MOLECULAR CHARACTERIZATIONS OF SOME HAZARD BACTERIA ISOLATED FROM TABLE EGGS

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MOLECULAR CHARACTERIZATIONS OF SOME HAZARD BACTERIA ISOLATED FROM TABLE EGGS

ABDOU, M.S.¹; NAHLA A. EBIED²; EMAN, T. ALSOKARY³

¹ Bacteriobiology Unit., Kafr El-Sheikh Lab., Animal Health Research Institute, Egypt.

² Food Hygiene Unit., Kafr El-Sheikh Lab., Animal Health Research Institute, Egypt.

³Biochemistry and Toxicology Unit., Kafr El-Sheikh Lab., Animal Health Research Institute, Egypt.

Received: 26 March 2017;       Accepted: 23 April 2017

INTRODUCTION

Chicken table egg is considered as the most valuable and perfect foodstuffs to human specially infants and elderly (Paskal et al., 2014). At the same time, Eggs have been described as the most critical food vehicles of pathogenic microorganisms participating in the etiology of food borne diseases in humans (Stepien-Pysniak, 2010).

Corresponding author: Dr. ABDOU, M.S.

E-mail address: dr.mostafaabdou@yahoo.com

Present address: Bacteriobiology Unit., Kafr El-Sheikh Lab., Animal Health Research Institute, Egypt.

Contamination of eggs and egg products with microorganisms can affect egg quality, which may lead to spoilage and pathogen transmission. This may induce cases of food-borne infection or intoxication to consumers, which constitute public health hazards. Several pathogenic microorganisms have been isolated from the surface of chicken egg shells and contents, among them, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella and Campylobacter (Moore and Madden, 1993; Schoeni and Doyle, 1994; Hope et al., 2002; Adesiyun et al., 2005).

Eggs are liable to contamination either before laying (congenitally) or after laying when the microorganisms reach the egg contents through penetration the shell and cause low egg quality, low shelf life and safety inducing public health hazards (Board and Fuller, 1994), in addition, fecal matter, improper washing, using of contaminated water and bad handling are the common sources of contamination. Egg can be contaminated by a variety of microbes such as E. coli and Salmonella (Ricke et al., 2001). Contamination of the shell occurs from nest material, floor litter, avian fecal matter, collector's hands, packing materials and improper washing (Moats, 1980).

E.coli is a normal inhabitant of the intestinal tract of both man and animals and can penetrate the shell contaminating the egg contents (Mayes and Takeballi, 1983).

E. coli constitutes a major economic menace to poultry industry and consequently is of public health importance for human causing profuse watery diarrhea which is varying in its severity and persistence due to inflammation of the intestinal mucosa(Schiavoni and Vergora, 2000). The organism is taken as index of recent fecal contamination. Quiroga et al. (2000) stated that diarrhoegenic E. coli is the major agent involved in diarrhoeal disease in developing countries, could isolate E. coli from 35% of diarrhoeal and 26% of non diarrhoeal cases.

E. coli can multiply in egg content and cause infection when the number of the organism reaches 10⁵ – 10⁷ organisms/g (Eley, 1996). Bacterial contamination can happen at three main part of egg (egg yolk, albumen and shell memberan\egg shell)Bagrouz and Al-Jaff, (2005).

Escherichia coli can cause diarrheal disease in humans, referred to as diarrheagenic E. coli. These including enteropathogenic (EPEC), enterohaemorrahgic (EHEC), enteroinvasive (EIEC), enterotoxigenic (ETEC), Shiga toxin-secreating (STEC), diarrhea-associated haemolytic (DHEC), entero- aggregative (EAAggEC) and cytolethal distending toxin- recreating (CDTEC) E. coli strains. WHO (2009) reported also that each year, infections and persistent diarrhea in children in developing countries are not rare, as observed in infants living in the Nile delta area, who experienced between 4.6 and 8.8 diarrheal episodes, with ETEC accounting for 66% of these episodes. The production of cytotoxin in E. coli isolates have been extensively studied among isolates from humans and several animal species including poultry (Zahraei Salehi et al., 2007).

Salmonella enteritidis is able to invade the cells of the follicles before ovulation and multiply themselves after 2 h of infection Howard et al. (2005). Moreover, Salmonella spp. remains a potential threat to human health, as well as, broiler chickens. Avian salmonellosis has public health importance associated with gastroenteritis in man resulting from consumption of infected hen's and duck's eggs (Abouzeed et al., 2000). Different Salmonella spp. could be isolated from human diarrhoeal swapsby several authers (Urio et al., 2001 and Biendo et al., 2003).

Salmonella human infection resulting from the consumption of contaminated eggs is still a major public health problem (Koen et al., 2006).

Staphylococci are most common bacteria contaminating egg shells and egg contents from external or during formation and laying process(Abdullah, 2010).

Furthermore, among the pathogenic food poisoning organisms that affect the public health of humans due to consumption of eggs is Staph. aureus which is of serious concern to public health(Wyah, 1992). Its thermostable enterotoxins elaborated in large numbers of foods and animal products including eggs causing rapid onest of nausea, vomiting and diarrhea within 6 hours of ingestion of food. Several outbreaks of Staph. aureus food poisoning have been recorded, involving large number of individuals throughout the world (Ko and Chang, 1995). Also, there have been many research works that deal with Staph. aureus in hen's eggs (Sabreen, 2001 and Bastawrows et al., 2002).

Staphylococcal food-borne disease (SFD) is one of the most common food-borne diseases worldwide resulting from the contamination of food by preformed S. aureus enterotoxins.

MATERIALS AND METHODS

1. Collection of samples:

A total of 50 random samples of Baladi eggs and Farm eggs, 25 samples of baladi eggs (125 intact eggs) and 25 samples of farm eggs (125 intact eggs) were collected from different supermarkets in Kafrelsheikh governorate. The eggs were transported to the laboratory and each sample was placed in a sterile plastic bag and carried to the laboratory without undue to be examined microbiologically (Cultured within 6 hours of collection).

2. Preparation of samples:

The egg was prepared for evacuation of its content according to APHA (2004)recommended by El-Kholy et al. (2014). Each egg was washed with warm water (32ºC) using a brush and soap, the egg was drained and immersed in 70% Alcohol for 10 min, then flamed after it has been removed from alcohol. A hole was made in the blunt end of the egg by using sterile scalpel. The content of each egg sample(content from 5 eggs were pooled to form one sample) was removed aseptically and received into a sterile mixer until the sample became homogenous and from the pooled egg content ten-fold serial dilutions up to 10⁵ were aseptically prepared using 0.1% sterile peptone water. The prepared samples were subjected to the following examinations:                                                                                           

2.1. Aerobic Plate Count (APC) (APHA 2004): The technique was applied by using surface plating method on standard plate count agar. Plates showing colonies between 30 and 300 were selected and counted.

2.2. Total coliform count using three tubes most probable number(MPN) method: (FDA, 2002)            

Lauryl sulphatetryptose broth (LST) and brilliant green lactose bile (2%) broth (BGLB broth) were used for presumptive and confirmed tests for total coliform, respectively.

2.3. Staph aureus count (APHA 2004): The surface plating technique of Baird–Parker ager plates was followed. Further, the suspected colonies were picked up and identified biochemically.          

3. Isolation and identification of E. coli (Bailey and Scott, 1990) and (ISO, 2004):

For the isolation and identification of E. coli, 1 ml of homogenized egg contents was inoculated into MacConkey enrichment broth and incubated at 37°C for 24 hours, the positive enriched sample with gas production was cultured on Eosin Methylene Blue (EMB) Agar plates and incubated at 37 °C for 24 hours. Morphologically, typical colonies (at least 4 / plate) producing metallic sheen on EMB were stabbed into semisolid agar tubes for further identification. Biochemical tests according to Macfaddin (2000) were performed to confirm E. coli using Catalase, Indole, Methyl red, Voges- Proskauer, Nitrate reduction, Urease production, Simon citrate agar and various sugar fermentation tests. The purified colonies were confirmed by using API strips (BioMerieux, Mary- I'Etoile, France). The positive isolates were serologically identified according to Kok et al. (1996) by using rapid diagnostic E.coli Set1: O and Set 2: H antisera sets (DENKA SEIKEN Co., Japan).

4. Isolation and identification of Salmonellae (Mackie and McCartney, 1989 and Quinn et al. 2004):

For the isolation and identification of Salmonellae, 1 ml of homogenized egg contents was inoculated into 10 ml of Rappaport Vassilidis broth (enrichment broth) followed by S.S as selective plating media. The suspected colonies appeared as red colonies with or without black center were identified biochemically and serologically. The purified colonies were confirmed by using API strips (BioMerieux, Mary- I'Etoile, France).

In general, serological identification of Salmonellae was carried out according to Kauffman – White scheme (Kauffman, 1974) for the determination of somatic (O) and flagellar (H) antigens using Salmonella antiserum (DENKA SEIKEN Co., Japan).

5. Isolation and identification of Staph. aureus as recommended by Mekonnen et al. (2011): Isolation of Staph. aureus was done by streaking (0.1ml)  of the enriched egg contents samples on mannitol salt agar  and Baird Parker agar supplemented with egg yolk and potassium tellurite and the plate was incubated at 37 °C for 24–48 hours. Typical coagulase-positive Staph.aureus colonies are yellow colonies surrounded with halo zone on  mannitol salt agar or jet black shining convex colonies surrounded by white halo zone, 1-1.5 mm in diameter on Baird Parker agar were considered to be presumptive Staph. aureus. Characteristic colonies were stabbed into semisolid agar tubes for further identification by conventional methods including Gram’s stain and various biochemical tests including coagulase test with rabbit plasma, anaerobic utilization of glucose, catalase test, oxidase test, indole, nitrate reduction and hemolysis on sheep blood agar. The purified colonies were confirmed by using API strips (BioMerieux, Mary- I'Etoile, France).

Multiplex Polymerase Chain Reaction (multiplex PCR)

1. Primer sequences used in the study:

1.1. Primer sequences of antibiotic resistance genes for E. coli:

The molecular characterization of tetracycline resistance genes (tetB, tetC and tet D) of E.coli was applied using the following primers (Pharmacia Biotech):

1.2. Primer sequences of S. aureus used for PCR identification system:

The demonstration of virulence factors including enterotoxin genes (A, B, C & D) of S. aureus was adopted by using the following primers:

1.3. Primers sequences of B-lactamase resistance genes of Salmonella species:

Accurately, the molecular characterization of B-lactamase resistance genes as virulence factors of Salmonellae were represented by cefotaxime (blaCTX) and cephalosporine (blaCMY) as follow:

2. DNA Extraction using QIA amp kit (Shah et al., 2009):

After overnight culture on nutrient agar plates, one or two colonies were suspended in 20 ml of sterile distilled water, and the suspension was then heated at 100ºC for 20 minutes. Accurately, 50-200 µl of the culture were placed in Eppendorf tube and frozen at -20ºC till use.

3. Amplification of DNA:

3.1. Multiplex PCR detection of tet genes for E. coli (Karami et al., 2006):

All isolates of tetracycline-resistant E. coli strains were assessed for carriage of the tetracycline resistance genes tetB, tetC and tetD. The method was optimized using the following reference E. coli strains with known tetgenes. The following procedure was used: a small amount of biomass from a bacterial colony was added to a sterile thin-walled reaction tube containing Taq Master Mix, primers, MgCl₂, and distilled water. The multiplex PCR included primers for tetB, tetC (0.25 μM each) and tet D (3 μM) and 1.5 mM MgCl₂. The tubes were sealed with a drop of mineral oil and heated to 95°C for 15 min in a thermocycler to activate the Taq DNA polymerase. After 5 min DNA template denaturation at 94°C, 25 PCR cycles followed, with DNA denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min, and primer extension at 72°C for 1.5 min. Finally, PCR products were separated electrophoretically in a 2% agarose gel, visualized by staining with 0.5 μg/ml ethidium bromide, and examined in UV light. A 100 bp plus DNA Ladder (Qiagen, Germany, GmbH) was used to determine the fragment sizes.

3.2. Amplification of B-lactamase resistance genes of Salmonellae (Roschanski et al., 2014):

PCR amplifications were performed in 50 µL reactions containing 25 µL DreamTaq Green PCR Master Mix (Thermo Scientific, St. Leon Roth, Germany), 2 µL of each forward and reverse primer (10 pmol), 20 µL of sterile water and 1 µL of DNA-mixture. The PCRs were performed with an initial denaturation step of 95°C followed by 35 cycles – denaturation at 95°C for 30 sec, annealing for 30 sec and elongation for 1 min. at 72°C – before finishing the run a final elongation step at 72°C for 5 min was attached. Similar multiplex PCR conditions were applied to the DNA templates of negative control isolates. The amplified products were electrophoresed on 2% agarose gel, stained with ethidium bromide (5 μg /100 ml) and captured as well as visualized on UV transilluminator.

3.3. Amplification of enterotoxin genes of S. aureus (Mehrotra et al., 2000):

Ten µl of DNA sample was diluted in 990 µl of nuclease free water for PCR. The genomic DNA samples were amplified by PCR in a reaction mixture (25µl) containing 13.25 sterile dH₂O, 2.5ml 10 x buffer, 0.63ml 10mMNTPs, 1ml 25Mm Mgcl₂ , 1.25 µl primer F(20pmol/ml), 1.25 µl primer R (20pmol/ml) and fill up to 25 µl PCR grade water. Concerning the primers used for demonstration of S. aureus enterotoxins (sea, seb, sec &sed), the amplification was performed on a Thermal Cycler (Master cycler, Eppendorf, Hamburg, Germany). DNA amplification was performed using the following conditions: initial denaturation for 5 min at 95°C followed by 30 cycles of denaturation (94°C for 2 min), annealing (55°C for 1 min), and extension (72°C for 2 min).A final extension step (72 °C for 5 min) was performed after the completion of the cycles. Amplified products were analyzed by 3% of agarose gel electrophoresis (Applichem, Germany, GmbH) in 1x TBEbuffer stained with ethidium bromide and captured as well as visualized on UV transilluminator at 254 nm.

RESULTS

Table 1: Statistical analytical results of different bacterial count (/ml) in the examined samples of egg contents (n=25).

The different superscripts in the same row indicated significant differences

S.E*= Standard error of mean       

Table 2: Incidence of E. coli isolated from the examined samples of egg contents (n=25).

EPEC = Enteropathogenic E.coli ETEC = Enterotoxigenic E.coli

EIEC = Enteroinvasive E.coli

Table 3: Incidence of Salmonellae isolated from the examined samples of egg contents (n=25).

Table 4: Incidence of Staph. aureus in the examined samples of egg contents  (n=25).

Photograph (1): Agarose gel electrophoresis of multiplex PCR for identification of tetB (659 bp), tetC (418 bp) and tetD (787 bp)

Resistance genes of E. coli.

Lane M: 100 bp ladder as molecular size DNA marker.

Lane C+: Control positive E. coli for tetB, tetC and tetD genes.

Lane C-: Control negative.

Lanes 1 & 4 (O78 and O1): Positive E. coli strains for tetB gene.

Lanes 2 & 6 (O78 and O44): Positive E. coli strains for both tetB and tetD genes.

Lane 3 (E. coli O78): Positive E. coli strain for tetC gene.

Lane 5 (E. coli O2): Positive E. coli strain for tetD gene..

Photograph (2): Agarose gel electrophoresis of multiplex PCR for identification of B-lactamase resistance genes of Salmonella species represented by blaCTX (550 bp) and blaCMY (1007 bp).

Lane M: 100 bp ladder as molecular size DNA marker.

Lane C+: Control positive for blaCTX and blaCMY genes.

Lane C-: Control negative.

Lane 1 (S. Enteritidis): Positive strain for blaCMY gene.

Lanes 2, 3 (S. Enteritidis), 6 and7 (S. Kentucky): Positive strains for blaCTX gene.

Lane 5 (S.Typhimurium): Positive strain for blaCTX and blaCMY genes.

Lanes 4 (S. Enteritidis) and 7 (S. Virchow): Negative strains for both genes.

Photograph (3): Agarose gel electrophoresis of multiplex PCR of sea (120 bp), seb (478 bp), sec (257 bp) and sed (317 bp) enterotoxin genes for characterization of S. aureus.

Lane M: 100 bp ladder as molecular size DNA marker.

Lane C+: Control positive for sea, seb, sec and sed genes.

Lane C-: Control negative.

Lane 1: Positive S. aureus strain for sea and seb genes.

Lane 2: Positive S. aureus strain for sec gene.

Lane 4: Positive S. aureus strain for sea gene.

Lane 3: Negative S. aureus strain for enterotoxins.

DISCUSSION

In Egypt eggs most commonly used unwashed. Special attention has been paid for raw or undercooked eggs because the hens act as natural reservoirs of a variety of pathogens. The contamination occurs through the shell; But humidity, temperature and storage time are critical for migration of bacteria from the surface of the shell to the inner structures of the egg (Evencio et al., 2012).

Food-borne diseases caused by microorganisms are a large and growing public health problem. Most common foodborne pathogens associated with food of animal origin are Salmonella, Campylobacter, Listeramonocytogenes, Staphylococcus  aureus and E.coli (Akbar and Anal 2011, Ghasemian, (2011) and Akbar and Anal, (2013a). Freshly laid eggs are generally devoid of organisms. Exposure to environmental conditions as temperature and humidity influence the bacterial penetration and thus enhance the infection and spoilage (Theron et al., 2003).

Foodborne illness is a major public health problem and the main cause of diarrheal diseases affecting all developed and developing countries (Akbar and Anal, 2014).

In table (1)results ofAerobic plate count showed mean values of baladi eggs 2.48x10⁴± 0.39x10⁴ and for farm eggs 6.15x10³± 1.41x10³, coliform count showed a mean value of baladi eggs 9.53x10²± 2.16x10²   and for farm eggs 4.99x10²± 0.72x10² and staphylococcal count showed mean values of baladi eggs 2.05x10³± 0.91x10² and for farm eggs 5.00x10²± 0.30x10²and it was lower than the accepted 10x10⁵ gm/cfu as recommended by the International Commission on the Microbiological Specification for Food (ICMSF 1996). Statistical analytical results between baladi eggs and farm eggs was highly significant (p < 0.01) and these results were lower than Eman et al. (2015) they recorded that the mean value of total colony count was 5.36x10⁶ ± 1.07x10⁵cfu/gm., while the mean values of coliforms and Coagulase Positive Staphylococci were 2.61x10⁷ ± 5.22x10⁵ MPN/gm. and 1.94x10⁷ ± 3.88x10⁵cfu/gm., respectively, from chicken table eggs.

El-kholy et al. (2014) Evaluate the microbiological quality of poultry farms eggs in Beni-Suef city and results were The aerobic plate count (present in 50% of egg contents) and coliform count(present in 47.06 % of egg content) were 1.1x10³ ± 3x10² and 1.5x10³± 8.4x10²cfu/ml for egg content respectively, the high counts of coliforms may be due to bad sanitary conditions and/or delay in eggs collection from nests which were contaminated with fecal matters, Jull (1984).

Results in Table 2 revealed that incidence of E. coli isolated from the examined samples of baladi and farm egg contents (25 for each) were 4 isolates (16%) and 2 isolates (8 %) respectively. Serotypes of isolated E. coli were O1: H7 (1 isolate from farm eggs), O2 : H6(1 isolate from baladi eggs), O44: H18 (1 isolate from baladi eggs), O78(2 isolate from baladi eggs and 1 isolate from farm eggs).

E.coli is one of the major problems in chicken production influencing heavier losses and sever drop in egg production, about 5.5 % mortality and 10-20% drop in eggs was observed with E.coli infections Qu et al. (1997). Samah et al. (2015) declared a total of 36 E. coli isolates with prevalence rates of 18%. Among the total isolates, 21 (10.5%) isolates, 9 (4.5 %) isolates, and 6(3.0%) isolates were detected from over the shell, egg contents, and both shell and contents, respectively. Arathy et al. (2011) could detect an overall isolation rate of 12.2%, while 8% of the isolates were detected from shell and 5% from yolk samples, respectively. Isolation rate was reported by (Adesiyun et al., 2005)who recorded (37.0%) as an overall isolation rate, (28.3%) as an isolation rate from egg shell, while they recorded isolation rate from egg content samples (3.8%). Lower isolation rates were recorded by (Saitanu et al., 1994) who isolated E. coli from egg contents with a rate of 1.2%.Ansah et al. (2009) Isolated Escherichia coli from egg content. Resistant E. coli strains from the gut often cause contamination of eggs during lay with multi resistant E. coli (Turtura et al., 1990) El-kholy et al. (2014)recorded that E.coli could be detected in 11.76% of contents of poultry farm eggs.

Jehan et al. (2014) Isolated E.coli, in both baladi and farm eggs were in the following percentage (22) and (19) respectively. Egg shell contamination is the main reason for E. coli infection. Poor hatcher sanitation can leave a residue of E. coli from the previous hatch leads to yolk infections which occur during hatching process (Eric, 2011) and (Maha 2013) reported that Esherichia coli population can be used as measures of quality and sanitary processing condition (Kornacki and Johnson, 2001). Also, it is an ideal indicator organisms of fecal contamination, in human and animal feces, 90-100% of coliform organisms isolated are E.coli (Hurst et al., 2002). The conventionally identified E.coli strains isolated from different types of table eggs were serotyped into 7 different serotypes included O44, O111, O114, O125, O126, O127 and O128. E. coli serotyping is an important technique for making the proper diagnosis and epidemiological investigations during food borne outbreaks. Thus serotyping alone cannot be relied on for categorizing a strain of E. coli and the identification of specific virulance characteristics/genes must also be performed (Barlow et al., 1999). Ansaruzzaman et al. (2007) reported that classification of ETEC strains, largely based on O-antigen type, Colonization Factor (CF) expression pattern and toxin profile.  Escherichia coli is the major micro-organism isolated both from the surface and in the content of the egg, this may be attributed to the fact that Escherichia coli are normal inhabitants of intestinal tracts of birds (Singleton and Sainsburg, 1981). They have also been known to contaminate the surface of egg while the mechanical process can spread the bacteria through the eggs. Contaminations with the pathogen while in the field occur through improperly decomposed manure and poor hygienic practice of farm workers. Escherichia coli can bring about urinary tracts infections, pneumonia meningitis and peritonitis in humans (Schoeni and Doyle, 1994). Eman et al. (2015) isolatedE. coli from chicken table eggs 12 isolates(28.58%).

Photo1 showed that Multiplex PCR for identification of tetracycline resistance genes (tetB, tetC  and tetD) of E. coli were (O78 and O1) Positive E. coli strains for tetB gene ,(O78 and O44) Positive E. coli strains for both tetB and tetD genes, (O78) positive E. coli strain for tetC gene, (O2) positive E. coli strain for tetD gene. Diarrassouba et al. (2007) Recorded that Fifty-two E. coli isolates (70.3%) and nine Salmonella isolates (14.52%) were multi-resistant to at least nine antibiotics. The multi-resistant isolates were evaluated for the presence of tetracycline resistance, integron class 1, and bla CMY-2 genes by PCR. Of the 74 E. coli isolates, 55 isolates were resistant to amoxicillin and ceftiofur. Among these 55 resistant E. coli isolates, 45 (81.8%) and 22 (40.0%) were positive for bla CMY-2 and qacEΔ1-SulI genes, respectively. Tetracycline resistance was found in 56 isolates (75.8%) among which 12 (21.4%) and 24 (42.9%) gave positive results for tetA and tetB, respectively.

Musgrove et al. (2008) isolated E. coli from eggs and analyzed for their antibiogramagainst 16 antibiotics including ampicillin, tetracycline, gentamicin, and kanamycin. They reported that E. coli (73.20%) were susceptible to all antimicrobial used. They reported that some E. coli (29.90%) isolates exhibited resistance against tetracycline. Adesiyun et al. (2005) Isolated E. coli in 4.3% of egg contents of farm eggs also, they added that the frequency of E. coli founded in eggs depending on their rearing sources. Study presence of 14 tetracycline resistance determinants from E. coli isolated from animal origin  and analyses indicated that 97% of these strains contained at least 1 of 14 tetracycline resistance genes [tetA, tetB, tetC, tetD, tetE, tetG, tetK, tetL, tetM, tetO, tetS, tetA(P), tetQ, and tetX] examined, while the most common genes found in these isolates were tetB (63%) and tetA (35%), tetC, tetD, and tetM were also found, E. coli isolates from chickens were the only strains to have tetM, 31% of e.coli were highly resistant to tetracycline. Samah et al. (2015) reported the following results from baladichicken a total of 36 E. coli strains were isolated with an overall isolation rate of 18%, were judged by PCR, for E. coli strains, the blaTEM, sul1 and tetA(A) genes were tested for the B-lactams, trimethoprim sulphamethazone and tetracyclines, respectively and  E. coli virulence was tested by PCR through the testing ofiss, eaeA, stx1, stx2, tsh and papC genes.

Results in Table 3 and photo 2 revealed Incidence of salmonellae isolated from the examined samples of baladi and farm eggs contents (25 for each) were 5 isolates (20%) from baladi eggs (2 isolates for S. enteritidis and 1 isolate for S. typhimurium, S. kentucky and S. virchow) and 3 isolates (12%) from table eggs (2 isolates for S. enteritidis and 1 isolate for S. kentucky). Multiplex PCR for identification of B-lactamase resistance genes of Salmonella species were (S. enteritidis) Positive strains for blaCMY gene, (S. enteritidis and S. kentucky) Positive strains for blaCTX gene, (S. typhimurium) Positive strain for blaCTX and blaCMY genes, (S. enteritidis) and (S. virchow) Negative strains for both genes. Salmonella can be regarded as two types of infections. The first is primarily of importance for public health by causing food borne illness. The other type causes severe disease leads to great economic losses in poultry industry (Anbessa and Shiferaw, 2013). Chousalkar et al. (2010) and Samah et al. (2015) detected no Salmonella neither from on the shell nor from the egg contents. Salmonella spp. was isolated by other researcher as (Mona et al., 2014) who detected Salmonella in eggs at a rate of (1.5%). However, (Camilleri, 1992) stated that failure to detect salmonella spp. from eggs does not imply that local flocks are not infected by salmonella. Other researcher detected lowest incidence of Salmonella in table eggs with 0.07% in egg content by De Reu et al. (2006)who found 0.18% and Begum et al. (2010)who reported variable and very low incidence of Salmonella. On contrary, Abdul Aziz et al. (2012) failed to isolate Salmonella spp. these results variation could be attributed to different control measures applied against these bacteria.

Egg-associated Salmonellosis is a public health problem, the use of antibiotics in animals disrupts normal flora of intestine, resulting in emergence of antibiotic-resistant Salmonellae and their prolongedfecal shedding into the environment (Ahmed et al., 2011). Resistance genes may be transferred from microorganisms to others, or may be due to mutations and selection pressure in microorganisms. Salmonella are potentially dangerous because of their ability to producing potent enterotoxin (Singh et al., 2010). Soo Jin et al., (2001) Detected   β – lactamase gene distributed among salmonella isolates from animal origin using multiplex PCR. Eman et al. (2008) isolated salmonellae from baladi in percentage (3.3) salmonella from farm eggs in percentage (3) respectively. Salihu et al. (2015) Isolated salmonella (13.5%) from contents of farm eggs at retail outlets in Sokoto metropolis Nigeria, and they recorde that the high percentage of isolation of salmonella from the egg contents may be due to the organism's presence in the ovary or oviducts before the shell forms around it. Our results agrees with (Kinde et al., 2000). Van et al. (2005) reported that the bacterium infects the eggs by either vertical transmission during development of these eggs within the ovary or horizontal transmission through trans-shell contamination.

InTable 4 and photo 3 Incidence of s.aureus isolated from the examined samples of baladi and farm egg contents (25 for each) were 2 isolates (8%) isolated from each type. Multiplex PCR of enterotoxins genes (sea, seb, sec and sed) for characterization of s. aureus, 1 isolate positive s. aureus strain for sea and seb genes, 1 positive isolate s. aureus strain for sec gene, 1 isolate positive s. aureus strain for sea gene and 1 isolate negative s. aureus strain for enterotoxins genes. Table eggs were bacteriologically better than baladi eggs. Abdullah (2010) reported the highest degree of egg contamination with Staphylococcus spp and eggs laid in dirty environment contained more bacteria than eggs lay in clean environment. The surrounding environment and storage condition including temperature and storage duration can influence the level of bacterial contamination (Stepien 2010). However, some of the common forms of Staphylococci are associated with poultry infections. Increasing attention has been given to the role of poultry and poultry products, including eggs, as a potential source of infections in humans induced by antibiotic-resistant Staphylococcus strains (Abulreesh and Organji 2011). Staphylococcus aureus and other spp. are important Pathogens in human and veterinary medicine, beside their importance in regard to food hygiene because of their ability to form staphylococcal enterotoxins (SEs).Jannatul Fardows et al. (2016) Recorded that out of 150 egg shells, 120 (80%) yielded growth of different bacteria, of them, Staphylococcus spp. were 80 (66.67%), out of 80 Staphylococcus spp., 30 (25%) were Staphylococcus aureus and  sensitive to ciprofloxacin. Staphylococcus is considered to be a normal flora of chickens, isolated from the skin and feathers as well as in the respiratory and intestinal tracts Andrew Bryan et al. (2004) and Casey et al. (2007). Goto et al. (2007) recorded that the enterotoxin genes of 30 strains of Staph. aureus were detected with LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes and the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. Lee et al. (2007) reported that verifying the actions of toxin genes in humans is very important. Therefore, the expression of enterotoxin genes in S. aureus was confirmed by a reverse transcription real-time PCR that facilitates detection on an RNA level. Consequently, comparing the expression of various staphylococcal enterotoxin genes can provide a more effective evaluation of the toxic hazards of S. aureus. Siriporn et al. (2015) Isolated Staphylococcus aureus (18.4%), from table eggs soled in markets. Arathy et al. (2011) reported bacteria of 15 different genus, included Staphylococcus spp., Staphylococcus spp. (18.40% in market and 28.40% farm layer) was predominantly found associated to egg.

The degree of contamination with bacteria depending on the source of eggs (Stepien-Pysniak et al., 2009; Shareef et al., 2009)the safety of eggs depends on the number of bacterial cells on shell and content of eggs for presence of factors that initiate pathogen multiplication (Ricke et al., 2001). The risk of illness resulting from consumption of contaminated eggs depends not only on the number of bacterial cells in eggs, but also on the type of bacteria (Bradshaw et al., 1990). Staphylococcus aureus has been reported by Stepien-Pysniak et al. (2009) demonstrated that Staphylococcus spp. dominated in the yolks 38.8%, on the shells 58.9 and 2.5% in white of table eggs. Variance in prevalence of bacterial contamination from shell and content might attributed to penetration rate. These results supported by Al-Ali et al. (2012)who found that Salmonella spp. were the highest in penetration rate followed by Stapylococcus aureus and Escherichia coli through egg pours.Samah et al. (2015) reported the following results from baladichicken egg, eighty (40%) Coagulase Positive Staphylococci isolates were detected and the antibiotic susceptibility results were judged by PCR, for mecA, blaZ and tetK genes were tested for the evaluation of the susceptibility of Coagulase Positive Staphylococci against oxacillin, B-lactams, and tetracyclines, respectively and the virulence of Coagulase Positive Staphylococci was estimated through testing of the enterotoxins genes in addition to coagulase, spa and hlg genes

CONCLUSION AND RECOMMENDATIONS

From results obtained in this study we can concluded that farm table eggs were more hygienic than baladi table eggs (Aerobic plate count, coliform count, staphylococcal count and Incidence of E. coli and Salmonellae were higher in baladi eggs than farm eggs). Isolation of E.coli, Salmonellae and Staph.aureus from both baladi and farm eggs may be attributed to lack of sanitary conditions, so eggs should be considered as a vehicle for transmission of potentially pathogenic bacteria. It is important to handle eggs in such a way that microorganisms present do not have chance to multiply and to prevent eggs from becoming contaminated with other microorganisms (WHO, 2009). Improving hygienic practices during routine handling of eggs should be followed to limit the spread of such bacteria to humans. While less than 100 cell/ml of bacterial counts can be achieved if some better hygienic practices implemented, such as introduction of cooling system for eggs during production, transportation and during distribution process. Increase awareness of public health of producers, sellers and consumers. Also, antibiotic sensitivity tests should be done to the isolated bacteria to detect effective antibiotic in treatment and for saving our time, costs of treatment and decreasing our losses. It is concluded that table eggs should not be consumed raw. Also we can recommend strict hygienic measures to safe guard eggs from being deteriorated should be adopted in the farms and during handling and processing of eggs.

REFERENCE

Abdul Aziz, S.; Sabry, A.H. and Bahig, A.E. (2012): Microbial quality and aflatoxins content of commercially available eggs in Taif, Saudi Arabia. African J. Microbiol. Res., 6: 3337-3342.                                 

Abdullah, I.N. (2010): Isolation and identification of some bacterial isolates from table eggs. Journal of Veterinary Science.3 (2): 59-67.

Abouzeed, Y.M.; Hariharan, H.; Poppe, C. and Kibenge, F.S. (2000): Characterization of Salmonella isolates from beef cattle, broiler chickens and human sources on Prince Edward Island. Comp. Immunol. Microbiol. Infect. Dis., 23 (4): 253–266.

Abulreesh, HH. and Organji, SR. (2011):The Prevalence of Multidrug-resistant Staphylococci in Food and the Environment of Makkah, Saudi Arabia. Res. J. Microbiol. 6: 510-523.

Adesiyun, A.; Offiah, N.; Seepersadsingh, N.; Rodrigo, S.; Lash-ley, V. and Musai, L. (2005): Microbial health risk posed by table eggs in Trinidad. Epidemiol Infect., 133: 1049-1056.

Ahmed, M.M.; Rahman, M.M.; Mahbub, K.R. and Wahiduzzaman, M. (2011):Characterization of Antibiotic Resistant Salmonella spp Isolated from Chicken Eggs of Dhaka City. J. Sci. Res. 3 (1), 191-196.

Ahmed, A.; Motoi, Y.; Sato, M.; Maruyama, A.; Watanabe, H.; Fukumoto, Y. and Shimamoto, T. (2007): Zoo animals as a reservoir of Gram-negative bacteria harboring integrons and antimicrobial resistancegenes. Appl. Environ. Microbiol. 73: 6686-6690.

Akbar, A. and Anal, A.K. (2011): Food safety concerns and foodborne pathogens, Salmonella, E.coli and Campylobacter.

Akbar, A. and Anal, A.K. (2013a): Prevalence and antibiogram study of Salmonella and Staph. aureus in poultry meat. Asia Pacific Journal of tropical Biomedicine. 3(2): 163-168.

Akbar, A. and Anal, A.K. (2014): Zinc oxide nanoparticles loaded active packing a challenge study against S.typhimurium and staph. aureus in ready to eat poultry meat. Food Control Journal. 38:88-95.

Al-Ali, M.A.; Al-Bahry, S.N.; Mahmoud, I.Y. and Al-Musharafi, S.K. (2012): Penetration of food spoilage and food poisoning bacteria into fresh chicken egg: A public health concern. G.J.B.B., 1: 33-39.

Anand, S.K.; Pondey, N.K.; Mohapotra, C.M. and Verma, S.S. (1994): Microbial studies on some convenient egg products. Indian - Journal of Poultry Science, 29 (2): 198-200.

Anbessa, D. and Shiferaw, D. (2013): Correlation of traditional knowledge with laboratory bases salmonella detection on egg shell and content of market sold egg. Asian J. Biological Sci., 6: 168-174. Anon, 2004. Report of the survey of Salmonella contamination of UK produced shell eggs on retail sale. Food Standard Agency, London.

Andrew Bryan, NirShapir, and Michael J. Sadowsky (2004):Frequency and Distribution of Tetracycline Resistance Genes in Genetically Diverse, Nonselected, and Nonclinical Escherichia coli Strains Isolated from Diverse Human and Animal Sources Journal List Appl Environ Microbiol. Apr; vol.70 (4); 2503–2507.

Ansah, T.; Dzoagbe, G.S.K.; Teye, G.A.; Adday, S. and Danquah, J.K. (2009): Microbial quality of table eggs sold on selected markets in the Tamale municipality in the Northern Region of GhanaLivestock Research for Rural Development 21 (8).

Ansaruzzaman, M.; Bhuiyan, N.A.; Begum, Y.A.; Kuhn, I.; Nair, G.B.; Sack, D.A.; Svennerholm, A.M. and Qadri, I.F. (2007): Characterization of enterotoxigenic Escherichia coli from diarrhoeal patients in Bangladesh using phenotyping and genetic profiling. J. Med. Microbiol., 56: 217-222.

Arathy, S.; Vanpee, G.; Belot, G.; Vanessa, M.; Claude, D. and Ravindra, N.S. (2011): Bacterial contamination of commercial chicken eggs in Grenada, West Indies. West Indian Veterinary Journal. 9 (2): 4-7.

APHA (American Public Health Association) (2004): Compendium of Methods for the Microbiological Examination of Food. 17th ed. Washington D. C. USA.

Bailey, W.R. and Scott, E.G. (1990): Diagnostic Microbiology. A textbook for isolation and identification of pathogenic microorganisms. The C.V. osby Company Publishing.St. Louis, Tonasto.

Bagrouz, M.A. and Al-Jaff. (2005):The risk of bacterial contamination in hen eggs of Sulaimani  poultries. Journal of Zankoy Sulaimani. 8A(1):63-71.                                                    

Barlow, R.S.; Hirst, R.G.; Norton, R.E.; Asshhurst- Smith, C. and Bettelheim, K.A. (1999): Novel serotype of enteropathogenicEcherichia coli (EPEC) as a major pathogen in an outbreak of infantile diarrhoea. J. Med. Microbiol, 48: 1123-1125.

Bastawrows, A.F.; Sayed, A.M.; Makar, N.H. and Thabet, A.R. (2002): Microbiological profile of commercial hen's eggs in Assiut governorate. Part 2: Campylobacter jejuni and S. aureus organisms in hen's eggs. Assiut Vet. Med. J. 45 (89).

Begum, K.; Reza, T.A.; Hague, M.; Hossain, A. and Hassan, F.M.K. et al. (2010): Isolation, Identification and antibiotic resistance pattern of Salmonella spp. from chicken eggs, intestines and environmental samples. Bangladesh Pharmaceut. J., 13: 23-27.

Biendo, M.; Laurans, G.; Thomas, D.; Dechepy, O.; Hamdad – Daoudi, F.; Canarelli, B. and Fb, F. (2003): Regional dissemination of Salmonella entericaserovarenteritides in season dependent. Clin. Microbiol. Infect. 9 (5): 360–369.

Board, R.G. and Fuller, R. (1994): Microbiology of avian egg. 1st ed. Chapman and Hall p. 112-128.

Bradshaw, J.G.; Shah, D.B.; Forney, E. and Madden, J.M. (1990): Growth of Salmonella enteritidis in yolk of shell eggs from normal and seropositive hens. J. Food Prot., 53: 1033-1036.

Camilleri, F. (1992): Incidence of salmonellosis in local hens' eggs. J R Soc Health. Oct; 112(5): 212-3.

Casey, AL.; Lambert, PA. and Elliot, T.S.J. (2007):Staphylococci. International Journal of Antimicrobial Agents, 29, 23-32.

Chousalkar, KK.; Flynn, P.; Sutherland, M.; Roberts, JR. and Cheetham, BF. (2010): Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs. Int. J. Food Microbiol. 2010 Aug 15; 142(1-2).

De Reu, K.; Heyndrickx, M.; Grijspeerdt, K.; Rodenburg, B. and Tuyttens, F. (2006): Assessment of the vertical and horizontal aerobic bacterial infection of shell eggs. J. World's Poutry Sci., 62: 564.

Diarrassouba, Fatoumata; Diarra, Moussa Sory; Bach, Susan; Delaquis, Pascal; Pritchard, Jane; Topp, Edward; Skura, Brent J. (2007): Antibiotic Resistance and Virulence Genes in Commensal Escherichia coli and Salmonella Isolates from Commercial Broiler Chicken Farms Journal of Food Protection, Number 6, June, pp. 1316-1526 (12).

Eley, A.R. (1996): Microbiol Food Poisoning. 1^st Ed. Chapman and Hall publisher, London, New York.

El-kholy, A.M.; Hassan, G.M. and Dalia, M.A. (2014): Microbiological quality of poultry farm table eggs in Beni-Suef city, Egypt. Assiut Vet. Med. J. Vol. 60 No. 142:10-13.

Eman Korashy, A.; Nahed M. Wahba and Hassanein, R. (2008): Public health hazards of some bacterial pathogens associated with consumption of eggs and studying the best cooking methods for their destruction Assiut Vet. Med. J. April. Vol. 54.  No. 117.

Eman, F. Abdel-Latif and Saad, M.F. (2015): Microbiological profile of leaking chicken table eggs. International Journal of science and Nature, vol. 6(1)51-55.

Eric, G. (2011): E. coli infections in poultry. Nutrition on line. Diamond V, pp: 1-4.

Evencio-Luz, L.; Lima-Filho, J.V. and Evencio-Neto, J. (2012): Occurrence of Salmonella sp. and coagulase-positive staphylococci in raw eggs and Coalho cheese: comparative study between two cities of Brazil's northeast. Braz J Microbiol. 2012 Oct-Dec; 43(4): 1463-1466.

FDA “Food and Drug Administration” (2002): Isolation and Identification of Staph. aureus.Acumedia PI 7112 rev 6, March.

Ghasemian, S. (2011): The prevalence  of bacterial contamination of table eggs from retails markets by Salmonella spp., Listeria monocytogenes, Campylobacter Jejuni and E.coli in Shahrekord, Iran, Jundishapur Journal of Microbiology.4(4): 249-253.

Goto, M.; Hayashidani, H.; Takatori, K. and Hara-Kudo, Y. (2007): Rapid detection of enterotoxigenic Staphylococcus aureusharbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay. Yukiko Hara-Kudo, Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.

Hope, BK.; Baker, R.; Edel, ED.; Hogue, AT.; Schlosser, WD.; Whiting, R.; McDowell, RM. and Morales, RA. (2002). An overview of the S. enteritidisrisk assessment for shell eggs and egg products. Risk Anal., 22: 203­218.

Howard, Z.R.; Moore, R.W.; Zabala-Diaz, I.B. and Landers, K.L. (2005): Ovarian laying hen follicular maturation and in-vitro Salmonella internalization. Veterinary Microbiology,108: 95-100.

ICMSF (International Commission on Microbiological Specifications for Foods) (1996): Microorganisms in Food 5. Microbiological Specification of Food Pathogen. London: Blackie Academic and Professional.

ISO (2004):International Organization for standardization. No.11291-1. Microbiology of food and animal feeding stuffs - Horizontal   methods for the detection and enumeration of Enterobacteriaceae.

Jannatul Fardows; Abu Bakar Siddique; Adneen Moureen; Tasmin Afroz; Binte Farhana and Chowdhury Rafia Naheen (2016): Isolation and identification of pathogenic gram-positive bacteria from egg shell of hen and to see their antimicrobial susceptibility pattern. Journal of Enam Medical College Vol 6, January No 1.

Jehan Ismail Ibrahim; Dalia Mansour, H. and Hosny Abdellatief Abdelrahman (2014): Prevalence and inhibition of microbial load on chicken eggs with special references to egg quality and hatchability. American Journal of Animal and Veterinary Sciences, 9 (4): 294. 302.

Jhalka Kadariya, Tara C. Smith, and Dipendra Thapaliya (2014): Staphylococcus aureus and Staphylococcal Food-Borne Disease: An Ongoing Challenge in Public Health. Bio-Med Research International. Volume 2014, Article ID 827965, 9 pages.

Jull, M.A. (1984): Poultry Husbandry. Tata McGraw - Hill Book Company, Inc. New York.

Karami, N.; Nowrouzian, F.; Adlerberth, I. and Wold, A. (2006): Tetracycline resistance in Escherichia coli and persistence in the infantile colonic microbiot. Antimicrob. Agents Chemother, 50 (1): 156-161.

Kinde, H.; Shivaprasad, HL.; Daft, BM.; Read, DH.; Ardans, A. and Breitmeyer, R. (2000): Pathologic and bacteriologic findings in 27-week-old commercial laying hens experimentally infected with Salmonella enteritidis, phage type 4.Avian Disease,44(2): 239-48.

Ko, H.C. and Chang, T.Y. (1995): Using the reversed passive latex agglutination method to detect enterotoxigenic Staphylococcus areus and enterotoxin in foods. J. Food & Drug Analysis. 3: 57–63.

Koen De Reu, Grijspeerdit; Heyn Drickxm; U. Yttendaele; M. Debevene and Hermnal (2006): Bacterial contamination in the egg collectionchains of different housing systems of laying hens. Br. Poultary Science, 2006, Apr; 47(2): 163-172.

Kauffman, G. (1974): Kauffmann white scheme. J. Acta. Path. Microbiol. Sci., 61:385.          

Kok, T.; Worswich, D. and Gowans, E. (1996): Some serological techniques for microbial and viral infections. In Practical Medical Microbiology (Collee, J.; Fraser, A.; Marmion, B. and Simmons, A., eds.), 14^th ed., Edinburgh, Churchill Livingstone, UK.                                                                                      

Kornacki, J. and Johnson, J. (2001): Enterobacteriacae, coli-forms and Escherichia coli as quality and safety indicators. In F.P. Downes and K Ito (Eds.), Compendium methods microbial. exam.food. (pp.69-80). Washington DC: American Public Health Association.

Lee, Young-Duck; Bo-Youn Moon; Jong-Hyun Park; Hyo-Ihl Chang, and Wang June Kim (2007): Expression of Enterotoxin Genes in Staphylococcus aureus Isolates Based on mRNA Analysis Microbiol. Biotechnol. Journal, 17(3), 461-467.

Macfaddin, J.F. (2000): Biochemical testes for identification of medical bacteria. Lippincott Williams & Wilkins; Philadelphia.

Mackie, T.J. and McCartney, J.E. (1989):Practical Medical Microbiology 13^th ed. Churchill Living stone Publishing, Edinburg, London, Melbourne and New York.

Maha, A.M. AL-Ashmawy (2013): Prevalence of Enterobacteriaceae in Table Eggs with Particular Reference to Enterovirulent Escherichia coli Strains. International Journal of Poultry Science 12 (7): 430-435.

Mayes, F.J. and Takeballi, M.A. (1983): Microbial contamination of the hen's egg. A review. J. food port. 46: 1092-1098.

Mehrotra, M.; Wang, G. and Johnson, W. (2000): Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance. J. Clin. Microbiol., 38: 1032–1035.

Mekonnen, A.; Mahindra, P. and Moses, N. (2011):Isolation and identification of Staphylococcus species from raw bovine milk in Debre Zeit. Ethiopia Medwell J., 4(2): 45-49.

Moats, W.A. (1980): The effect of washing eggs under commercial conditions on bacterial load on egg shells. Poult. Sci. 58: 1228-1233.

Mona, A.A. Abdel Rahman; Jakeen, Are. El-Jakee and Soad, A. Nasef, ( 2014):Prevalance of salmonellae from layers and layer breeders farms, 2^nd Conference of Scientific Association of Animal Health Research Institute, 2-6/2/2014.PP 420: -432.ISSN: 2356-7767.

Moore, J. and Madden, RH. (1993): Detection and incidence of Listeria species in blended raw eggs. J. Food Protect, 56: 652-654.

Musgrove, M.T.; Northcutt, J.K.; Jones, D.R.; N.A.; Cox, D.R. and Harrison, M.A. (2008):Enterobacteriaceae and Related Organisms Isolated from Shell Eggs Collected During Commercial Processing. Poult. Sci., 87: 1211-1218.

Ng, L.; Martin, I.; Alfa, M. and Mulvey, M. (2001): Multiplex PCR for the   detection of tetracycline resistant genes. Mol. Cell. Probes 15: 209-215.

Paskal, F.; Decesaro, A. and Manfreda, G. (2014): Improvement of sampling plan for salmonella detection in pooled table eggs by use of real time PCR. Int J. Food Microbiol. 84(1), 84-131.

Petrak, B.K.; Petrak, T.; Medic, H. and Novakovic, P. (2000): Bacteriological contamination of egg products after thermal preservation processes. Acta- Alimentoria- Budapest., 29(4): 315-322.

QU, F.Q.; Yang, S.Q.; Jiu, B.; Hang, D.Z, Z.; Li, H.W.; Liu, S. and Chen, S.Y. (1997): Diagnosis and control of swollen head syndrome in layers. Chinase J. Vet. Medicine. 23(7): 23.

Quinn, P.; Carter, M.; Markey, B. and Carter, G. (2004): Clinical Veterinary Microbiology. 6^th ed., Mosby, Edinburgh, London, NewYork, Philadelphia, St. Louis, Sydney, Toronto.                         

Quiroga, M.; Oviedo, P.; Chinem, I.; Pegels, E.; Husulak, E.; Binztein, N.; Rivas, M.; Schiavoni, L. and Vergora, M. (2000):A symptomatic infections by diarrheagenic Escherichia coli in children from Misiones, Argentina, during the first twenty months of their lives. Rev. Inst. Med. Trop. Sao Paulo, 42 (2): 9–15.

Rall, V.; Vieira, F.; Rall, R.; Vieitis, R.; Fernandes, A.; Candeias, J.; Cardoso, K. and Araujo, J. (2008): PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk. Vet. Microbiol., 132: 408–413.          

Refaat, B.M. (2009): Microbiological evaluation of commercial hens and ducks' eggs in Assiut Governorate. M.V.Sc. of milk Hygiene. Faculty of Veterinary Medicine. Assuit University.

Ricke , S.C.; Birkhold, S.G. and Gast, R.K. (2001):Eggs and eggs products in compendium of methods for the microbiological examination of food, 4 thed. Downes F.P and itok.eds American Public Health Association,Washington, D-C.473-479.

Roschanski, N.; Fischer, J.; Guerra, B. and Roesler, U. (2014): Development of a multiplex real-time PCR for the rapid detection of the predominant Beta-lactamase genes CTX-M, SHV, TEM and CIT-Type AmpCs in Enterobacteriaceae. Plos One, 9 (7): 1-9.

Sabreen, M.S. (2001): Search for some pathogenic bacteria in commercial hens and ducks' eggs sold in Assiut Governorate. Assiut Vet. Med. J. 45 (89): 91–103.

Saitanu, K.; Koowatananukul, C.; Jerngklinchan, J. and Sasipreeyajan, J. (1994): Detection of salmonellae in hen eggs in Thailand. Southeast Asian J Trop Med Public Health. 1994 Jun; 25(2): 324-7.

Salihu, M.D.; Garba, B. and Isah, Y. (2015): Evaluation of microbial contents of table eggs at retail outlets in Sokoto metropolis, Nigeria Journal of Veterinary Sciences 13(1): 22-28.

Samah Eid; Soad Nasef and Ahmed M. Erfan (2015): Multidrug resistant bacterial pathogens in eggs collected from backyard chickens Assuit. Vet. Med.J.Vol.61 No 144.

Schiavoni, L. and Vergora, M. (2000):A symptomatic infections by diarrheagenic Escherichiacoli in children from Misiones, Argentina, during the first twenty months of their lives. Rev. Inst. Med. Trop. Sao Paulo, 42 (2): 9–15.

Schoeni, JL. and Doyle, MP. (1994): Variable colonization of chickens perorally inoculated with Escherichia coli O157: H7 and subsequent contamination of eggs. Appl. Environ. Microbiol. , 60: 2958-2962.

Shah, D.; Shringi, S.; Besser, T. and Call, D. (2009):Molecular detection of foodborne pathogens, Boca Raton: CRCPress, In Liu, D. (Ed). Taylor & Francis group, Florida, USA, Pp. 369-389.

Shareef, A.M.; Mansour R.S. and Ibrahim, K.K. (2009):Staphylococcus aureus in commercial breeder layer flocks. Iraqi J. Vet. Sci., 26: 63-68.

Singh, S.; Yadav, AS. and Singh, SM. (2010): Prevalence of Salmonella in chicken eggs collected from poultry farms and marketing channels and their antimicrobial resistance.Food Research International43(8): 2027-2030.

Singleton, P. and Sainsburg, D. (1981): Dictionary of microbiology. International edition. John Willey and Sons Limited Publication, New York. Pap 425-426.

Siriporn Chaemsanit, Ali Akbar and Anil Kumar Anal (2015): Isolation of total aerobic and pathogenic bacteria from table eggs and its contents .Food and applied Bioscience Journal, 3(3): 1-9.

Soo Jin Yang, Kyoung Yoon Park and Keun Seok Seo (2001): Multidrug-resistant Salmonella typhimurium and Salmonella enteritidis identified by multiplex PCR from animals J. Vet. Sci. 2(3), 181-188

Stepien-Pysniak, D. (2010): Occurrence of Gram-negative bacteria in hens' eggs depending on their source and storage conditions. Polish Journal of Veterinary Sciences. 13(3): 507-513.

St^pien-Pysniak, D.; Marek, A.R. and Rzedzicki, J. (2009): Occurrence of bacteria of the genus Staphylococcus in table eggs descended from different sources. Pol. J. Vet. Sci., 12: 481-484.

Theron, H.; Enter, P.V. and Lues, J.F.R. (2003): Bacterial growth on chicken eggs in various storage environments. Food Res. Int., 36: 969-975.

Turtura, GC.; Massa, S. and Chazvinizadeh, H. (1990):Antibiotic resistance among coliform bacteria isolated from carcasses of commercially slaughtered chickens. Int. J. of Food Microbiol.11: 351-4.

Urio, E.M.; Collison, E.K.; Gashe, B.A.; Sebunya, T.K. and Mpuchane, S. (2001): Shigella and Salmonella strains isolated from children under 5 years in Gaborone, Botswana and their antibiotic susceptaibility patterns. Trop. Med. Int. Health. Jap. 6 (1): 55–59.

Van, IlF.; De Buck, J.; Boyen, F.; Pasmans, F.; Bertrand, S. and Collard, JM. (2005): Salmonella dans la viande de volaille et dans les oeufs, un danger pour le consommateur qui demande la mise en place d'un programme de lutteefficace. Journal of Veterinaries Medicine.149(1):34-48.

W.H.O "World Health Organization ", (2009): Diarrhoeal Diseases, Enterotoxigenic Escherichia coli (ETEC). ases /diarrheal/en/index4.html#.

Wyah, G.M. (1992): Immunoassays for Food Poisoning Bacteria and Bacterial toxins. 1^st Ed., Chapman & Hall, PP: 5–13.

Zahraei Salehi, M.T.; Safarchi, A.; Peighambari, S.M.; Mahzounieh, M. and Rabbani Khorasgani, M. (2007): Detection of stx1, stx2, eae, espB and hly genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction. J. Vet. Res., 62: 37-42.