Prevalence of some antibiotic resistant strains of Salmonellae in commercial table eggs in Port-Said City.

Gihan M.O. Mohammed^*, I.M. Helal^* and Hanan A. El-Ghiaty^**

*Dept. of Bacteriology, Animal Health Research Institute, Port-Said branch.

**Dept. of Food Hygiene Animal Health Research Institute, Port-Said branch.   

___________________________________________________________________________

                                           ABSTRACT

___________________________________________________________________________

تواجد بعض عترات السالمونيلا المقاومة للمضادات الحيوية في بيض المائدة التجاري

في مدينة بورسعيد

جيهان محمد عمر محمد، إيهاب محمود هلال ، حنان عباس الغياتى

 يعتبر البيض من أهم العناصر الغذائية التي تدخل في العديد من أصناف الطعام مثل الحلويات. ولما کان البيض النيئ يستخدم في تحضير بعض أصناف الطعام مثل المايونيز، المارنج والأيس کريم وبذلک قد يؤدي إلي العدوى بميکروب السالمونيلا في بعض الأحيان، خاصةﹰ وأن معظم الدراسات تشير أن هناک ارتباط وثيق بين تناول البيض النيئ والغير مطهو جيدا والإصابة بذلک الميکروب. لذلک تم تجميع 140 عينة من بيض المائدة المعد للتسويق بمدينة بورسعيد بهدف عزل وتصنيف عترات السالمونيلا المقاومة للمضادات الحيوية التي تظهر نتيجة للاستخدام العشوائي للمضادات الحيوية والوصول لأفضل مضاد حيوي للتخلص من ميکروب السالمونيلا مما يشکل استخدام آمن للبيض النيئ الذي يدخل في العديد من الأطعمة وما له من أثر علي الصحة العامة. وقد أفضت النتائج إلي أن نسبة العينات الايجابية لبکتيريا السالمونيلا کانت 3.6% وتم تصنيف عترات السالمونيلا المعزولة من بيض المائدة سيرولوجيا وقد أوضحت النتيجة بأنها سالمونيلا تيفيميوريم (40%), سالمونيلا أجونا (20%) وسالمونيلا ديربي (40%). أما بالنسبة لنتائج اختبار الحساسية, فقد تبين أن کل العترات في غاية الحساسية لکل من دوکسي سيکلين هيدروکلورايد، کلورامفينکول وترايميثوبريم سالفميثاکسازول. بينما أظهرت مقاومة عالية للإيريثروميسن. وقد کان 50% من معزولات سالمونيلا تيفيميوريم مقاومة لکل من ستربتوميسين, ناليدکسيک أسيد, أوکسي تتراسيکلين وبنسيلين-ج على العکس من ذلک, کان باقي معزولات سالمونيلا تيفيميوريم حساسة لهذه المضادات الحيوية. کذلک کانت سالمونيلا ديربي حساسة لکل من نيومايسين وأوکسي تتراسيکلين على العکس من ذلک, کان باقي معزولات سالمونيلا ديربي متوسطة الحساسية لهذه المضادات الحيوية. هذا وقد تم مناقشة الأهمية الصحية وکذلک الاحتياطات التي يجب مراعاتها للحد من خطورة ميکروب السالمونيلا.

__________________________________________________________________________

Key words:  table chicken eggs, Salmonellaspecies, antibiotic resistance, public health hazard.

_________________________________________________________________________________

INTRODUCTION

Table eggs are one of the most economic and balanced source of protein, with a relatively lower cost than chicken or meat (per kg) in most countries. In addition, eggs contain unsaturated fatty acids, iron, phosphorus, trace minerals, and vitamins (Watkins, 1995). Due to its exceptional nutritive value, eggs remain a potential host for pathogens like Salmonella spp. Gast and Beard (1992) suggested that, outbreaks of human salmonellosis, related to consumption of eggs, occurred as a consequence of three independent events; (i) contamination of eggs with Salmonella spp. by infected hens, (ii) improper handling of eggs or products allowing proliferation of the microorganisms to infectious levels, and (iii) ingestion of raw or undercooked contaminated eggs. It is apparent from the voluminous reports that Salmonella spp.were more oftenly associated with food borne    disease     outbreaks      than     other

pathogens particularly those associated with egg and egg products (Shirota et al., 2001). Unlike other pathogens, Salmonella serovars can infect the egg before it is laid or may contaminate the shell during the laying process (Humphrey, 1999). More than 90% of food borne salmonellosis, were due to contaminated egg shells (Woodward et al., 1997). Food borne Salmonellae were estimated to cause 1.3 million illnesses, 15,000 hospitalizations and 500 deaths per year (Schroeder et al., 2005).

Generally, Salmonella spp. are the causative agent of human gastroenteritis,an infection that results in a clinical syndrome generally known as salmonellosis which may manifested by severe abdominalpain, non-bloody diarrhea, myalgia, chills, nausea, headache, fever, vomiting, and prostration. In addition, other medical conditions such as pericarditis, neurological and neuromuscular diseases and reactive arthritis may result in some individuals after the infection (D^, Aoust, 1989). Clinical signs occur 12-72 h after consumption ofSalmonellaspp. the microorganism multiplies and colonizes the

small intestine, producing an enterotoxin that causes inflammatory reaction and diarrhea, and in some cases it can invade the blood stream to cause more severe illness (Poppe, 1999; D^, Aoust, 2001 and Bell and Kyriakides, 2002). Duration of gastroenteritis syndrome generally varied from 4 -10 days; during this period, microbial invasion of the small intestine and colon could affect absorption of nutrients in the patient (Poppe, 1999). Moreover, susceptibility of humans to salmonellainfections depend on a series of factors that include the dose of the pathogen, the type of contaminated food, and the age and immune condition of the host (D^, Aoust, 1989 and Poppe, 1999). The newborn, elderly, and immune compromised patients are more susceptible to infection by Salmonella spp. In these risk groups, salmonellosis could result in serious systemic infections with sporadic cases of death (D^, Aoust, 2001). On the other hand, healthy individuals rarely die from salmonellosis, and they normally recover from the disease after treatment with fluid and electrolyte replacement, while antibiotic therapy is not usually recommended in developing countries (Bell and Kyriakides, 2002). Eggs are one of the major animal foods that mostly marketed raw and frequently consumed raw. Many of the dishes like caesar salad, mayonnaise, eggnog, mousse and home-made ice cream, which form an important part of meals, contain raw eggs as an essential ingredient. These dishes are not heated up to the Food and Drug Administration recommended temperatures of 155°C for at least 15 seconds (Mermelstein, 2001).

Salmonellae are gram negative, non-spore forming rod-shaped bacteria belonging to the family Enterobacteriaceae. However, Salmonella is not included in the group of organisms referred to as coliforms. Salmonella is one of the principal causes of foodborne gastroenteritis worldwide and is also an important zoonotic pathogen of livestock (Scallan   et al., 2011). Salmonella

food poisoning is one of the most common and widely distributed diseases in the world (Gomez et al., 1997). Outbreaks were usually associated with ingestion of contaminated food of animal origin like, poultry, meat and milk
(Swartz, 2002). Although the majority of infections resulting in asymptomatic or self-limited disease; however, in immuno-compromised patients, neonates and elderly, it requires antibiotic treatment (Van et al., 2007). Recently, multi-drug resistant (MDR) strains have emerged, presumably due to the extensive use of antimicrobial agents both in human and animals. In veterinary practice, antibiotics are used in livestock production, disease prevention and as growth-promoting feed additives (Swartz, 2002). The use of antibiotics in animals disrupts the normal flora of intestine, resulting in the emergence of antibiotic-resistant salmonellae and their prolonged faecal shedding into the environment (Threlfall, 2002). The fatality rate in people infected with antibiotic-resistant salmonellae is 21 times greater than that infected with non-antibiotic resistant Salmonella strains (Altekruse et al., 1999).

 Egg-associated salmonellosis is a public health problem. Salmonellae infect ovaries of healthy hens and contaminate eggs before shell is formed and illness sometimes occurs, if such eggs are consumed raw or undercooked. The present study aimed for isolation and identification of different species of Salmonellae in chicken eggs of Port-SaidCity in addition to the resistance pattern to different antibiotics.

MATERIALS and METHODS

1-Collection of samples:

A total of 140 samples (70 egg shells and 70 egg contents) were collected from different markets inPort-SaidCity. Each individual sample was placed separately into sterile plastic bag, identified and delivered to laboratory in a refrigerated container. All specimens were processed within 24 hours of collection.

2- Bacteriological examination:

2-1 Preparation and enrichment of the samples:

Enrichment of egg samples was done according to Official Methods of Analysis of AOAC International(2005). Egg shells were washed with 10ml sterile Ringer’s solution. Both egg shells and Egg contents (yolk and white) 10 milliliter were enriched in 90ml Buffered peptone water and homogenized in a stomacher for two min. and incubated at 37°C ± 1°C for 18±2 h. After incubation 0.1 ml was inoculated into tube containing sterile 10 ml Rappaport-Vassiliadis Soy broth (RVS), and 1 ml of the same culture was inoculated into a tube containing 10 ml sterile Muller-Kauffmann tetrathionate/ novobiocin broth (MKTTn). The RVS broth was incubated at 41.5 °C ± 1°C for 24h±3h, and MKTTn broth at 37°C ± 1°C for 24h±3h. Isolation and identification was done according to the method recommended by ISO (2002). 

2-2 Serotyping of Salmonella isolates:

The antigenic formula of Salmonella serotypes are defined and maintained by  the

World Health Organization.

3-Antimicrobial susceptibility test:

The antimicrobial susceptibility test was performed using agar disc diffusion assay as described by National Committee for Clinical and Laboratory Standards Institute (NCCLS, 2004). Antimicrobials used were streptomycin 10 μg, neomycin 30 μg, penicillin 10 U, chloramphenicol 30 μg, erythromycin 15 μg, nalidixic acid 30 μg, oxytetracycline 30 μg, trimethoprim/sulphamethoxazole (1.25+23.75) and doxycycline hydrochloride 30 μg. Pure colonies of isolated Salmonellaewere emulsified in normal saline and turbidity was matched with 0.5 McFarland turbidity standards. Selected antimicrobial discs were placed on Mueller Hinton Agar plates seeded with bacteria. These plates were incubated at 37°C for 24 hours. The organisms were observed for antimicrobial sensitivity based on diameters of zones of inhibition on petridishes. Susceptible and resistant isolates were defined according to the criteria suggested by the NCCLS (2004).

RESULTS

Table 1: Prevalence of differentSalmonellaserovars isolated from chicken commercial table eggs:-

Table 2: Prevalence of different Salmonella serovars in the total isolates (5):-

Table 3: Antigenic structure of different Salmonellae isolated from chicken commercial table eggs:-

Table 4: Antibiotic resistance pattern of Salmonellae (5) isolated from chicken commercial table eggs:-

               S= susceptibility, I= Intermediate, R= Resistant

Table 5: Antibiotic resistance pattern of different Salmonella isolates from chicken commercial table eggs:-

DISCUSSION

Little attention has been given to antimicrobialresistance of bacteria isolated from commercial shell eggs orthe egg-processing environment. Salmonella may colonize the ovaries and peri-ovarian tissue of laying hens, and thus it has the potential for vertical transmission from breeders to layers and then to eggs sold for human consumption. The results of examination of 140 commercial chicken eggs showed that, Salmonella spp.wasobservedin (3.6%), (Table1).

Serological identification of the isolated Salmonella serovars obtained from commercial chicken table eggs, (Table 2), revealed the identification of Salmonella Typhimurium (40%), Salmonella agona (20%) and Salmonella derby (40%). The antigenic structures of the isolated serovars were shown in Table (3). Akhtar et al. (2010)isolatedSalmonella Typhimurium in a percentage of (14.58%) from Egg-shell, while Selvaraj et al. (2010) could detect Salmonella Typhimurium in a percentage of (5.88%) from egg wash. The difference in the percentage for isolation of Salmonella Typhimurium may attributed to difference in localities and methods of sampling.

Salmonellaantibiogram results were summarized in Tables (5) & (6). Highest resistance was observed for erythromycin (100%) then penicillin G. (40%). Resistance was also noted for streptomycin (20%), oxytetracycline (20%), and nalidixic acid (20%). Akhtar et al. (2010) demonstrated very high level of resistance to erythromycin by Salmonella enteritidis. Salmonella resistance at varying concentrations of penicillin, streptomycin, spectinomycin and erythromycin has also been reported by Sultana et al. (1995). High susceptibility was shown to chloramphenicol, doxycycline hydrochloride and trimethoprim-sulphamethoxazole by all isolated serovars. One isolate of theSalmonella Typhimurium serovars (50%) showed resistance for streptomycin, nalidixic acid, oxytetracycline and penicillin G., while the other Salmonella Typhimurium isolates (50%) showed high susceptibility for the same 4 antimicrobial drugs. Nearly similar results were shown for the isolated Salmonella derby, one isolate (50%) showed high susceptibility for neomycin and oxytetracycline, while it was resistant to penicillin G. The otherSalmonella derby isolates (50%) showed intermediate susceptibility for neomycin and oxytetracycline, while it was highly sensitive for penicillin G. This difference - for the same serovar - may be due to the empirical use of different antimicrobial drugs during the chicken production. Few studies have reported on antimicrobial resistance of Salmonella isolates collected from eggs or the egg-processing environment. In a study conducted in India, Bajaj et al. (2003) indicated that all of the 66 Salmonella isolates were susceptible to at least one of the compounds tested however, they did not report on the serotypes of Salmonellae. Verma and Gupta (1992) demonstrated the susceptibility of various Salmonella serovars to several antimicrobial drugs and reported high resistance to kanamycin, followed by trimethoprim-sulphamethoxazole and tetracycline. High susceptibility was shown to chloramphenicol, ampicillin and tetracycline.

Conclusion

This study focused on the indirect danger of the empirical use of antimicrobial agents in poultry industry, which leads to the release of resistant Salmonella serovars to the environment thus, creating a major health hazard problem. It also proved the importance of periodical antibiotic sensitivity tests for the Salmonella serovars from chicken to find the proper antibiotic which could be used in treatment of salmonellosis.

REFERENCES

Akhtar, F.; Hussain, I.; Khan, A. and Rahman, S.U. (2010):Prevalence and antibiogram studies of Salmonella enteritidis isolated from human and poultry sources. Pakistan Vet. J., 30, 1: 25-28.

Altekruse, S.F.; Cohen, M.L. and Swerdlow, D.L. (1999):Centers for Disease Control and Prevention (CDC), http://www.cdc.gov/.

AOAC (Association of Official Analytical Chemists) International (2005): Official Methods of Analysis of AOAC International, 18^th Edition. Gaithersburg, Maryland, USA, AOAC International.

Bajaj, B.K.; Sharma, V. and Thakur, R.L. (2003):Prevalence and antibiotic resistance profiles of Salmonella spp. in poultry eggs. J. Food Sci. Technol. 40: 682–684.

Bell, C. and Kyriakides, A. (2002): SalmonellaIn: Foodborne Pathogens: Hazards, Risk Analysis and Control. Eds Blackburn C de W., McClure P.J. Cambridge. Wood head Publishing Ltd., : 307-5.

D^, Aoust, J.Y. (1989): "Salmonella". In: M.P. Doyle (Ed.), Foodborne Bacterial Pathogens, Marcel Dekker, New York pp.: 327-445.

D^, Aoust, J.Y. (2001): "Salmonella". In: R.G. Labbé, and S. Garcia (Eds.), Guide to Foodborne Pathogens, Wiley-Interscience, New York, pp.: 163-191.

Gast, P.K. and Beard, C. W. (1992): "Detection and enumeration of Salmonella enteritidis in fresh and stored eggs laid by experimentally infected hens". J. Food Prot., 55: 152-156.

Gomez, T.M.; Motarjemi, Y.; Miyagawa, S.; Kaferstein, F.K. and Stohr, K. (1997): "Foodborne Salmonellosis". World Health Stat Q. 50, 81.

Humphrey, T.J. (1999): Contamination of eggs and poultry meat with Salmonella enterica serovar enteritidis. In: A.M. Saeed, P.K. Gast, M.E. Potter, and P.G. Wall (Eds.), Salmonella enterica serovar enteritidis in humans and animals: Epidemiology, pathogenesis, and control. IowaStateUniversity Press, Ames, IA, pp. : 183-192.  

ISO (2002): Microbiology of food and animal feeding stuffs- Horizontal method for detection of Salmonellaspp. ISO 6579 Organisation for Standardization.

Mermelstein, N.H. (2001): Pasteurization of shell eggs. Food Technology, 9:49-53.

National Committee for Clinical Laboratory Standards "NCCLS" (2004): Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; 2nd ed., NCCLS M100-S14. Clinical Laboratory Standards Institute (formerly NCCLS), Wayne, PA.

Poppe, C. (1999):  EpidemiologyofSalmonella enterica serovar enteritidis. In A.M. Saeed, P.K. Gast, M.E. Potter, andP.G. Wall(Eds.),Salmonella enterica serovar enteritidis in humans and animals.IowaStateUniversity Press, Ames, IA, pp.: 3-18. 

Scallan, E.; Hoekstra, R.M. and Angulo, F.J. (2011): Foodborne illness acquired in the United States--major pathogens. Emerging Infectious Diseases17(1):7- Emerging Infectious Diseases 15.

Schroeder, C.M.; Alecia, L.N.; Wayne, D.S.; Allan, T.H.; Fredrick, J.A.; Jonathon, S.R.; Eric, D.E.; Terry, D.W.; Kristin, G.H. and David, P.G. (2005): Estimate of illnesses from Salmonella enteritidis in eggs, United States, 2000. Emerging Infectious Diseases, 11(1): 113-115.

Selvaraj, R.; Das, R.; Ganguly, S.; Ganguli, M.; Dhanalakshmi, S. and Mukhopadhayay, S. K. (2010): Characterization and antibiogram of Salmonella spp. from poultry specimens. J. of Microb. and Antimicrobials, 2(9): 123-126.

Shirota, K.; Katoh, H.; Murasa, T.; Ho, T. and Otsuki, K. (2001):
Monitoring of layer feed and eggs for Salmonella in eastern Japan 1993 and 1998. J. Food Prot., 64, 5: 734-737.

Sultana, K.; Bushra, M.A. and Nafisa, I. (1995): Evaluation of antibiotic resistance in clinical isolates of Salmonella typhifrom Islamabad, Pakistan. Zoology, 27, 2: 185-187.

Swartz, M.N. (2002): Human diseases caused by foodborne pathogens of animal origin. Clin. Infect. Dis., 34, 3: 111–122.

Threlfall, E.J. (2002): Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections. FEMS Microbiol. Rev., 26, 2:        141- 149.

Van, T.T.H.; Moutafis, G.; Istivan, T.; Tranand, L.T. and Coloe, P.J. (2007): Detection of Salmonella spp. in retail raw food samples from vietnam and characterization of their antibiotic resistance. Appl. Environ. Microbiol., 73:6885.

Verma, J.C. and Gupta, B.R. (1992): Determination of minimum inhibitory concentration of antibiotics against Salmonella organisms from animal sources by using multiple inoculation. Indian J. Anim. Sci., 62: 793-796.

Watkins, B.A. (1995): The nutritive value of the egg. In: W.J. Stadelman, and O. J. Cotterill (Eds.), Egg Science and Technology. The Haworth Press, New York, pp.: 177-194.

Woodward, D.L.; Khakhria, R.L. and Johnson, W.M. (1997):Human Salmonellosis associated with exotic pets. J. Clinical Microbiology, 35, 11: 2786-2790.