World Journal of Pharmaceutical Research Danish et al ...

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Danish et al. World Journal of Pharmaceutical Research

AGE AND GENDER WISE DISTRIBUTION PATTERN OF TYPHOID

CAUSING BACTERIA SALMONELLA SEROVARS IN

MAHAKAUSHAL REGION

*Mohammad Danish Siddiquie and Ravi Prakash Mishra

Department of Post Graduate Studies and research in Biological Sciences Rani Durgavati

University Jabalpur, India.

ABSTRACT

The present study was undertaken to determine the diversity and

occurrence of serovars of genus Salmonella, in the clinical samples of

typhoid positive patients from different geographical area in and

around Mahakaushal region of Madhya Pradesh. In this study, the

isolation of Salmonella from clinical samples taken from various

districts indicates the wide spread distribution of Salmonella in

different age groups at Mahakaushal regions. The occurrence of

Salmonella was not confined to a particular location as Salmonella was

recovered from all areas sampled. Salmonella is widespread and many

of the serotypes can infect animals and humans. Infection may cause

pathological conditions resulting in morbidity and mortality. The

detection of Salmonella in food and water is therefore important in the prevention of such

infections. Investigation was aimed to explore the diversity of Salmonella Serovars by means

of cultural and biochemical characteristics.

KEY WORDS: Salmonella serovars, Mahakaushal region, typhoid.

INTRODUCTION

Microbial diseases constitute a major cause of death in many parts of the world particularly in

developing countries. The new organisms emerge from the dynamic interaction between the

classical epidemiological triad of agent, host and the environment. A large number of factors

are thought to be playing a role in this, with varying degrees of contribution in emergence of

each infection. Somolinski et al., 2003. Infections by Salmonella enterica are a significant

World Journal of Pharmaceutical Research SJIF Impact Factor 5.045

Volume 3, Issue 4, 1183-1203. Research Article ISSN 2277 – 7105

Article Received on

15 April 2014,

Revised on 10 May 2014,

Accepted on 03 June 2014

*Author for Correspondence

M. Danish Siddiquie

Department of Post Graduate

Studies and Research in

Biological Sciences Rani

Durgavati University Jabalpur,

Jabalpur, Madhya Pradesh,

India.


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public health concern around the world. Salmonella infections are the second leading cause of

bacterial food-borne illness in the United States and Europe (EFSA, 2009 and McNabb et al.,

2008). An estimated 95% of these Salmonellosis cases are associated with the consumption

of contaminated food products (Mead et al., 1999). The impact of Salmonella infections on

the economy in the United States has been estimated at approximately $3.6 billion due to loss

of work, medical care and loss of life (Frenzen, et al., 1999). Based on such economic impact

and statistics there is a worldwide interest in lowering Salmonella infections. Within the last

decade a number of various phenotypic and genotypic methods have been developed to

distinguish Salmonella from each other to understand their epidemiology, pathogenicity,

resistance and spread in animals, humans and their environment.

Typhoid fever is caused by Salmonella enterica serovar typhi (S. typhi), a gram negative

bacterium (Altekruse et al., 1998; Eleyr 1996; Miller et al., 1998; Gomez et al., 1997). It

continues to be a global public health problem with over 21.6 million cases and at least

250,000 deaths occurring annually (Khakhriar et al., 1983; Lax et al 1995; Tletjen, 1995).

Almost 80% of the cases and deaths are in Asia; the rest occur mainly in Africa and Latin

America (D'aoust, 1991).

MATERIAL AND METHOD

The present study was conducted during the period from August 2008 to December 2012 at

the Department of Post-Graduate Studies and Research in Biological Sciences Rani

Durgavati University, Jabalpur, M.P. The stool and urine samples of typhoid positive patients

of different hospitals in and around Mahakaushal region viz., Jabalpur, Katni, Mandla,

Dindori, Narsinghpur, Kareli, Katangi, Sehpura Bhitoni, Damoh, Sehora, Sagar, Satna, Rewa

were investigated.

The isolation of Salmonella was performed following the standard laboratory methods (ISO,

2002; Iwade et al., 2006).

Biochemical Examination

Different media and reagent were used for the biochemical identification of Salmonellae in

addition to this HiSalmonella kit KB011 (Span Diagnostics Ltd. India) was also used as a

cross reference test, this kit contains Methyl red, Voges Prosuker, Urease, H2S production,

Citrate, Lysine, ONPG, Lactose, Arabinose, Maltose, Sorbitol and Dulcitol.


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I) Triple Sugar Iron Agar (TSI) Test

This test was performed to determine the ability of an organism to attack a specific

carbohydrate incorporated in a basal growth medium, with or without the production of gas,

along with the determination of possible hydrogen sulphide (H2S) production. The TSI agar

slant (long butt and short slant) containing three types of sugars (Dextrose, Lactose and

Sucrose) was stabbed, streaked with inoculums (18-24 hours old) and incubated at 35 ± 20C

for 18 hours.

a) Acid production: Fermentation of glucose was indicated by red color of slant surface and

yellow color of butt. Fermentation of glucose, lactose and sucrose was indicated by yellow

color of slant surface and butt. Fermentation of neither glucose nor sucrose was indicated

by red color of slant and butt.

b) Gas production: Splitting of medium or displacement of medium from the bottom of the

tube constituted a positive test for the gas production.

c) H2S production: Blackening of the medium indicated a positive test while no change in the

color of the medium constituted a negative test.

II) SIM Agar Test

The ingredients in SIM Medium enable the determination of three activities by which enteric

bacteria can be differentiated. Sodium thiosulfate and ferrous ammonium sulfate are

indicators of hydrogen sulfide production. The ferrous ammonium sulfate reacts with H2S gas

to produce ferrous sulfide, a black precipitate. The casein peptone is rich in tryptophan,

which is attacked by certain microorganisms, resulting in the production of indole. The indole

is detected by the addition of kovac’s reagents following the incubation period. Motility

detection is possible due to the semisolid nature of the medium. Growth radiating out from

the central stab line indicates that the test organism is motile. A pure culture of the

microorganism was introduced into the butt till the bottom by punchring. The tubes were

incubated for 18-24 hours at 370C aerobically.

Motility was indicated by diffuse growth outward from the stab line or turbidity throughout

the medium. H2S production was shown by blackening of the medium. The medium was

covered with a layer of Kovac’s indole reagent. Production of indole caused the reagent layer

to become purple in color and indole negative organism caused the black color to disappear.


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III) Urease Test

Christensen’s medium was used to test the urease activity of the isolates. The hydrolysis of

urea releases ammonia which increases the pH of the medium that changes the color of

phenol red (pH indicator) from red to pinkish red. The basal medium was sterilized by

autoclaving at 1210C for 15 minutes. When it cool to about 50

0C, a sterile solution of glucose

was added to give final concentration of 0.1% after that 100 ml of a 20% solution of urea

previously sterilized by filtration was added. The medium was poured in test tubes as deep

slopes. It may, however, be used in fluid form without agar. The isolates giving acidic butt

and alkaline slant with H2S and gas production were inoculated into the urea broth to

determine urease production. The inoculated tubes were incubated at 370C for 96 hours. The

observations were made at an interval of 4, 24, 48 and 96 hours. Urease positive cultures

changed the colour of the indicator to red. The isolates showing negative reaction were used

for biochemical typing (Jaffer, 1984).

IV) Methyl Red Test

Some bacteria ferment glucose and produce sufficient amount of acid as end products.

Peptone and phosphate was dissolved in distilled water, pH adjusted to 7.6 filtered, dispensed

in 5ml amounts and sterilized at 1210C for 15 minutes. After that 0.25 ml of sterile glucose

solution was added to each tube to make a final concentration of 0.5% glucose. The test was

performed as follows.

The glucose phosphate peptone water tubes were inoculated with the young culture. Then, the

inoculated and non inoculated controls were incubated at 37oC for 96 hours. At the end of 96

hours of incubation, 5 drops of methyl red reagent was added to each tube and the results

were recorded. Positive result was indicated by bright red color and negative by yellow color.

V) Voges Proskaur Test

Some bacteria produce acetonin and 2, 3 butanediol from glucose fermentation under alkaline

condition this compound oxidize to diacetyl which gives pink color with creatine. Under

alkaline condition this compound oxidizes to diacetyl, which give pink colour with creatine.

The cultures were inoculated into tubes containing 5ml of glucose phosphate peptone wate,

incubated at 370C for 48 hours and to this broth about 0.5 ml Omera’s reagent was added.

The tubes were placed at 370C in water bath for 4 hours and the observation was recorded. A

pink colour indicated a positive test and absence of any colour indicated negative reaction.


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VI) Malonate Test

This test was performed to determine an organism’s ability to utilize sodium malonate as the

sole source of carbon with resulting alkalinity. Malonate broth was prepared and autoclaved

at 1210C for 15 minutes, inoculated with a loopful culture of test organism, and incubated at

37 ± 20C for 48 hours. Positive test was indicated by the development of light blue to deep

prussian blue color throughout the medium while no change in the color indicated a negative

test.

VII) Citrate Utilization Test

Ability to utilize citrate as a sole source of carbon and energy distinguishes certain Gram-

negative organisms. Simmon’s citrate agar contains citrate as its only carbon and energy

source. Growth on this medium is a positive test for citrate utilization. Certain organisms that

give a positive test increase the pH of the agar, changing bromothymol blue indicator in the

medium from green to blue. The tubes containing inoculum in Simmon’s citrate medium

were incubated at 370C for three days. Opacity and change in colour of bromothymol

indicated a positive reaction i.e., from pale green to blue.

VIII) Nitrate Reduction Test

Young culture was inoculated in 5 ml of nitrate broth and incubated at 370C for 96 hours. 0.1

ml of the test reagent was added and results were recorded. Development of pink, red or

maroon colour was indicative of positive test. If no color develops it was considered as

negative.

IX) Lysine Decarboxylase Test

Decarboxylation of an amino acid is splitting off, of its carboxyl group to yield an amine and

CO2. Bacterial decarboxylation can be demonstrated by showing the disappearance of the

amino acid or by formation of amine carbon dioxide. The reaction resulted into accumulation

of an amine, which is basic. Therefore, decarboxylation can be demonstrated by measuring

the rise in pH. All the components of lysine decarboxylase broth were dissolved, adjusted to

pH 6.0, and distributed in four portions and dissolved the amino acid lysine at the rate of 1%.

The medium was distributed into 1ml amount and overlayed with 5mm thickness of liquid

paraffin and autoclaved at 1210C for 15 minutes. Lysine decarboxylase broth was inoculated

with the loopful culture of the test organism and one was kept uninoculated as control. Broth

tubes were incubated for 24 hours at 370C. A change in color of the tube containing the


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amino acid to bluish purple indicated positive test while no change in the color of the medium

indicated a negative test.

X) Gelatin Liquefaction Test

Using an inoculums (18-24 hours old culture), the tubes of nutrient gelatin were stabbed with

an inoculating needle directly down the center of the medium to a depth of approximately one

and and a half inch from the bottom of the tube. Tubes were incubated, including an

uninoculated control, at room temperature (about 200C) for 24-48 hours and up to 14 days. At

various intervals during the incubation process, the tubes were examined for growth

(turbidity) and liquefaction. Uninoculated control tubes were used for comparison. At each

interval, the tubes were transferred to a refrigerator or ice bath for a sufficient time period to

determine whether liquefaction occurred or not. It is important that the tubes should not be

shaken during the transfer from incubator to refrigerator. At the time of reading results, the

chilled tubes were inverted to test for solidification or liquefaction. If refrigerated gelatin

remains liquid within 7 days of incubation it indicates a Positive result and if refrigerated

gelatin is solid after 7 days of incubation it indicates negative result.

XI) Carbohydrate Utilization Test

This test was performed to determine the capability of an organism to ferment (degrade) a

specific carbohydrate incorporated in a basal medium producing acid or acid with visible gas.

Phenol red broth base was prepared (pH 7.4) and autoclaved at 1210C for 15 minutes and

inoculated from an overnight grown culture and incubated at 35 ± 20C for 24 hours. Sterile

glucose, arabinose, dulcitol, mannitol, sorbitol, maltose, trehalose, xylose, mellibiose each

were added at concentration of 1% and salicin at concentration of 0.5%. After 48 hours of

incubation at 35 ± 20C, change in color of broth from reddish orange to yellow indicated a

positive test while formation of reddish pink color indicated a negative test.

XII) ONPG Test

This test demonstrates the presence or absence of the enzyme β-galactosidase in bacterial

species on the basis of their availability to utilize o-nitrophenyl-β-d-galactopyranoside

(ONPG). The broth base was prepared and autoclaved at 1210C for 15 minutes. A loopful of

inoculums was added into 2.5 ml of sterile ONPG-peptone broth and incubated at 370C for 24

hours. After incubation change in the color of the broth from colorless, to yellow indicated a

positive test while no change in color after 24 hours, indicated a negative test.


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XIII) Oxidase Test

The Oxidase reaction is due to the presence of cytochrome oxidase system. Oxidase catalyzes

the removal of hydrogen from substrate, but uses only oxygen as a hydrogen acceptor. The

various reagent dyes used in oxidase test are artificial electron acceptors, these artificial

substrate are either colorless or colored depending upon the state they exhibit, the final

oxidase reaction shows a colored product. A small piece of filter paper was soaked in 1%

aqueous solution of tetramethyl-p-phenylene-diamine dihydrochloride and placed on a Petri

dish. A small portion of growth was smeared on the filter paper with the help of a sterile

toothpick. Appearance of blue color within 1-2 minutes indicated a positive test while no

change in the color indicated a negative test.

XIV) Potassium Cyanide Test

The basic principle behind this test is to determine the organism’s ability to live and

reproduce in presence of potassium cyanide. In this test, potassium cyanide (KCN) broth base

(pH 7.6) was prepared and autoclaved at 1210C for 15 minutes. The medium was allowed to

cool and the aqueous KCN solution (0.5%) was added to the broth base, inoculated with an

overnight grown culture and incubated at 35 ± 20C for 24 hours. An uninoculated tube

without KCN served as control. Growth of the organisms in KCN broth base indicated a

positive test.

RESULT AND DISCUSSION

A total of 146 clinical samples (stool and urine) from typhoid positive patients were collected

from different hospitals located in and around Mahakaushal region viz. Jabalpur, Katni,

Mandla, Narsinghpur, Sehora, Shehpura Bhitoni, Kareli, Dindori, Balaghat, Damoh, Katangi,

Sagar, Satna and Rewa and analyzed for the presence of Salmonella species as shown in

Table 4.1A, 4.2A and Figure 4.1A. The clinical symptoms in these patients included

abdominal pain fever, diarrhea, vomiting, chills, headache, joint pain, nausea and blood in

stool. Investigation of food consumed by gastroenteritis patients during the period of study

revealed that unhygienic food and contaminated water were the major cause of disease

among peoples. The major unhygienic food included poultry eggs, poultry meat, bakery

products and contaminated milk. Poor household cooking conditions are also lead to

Salmonella infections. It was also analyzed that some unknown causes were also responsible

for typhoid. Out of 146 samples collected, 103 samples (stool and urine) showed successful

isolation of Salmonellae after primary screening (Table 4.3). Cultural characteristics of the


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isolates were recorded after the incubation of 24-48 hours. The colonies which showed

typical Salmonella characterstics (Table 4.4A and Plates I, II and III) were selected for

confirmation by biotyping and the colonies showing any other color pattern were discarded.

All the samples were pre-enriched into trypticase soy broth and enriched into the

Tetrathionate broth. The samples enriched in tetrathionate broth showed turbidity in

maximum samples. These turbid tubes was streaked on bile salt brilliant green starch agar

plates and incubated for 24 to 48 hours at 370C. After incubation green color colonies were

selected as Salmonella colonies. The colonies of possible Salmonella isolates were streaked

on Salmonella-Shigella agar (SSA) plates and incubated at 370C for 24 hours. Smooth, small

and colorless with dark center colonies were confirmed to be Salmonella. The isolates from

Salmonella-Shigella agar were further streaked on MacConkey agar to ensure their purity

from other Enterobacteria.

All isolates produced circular, 1-3 mm in diameter and colorless, lactose non-fermenting

colonies. The isolates were further streaked on blood agar, where Salmonella colonies

appeared 2-3 mm in diameter, grey-white, and non-hemolytic. In nutrient agar Salmonella

isolates produced small, discrete, smooth and colorless colonies. Where as on xylose-lysine-

deoxycholate (XLD) agar the colonies appeared as smooth red with black centered. For the

isolation of Salmonellae from different sources, bile salt brilliant green agar, Salmonella-

Shigella agar (SSA), macconkey agar and xylose-lysine-deoxycholate (XLD) agar were used,

on these media morphologically distinct colonies of bacteria were produced, which would be

picked up easily. Therefore, samples were firstly cultured in tetrathionate broth, (which

enrich small number of Salmonella in contaminated samples) and were incubated for 24

hours, before plating on solid media.

Gram Staining

Gram staining of the 103 isolates indicates that bacterial cells of all the isolates appeared

pinkish rods of varying size occur singly or in short chains which indicate the gram negative

nature of the organism.

ii) Biochemical Test

The characterization of 103 isolates of Salmonella isolates were carried out by using

biochemical test resulted total of two species viz. Salmonella enterica and Salmonella

bongori and 10 subspecies/serovars viz. S. arizonae, S. diarizonae, S. houtenae, S. salmae, S.


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indica, S. typhi, S. enteritidis, S. typhimurium, S. virchow and S. bongori as presented in

(Table 4.5 to 4.12 and Plates IV and V).

All biochemical tests were found to be consistently similar in all the isolates of particular

strains of Salmonella. The biochemical test confirmed Salmonella strains. Variation in

biochemical reaction has been reported to be very low in Salmonella at serovars level,

however, biochemical tests showed variation at Salmonella sub-species. The presence of

diverse biochemical patterns in Salmonella were observed for malonate, lactose and dulcitol

utilization. All isolates fermented glucose on triple sugar iron media by changing the color of

the butt from red to yellow and gas produced by these isolates broke the agar column. To

assess the typical fermentation characteristics by Salmonella, TSI agar test was performed,

which have the ability to describe the fermentation characteristics of three sugars (glucose,

lactose and sucrose). Some specific serovars of Salmonella was not able to ferment lactose

and sucrose and therefore produce: acidic butt, alkaline slant with gas production black butt

and accumulation of gas at the bottom.

The slant of Triple Sugar Iron (TSI), from which these strains were isolated, and were red,

suggested no fermentation of saccharose or lactose or both. Iwade et al. (2006) characterized

an outbreak derived Salmonella enteritidis strains with atypical triple sugar iron and

Simmons citrate reactions. Similarly, all Salmonella serovars blackened SIM medium

representing hydrogen sulfide production. The diffused turbidity around the punctured line

was also observed which inferring to motility of the isolated organisms. No change in color

of the medium was observed with disappearance of black color thereby conferring the

serovars to be Indole negative.

In the present study the isolates failed to change the color of urea broth and were

characterized as Urease negative. All the isolates were positive to methyl red, nitrate

reduction and citrate utilization whereas all the isolates were negative for urease, lysine

decarboxylation, gelatin liquefaction, indole and voges-proskaur. Acid and gas was produced

from glucose, mannitol, maltose, arabinose, xylose, dulcitole, Inositol and mannose. Either no

or variable reaction in lactose was observed. Urease activity, nitrate reduction, citrate

utilization, gelatin liquefaction, methyl red, voges-proskaur’s and indole production tests. All

the tests were found similar as compare to the chart provided by the manufacturer in Hi

Salmonella kit (KB O11 HiMedia) (Table 4.4 to 4.12).


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The prevalence of different species and serovars of Salmonella in typhoid positive patients of

the Mahakaushal region is presented in Table (4.14). The results of present study

revealed Salmonella enterica as the principal etiological agent occurring 98.1% of all the

cases analyzed, whereas S. bongori occurred only 1.94% of the total isolates. Only three

Salmonella serovars were targeted for study as they are the main focus of the present study

i.e., Salmonella enteritidis (35.9%), Salmonella typhimurium (24.3%) and Salmonella

virchow (3.9%). The other species, namely Salmonella typhi, S. salamae, S. bongori, S.

arizona, S. diarezona, S. houtenae, S. indica occurred 35.9% .

The results of present study revealed Salmonella enterica as the principal etiological agent

occurring 98.1% of all the cases analyzed, whereas S. bongori occurred only 1.94% of the

total isolates. Only three Salmonella serovars were targeted for study as they are the main

focus of the present study i.e., Salmonella enteritidis (35.9%), Salmonella typhimurium

(24.3%) and Salmonella virchow (3.9%). The other species, namely Salmonella typhi, S.

salamae, S. bongori, S. arizona, S. diarezona, S. houtenae, S. indica occurred 35.9% were

excluded from the molecular study.

Age-wise distribution pattern among targeted Salmonella serovars (Table 4.16 and Figure

4.16) i.e., S. enteritidis, S. typhimurium and S. virchow showed a greater prevalence amongst

infants, children and adults as compared to veteran counterparts. Among the Salmonella

positive patients 9.1% are in less than 5 years age group, 27.3% were among the 5-14 years

age group, another pattern was reported among the specific isolates as well, in that 24.2% of

the Salmonella cases were reported in age group of 15-24 years and 16.7% in adults aged

between 25-34 years, whereas only 3% were observed in 45-54 year age group and 10.6%

were observed in >55 years age group. The age wise distribution among all the serovars were

recorded separately and presented in (Table 4.15A and Figure 4.2A). As The highest risk of

infection in lower age may be attributed to poor immune response to infection, bad quality of

drinking water especially among school going children, with a continuous exposure to

contaminated ready-to-eat food items available in open-air cafeteria, especially during out-

door activities associated with extra-curricular school activities.

Sex-wise distribution pattern of the Salmonella positive cases showed marked differentiation;

in the present study the typhoid incidence was higher among females (72%) as compared to

males (28%), (Table 4.17A and 4.18A; Figure 4.17A and 4.18A). Most of the women,

positive for typhoid, were pregnant, which may help to explain the high incidence as during

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pregnancy a women’s immune response weakens and they become more susceptible to

infections.

The high incidence among young children also indicated to a marked susceptibility of young

children to Salmonellosis, coupled with lack of good hygiene practices and level of health

awareness both among the children and their parents. Another potential factor may be the

intimate association of children with pets, which act as carriers of Salmonellosis.

The reason of high incidence of typhoid in children, as reported in this study, lays credence to

the fact that there is a lack of knowledge on the practices of good personal hygiene,

correlating to unhygienic conditions dirty hands which spread infectious microbes

like Salmonella through the preferred fecal-oral route. Furthermore, the common practice of

consuming unwashed and partially cooked vegetables by these people enhanced the

possibility of spread of infection. This study revealed that fever was the most common

symptom followed by chills, abdominal pain, blood in stool, vomiting, diarrhea, headache

(Table 4.14).

The overall prevalence pattern of Salmonellosis, based on the locality of the incidence,

showed interesting results. In Mahakaushal region, the highest incidence of Salmonellosis

occurred among patients residing near slum areas surrounding a large waste water drain

running through the city. The second highest incidence was reported among patients living in

the surrounding rural areas, followed by those residing in town.

In present study, the incidence of Salmonella virchow was quite lower, as only a total of 4

cases of Salmonella virchow were reported (Table 4.14). However, the distribution pattern

highlighted the fact that most of the cases among Salmonella enteritidis (35.9%) and

Salmonella typhimurium (24.3%) were reported in patients residing in the poorer and more

congested sectors in and around Mahakaushal region.

Causes of typhoid in some areas of Mahakaushal regions may be associated with the densely

populated urban centers with adjoining suburbs, coupled with poverty and poor infrastructure

development, like sanitation and waste disposal, lack of quality education, inadequate

primary health care facilities, cumulatively resulting in the contamination of food product and

water. The lack of adequate health care facilities with affordable diagnostic laboratories and

proper counseling of parents on good hygiene practices has lead to high prevalence

of Salmonellosis in this region.


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Without doubt, like many infectious diseases in developing countries, Salmonellosis may also

be labeled as a disease of the poor and under privileged. Countries that have inadequate

infrastructure to support a growing population, coupled with poor waste water treatment

facilities and improper sanitation mechanisms or systems, are prone to gastrointestinal

infections, particularly those caused by enteric gram-negative bacilli. To combat such

endemic diseases it is essential to prioritize political commitments for sustained socio-

economic development, through adequate allocation of resources, quality education, properly

managed implementation of urban and rural planning and use of affordable, preferable

indigenous, cost-effective technologies to combat the plight of water contamination and

waste water treatment.

It has been reported that 13 million cases of Salmonellosis occur worldwide annually, of

which 70% reports comes from India, China and Pakistan (Bhat and Macadan, 1983). The

present study is the first attempt to observe prevalence and molecular diversity study of

Salmonella serovars in clinical samples at Mahakaushal region. Reports suggests that

contaminated water, contaminated or unhygienic food and food products, poultry and meat

products are also responsible for increased bacterial contamination, leading to human

Salmonellosis (Mead, 1999).

Table 4.3. Number of Samples Showing Successful Isolation of Salmonella.

Name of the City Total no. of Sample

Isolated

Type of samples

Stool Urine

Jabalpur 63 39 24

Narsinghpur 14 08 06

Mandla 04 02 02

Sagar 02 02 00

Satna 04 03 01

Rewa 02 01 01

Katni 01 01 00

Balaghat 04 03 01

Damoh 02 00 02

Katangi 01 00 01

Sehora 01 01 00

Shehpura Bhitoni 03 02 01

Kareli 01 01 00

Dindori 01 01 00

Total 103 64 39


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Table 4.17 Gender Wise Distribution of Typhoid Positive Patient in Reference to

Selected Serovars

Number of Patients Positive For

S. enteritidis

S. typhimurium

S. virchow

Total Male Female Total Male Female Total Male Female

37 09 28 25 11 14 04 03 01

Figure 4.17 Graphical Representation of Table 4.17

Table 4.18 Gender Wise Distribution of Other Salmonella Serovars

Salmonella

species/serovars Male Female Total

typhi 03 10 13

salamae 00 08 08

bongori 00 02 02

arezonae 01 03 04

diarezonae 00 01 01

hautanae 01 02 03

indica 01 05 06

TOTAL 06 31 37


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Figure 4.18 Graphical Representation of Table4.18A

Table 4.15 Age Wise Distribution of Salmonellosis among All Isolates

S/no Age Group Isolates

Number Percentage

1 <5 6 8.7

2 5-14 29 27.2

3 15-24 27 24.3

4 25-34 18 17.5

5 35-44 10 9.7

6 >55 13 12.6



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Table 4.16 Age Wise Distribution of Typhoid Positive Patients in Reference to Selected

Serovars.

Age

group(years)

Number of patients positive for Total patients

S.enteritidis S. typhimurium S. virchow Number Percentage

<5 02 01 03 06 9.1

5-14 07 10 01 18 27.3

15-24 11 05 00 16 24.2

25-34 09 02 00 11 16.7

35-44 02 04 00 06 9.1

45-54 00 02 00 02 3.0

>55 06 01 00 07 10.6



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Plate- I Salmonella on Nutrient Agar Media

Plate- II Salmonella on XLD


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Plate- III Salmonella on Different Medias

A

B C

D

1

5 2

4 3

A- 1 Brilliant Green Agar; 3 and 5- Blood Agar, 2 and 4-Salmonella Shigella

Agar plates, B-Large view of Blood Agar plate, C- Xylose Lysine Deoxycholate

Agar plates with control; D- Another view of plate D.


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Plate- IV Biochemical Identification on KB011 Hi-Media Kit


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PLATE - V Biochemical Identification on KB011 Hi-Media Kit

CONCLUSION

The prevalence of different species and serovars of Salmonella in typhoid positive patients of

the Mahakaushal region, revealed that Salmonella enterica is the principal etiological agent

and occurring 98.1% of all the cases analyzed, whereas S. bongori occurred only 1.9% of the

total isolates. Only three Salmonella serovars were targeted, as they are the main focus of the

present study viz., Salmonella enteritidis occurs (35.9%), Salmonella typhimurium occurs

(24.3%) and Salmonella virchow occurs (3.9%). The other subspecies/serovars,

namely Salmonella typhi, S. salamae, S. bongori, S. arizona, S. diarezona, S. houtenae, S.

indica in total occurred 35.9% were excluded from the molecular study.

Age-wise distribution pattern among targeted Salmonella serovars i.e., S. enteritidis, S.

typhimurium and S. virchow showed a greater prevalence amongst infants, children and adults

as compared to veteran counterparts. Therefore 9.1% of the Salmonella positive patients were

found in less than 5 years age group, 27.3% were among the 5-14 years age group, another


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pattern was reported among the specific isolates as well, in that 24.2% of

the Salmonella cases were reported in age group of 15-24 years and 16.7% in adults aged

between 25-34 years, whereas only 3% were observed in 45-54 year age group and 10.6%

were observed in >55 years age group. The highest risk of infection in lower age may be

attributed to poor immune response to infection, bad quality of drinking water especially

among school going children, with a continuous exposure to contaminated ready-to-eat food

items available in open-air cafeteria, especially during out-door activities associated with

extra-curricular school activities. Sex-wise distribution pattern of the Salmonella positive

cases showed marked differentiation; in the present study the typhoid incidence was higher

among females (72%) as compared to males (28%). Most of the women, positive for typhoid,

were pregnant, which may help to explain the high incidence as during pregnancy, a

women’s immune response weakens and they become more susceptible to infections.

ACKNOWLEDGEMENT

Authors are thankful to Head, Dept. of Post Graduate Studies and Research in Biological

Science, R.D. University, Jabalpur, 482001 (M.P.) for providing laboratory facilities.

REFERENCES

1. Altekruse SF, Swerdlow DL and Wells SJ (Factors in the emergence of food-borne

diseases) Vet. Clin. North Am. Food Anim. Pract, 1998; (14): l-15.

2. Bhat PR and Macaden (1983) Outbreak of gastroenteritis due to multidrug resistant

Salmonella typhimurium phage type 66/122/UT in Bangalore. Indian Journal of Medical

Research, 1983; (78): 329-349.

3. EFSA, (Trends and Sources of Zoonoses And Zoonotic Agents in the European Union)

The Community Summary Report on in 2007. The EFSA Journal 223. 2009.

4. Eleyr A (Infective bacterial food poisoning) In: Eley RA, Ed. Microbial food Poisoning.

Chamman and Hal l, 2-6 Boundary Row, London S E I., 1996; (8): 15-33.

5. Frenzen PD, Riggs TL, Buzby JC, Breuer T, Roberts T, Voetsch D and Reddy S

(Salmonella cost estimate updates using Food Net Data) Food. Rev, 1999; 22 (2): 10-15.

6. Gomez TM, Motarjemi Y, Miyagawa S, Kaferstein FK and Stohr K (Food bore

Salmonellosis) World Health Stat Q, 1997; (50): 81-89.

7. ISO 6579 Microbiology general guidance on methods for the detection of Salmonella.

(4th ed.) International organization for standardization, Geneva, Switzerland. 2006.



1203


8. Iwade Y, Tamura K, Yamauchi A, Kumazawa NH, Ito Y and Sugiyama A

(Characterization of the outbreak-derived Salmonella enterica serovars enteritidis strains

with atypical triple sugar iron and Simmon’s citrate Reactions) Japanese Journal of

Infectious Diseases, 2006; (59): 512-513.

9. Jaffer MA (Studies on isolation and serotyping of Salmonella from mesenteric lymph

nodes of cattle slaughtered at Lahore abattoirs) M.Sc. (Hons) Thesis, University of

Agriculture, Faisalabad. 1984.

10. Khakhria R, Woodward D, Johnson WM and Poppe C (Salmonella isolated from humans,

animals and other sources in Canada) Epidemiol. Infect., 1983; (119): 15–23.

11. Lax AX, Barrow PA, Jones PW and Wallis TS (Current perspectives in Salmonellosis)

Br. Vet., 1995; (1): 151: 351-377.

12. McNabb SJ, Jajosky RA, Hall-Baker PA, Adams DA, Sharp P, Worshams C, Anderson

WJ, Javier AJ, Jones GJ, Nitschke DA, Rey A and Wodajo MS (Summary of notifiable

diseasesUnited States 2006 MMWR) Morb. Mortal. Wkly. Rep, 2008; (55): 1-92.

13. Mead PS., Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM and Tauxe

RV (Food-related illness and death in the United States) Emerg. Infect. Dis, 1999; (5):

607-625.

14. Miller M and Page JC, (Other food borne infections). Vet. Clin. North Am. Food Anim.

Pract, 1998; (14): 71-89.

15. Smolinski M, Hamburg A and Leaderberg J, (Committee on emerging microbial threats

to health in the 21st century: microbial threats to health in the United States: emergence,

detection and response. National Academy Press, Washington DC. 2003.

16. Tletjen M and Fung D. Y (Salmonellae and food safety). Crit. Rev. Microbiol, 1995; (21):

53-83.

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