World Journal of Pharmaceutical Research
Danish et al.
World Journal of Pharmaceutical
Research
SJIF Impact Factor 5.045
Volume 3, Issue 4, 1183-1203.
Research Article
ISSN 2277 – 7105
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.
Article Received on
15 April 2014,
Revised on 10 May 2014,
Accepted on 03 June 2014
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
*Author for Correspondence
M. Danish Siddiquie
isolation of Salmonella from clinical samples taken from various
Department of Post Graduate
districts indicates the wide spread distribution of Salmonella in
Studies and Research in
different age groups at Mahakaushal regions. The occurrence of
Biological Sciences Rani
Salmonella was not confined to a particular location as Salmonella was
Durgavati University Jabalpur,
Jabalpur, Madhya Pradesh,
India.
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
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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 500C, 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 Gramnegative 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-lysinedeoxycholate (XLD) agar the colonies appeared as smooth red with black centered. For the
isolation of Salmonellae from different sources, bile salt brilliant green agar, SalmonellaShigella 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 outdoor 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 socioeconomic 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
Jabalpur
Narsinghpur
Mandla
Sagar
Satna
Rewa
Katni
Balaghat
Damoh
Katangi
Sehora
Shehpura Bhitoni
Kareli
Dindori
Total
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Total no. of Sample
Isolated
63
14
04
02
04
02
01
04
02
01
01
03
01
01
103
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Type of samples
Stool
Urine
39
24
08
06
02
02
02
00
03
01
01
01
01
00
03
01
00
02
00
01
01
00
02
01
01
00
01
00
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. typhimurium
S. enteritidis
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
typhi
salamae
bongori
arezonae
diarezonae
hautanae
indica
TOTAL
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Male
Female
Total
03
00
00
01
00
01
01
06
10
08
02
03
01
02
05
31
13
08
02
04
01
03
06
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
1
2
3
4
5
6
<5
5-14
15-24
25-34
35-44
>55
Isolates
Number
Percentage
6
8.7
29
27.2
27
24.3
18
17.5
10
9.7
13
12.6
Figure 4.15 Graphical Representation of Table 4.15
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Table 4.16 Age Wise Distribution of Typhoid Positive Patients in Reference to Selected
Serovars.
Age
group(years)
<5
5-14
15-24
25-34
35-44
45-54
>55
Number of patients positive for
S.enteritidis
02
07
11
09
02
00
06
S. typhimurium
01
10
05
02
04
02
01
S. virchow
03
01
00
00
00
00
00
Total patients
Number
06
18
16
11
06
02
07
Percentage
9.1
27.3
24.2
16.7
9.1
3.0
10.6
Figure 4.16 Graphical Representation of Table 4.16
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Plate- I Salmonella on Nutrient Agar Media
Plate- II Salmonella on XLD
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1
2
5
4
3
A
B
C
D
Plate- III Salmonella on Different Medias
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.
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