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Original Article | Open Access | Am. J. Pure Appl. Sci., 2019; 1(1), 1-11 | doi: 10.34104/ajpab.019.019111

Isolation, Identification, and Antibiotic Sensitivity Pattern of Salmonella spp from Locally Isolated Egg Samples

Md. Atikur Rahman

Azizul Haque*

Tasnim Ahmad

Shahriar Mahmud

Shaharuq Nahid Sohana

Md. Rajib Hossain

Nirmal Chra Barman

Md. Badiruzzaman

Tofajjal Hossain

Md. Sherajul Haque

Md. Ekhlas Uddin

Rokeya Ahmed

Abstract

Salmonella is the major foodborne disease in the world with a serious public health problem. The presence of any serotype of Salmonella in food under that food unfit for human consumption. Poultry eggs are considered major sources of these pathogenic microorganisms. The current study was carried out to isolate and identify Salmonella spp from egg samples collected from different retailer markets and farms in the Savar area, Bangladesh. An antibiotic susceptibility test was also done to determine the resistance pattern. A total of 50 poultry eggs were examined. Salmonella spp were isolated by culturing on the selective plate and characterized by biochemical tests. In the present study, the average prevalence of Salmonella was found to be 100% from the outer surface of eggs and 20% from egg yolk. All identified isolates were tested for antibiotic susceptibility to six commonly used antimicrobials by the disk diffusion technique. The highest percentage of resistance (60%) was found in Chloramphenicol, Ampicillin, Gentamicin, and Tetracycline. Salmonella isolated from the egg surface were found more antibiotic-resistant than that of egg yolk. The present study suggests that poultry eggs are a potential reservoir of antibiotic-resistant Salmonella.

Keywords

INTRODUCTION

Salmonellosis is primarily a food poisoning syndrome, which occurs when ingesting pathogenic Salmonella serotypes. Salmonella have been known to cause illnesses for more than 100 years when it was discovered by Dr. Daniel Salmon (Yan et al., 2003). Eggs are laid by female animals of many different species, including birds, reptiles, amphibians, mammals and fish (Kenneth et al., 2017). The egg is comprised of 32-35% yolk, 52-58% albumen and 9-14% shell. The yolk contains about 60 Calories; three times the energy content of the egg white, Egg white consists primarily of about 90% water into which is dissolved 10 percent proteins. Due to their protein content, the United States Department of Agriculture categorizes eggs as Meats within the Food Guide Pyramid (Kenneth et al., 2017).

Despite the nutritional value of eggs, there are some potential health issues arising from cholesterol content, Salmonella contamination and allergy to egg proteins. Salmonella is capable can cause a variety of disease syndromes: enteric fever, bacteremia, enter colitis, and focal infections. Salmonella enteric is a bacterial pathogen that causes enteric fever and gastroenteritis in humans and animals (Didelot et al., 2011). Typhoid or enteric fever is an acute illness with incubation period 2 or 3 weeks, often characterized by high fever of about 40°C, malaise and abdominal pain (Gonzalez-Escobedo et al., 2011), continual headache, diarrhea appears only during the second or third week (Tortora et al., 2011). There are two possible routes of bacterial contamination of egg; vertical and horizontal (De Reu et al., 2008). Vertical transmission takes place in the Trans ovarian route where the yolk, albumen and membranes are directly contaminated as a result of bacterial shedding from the infection of hens reproductive organs, which takes place before the shell covers the eggs (Messens et al., 2005). Horizontal contamination begins with the passage of the eggs through the highly contaminated cloaca area at the moment of lay and leads to the shells be penetrated by microorganisms (De Reu et al., 2008; Md et al., 2014).

In recent years, the prevalence of Salmonellosis in breeder flock, commercial broiler, and layer flocks is increasing day by day. Egg shells may additionally become contaminated from any surface with which it comes into contact. The hens are released in an environment with a temperature of approximately (68°F), where they cool immediately and their content contracts creating a negative pressure inside which can allow for the contaminant to move through the shell (De Reu et al., 2008). To avoid the contamination, eggs should be refrigerated at 4°C and cooked to an internal temperature of 71°C or hotter. Salmonella is one of the major bacterial agents that cause food borne infection in humans leading food borne disease worldwide (Herikstad et al., 2002).

The human health concern is the risk of direct transfer of resistant human pathogens via the food chain. S. Enteritiss came to prominence as a major food borne pathogen in Europe and America during the 1980s (Cogan and Humphrey, 2003). Human infections are usually associated with animal contact and the consumption of contaminated food products such as poultry egg, meat and other dairy products are food poisoning by Salmonella (William et al., 2003). Salmonella infection is one of the major constraints of poultry farming that hindered its development in Bangladesh (Kamaruddin et al., 2012). Therefore, Salmonellosis status of a farm needs to be determined for its proper control and management (Ahmed et al., 2008). The main objective of this study is to isolate and identify and determine the antibiotic resistance pattern of isolated Salmonella spp from poultry eggs of local farms and retailers in Savar.

MATERIALS AND METHODS

Sampling, Samples Collection and Storage

Fig 1: Sampling site at different areas in Savar Upazila of Dhaka city.

The samples were collected during the study period from July to December 2017, at different region of Savar, Dhaka. The samples were collected by aseptic technique using sterile zipper bags, gloves, etc. The microbiological analysis was conducted in the Department of Microbiology, Gono Bishwabidyalay, Savar, Dhaka. Total 50 samples were collected from different egg retailers and farm house. During collection of the eggs, it was ensured that cross contamination was minimized by taking precautions at all stages such as sampling, transportation and storage of the eggs. Samples are stored at 4°C temperature.

Isolation and Identification of Salmonella spp on Selective Agar

After collection of samples, outer shell surface washed with 70% alcohol and air dried then crack with a sterile knife. Egg yolk were also prepared and enriched in selenite cystein broth. Each eggs content was mix thoroughly and homogenized and incubates at 37°C for 24 hours. At the end of selective enrichment, sample was streaked on selective media such as Xylose lysine deoxycholate (XLD) agar, Salmonella-Shigella (S-S) and Rappaport Vassiliadis agar. Solidification of the medium was tested their sterility by incubation at 37°C for overnight to cheek their sterility and then in refrigerator for future use (Cappuccino et al., 2005). The agar itself will turn red due to the presence of Salmonella type colonies and No heavy greyish swarming zone is visible in a Salmonella negative sample, whereas Salmonella positive samples give a very heavy swarming, at MSRV agar.

Table 1: Details of the drug, reference zone of inhibition based on clinical laboratory standards.

Microscopic Study by Grams Staining Method

Gram staining method was used to distinguish and classify bacterial species into two large groups. Grams staining of the pure culture was performed according to method described by Aneja et al. (2003).

Biochemical Tests for the Confirmation of Salmonella spp

Indole Test - Indole test demonstrates the ability of certain bacteria to decompose amino acid tryptophan to indole, which accumulates the medium. Tryptophan is hydrolyzed by tryptophans to produce three possible end products, one of which is indole. Indole production is detected by Kovacs reagent which contains 4(p)-dimethyl amino Benz aldehyde; this reacts with indole to produce a red color.

Methyl Red-Voges Proskauer (MR-VP) Test – MR-VP test detects the production of sufficient acid during the fermentation of glucose. Mixed acids fermentation results in accumulation of a variety of acids and a significant drop in the pH of the medium. The medium used is MR VP broth. A nutrient medium with 0.5% Glucose added. This medium is also used for the Voges-Proskauer test, which determines acidic fermentation products results for growth.

Citrate Test - Citrate test is used to test an organisms ability to utilize citrate as a source of energy. The pH indicator is Bromo thymol blue, which is green at neutral pH, yellow at acidic pH<6.0 and turns blue at alkaline pH>7.6. Oxidase Test - This detects the presence of a cytochrome oxidase system that will catalyze the transport of electrons in the bacteria. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple. This is considered a positive result.

Catalase Test - This test demonstrates the presence of catalase, an enzyme that catalyzes the release of oxygen from hydrogen peroxide. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas. The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result.

TSI Test - The triple sugar Iron (TSI) is a microbiological test roughly named, which is used to differentiate among the different groups of Enterobacteriaceae, based on their ability to ferment glucose, lactose and/or sucrose and to produce hydrogen sulfide. It is often used in the selective identification of enteric bacteria.

Urease Test - This test is used to determine the ability of an organism to split urea, through the production of an enzyme Urease. It identifies those organisms that are capable of hydrolyzing urea to produce ammonia and carbon dioxide. Urease is a constitutively expressed enzyme that hydrolyzes urea to carbon dioxide and ammonia (Cappuccino et al., 2005)

(NH2)2 CO + H2O = CO2 + 2NH3

Antibiotic Sensitivity test for Salmonella Spp

Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo, testing for antibiotic sensitivity is often done by the Kirby-Bauer method. Place agar plates right side up in an incubator heated to 37°c for 10 to 20 minutes with the covers adjusted so that the plates are slightly opened. Inoculate all agar plates with their respective test organisms as follow; dip a sterile cotton swab into a well-mixed saline test culture and removes excess inoculated by processing the saturated swab against the inner wall of the culture tube. Allow all culture plates to dry for about 5 minutes. Gently press each disc down with the wooden end of a cotton swab or sterile, forceps to ensure that the discs adhere to the surface of the agar. Finally incubate all plate cultures in an inverted position for 24 hours at 37°C. In this study, all the samples were tested for the susceptibility against five different commercially available antibiotics.

RESULTS AND DISCUSSION

Isolation and identification of Salmonella spp

Fig 2: (a) Growth of Salmonella in Salmonella-Shigella agar medium; (b) Growth of Salmonella in XLD agar medium; and (c) Growth of Salmonella Spp in Selenite crystal broth.

Eggs and egg products are nutritious foods and they form an important part of the human diet. In this present study 50 poultry eggs were examined for the isolation and identification of Salmonella spp. Inoculation was done from both shell and internal content of each egg sample. Among them 50 (100%) samples from eggshell were found positive for Salmonella spp and only 10 (20%) samples from internal content were found positive for Salmonella spp (Table 2).

Selenite cysteine broth

Table 2: Percentage of Salmonella in total collected sample

Selenite broth is used as a selective medium for the isolation of Salmonella species. Selenite Broth is used as a selective enrichment for the cultivation of Salmonella spp. Selenite broth gives pale or colorless colonies. The growth of Salmonella Spp in Selenite crystal broth was characterized by diffused turbidity of the broth (Fig 4).

Salmonella-Shigella agar: Salmonella-Shigella agar is a differential, selective medium for the isolation of Shigella and Salmonella species from pathological specimens, suspected foodstuffs, etc. Salmonella spp to grow sodium Thiosulfate and Ferric Citrate permit detection of hydrogen sulfide by the production of colonies with black centers and Shigella produced colorless smooth colony in SS agar.

XLD agar

XLD agar is a selective growth medium used in the isolation of Salmonella and Shigella species from clinical samples and from food. Two agar media Salmonella-Shigella agar, Xylose Lysine Deoxycholate agars were used for the identification and isolation of Salmonella species from egg samples.

Result of Gram Staining

In grams staining under microscope, the organisms revealed gram negative, pink color; small rod shaped appearance, arranged in single, paired or chain form. (Fig. 3)

Biochemical characterization of organisms

The biochemical identification of the organism was done by performing the biochemical tests. The biochemical tests were done as stated on Berges Manual of Determinative Bacteriology.

Fig. 3: Gram Negative rod shape bacteria.

Results of different biochemical tests are given below

TSI test: A bacterium that is non-lactose fermenter and ferments glucose, initially causes a yellow slant/yellow bottom (acid/acid reaction) after 8 hours but then converts to a red slant/yellow bottom after 24 hours. The whole sample tubes covered with black color due to excess production of H2S. Citrate Test: Bacteria are inoculated on a medium containing sodium citrate and a pH indicator such as bromothymol blue. Use of citrate involves the enzyme citrase, which breaks down citrate to oxaloacetate and acetate. The isolated bacteria Salmonella will be citrate positive. All of the isolated Salmonella utilize citrate and change the color of the media green to blue (Alkaline).

Fig 4: (a) TSI test (Right showing negative control); (b) Citrate test (Left showing control) (c) Methyl Red test (Left showing negative control).

Methyl Red test

The MR test, the "M" portion of the four IMViC tests, is used to identify enteric bacteria based on their pattern of glucose metabolism. All enterics initially produce pyruvic acid from glucose metabolism. The isolated bacteria Salmonella were MR positive.

Voges-Proskauer test

Voges-Proskauer is a test used to detect acetoin in a bacterial broth culture. A cherry red color indicates a positive result, while a yellow-brown color indicates a negative result. The isolated bacteria Salmonella were VP negative.

Indole test

The indole test is a biochemical test performed on bacterial species. A positive result is shown by the presence of a red or red-violet color in the surface alcohol layer of the broth. A negative result appears yellow. The isolated bacteria Salmonella were indole negative.

Motility test

Non-motile bacteria generally give growths that are confined to the stab-line, have sharply defined margins. Motile bacteria move about with structures called flagella. The isolated bacteria Salmonella were motility positive.

Table 3: Biochemical characterization of isolates.

Table 4: Biochemical tests Result: (For confirmation of Salmonella spp.)

Fig 6: Catalase test.

Fig 7: Oxidase test.

Table 5: Antibiotic sensitivity test result: Zone Diameter, mm

Oxidase test

This test depends on the presence of cytochrome oxidase in bacteria that will catalyze the transport of electrons between electron donors and redox dye. The isolated bacteria Salmonella were oxidase negative.

Catalase test

The catalase test is one of the three main tests used by microbiologists to identify species of bacteria. If the mixture produces bubbles or froth, the organism is said to be catalase-positive. If not, the organism is catalase-negative. The isolated bacteria Salmonella were catalase positive.

Fig 8: Antibiotic sensitivity test of Salmonella.

Table 6: Percentages of resistances patterns of Salmonella.

Fig 9: Percentage of sensitivity, intermediate resistance and resistance of Salmonella spp.

Antibiotic sensitivity test result

In this study, the size of zone of inhibition of every antibiotic disc was measured in millimeter and while those zones of inhibition compared with zone diameter interpretive standards from C LSI (2000) (Table 5). The highest resistance rates were recorded against ampicillin (80%), chloramphenicol (60%) and tetracycline (46.66%), while the highest sensitivity rates (100%) were recorded against co-trimoxazole and ciprofloxacin.

The resistance rate against tetracycline (46.66%) was similar to the result of Mahammad et al., who reported approximately 52% of resistance. However, higher resistance rate was reported by Samah who recorded resistance rate of 92.9%. The resistance rate against chloramphenicol (60%) was in accordance with the result of Manie et al., who reported a rate of approximately 62% resistance against antimicrobial drugs. In the present study ciprofloxacin & co-trimoxazole works much better than other antibiotics used.

CONCLUSION

Salmonella contamination of eggs is a complex issue affected by variables at each stage of the food production process. Eggs associated Salmonellosis is an important public health problem in the world we have to consider some of the point that eggs offered for sale must be free of faces, dirt and stains. Enteric fever is mostly caused by members of serovar Typhi and Paratyphi A, both of which only infect humans (Uzzau et al., 2000). Gastroenteritis on the other hand is most often caused by Enteritidis in humans and Typhimurium in animals (Galanis et al., 2006), although both serovars can infect a wide range of hosts (Uzzau et al., 2000). However, the usefulness of the serological classification of S. enterica is undermined by the fact that unrelated strains sometimes belong to the same serovar. Egg farms must be regularly visited by field inspectors to monitor bird health by recording feed and appearance. In particular the risk of air pollution with bacteria, fungi, dust, endotoxins and ammonia, this is significantly higher in alternative than in conventional systems (Rodenburg et al., 2005; Vučemilo et al., 2008). Efforts including critical control point programs in food production are needed to reduce the incidence of Salmonella in food. Consumers-awareness efforts would protect public health from food borne and Salmonellosis.

ACKNOWLEDGMENTS

This research was financially supported with proper guidance and helping for data analysis in the Department of Microbiology of the Gono Bishwabidyalay, Savar, Dhaka, Bangladesh. Heartiest thanks to the authority of Gono Bishwabidyalay for helping us to conduct the research work.

CONFLICTS OF INTEREST

The author (s) declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

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Article References:

Ahmed F., Ahmad I., Khan M.S. (2008). Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol. Res., 163: 173-18. https://doi.org/10.1016/j.micres.2006.04.001
Aneja K.R. (2003). Staining and biochemical techniques. In: Experiments in microbiology, plant pathology and biotechnology, New Age International Ltd, Publishers, New Delhi.
Cappuccino J. C., Sherman N. (2005). In: Microbiology: A Laboratory Manual, 3rd Ed. Benjamin Pub. Co. New York, 125-179.
Cogan T., Humphrey T. (2003). The rise and fall of Salmonella Enteritidis in the UK. J. of Appl. Microbiol. 94: 114–119. https://doi.org/10.1046/j.1365-2672.94.s1.13.x
De Reu K., Messens W., Herman L. (2008). Bacterial contamination of table eggs and the influence of housing systems. Worlds Poultry Science Journal, 64: 6-19.
Didelot X., Bowden R., Street T., Golubchik T., McVean G. (2011). Recombination and Population Structure in Salmonella enterica. PLoS Genet., 7(7): e1002191. https://doi.org/10.1371/journal.pgen.1002191
Galanis E., Lo Fo Wong DM, Patrick ME., Cieslik A. (2006). Web based surveillance and global Salmonella distribution, 2000-2002. Emerg Infect Dis., 12: 381-388
Gonzalez-Espada W., Zaras, D. (2011). Evaluation of the impact of the National Weather Center REU program compared with other undergraduate research experiences. 15th Symposium on Education, Amer Meteorolog Soc. Atlanta.
Herikstad H., Motarjemi Y., Tauxe R. V. (2002). Salmonella surveillance: a global survey of public health sero-typing. Epidemiol. Infect, 129: 1-8. https://doi.org/10.1017/s0950268802006842
Kamaruddin R., Shabudin A., Iberahim H. (2012). Halal Compliance Critical Control Point (HCCCP) Analysis of Processed Food. Proceedings of 2012 IEEE Business, Engineering & Industrial Applications Colloquim.
Kenneth F. Schaffner. (2017). University of Pittsburgh Department of History and Philosophy of Science website. University of Pittsburgh. Retrieved, 03-11.
Md. Ekhlas Uddin, H. M. Faruquee, Md. Firoz Alam. (2014). Isolation and characterization of proteases enzyme from locally isolated Bacillus sp. American J. of Life Sciences. 2(6): 338-344. https://doi.org/10.11648/j.ajls.20140206.12
Messens W., Grijspeerdt K., Herman L. (2005). Egg shell penetration by Salmonella: A review. Worlds Poult. Sci. J., 61: 71-85
Rodenburg T.B., Tuyttens F.A.M., Sonck B., Reu De K., Herman L. and Zoons J. (2005). Welfare, health and hygiene of laying hens housed in furnished cages and in alternative housing systems. J. of Appl Animal Welfare Sci. 8: 211-226. https://doi.org/10.1207/s15327604jaws0803_5
Tortora GA. (2008). Microbiology: An Introduction, 9th ed. Pearson. pp. 323-324. ISBN 8131722325.
Uzzau S., Brown DJ., Wallis T., Rubino S., Leori G. (2000). Host adapted serotypes of Salmonella enterica. Epidemiol Infect. 125: 229-255-
Vučemilo M., Granić K., Orct T. (2008). Effect of microclimate on the airborne dust and endotoxin concentration in a broiler house. Czech J. of Animal Sci. 53: 83-89. https://www.agriculturejournals.cz/publicFiles/00745.pdf
William N., Wilson J., Hazel S., Goetzmann (2003). Non-typhoidal Salmonella in United Kingdom Badgers: Prevalence and spatial distribution. Appl and Environmental Microbiol. 69: 4312-4315.
Yan S., Pendrak M., Abela-Ridder B. (2003). An overview of Salmonella typing public health perspectives. Clin and Appl Immunol Rev. 4: 189-204. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004630/

Article Info:

Academic Editor

Md. Ekhlas Uddin, Department of Biochemistry and Molecular Biology, Gono Bishwabidalay, Dhaka, Bangladesh.

Received

January 1, 2019

Accepted

February 23, 2019

Published

February 31, 2019

Article DOI: 10.34104/ajpab.019.019111

Corresponding author

Azizul Haque*

Department of Microbiology, University of Chittagong, Bangladesh.

Cite this article

Rahman MA, Haque A, Ahmad T, Mahmud S, Sohana SN, Hossain MR, Barman NC, Badiruzzaman M, Hossain T, Haque MS, Uddin ME, and Ahmed R. (2019). Isolation, identification, and antibiotic sensitivity pattern of Salmonella spp from locally isolated egg samples. Am. J. Pure Appl. Sci., 1(1), 1-11. https://doi.org/10.34104/ajpab.019.019111