar is often employed. To manufacture the MHA
plates, autoclaved MHA was placed into plates under sterile conditions. To assure sterility, the
plates were incubated. The MHA composition is shown in Table 3.4.3.5.7. Preparation and
storage of media
All The media will be prepared as directed by the manufacturer and autoclaved at 121°C
for 15 minutes. After autoclaving, the media will be chilled to about 50°C and placed into
sterile plates and tubes (in the case of broth) in a totally sterile environment to avoid
contamination. At the conclusion, the plates will be stored at 4°C.
5.6. Culturing Methodologies:
BHI broth will be left outside to cool to room temperature at first. After that, the blood
will be blended in. Blood containing BHI broth will be sent to the hospital's microbiology lab
right away. This reduced bacterial overgrowth and increased the likelihood of growth
positivity for Salmonella typhi. In a lab incubator, BHI broth was incubated for 24 hours at 37
°C.
5.6 Culturing in Subcultures:
After that, the bacteria were sub cultured in MacConkey agar and salmonella shigella
agar, a specific media for S.typhi. After an overnight incubation at 37 °C, the pale smooth,
non-lactose fermenting colonies were sought. If S.typhi was discovered, a series of tests were
carried out to establish its existence. Antibiotic sensitivity was examined in Muller-Hinton
agar using the Kirby-Baur technique after growth was confirmed.
5.7. Identification of Bacteria:
Gram's Staining for Morphological Identification:
Gram's staining is a technique for identifying bacteria based on their capacity to absorb
particular dyes according to their cell membrane makeup. Bacteria can be classified into two
groups using this method: gram positive and gram negative.
5.8. Procedure for Gram Staining:

8
 After a drop was placed on a clean slide, a single colony was emulsified in distilled
water. The slide had been heated and was in good working order. The slide was drenched with
crystal violet solution for one minute before being washed with tap water. After one minute,
the slide was treated with iodine and washed. Before being gently cleaned with tap water, the
smear was decolorized for 10 seconds with 95 percent ethyl alcohol. The counter stain
safranin was applied and rinsed with tap water after 1 minute. After air drying, the slide was
viewed using a 100X objective.
5.9. Test for Catalase:
A bacterium's ability to convert hydrogen peroxide into water and oxygen is measured
using the catalase test. The bacterial test organism is emulsified in a 3 percent H2O2 solution
in a clean test tube. The appearance of bubbles denotes a favorable outcome. Catalase is found
in the Staphylococcus and Micrococcus genera, but not in the Enterococcus and Streptococcus
genera.
5.9.1 Procedure:
1. A drop of catalase reagent was placed on clean slides.
2. From the MacConkey culture plates, selected and combined a pure colony.
5.10 Tests for Citrates:
Simmon's Citrate test uses sodium citrate as the single carbon source and ammonia as the
sole nitrogen source in the medium to differentiate Enterobacteriaceae members.
5.10 .1 Procedures:
1. A sterile loop was used to streak a bacterial colony on the medium.
2. The tubes were incubated for 24 hours at 37°C.

5.11 Test of Widal:

Donor sera will be screened by slide agglutination with Salmonella enterica serotype
Typhi O and H antigens (Difco). Positive donor sera and sera from all patients will be serially
diluted from 1/50 to 1/6,400 in tubes containing 08.5% NaCl, and antigens (H and O) will be
added. Before being inspected for agglutination with an agglutinoscope overnight, all tubes
will be incubated at 37°C for 2 hours at room temperature before being incubated at 37°C for
2 hours at room temperature. Widal retesting will be done when the patient is asymptomatic.
Serology (follow-up) is sometimes required to acquire accurate findings, thus testing will be
performed in both vaccinated and non-vaccinated patients after 7 to 10 days. (13)

9
5.12 Disc Diffusion Method for Antimicrobial Susceptibility Testing:
Antibiotic sensitivity testing was performed using the Kirby-Bauer disc diffusion method
after bacterial isolates were identified. To begin, pure culture was chosen and emulsified in
sterile water, and turbidity was tested to the 0.5 MacFarland standards and corrected
accordingly. A bacterial grass was created on an MHA plate. A positive and negative control
run was also performed. After that, the antibiotic discs were applied aseptically and incubated
for 24 hours at 37 °C. The zone(s) were measured with a ruler the next day and classified as
sensitive, moderate, or resistant, with the results interpreted according to the Clinical and
Laboratory Standard Institute's guidelines (CLSI, 2019).
5.13 Susceptibility Testing for Antibiotics:
In cases of resistance, antibiotic susceptibility tests will be performed so that effective
antibiotics can be administered to treat S-Typhi and S-Paratyphi.S-Typhi and S-Paratyphi:
Isolation and Identification in the hospital-based cross-sectional study is being conducted.
5.14 Isolation and Identification Of S-Typhi And S-Paratyphi:
This is hospital base cross-sectional study is performed in Microbiology research
laboratory in Shifa International Hospital Islamabad and in Qazi Husain Ahmad Medical
Complex Nowshera. Samples should be collected from outpatient department and in patient
department of the Nowshera hospitals having clinical symptoms of typhoid of both sexes male
and female and in different age group. Blood samples are to be collected from different area
of district Nowshera during specific time period .this process is being done by manual method
minimum blood 5-10 ml blood are required from adults and 1-4 ml blood are required from
peeds in strial blood culture vils after the this blood are incubated for 24 hrs if growth occur
then antibiotic tests are doing for subscubality of drugs.
5.15 Antibiotic Subscibality Of Salmonella Isolated:
Ager plates inoculated with 0.5 MC Farland standers of the isolates antibiotic disks are to
be placed in incubated at 37c for 24 hrs if not diagnose bacteria during this period then it
should be incubated as long for 5 days . Zone of inhibition in diameters, measured and
interpret according to the guidelines of the clinical laboratory standard institute resistance
isolates to at lest one member of three different antimicrobial groups is considered as multi
drug resistance .the antibiotic contrimoxazole among S.Typhi population in the same study.

10
 For this resin antimicrobial susceptibility tests is essential to see changing trend of
antibiogram of circulating strains in community. Therefore we performed the present study to
investigate the resistance of S.Typhi and S.Paratyphi samples to commonly used antibiotic in
different hospitals in district Nowshera and also to determine the prevalence of typhoid fever
among different age group and sexes (14)
TIME FRAME:

S.N Du
O Activity ration
1
Work plan and sample collection 2 Months

2
Lab work 6 Months
3
Data interpretation and thesis writing 3 Months

4. FLOW CHART DIAGRAM

Isolation of Bacteria
Samples collection from
culturing
different flea markets

Samples washing with

Gram staining
Detergentss

CFU after washing

Identification
samples with detergents

Antibiotic suseptibility
Biochemical Tests
Tests

Results interpretation

11
5. EXPECTED OUTCOMES:
The importance of the current study is to evaluate the occurrences of typhoid fever in district
Nowshera. These contextual data provide important information about typhoid fever to the social
,economic and also about the physical cost of illness and vaccine demonstration projects to
ensure that household and community experiences are an integral part of future
policies ,treatment and prevention programs .

6. REFERENCES:
1. Kidgell, C., Reichard, U., Wain, J., Linz, B., Torpdahl, M., Dougan, G., & Achtman, M.
(2002). Salmonella typhi, the causative agent of typhoid fever, is approximately 50,000
years old. Infection, Genetics and Evolution, 2(1), 39-45.
2. Merrell, D. S., & Falkow, S. (2004). Frontal and stealth attack strategies in microbial
pathogenesis. Nature, 430(6996), 250-256.
3. Bhan, M. K., Bahl, R., & Bhatnagar, S. (2005). Typhoid and paratyphoid fever. The
Lancet, 366(9487), 749-762.
4. Kumar, A., Walia, B. S., & Mohan, J. (2006). Compressive strength of fiber reinforced
highly compressible clay. Construction and building materials, 20(10), 1063-1068.
5. Crump, J. A., Luby, S. P., & Mintz, E. D. (2004). The global burden of typhoid
fever. Bulletin of the World Health Organization, 82, 346-353.
6. Kalra, E. K. (2003). Nutraceutical-definition and introduction. Aaps Pharmsci, 5(3), 27-
28.
7. Mweu, E., & English, M. (2008). Typhoid fever in children in Africa. Tropical Medicine
& International Health, 13(4), 532-540.
8. Wasfy, M. O., Oyofo, B. A., David, J. C., Ismail, T. F., El-Gendy, A. M., Mohran, Z.
S., ... & Peruski Jr, L. F. (2000). Isolation and antibiotic susceptibility of Salmonella,
Shigella, and Campylobacter from acute enteric infections in Egypt. Journal of Health,
Population and Nutrition, 33-38.
9. Ivanoff, B., Levine, M. M., & Lambert, P. H. (1994). Vaccination against typhoid fever:
present status. Bulletin of the World Health Organization, 72(6), 957.
10. Naheed, A., Ram, P. K., Brooks, W. A., Hossain, M. A., Parsons, M. B., Talukder, K. A.,
... & Breiman, R. F. (2010). Burden of typhoid and paratyphoid fever in a densely

12
 populated urban community, Dhaka, Bangladesh. International Journal of Infectious
Diseases, 14, e93-e99.
11. oul, O., & Isman, M. B. (1991). Effects of azadirachtin on the dietary utilization and
development of the variegated cutworm Peridroma saucia. Journal of Insect
Physiology, 37(8), 591-598.
12. Centers for Disease Control (US), Centers for Disease Control, & Prevention (US).
(2004). Morbidity and mortality weekly report: MMWR. US Department of Health,
Education, and Welfare, Public Health Service, Center for Disease Control.
13. Parry, C. M., Thuy, C. T., Dongol, S., Karkey, A., Vinh, H., Chinh, N. T., ... & Baker, S.
(2010). Suitable disk antimicrobial susceptibility breakpoints defining Salmonella
enterica serovar Typhi isolates with reduced susceptibility to
fluoroquinolones. Antimicrobial agents and chemotherapy, 54(12), 5201-5208.
14. Troeger, C. E., Blacker, B. F., Khalil, I. A., Zimsen, S. R., Albertson, S. B., Abate, D., ...
& Rai, R. K. (2019). Mortality, morbidity, and hospitalisations due to influenza lower
respiratory tract infections, 2017: an analysis for the Global Burden of Disease Study
2017. The Lancet Respiratory Medicine, 7(1), 69-89.

13