Advances in Bioresearch

Adv. Biores., Vol 15 (3) May 2024: 136-151 Advances

©2024 Society of Education, India
Print ISSN 0976-4585; Online ISSN 2277-1573 in
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CODEN: ABRDC3
DOI: 10.15515/abr.0976-4585.15.3.136151
Bioresearch
ORIGINAL ARTICLE

Prevalence and Characterization of Multidrug-Resistant Non-

Typhoidal Salmonella in Poultry: Implications for Public Health
and Food Safety
T. Sathiamoorthi1, HP Jai Shanker Pillai 2, K. Saranya Devi3, R. Rajalakshmi4, B. Rajalinga Malathi5,
R. Rajesh Kumar6,7*
1Department of Microbiology, Alagappa University, Karaikudi- 630003, Tamilnadu, India
2Department of Medical Laboratory Science, Komar University of Science and Technology, Sulaimaniyah,

Kurdistan Region, Iraq

3Department of Microbiology, Government Arts and Science College Kozhinjampara, Palakkad, Kerala,

India
4PG & Research Department of Botany, V. O. Chidambaram College, Thoothukudi, Affiliated to

Manonmaniam Sundaranar University, Tirunelveli - 627012, Tamilnadu, India

5Department of Microbiology, Kamaraj College, Thoothukudi, Affiliated to Manonmaniam Sundaranar

University, Tirunelveli - 627012, Tamilnadu, India

6Pondicherry Institute of Agricultural Sciences, Puducherry- 605 007, India
7All India Institute of Training and Education, New Delhi – 110 097, India

Corresponding author’s Email: Dr. R. Rajesh Kumar

Email: iamramrajesh@gmail.com

A BSTRACT
This study investigates the prevalence of multidrug-resistant non-typhoidal Salmonella species in poultry meat sourced
from Karaikudi, Tamil Nadu, India, aiming to address the escalating global issue of foodborne infections associated with
antibiotic-resistant pathogens. Through biochemical tests, Salmonella was isolated and identified from flesh, intestine,
liver, and spleen samples obtained from fresh retail chicken. Among the 200 samples examined, positive Salmonella
isolates were found, with varying prevalence across different poultry organs: 28 from flesh, 42 from intestine, 15 from
liver, and 25 from spleen. The results underscore the widespread occurrence of Salmonella in poultry, highlighting a
significant public health concern. Moreover, the study reveals a concerning rate of multidrug resistance among the
isolated Salmonella strains, exacerbating the gravity of the situation. Urgent measures are required to regulate
antibiotic usage in the poultry industry and mitigate the emergence of resistant strains. Effective control strategies for
salmonellosis necessitate comprehensive management systems, accurate identification of carrier birds, and precise
medication protocols. By addressing these aspects, stakeholders can work towards reducing the prevalence of antibiotic-
resistant pathogens in poultry meat, thereby safeguarding public health and promoting food safety.
Keywords: Salmonella, multidrug resistance, poultry meat, non-typhoid, foodborne infections, antibiotic usage, public
health,

Received 24.12.2023 Revised 01.01.2024 Accepted 23.04.2024

How to cite this article:
T. Sathiamoorthi, HP Jai Shanker Pillai, K. Saranya Devi, R. Rajalakshmi, B. Rajalinga . Malathi, R. Rajesh Kumar.
Prevalence and Characterization of Multidrug-Resistant Non-Typhoidal Salmonella in Poultry: Implications for Public
Health and Food Safety. Adv. Biores., Vol 15 (3) May 2024: 136-151

INTRODUCTION
In last three decades, Salmonella spp. has become one of the major food borne pathogenic bacteria leading
to high morbidity and mortality worldwide. Salmonella spp. causes an intestinal infection in humans
known as Salmonellosis (1). The disease is endemic in most developing countries and may cause
occasional outbreaks in industrialized countries (2). The global burden of food borne disease is currently
unknown but the World Health Organization (WHO) has responded to this data gap by launching a new

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initiative to provide better estimates. In last five years it was reported that 600 million, or almost 1 in 10
people in the world, fall ill after consuming contaminated food according to WHO. Poultry is one of the
fastest growing agricultural sectors in India. Poultry meat and its products are very popular food in India
as well as throughout the world. No wonder since it is delicious, nutritious and considered as a good and
cheap source of protein characterized by good flavour and easily digested (www.apeda.gov.in). Poultry is a
natural reservoir of Salmonella species, constituting the most important source of human infection. There
is scope of transmission of Salmonella infection from poultry to human through food chain. Several studies
have shown that sources of Salmonella infection in poultry and poultry products. In developing countries,
most chicken meat is produced by integrated broiler operations. These broiler operations control and
operate through all phases of the chicken industry, such as breeder flock management, hatchery operation,
feed management, broiler slaughter, retail distribution and also handling by consumers (3,4,5). The
current emergence of antimicrobial food borne pathogens is a major challenge in both human and
veterinary medicine due to over- dependency of antimicrobial agents in the food and livestock sector (6).
Poultry, especially broiler chicken, can use antibiotics used for extensively to prevent or treat microbial
infections to the birds. Particularly Salmonella Spp. has become a concern because of the development of
multiple antimicrobial resistance strains, emphasizing the importance of continuous monitoring of the
pathogen according to CDC, 2014 (7). In light of their importance, the World Health Organization (WHO)
has established risk assessments on Salmonella in broiler chickens. Although in India, data regarding food
borne diseases associated with resistant pathogenic strains in poultry is lacking, it is considered to be
significant. These drugs are used routinely in humans for the treatment of acute and severe diarrhoea, but
recent studies have shown that infections with quinolone resistant Salmonella tend to be more severe and
more often fatal compared with infections with sensitive strains.

MATERIAL AND METHODS

Sampling
The specimens were collected from retail broiler chicken shop in Karaikudi city, Tamilnadu, India. The
selected parts of the Samples are intestine flesh liver and spleen of retail chicken were collected from
different zones of Karaikudi (East, West, South and North) during the period between December 2022 to
April 2023. Sites in the area from which samples were collected include Mathur, Iluppakkudi, Ariyakudi,
Kattuthalaivasal, Kalanivasal, Water tank, Five lamps, corporation street and Koviloor.
Cultivation of samples
Bacteriological examine of different organs of retail chicken meat
Using sterile scissors, organs were individually cut into small pieces and enriched with Selenite broth in a
ratio of 1:10, the cultures were incubated at 37°C overnight. Twenty-five grams of each sample were pre-
enriched in 225 ml of phosphate buffered peptone water (Hi- Media, Mumbai) for 48-hr at 37oC. One-
millilitre pre-enriched sample was transferred into 10 ml of tetra thionate broth and selenite cysteine
broth and incubated for 48-hrs at 42°C, then a loopful culture was aseptically streaked on modified
brilliant green sulphadiazine agar and Xylose- Lysine Deoxycholate agar (XLD). The plates were incubated
at 37oC for 24 hrs (APHA 1992). At least five colonies were qualified as presumptive Salmonella colonies on
modified brilliant green agar and XLD agar plates, (red colonies and red colonies with black centres,
respectively) were then picked and sub-cultured on slants of nutrient agar.
Inoculation of plates
A loop of the inoculated selenite-f-broth was streaked on a plate of deoxycholate citrate agar and incubated
aerobically at 37º C for 24hours.
Purification and storage of isolates
Non- lactose fermented colonies was purified by repeated subculture on nutrient agar. Pure isolates was
stored on nutrient agar slopes in the refrigerator at 4º C.
Microscopic Examine:
Gram’s stain
Smears were prepared from the culture by emulsifying a part of a colony in a drop of normal saline on a
glass slide, dried and fixed by heating. Then the slides was flooded by crystal violet for 1 minute and then
washed with tap water. Iodine solution was applied for 1minute, and then the slide was washed with tap
water. The smear was then decolorized with few drops of acetone for seconds and washed immediately
with water. Then the smear was flooded with diluted carbolfuchsin for 30seconds and washed with tap
water. Slides were then blotted with filter paper and examined under oil immersion lens. Gram-positive
bacteria appeared violet in color while that of gram-negative bacteria appeared red.

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Biochemical tests for identified bacteria
Primary biochemical tests
Oxidase Test
Strips of filter paper were soaked in 10% solution of tetramethyle–phenylenediaminedihydrochloride in a
petridish and then left to dry. Then a fresh young test culture, on nutrient agar, was picked up with a sterile
glass rod and streaked on that filter paper. A dark purple color that developed within five to ten seconds
was considered positive reaction.
Catalase Test
A drop of 3% aqueous solution of hydrogen peroxide was placed on a clean microscope slide. A colony of
test culture, on nutrient agar was then placed on the hydrogen peroxide drop. The test was considered
positive when gas bubbles appeared on the surface of the culture material.
Glucose utilization Test
The sugar media were inoculated with the test organism and incubated at 37ºC over night. They were
examined daily for 7 days. Acid production was indicated by the development of pink color in the medium,
Gas production was indicated by air trapped in the Durham’s tube.
Oxidation-Fermentation (O/F) Test
The test was made by growing the test culture in tow tubes of Hugh and Lifeson’s medium. A layer of soft
paraffin was added to one tube to a depth of about 1 cm. Both tubes were incubated at 37ºC and examined
daily. Oxidizer organisms showed acid production in the upper part of medium in the paraffin-covered
tube and at the bottom in the open tube.
Motility (Hi-Media, Mumbai)
Motility medium (Semi-solid medium in U- shape tube) was inoculated at the top of one end of the tube
with tested organism and incubated at 37ºC for about 4 days. Positive test was indicated by presence of
growth in the other sides of the tube.
Secondary biochemical tests
Urease Test
Suspected Salmonella colonies were streaked on urea agar slope, incubated 37º C for 2 days. A positive
reaction was indicated by a change of color to pink.
Indole Test
The test culture was inoculated into peptone water medium and incubated at 37º C for 48 hours. 1 ml of
Kovacs’s reagent was rundown to the side of the tube. A pink ring which appeared on the surface within 1
minute indicated positive reaction.
Methyl Red (MR) Test
The test organism was inoculated in glucose phosphate peptone water, incubated 37º C for 2 days. Five
drops of methyl red reagent were added. A positive reaction was indicated by appearance of a red color.
Voges Proskauer (V.P) Test
The test organism was inoculated in glucose phosphate peptone water, and then 3 ml of 5% alcoholic
solution of α-naphthol and 1ml of 40% KOH aqueous solution was added. A positive reaction was
indicated by development of bright pink color within 30 minutes.
Citrate utilization
An isolate colony from nutrient agar was picked up with a straight wire, then inoculated in Simmon’s
citrate agar and incubated at 37º C and examined daily. A positive test was indicated by change of color
from green to blue.
Hydrogen sulphide (H 2 S) Production
The test culture was inoculated by stabbing the butt and streaking the slope of triple sugar iron agar in
McCarteny bottles and incubated at 37ºC for 2 days. A positive reaction was indicated by development of a
black color.
Sugar fermentation test
The sugar media were inoculated with the test organism and incubated at 37ºC overnight. They were
examined daily for seven days. Acid production was indicated by development of pink color in the medium,
Gas production was indicated by air trapped in the Durham’s tube. The sugars used in these tests were
lactose, salicin, sucrose, maltose, mannitol, rafinose, inositol, xylose and sorbitol.
Casein hydrolysis test
The skim milk agar plates was prepared and the test organism was streaked. The plates were incubated
over night at 37°C. After incubation the plates were observed. The clear zone was formed around the
colonies that indicated a positive result.

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Starch hydrolysis test
The starch agar plates were prepared and the test organism was streaked. The plates were incubated over
night at 37°C. After incubation iodine solution was added over the starch agar plates. The clear zone was
formed around the colonies that indicated a positive result.
Antimicrobial sensitivity test
Sensitivity of Salmonella isolates to a number of antimicrobial agents was determined by the standard
disk diffusion method. Each isolate was tested to 12 different antimicrobial agents used for Gram-
negative bacteria. Colonies from each isolate were emulsified in 2ml nutrient broth and shaken
thoroughly to obtain a homogenous suspension of the test culture. The plates were then flooded with the
bacterial suspension, tipped in different directions to cover the whole surface with the suspension. Excess
fluid was aspirated and the plates were left for 15minutes to dry. The antimicrobial disks were placed on
the agar medium by using sterile forceps. The plates were then incubated at 37ºC and examined after 24
hours for zones of inhibition which were measured in mm. The isolates were described as resistant,
intermediate and sensitive to different antimicrobial agents according to Bauer et al., method.
Table 1: Standard zone of inhibition to different antimicrobial Agents
Antimicrobial agent Disk potency Zone of Inhibition
(mcg) Resistant Intermediate Sensitive
Amikacin 10 14 or less 15- 16 17 or more
Amoxyclav 50/10 12 or less 14- 15 22 or more
Ampicillin 25 11 or less 12- 14 15 or more
Chloramphenicol 10 12 or less 13- 17 18 or more
Ciproflaxacin 10 15 or less 16- 20 21 or more
Co- Trimaxazole 25 13 or less 18- 20 21 or more
Gentamycin 10 13 or less 14- 15 16 or more
Tetracyclin 30 14 or less 15- 18 19 or more
Trimethoprim 10 13 or less 16- 17 20 or more
Nalidixic acid 30 15 or less 13- 14 19 or more
Nitrofurantoin 200 12 or less 14- 15 19 or more
Vancomycin 10 13 or less 15- 16 20 or more

Factor influencing of Salmonella isolates

Factor influencing of Salmonella isolates different pH
LB broth was prepared and adjusting the following pH 5, 6, 7 and 9 before sterilization. The test organisms
were inoculated into the LB broth and measured the OD value at 600 nm for the interval of time 0 hours, 4
hours and 18 hours during the incubation time.
Factor influencing of Salmonella isolates different temperature
LB broth was prepared and autoclaved. The test organisms were inoculated into the LB broth and
incubated at various temperatures 28°C, 37°C and 42°C. The test organism were measured the OD value at
600 nm for the interval of time 0 hours, 4 hours and 18 hours during the incubation period.
Factor influencing of Salmonella isolates different concentration of NaCl
LB broth was prepared with different concentration of sodium chloride 0.30%, 0.50% and 0.70%. The test
organisms were inoculated into the LB broth and incubated. The test organism were measured the OD
value at 600 nm for the interval of time 0 hours, 4 hours and 18 hours during the incubation period.

RESULTS AND DISCUSSION

Isolation of bacteria
A total of 200 samples were subjected to bacteriological examinations. Hundred and ten Gram-negative
Enterobacteriaceae were isolated from 200 samples. 20 samples showed no bacterial growth, 70 samples
did not give typical reactions of Enterobacteriaceae with oxidase, Catalase, and glucose fermentation so
they were not further identified. A total of 200 chicken samples, the positive isolates were identified. There
are 28 samples from flesh, 42 samples from intestine, 15 samples from liver and 25 samples from spleen
(Fig 1) and (Table 2).

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 S.N Sources No. of No. of Percentage
o samples isolates (%)
1 Flesh 50 28 56
2 Intestine 50 42 84
3 Liver 50 15 30
4 Spleen 50 25 50
Table 2. Isolation of salmonella sp from chicken

Figure 1. Number of salmonella isolates from chicken

In the present investigation, Out of 110 Salmonella, 33 (66%), 19 (38%), 41 (82%), and 17 (34%) were
isolated from East, West, South and North zones, respectively. The higher number of Salmonella observed
in the south zone of Karaikudi (Tab. 3, Fig. 2).
Table 3. Number of Salmonella from different Zone
S.No Selected zone No. of positive
1 East zone 33
2 West zone 19
3 South zone 41
4 North zone 17

Figure 2. Number of Salmonella from different Zone Prevalence of Salmonella species of retail
broiler chicken sample Site of isolation

The retail broiler chicken samples were collected from the East, West, South and North zones of Karaikudi.
Out of 200 samples examined, 55% were positive for Salmonella. The present investigation indicated the
occurrence of Salmonella in intestine (84%) at high levels when compared to the flesh (56%), spleen
(50%), and liver (30%) (Fig 3).

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 Figure 3. Percentage of Salmonella sp from chicken
In the present investigation, Out of 110 Salmonella, 33 (66%), 19 (38%), 41 (82%), and 17 (34%) were
isolated from East, West, South and North zones, respectively. The higher number of Salmonella observed
in the south zone of Karaikudi.
Biochemical reactions
Salmonella isolates were Methyl red positive, Indole, VP, urease and gelatinase negative (Fig 4). On triple
sugar iron agar Salmonella colonies produced hydrogen sulfide which was indicated by black discoloration,
gas production causes bubbles in the agar, and pH change was indicated by production of red color in the
slant (Fig 5). ONPG test were positive that indicate yellow color zone surrounding the disc (Fig 6), On
Oxidase test Salmonella isolates do not forms blue color in the disc that indicate the negative result (Fig 7)
and Catalase positive (Fig 8).Carbohydrate fermentation test produced gas from glucose and mannitol,
while sucrose and lactose were not fermented. Hydrogen sulfide was produced by the isolates (Fig 9). The
Table 4 and 5 shown Biochemical confirmation test for Salmonella isolates and Interpretation of TSI test.
Table 4. biochemical conformation test
Test S. typhi S. paratyphi S. enterica
Gram strain -ve -ve -ve
Motility +ve +ve +ve
Catalase +ve +ve +ve
Oxidase -ve -ve -ve
Indole -ve -ve -ve
Methyl red +ve +ve +ve
Voges- -ve -ve -ve
Proskauer
Citrate -ve -ve +ve
Nitrate +ve +ve +ve
Urease -ve -ve -ve
Ornithine -ve +ve +ve
Dulcitol -ve +ve +ve
Rhamnose -ve +ve +ve
Gelatinase -ve -ve -ve
ONGP -ve -ve +ve

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 Table 5. Interpretation of TSI test
Test Salmonella strain
S. typhi S. paratyphi A S. enterica
TSI Acid from glucose + + +
TSI gas from glucose - + +
TSI acid from lactose - - -
TSI acid from sucrose - - -
TSI H2S produced + - +

Figure 4. Imvic Test and Gelatinase Test

(Indole Negative, MR Positive, VP Negative, Citrate Positive, TSI - H2S production and Gelatinase
Negative (Left to Right)

Figure 5. TSI test - Black Discoloration Indicates The Production Of Hydrogen Sulfide

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 Figure 6. ONPG Disk test- Positive result – yellow color zone surrounding the disc

Figure 7. oxidase test -Negative Result – No blue color formation

Figure 8. catalase test- Positive result in air bubbles formation

Figure 9. carbohydrate fermentation test- Gas production in Durham’s tubes

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In Casein hydrolysis test formation of clear zone around the colonies that indicates test organism
hydrolysed the caseinolyase enzyme (Fig1.7). In Starch hydrolysis test formation of clear zone around the
colonies that indicates test organism hydrolysed the starch (Fig1.8).

Figure 10. Casein test- Positive result – clear zone was formed

Figure 11. Starch hydrolysis test - Positive result – clear zone was formed

Factors influencing the growth of Salmonella isolates

In this study, four highly drug resistant Salmonella isolates from Flesh, Intestine, Spleen and Liver samples
were used. The results indicated that different level of pH (5, 6, 7 and 9), and Temperature (28 ̊ C, 37 ̊ C and
42 ̊ C), different concentration of sodium chloride (Nacl) (0.30%, 0.50%, 0.70% and 0.90%) all are the
important factors influencing the survival of Salmonella isolates in laboratory media.
Influence of Salmonella growth at different pH
Table 6 shown the isolates exhibited a wide range of growth in different pH. In the present study
Salmonella isolates were grown well in different level of pH (Fig 12).
3.6 Influence of Salmonella growth at different temperature
The Salmonella isolates exhibited a wide range of growth in different level of temperature shown in Table 7
and Figure 13.
Influence of Salmonella growth at different concentration of Nacl
In the Salmonella isolates were tolerated and grown well in different concentration of sodium chloride
(NaCl) shown in Table 8 and Figure 14.
Table 6. Influence of Salmonella growth pattern at different pH range
S.No Time (hrs) pH Flesh Intestine Liver Spleen
1 0 5 0.043 0.051 0.024 0.032
6 0.241 0.907 0.612 0.717
7 0.243 0.817 0.021 0.121
9 0.372 0.467 0.421 0.248
2 4 5 0.123 0.289 0.321 0.021
6 0.415 0.424 0.200 0.321
7 0.251 0.383 0.172 0.167
9 0.132 0.241 0.211 0.210
3 18 5 0.216 0.245 0.278 0.311
6 0.823 0.588 0.286 0.211
7 0.621 0.518 0.821 0.721
9 0.543 0.409 0.621 0.217

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 Figure 12. Factors influencing of Salmonella species at different pH level

Table 7. Influence of Salmonella growth at different temperature

S.No Time Temperature Flesh Intestine Liver Spleen
(hrs) (C
̊ )
1 0 28 0.051 0.042 0.017 0.066
37 0.076 0.051 0.021 0.052
42 0.081 0.061 0.075 0.125
2 4 28 0.412 0.512 0.471 0.442
37 0.467 0.321 0.437 0.041
42 0.135 0.420 0.315 0.361
3 18 28 0.110 0.461 0.310 0.410
37 0.50 0.601 0.118 0.52
42 0.341 0.421 0.124 0.51

Figure 13. Factor influencing Salmonella species at different temperature

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 Table 8. Influence of Salmonella growth at different concentration of NaCl
S.No Time Concentration Flesh Intestine Liver Spleen
1 0 0.30% 0.053 0.071 0.072 0.070
0.50% 0.060 0.061 0.069 0.073
0.70% 0.052 0.072 0.065 0.019
0.90% 0.029 0.031 0.021 0.020
2 4 0.30% 0.22 0.28 0.24 0.26
0.50% 0.46 0.42 0.45 0.48
0.70% 0.31 0.23 0.31 0.32
0.90% 0.32 0.31 0.24 0.25
3 18 0.30% 0.20 0.10 0.25 0.14
0.50% 0.51 0.21 0.61 0.42
0.70% 0.32 0.20 0.25 0.25
0.90% 0.41 0.45 0.35 0.24

Figure 14. Factor influencing Salmonella species at different Nacl

Antimicrobial resistant pattern (%) of Salmonella isolated from flesh, intestine, Liver and spleen
of broiler chicken.
The pattern of resistance of Salmonella analyzed in this study is shown in figure 15a. Significant differences
were seen in the diffusion zone sizes for all agents. Resistance to tetracycline was present in 100% of the
all Salmonella isolates.
Antimicrobial resistant pattern in Flesh
Antimicrobial resistance of Salmonella isolates in Flesh sample were analysed shown on Table 9. The
following these are highly resistant to sensitive of antimicrobial resistant pattern in flesh. Vancomycin
(100%) is highly resistance of Salmonella isolates. Amoxyclav (95.23%), Nalidixic acid (90.9%), Ampicillin
(89.9%), Chloramphenicol (64.72%) are also resistance of Salmonella. The intermediate antibiotic
resistance of isolates are Tetracycline (45.51%), Trimethoprim (35.58%). Amikacin (17.12%), Gentamycin
(15.32%), Nitrofurantoin (15.11%), Co- Trimaxazole (10.9%) and Ciprofloxacin (2.99%) are sensitive to
Salmonella isolates.
Antimicrobial resistant pattern in Intestine
Antimicrobial resistance of Salmonella isolates in intestine sample were observed shown on Table 9. The
following these are highly resistant to sensitive of antimicrobial resistant pattern in intestine sample.
Vancomycin (100%) is highly resistance of Salmonella isolates. Amoxyclav (96.26%), Ampicillin (94.73%),
Nalidixic acid (83.1%), Chloramphenicol (72.5%), Tetracycline (70.83%), Trimethoprim (60.53%) are also
resistance of Salmonella. The intermediate antibiotic resistance of isolates are Co-Trimaxazole (20.83%),
Amikacin (20.3%). Nitrofurantoin (16.21%) Gentamycin (13.12%) and Ciprofloxacin (3.62%) are sensitive
to Salmonella isolate.

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Antimicrobial resistant pattern in liver:
Antimicrobial resistance of Salmonella isolates in liver sample were noted shown on Table 9. The
following these are highly resistant to sensitive of antimicrobial pattern in live r. Vancomycin
(100%) is highly resistance of Salmonella isolates. Amoxyclav (98.09%), Nalidixic acid (86.25%),
Chloramphenicol (80.02%), Tetracycline (65.32%), Trimethoprim (56.66%) are also resistance of
Salmonella. The intermediate antibiotic resistance of isolates are Ampicillin (50.01%), Ciprofloxacin(34%),
Co-Trimoxazole (25.23%), Nitrofurantoin (23.21%). Gentamycin (20.13%) and Amikacin (18.17%) are
sensitive to Salmonella isolates.
Antimicrobial resistant pattern in Spleen
Antimicrobial resistance of Salmonella isolates in spleen sample were observed shown on Table 9. The
following these are highly resistant to sensitive of antimicrobial pattern in spleen. Vancomycin (100%) and
Chloramphenicol (100%) are highly resistance of Salmonella isolates. Amoxyclav (99.09%), Ampicillin
(95.32%), Nalidixic acid (81.81%), Trimethoprim (78.28%), Tetracycline (72.56%) are also resistance of
Salmonella. The intermediate antibiotic resistance of isolates are Amikacin (50.12%), Nitrofurntoin
(21.83%) Gentamycin (16.17%), Co-Trimoxazole (14.56%) and Ciprofloxacin (3.21%) are sensitive to
Salmonella isolates.
Average of Antimicrobial resistant pattern
Resistance was most commonly observed Salmonella isolates from flesh, intestine, spleen and liver to
Amoxyclav (97.16%), Nalidixic acid (85.51%), Ampicillin (82.49%), Chloramphenicol (79.31%),
Tetracycline (63.55%), Trimethoprim (57.67%), Amikacin (26.42%), Nitrofurantoin(19.09%), Co-
Trimoxazole (17.88%), Gentamycin (16.18%) and Ciproflaxacin (3.30%). Vancomycin (100%) is highly
resistance of Salmonella isolates. In Salmonella isolates, Amoxyclav, Nalidixic acid, Ampicillin,
Chloramphenicol and tetracycline are highly resistance and very low resistance were observed to
Ciprofloxacin and Gentamycin (Fig 15a,b and Fig 16).

Figure 15a. Antimicrobial resistant pattern

Ampicillin (AMP)
Tetracycline (TE)

Nalidixic acid (NA) Chloramphenicol (C)

Co – Trimoxazole (COT)
Nitrofurantoin (NIT)

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 Vancomycin (VA)
Amikacin (AK)

Trimethoprim (TR) Amoxyclav (AMC)

Ciproflaxacin (CIP)
Gentamycin (GEN)

Figure 15b. Antimicrobial resistant pattern

Figure 16. Average of antimicrobial resistance

Table 9. Antimicrobial resistant pattern (%) of Salmonella isolated from Flesh, Intestine, Liver and
Spleen of broiler chicken
S.No Antibiotics Flesh (%) Intestine Liver Spleen Average
(%) (%) (%) (%)
1 Amikacin 17.12 20.3 18.17 50.12 26.4275
2 Amoxyclav 95.23 96.26 98.09 99.09 97.1675
3 Ampicillin 89.9 94.73 50.01 95.32 82.49
4 Chloramphenicol 64.72 72.5 80.02 100 79.31
5 Ciproflaxacin 2.99 3.62 3.4 3.21 3.305
6 Co- Trimoxazole 10.9 20.83 25.23 14.56 17.88
7 Gentamycin 15.32 13.12 20.13 16.17 16.185
8 Tetracycline 45.51 70.83 65.32 72.56 63.555
9 Trimethoprim 35.58 60.53 56.66 78.28 57.7625
10 Nalidixic acid 90.9 83.1 86.25 81.81 85.515
11 Nitrofurantoin 15.11 16.21 23.21 21.83 19.09
12 Vancomycin 100 100 100 100 100

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Salmonellosis is a major public health concern and continues to have a serious economic importance in the
poultry industry in all countries (8). With the great expansion of the poultry industry, the wide spread
occurrence of the salmonellosis has ranked it as one of the most important bacterial diseases of poultry.
The present study was conducted to investigate the prevalence of Salmonella from selected parts of retail
broiler chicken samples were collected from the East, West, North and South zones of Karaikudi city. In this
study Salmonella represented the most dominant isolate and 55% were positive. The present
investigation indicated the occurrence of Salmonella in intestine (84%) at high levels when compared to
the flesh (56%), spleen (50%), and liver (30%). The Salmonella isolation rate was comparable to that
reported in other studies. Yagoub and Mohammed (8) examined 1488 samples and isolated 58 Salmonellae
which comprise 3.9% of total isolates. In another study Ezdihar (9) examined 610 samples from poultry in
the Sudan and isolated 45 Salmonellae which counted for 7.4% of the total isolates. The higher isolation
rate was obtained from a south zone (82%) of Karaikudi city. This can be due to poor hygiene in this farm.
Among the examined samples, the highest rate of isolation was obtained from Intestine. This finding
indicates a high shedding of Salmonella from the intestinal tracts of birds in this farm. S. enteritidis is the
most important serovar in poultry flocks and recently it was of high occurrence worldwide (10). Phillips
and Optiz (11) showed that S. enteritidis could attach to granulose cells in the preovulatory membrane and
subsequently infect the ovum during the ovulation. On the other hand, S. enteritidis had the ability to
penetrate eggs through the shell pores and causes egg contamination. From the view point of public health,
human Salmonellosis was reported to increase in France and United States of America due to S. enteritidis
(12). It was reported to cause food poisoning due to consumption of under cooked egg dishes (13).
Isolation of this bacterium from some farms in Karaikudi represents a real threat to the public health. S.
arizonae was reported to cause arizonae infection in chickens (14). Numerous studies have evaluated the
effect of environmental factors such as temperature (15), pH and sodium chloride (16) on microbial
growth. Salmonella have an optimum growth temperature of 37°C, however they readily adapt to extreme
conditions. Salmonella growth temperatures low at 5.9°C and high at 54°C have been reported for specific
experimental conditions. The antibiogram of the Salmonella isolates were encountered in the present study
revealed that most of the strains had acquired resistance to more than 4-5 antibiotics. Multiple antibiotic
resistant Salmonella in chicken and fish in Indian markets has been reported earlier (17, 18,). The
resistance to antibiotics was much higher when compared to the resistance patterns from developed
countries. This is mainly because of the frequent abuse of antibiotics in our country. Most of the antibiotics
can be purchased over the counter without prescription as well as there is no proper follow up to ensure
that the patients are completing the course. This results in an increased selection pressure, which could
add to the emergence of resistant strains. Also, wide spread use of antibiotics in animal production systems
is contributing significantly to the increased antibiotic resistance among pathogenic bacteria of animal
origin (19). Tetracycline has been used to treat day old chickens, which might have resulted in the
emergence of tetracycline resistant Salmonella in the layer and broiler flocks (20). This is evident in the
results from our study, where we observed relatively higher levels of tetracycline resistance among the
Salmonella isolates from commercial layer hen eggs and non-commercial layer hen eggs. The resistance
level was much higher than the level reported by Hatha and Lakshmanaperumalsamy (18). Ampicillin
resistance among the Salmonella strains encountered in the present study were lower than those reported
by Suresh et al., (21). Ciprofloxacin is a fluoroquinolone antibiotic that is increasingly and successfully used
for the treatment of septicaemia in humans Ciprofloxacin resistance in human and veterinary Salmonella
isolates has occasionally been found. The Salmonella having similar level of resistance and resistance
pattern indicates their origin from a common source. The multiplicity of resistance pattern indicates the
large pool of resistance plasmids among these bacteria, which would pose a potential threat once they
released into the environment. Also the treatments of infections caused by these MAR forms are going to
be extremely difficult. A high incidence of multidrug resistant Salmonella in the poultry wastes was noticed
by with frequent resistance against tetracycline, streptomycin and colistin (22). Almost all strains from
different sources in the present study were resistant to Vancomycin. Other studies have also reported
similar antibiotic resistance of Salmonella strains from different sources against Vancomycin. All strains of
Salmonella were found sensitive to ciprofloxacin, Gentamycin, Co – Trimoxazole, Nitrofurantoin, Amikacin
and Trimethoprim. Resistance to Gentamycin has been reported by Lee et al., (23) which determined 10%
resistance to this agent from 105 Salmonella isolates. Also there was an increasing development of
quinolones resistance all over the world. Treatment failure due to a reduced susceptibility to Ciprofloxacin
in Salmonella is now well established (24). The logical interpretation of the results of the MAR index of all
Salmonella strains isolated in the study showed that they might have originated from high risk sources of
contamination. Poultry is identified as one of the major reservoirs of Salmonella species. There is a large
body of literature reviewed by Novick (19) demonstrating that the sub-therapeutic use of antibiotics in the

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mass production of poultry, eggs and pork has promoted the emergence and maintenance of MAR
pathogenic bacteria in the environments of these animals. In general, Salmonella is the most important
agent implicated in outbreaks in food-borne diseases around the world (25). Effective control or
eradication programs for salmonellosis depend on good management system, identification of carrier birds
and accurate medication. The prevalence of Salmonella in poultry was relatively high than the other
environmental factors. The multidrug resistance in most of these Salmonella strains adds to the gravity of
the problem. Based on the results we again reiterate the need for regulating control over the use of
antibiotics in poultry industry. Effective control or eradication programs for salmonellosis depend on good
management system, identification of carrier birds and accurate medication.

CONCLUSION
The main purpose of this study was to investigate the prevalence, distribution, factors affecting the
growth of Salmonella isolates, and antimicrobial activity of multidrug resistant (MDR) Salmonella were
isolated from chicken samples in different region (East, West, North and South) of Karaikudi city,
Sivaganga district, Tamilnadu, India, during the periods from December 2022 to April 2023. A total of 200
samples were examined, 55% were positive for Salmonella. The present investigation indicated the
occurrence of Salmonella in intestine (84%) at high levels when compared to the flesh (56%), and spleen
(50%), liver (30%). The higher number of Salmonella observed in the south zone (82%) of Karaikudi.
Besides, the result indicates the existence and diversity of Salmonella species vary temporally and is
strongly influenced by seasonal precipitation, turbidity, heterotrophic bacteria and pH. There is a clear
seasonal trend for salmonellosis cases, gradually in the winter. Although higher case rates coincide with
peak annual temperatures, it remains unclear which factors drive this seasonality regionally or how this
pattern might relate to the presence of the pathogen itself in the environment. Antimicrobial agents are
used to treat infected human or animals, to protect them from infectious diseases, and to provide a faster
growth rate. But, now most of the pathogens are resistant to commonly used drugs. The Salmonella genus
has been traditionally susceptible to antimicrobial agents. Resistance was most commonly observed
Salmonella isolates from flesh, intestine, spleen and liver to Amoxyclav (97.16%), Nalidixic acid (85.51%),
Ampicillin (82.49%), Chloramphenicol (79.31%), Tetracycline (63.55%), Trimethoprim (57.67%),
Amikacin (26.42%), Nitrofurantoin (19.09%), Co- Trimoxazole (17.88%), Gentamycin (16.18%) and
Ciprofloxacin (3.30%). In Salmonella isolates, Amoxyclav, Nalidixic acid, Ampicillin, Chloramphenicol and
tetracycline are highly resistance and very low resistance were observed to Ciprofloxacin and Gentamycin.

ACKNOWLEDGEMENT
The first author thanks the Vice-Chancellor and Registrar of Alagappa University for providing the
research facilities. This work was supported by the RUSA- phase 2.0 grant -No. F.24–51/ 2014-U, policy
TN multi-Gen), Dept of Education, Govt. of India-Dt 2021-2023

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