MULTIDRUG RESISTANT BACTERIAL PATHOGENS IN EGGS COLLECTED FROM BACKYARD CHICKENS

SAMAH EID ^*; SOAD A. NASEF ^** and AHMED M. ERFAN^***

^*Bacteriology Dept., ^**Poultry Diseases Dept., ^***Biotechnology Dept.

Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute,  Nadi El-Seid St., P.O. Box 246, Dokki, Giza 12618, Egypt.

Email: samaheid@ymail.com

INTRODUCTION

Table eggs are consumed worldwide and are considered the most nutritious inexpensive source of protein that can be part of a healthy diet. However poultry may carry bacteria that can cause illness, infected birds do not usually appear sick and even unbroken clean fresh shell eggs may contain harmful bacteria. (Barbara and RON, 2010).

There is now considerable evidence that transfer of antimicrobial resistance from food-producing animals to humans directly via the food chain is a likely route of spread, transmission by direct handling or close contact between infected animals and humans, transmission via contaminated animal products, particularly but not exclusively food products. The World  Organisation for Animal Health (OIE) has developed a list of antibiotics categorized by the need for their use in animal treatment; The category ‘veterinary critically important antimicrobials’ includes fluoroquinolones, cephalosporins  and  macrolides , as well as a number of other families of antibiotics. Thus these antibiotics may all affect bacteria in both animal and human treatment settings (Wooldridge, 2012).

Escherichia coli is one of the common microbial flora of gastrointestinal tract of poultry and human, resistant E. coli strains from the gut often cause contamination of eggs during lay with multi resistant E. coli (Turtura et al., 1990).

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 prolonged fecal shedding into the environment (Ahmed et al., 2011).

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 (Casey et al., 2007). 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).

Backyards flocks are reared under limited or no veterinary supervision. In such production systems, antimicrobials are freely used as feed or water additives (Otalu et al., 2011). These practices can facilitate the emergence and spread of antibiotic resistant pathogens among birds with possible transmission to humans. Backyard chickens are extensively reared in close proximity to human dwellings and therefore play an important role in environmental contamination, in addition to serving as significant vehicles for the transfer of pathogens to humans by way of handling of live birds or consumption of contaminated meat and other poultry products. (Suleiman et al., 2013).

Thus the present study aimed to investigate the prevalence of multidrug resistant bacteria in egg produced from backyard chicken.

MATERIAL and METHODS

SAMPLING:

A total of two hundred eggs were collected from house hold backyard balady chicken from several villages in Sharkia governorate during summer 2014. Each egg was uniquely identified, and transported in a sterile plastic bag to the reference laboratory for veterinary  quality control on poultry production, Sharkia branch, kept in refrigerator at 2-5 ˚C till examined.

Sample preparation:

From each egg, the contents were separated from the shell; Each of the contents and the shell was collected in a separate sterile flask. Non selective pre-enrichment was performed by adding BPW in 1:10 dilution rate, samples were well mixed and  incubated at 37˚C±1  for 18 hours ±2.

Isolation and Identification:

Samples were examined for the detection of Salmonella Spp., Coagulase Positive Staphylococci and E.coli  according to ISO 6579:2002-COR 2004, ISO 6888-1:1999-AM:2003 and(Kreig et al., 1984), respectively.

Serotyping: E. coli isolates were serotyped in Reference Laboratory for Veterinary Quality Control on Poultry Production using commercially available kits (Test Sera Enteroclon, Anti –Coli, SIFIN Berlin, Germany).

Antibiogram: Antibiotic sensitivity was performed using Mueller Hinton Agar plates (HIMEDIA) using antibiotic discs of 14 commonly used chemotherapeutic agents, for E.coli isolates, and 15 commonly used chemotherapeutic agents for Coagulase Positive Staphylococci isolates according to (Bauer et al., 1966). Interpretation of the results based on the diameter of the inhibition zones produced was done according to (CLSI, 2011).

DNA extraction:

DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with few modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample was added to 20 µl of proteinase K and 200 µl of AL  lysis buffer and incubated at 56^oC for 10 min in a Biometra Tsc thermal block. After incubation, 200 µl of 100% ethanol was added to the lysate and vortexed. The sample was then washed twice and centrifuged according to the manufacturer’s instructions. DNA was eluted with 100 µl of elution buffer supplied in the kit.

Oligonucleotide Primers:

Different primers used in PCR were supplied from Metabion (Germany) and Biobasic (Canada) and are listed in Table (1) and Table (2).

PCR amplification:

A 25- µl master mix reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of 20 pmol conc. of each primer, 4.5 µl of water, and 6 µl of template DNA. The reactions were performed in Applied biosystem 2720 thermal cyclers.

Analysis of the PCR Products:

Fifteen microliters of each PCR product were loaded in each gel lane of 1.5% agarose gel (Applichem). Electrophoresis was done in 1x TBE buffer using 5V/cm gradients. A 100 bp DNA Ladder (Fermentas) was used to determine the fragment sizes. The PCR photos were photographed and analyzed by using a gel documentation system (Alpha Innotech, Biometra, Germany) through its computer software.

RESULTS

Prevalence of bacterial isolates:

Out of the total examined 200 eggs, no Salmonella was detected neither from on the shell nor from the contents. Regarding E.coli  a total of 36 isolates were detected with an overall isolation rate of 18%. Out of  total E.coli isolates, 21(10.5%) isolates were isolated from on  the shell, a total of 9(4.5%) from the contents,  and 6 (3%) from both shell and contents, respectively.  Concerning the isolation rates of Coagulase Positive Staphylococci, a total of 80 (40%)   isolates were detected; Of which, 29 (14.5%) isolates were detected from the shell,15 (7.5 %) isolates from contents, and 36 (18%)  isolates from both shell and contents. Out of  total examined 200 eggs, 15 (7.5%) harbor both Coagulase Positive Staphylococci and E.coli., as shown in“Fig.1”,and “Table 3”.

In the present study 36 E.coli were isolated out of total 200 examined eggs with an overall prevalence rate of 18%. Serotyping of  the isolates by slide agglutination technique revealed the distribution of the detected isolates in 13 different serotypes, which belonged to 6 somatic “O” groups ”2,3,4,5,6 ,and 8” . Five isolates  belonged to Poly  2 were detected, of which 1 strain O91 isolated from on the shell, O 125 (3 isolates were detected, of which  2 isolates from on the shell, and 1 isolate from contents), respectively; And  O 166 (1 isolate) from on the shell. Poly 3  ; O 145 (1 isolate) from on the shell. Poly 4 (14 isolates), of which 3 strains were O6 (2,1) from on the shell and contents, respectively; Eight isolates were identified as O27 (2,2, 4) from on the shell, contents, and from both on the shell and contents, respectively; Also 3 strains O 159 (2,1) from on the shell and contents, respectively. Poly 5 (2 isolates) 1 strain O 25, 1 strain O 153 both  were isolated from on the shell. In addition to 4 strains identified as Poly 6 of which 1 strain O 115 from on the shell, 3 strains O 169 (2,1) from the contents and from both on the shell and contents, respectively.  Finally 5 isolates were identified as Poly 8 of which 4 strains belonged to O 152(2,1,1)  from on the shell, contents and from both on the shell and contents, respectively; One strain O 29 from on the shell. Five untypeable isolates were detected (4,1) from on the shell and contents, respectively as shown in Table 3”.

Antibiotic susceptibility pattern of E.coli  isolates was studied using agar disc diffusion technique against 14 commonly field used chemotherapeutic agents. The study detected the prevalence of multidrug resistant MDR  E.coli ; As 34 (94.1%) of isolates were resistant  to more than 5 chemotherapeutic agents, 11 (29.4%) of  the isolates were resistant to more than 9 chemotherapeutic agents. The highest resistance rates were recorded against trimethoprim sulphate , doxycyclin, tetracycline, and amoxicillin; 94.1%, 93.2%, 92.9%, and 92.3 %, respectively. While the highest sensitivity rates were detected to colistin, cefotaxim, gentamycin, and ciprofloxacin; 94.4.%, 76.5%, 59.8%, and  55.6, respectivelyas shown in ”Table 4”, and “Fig.2”.

Antibiotic susceptibility pattern of Coagulase Positive Staphylococci isolates was studied using agar disc diffusion technique against 15 commonly field used chemotherapeutic agents. The study detected the prevalence of multidrug resistant Coagulase Positive Staphylococci; As 72 (90%) of isolates were resistant to 3 and more chemotherapeutic agents, 29 (36.7%) of isolates were resistant against 9 and more chemotherapeutic agents. The most predominant resistance rates were recorded against oxytetracyclin (90 %), followed by trimethoprim-sulphamethazone, and oxacillin 86.7%, each. The least resistance rates were recorded against ampicillin, ciprofloxacin, and gentamycin; 13.1%, 26.7 %, and 30 %, respectively,as shown in”Table 5” and “Fig 3”.

(Table 6) showing the relation between the PCR results of the different antibiotic resistance genes and antibiotic resistance profile of Coagulase Positive Staphylococci.

(Table 7)showing the PCR results of the different virulence genes of coagulase positive staphylococci; Positive results were detected for all genes except for Sea and Sed genes.

(Table 8) showing the relation between the PCR results of the different antibiotic resistance genes and antibiotic resistance profile of E. coli.

(Table 9) showing the PCR results of the different virulence genes of E. coli. All the genes were positively amplified in some isolates except for stx1 gene.

                Figure 1: Prevalence rates of isolates.

Figure 2: Antibiotic susceptibility profile of E.coli isolates.

Figure 3:Antibiotic  susceptibility pattern of Coagulase Positive Staphylococci isolates.

Table 1: Sequences and cycling conditions of the different PCR primers used for amplification of different virulence genes of E. coli and CPS.

Table 2: Sequences and cycling conditions of the different PCR primers used for amplification of different antibiotic resistance genes of E. coli, and CPS.

Table 3: Isolation rates of detected E.coli serotypes:

Table 4: Antibiotic susceptibility pattern of E.coli isolates

Table 5: Antibiotic susceptibility pattern of Coagulase Positive Staphylococci isolates:

Table 6: PCR results of the different antibiotic resistance genes and antibiotic resistance profile of Coagulase Positive Staphylococci.

Table 7: PCR results of the different virulence genes of the CPS isolates.

Table 8: PCR results of the different antibiotic resistance genes and antibiotic resistance profile of E. coli.

Table 9: PCR results of the different virulence genes of the E. coli isolates.

DISCUSSION

Eggs produced from backyard house hold chickens in Egyptian villages are commonly used for own consumption or to be sold in local markets,( 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  (Evêncio       et al., 2012).

In the present study 200 eggs produced from backyard chickens were examined for the presence of Salmonella spp., E.coli, and Coagulase Positive Staphylococci.

The study detected no Salmonella neither from on the shell nor from the egg contents. Although many researchers have reported similar results as (Chousalkar et al., 2010), higher isolation rates of Salmonella spp. were reported 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.

The study declared a total of  36 E.coli isolates with an overall 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.  Almost similar results were reported by (Arathy et al., 2011) they could detect an overall isolation rate of 12.2%, while 8% of the isolates were detected from shell and 5% from yolk samples, respectively. Higher  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 almost similar isolation rate from egg content samples (3.8%). Lower isolation rates were recorded by (Saitanu       et al.,1994) who isolated E.coli from egg shells and in egg contents with a rate of  3.5% and 1.2%, respectively.

Coagulase Positive Staphylococci including 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). The present study applied (ISO 6888-1:1999, AM: 2003) which specifies enumeration and detection of Coagulase Positive Staphylococci “among which enterotoxigenic strains are encountered” in products intended for human consumption or feeding of animals. Rosa et al. (2001) Suggested that Baird Parker is sufficient to screen the presence of Staphylococcus aureus without the need for further identification,  resulting in saving time and money. The study investigated eggs for the presence of Coagulase Positive Staphylococci both on the shell and in the contents, 80 isolates were detected with an overall prevalence rate of 40%. Isolation rates were 29(14.5%), 15 (7.5%), and 36 (18%) from on the shell,  contents, and both shell and contents, respectively. Higher prevalence rates were recorded by (Stepień et al., 2009) when they  reported the isolation of  Coagulase Positive Staphylococci from eggs with a rate of 45.7%, of which 2.5%, 38.7%, and 58.8% were detected from white, yolk, and on the shell, respectively.

In the present study, out of the 200 examined eggs 15 (7.5%) harbored both E.coli and Coagulase  Positive Staphylococci. This result was in agreement with that stated by (Obi  and Igbokwe, 2009)  who were able to reveal that freshly laid and stored domestic fowl eggs were contaminated by consortia of microorganisms, which migrated and invaded the inner parts of the eggs due to primarily heavy contamination and then prevailing poor storage conditions.

Serotypes of E. coli are classified according to the Kauffmann scheme, currently there are approximately 180 O, 60 H and 80 K antigens; The numbers change as new ones are identified and previous ones that are duplicated or attributable to another bacterial species are removed, additional serotypes with O antigens that have not been recognized also are found in most surveys. Even though molecular methods for identifying specific virulence genes are available, serotyping remains a useful tool for epidemiologic studies. Serotyping provides a means of relating previous work with new work. Variations  in the distribution of  serotypes  according to geographic region occur. Many other serotypes have been found less frequently, while some APEC do not belong to known serotypes or are untypeable  (Swayne, 2013). In the present study,13 serotypes  belonged to 6 different O groups were  identified, of which O27 predominated with an isolation rate of (22%), followed by O152 (11%).While, O125, O6, O159 and O169  were isolated with a rate of (8%), each. Finally, each of  O91, O166, O145, O25, O153, O115, and O29 was isolated  with a rate of  (3%). Serotyping with the available kits failed to identify  5 isolates (14%). The result of serotyping  agreed with that of (Rosario     et al., 2004) who  failed to identify the serogroups of 15% of isolates.

In the present study,  antibiotic susceptibility patterns of E.coli and Coagulase Positive Staphylococci isolates were studied. The study revealed the prevalence of MDR isolates among both microorganisms. The study recorded that 34 (94.1%), and 72 (90%) of E.coli, and Coagulase Positive Staphylococci isolates , respectively were considered MDR ; As it was observed that the tested isolates were resistant  to 5 and more; And 3 and more antimicrobial drugs, respectively.  Also those drugs belonged to different antimicrobial categories. The result was in accordance with (CLSI, 2011) where it was reported that MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories.

The study recorded that 94.1% of  E.coli isolates were resistant to 5 and more antimicrobial drug from varying drug category. This result agreed with that of (Muhammad et al., 2009) who reported the detection of MDR E.coli with a rate of 100%. The highest resistance rates were recorded against Trimethoprim- sulphamethazole, doxycyclin, tetracycline, and  amoxicillin; 94.1%, 93.2%, 92.9%, and 92.3 %, respectively. While the highest sensitivity rates were detected to colistin, cefotaxim, gentamycin, and ciprofloxacin; 94.4.%, 76.5% 59.8%, and  55.6%, respectively.

The result for trimethoprim- sulphamethazole susceptibility was similar to that of (Li et al., 2007) as they recorded resistance rate of 100%. While lower resistance rate was observed by (Hasan et al., 2011) who reported resistance rate of 26.7%.

Concerning resistance rate against doxycyclin, the result of this study was almost similar to that detected by (Jiang et al., 2009) who reported resistance rate of 95.6%.

The results for resistance profiles against tetracycline were in agreement   with that of (Jiang et al., 2009) who reported resistance rate of 93.4%. Lower resistance rate was recorded by (Muhammad et al., 2009) who recorded 52%.

The study of resistance rate against amoxicillin was in agreement with that of (Sheikh et al., 2012) who reported a rate of  92.86%, lower resistance rate was recorded by (Motayo et al., 2013) who recorded a resistance rate of   16.8%.

The result of colistin sensitivity agreed with the result of (Maalej et al., 2011) who observed sensitivity rate to colistin of 100%.

The result of susceptibility concerning cefotaxim was in accordance with that of (Pérez et al., 2014) who recorded resistance rate of 84.5%. In the contrary Oteo et al. (2006) recorded resistance rate of 100%.

The result of gentamycin was in agreement with that of (Huang et al., 2009) who detected sensitivity rate of   55.96%.

Higher susceptibility rate to ciprofloxacin was detected by (Hasan et al., 2011) who reported a sensitivity rate  of 87.1%. On the other hand, lower resistance rate was recorded by (Li et al., 2007) who recorded 19%.

The study revealed that 72  ( 90%) of Coagulase Positive Staphylococci isolates were considered MDR. This result was in agreement with that of(Suleiman et al., 2013) who reported that 100% of  the detected  Coagulase Positive Staphylococci isolates were MDR.

The study revealed that the highest sensitivity rate of Coagulase Positive Staphylococci was observed against ampicillin 87%. This result disagreed with that of (Lee, 2003)who recorded resistance rate of 73.3%.

The antibiotic susceptibility profile of Coagulase Positive Staphylococci isolates revealed 73.3% sensitivity rate to ciprofloxacin. This result was in accordance with that of (Suleiman et al., 2013) who detected sensitivity rate of 100%.

The detected sensitivity rate to gentamycin was 70%. This result was in agreement with that of (Suleiman et al., 2013) who observed sensitivity rate of 100%.

The study detected that the highest resistance rate of Coagulase Positive Staphylococci was against oxytetracyclin 90%, this result agreed with that of (El-Jakee et al., 2008) who observed resistance rate against oxytetracyclin of 80%.

The study also observed 86.7% resistance rate of Coagulase Positive Staphylococci against trimethoprime-sulphamethazone. This result disagreed with that of (Lee, 2003) who succeeded to detect 100% sensitivity rate for trimethoprime-sulphamethazone. On the other hand, the result was in accordance with that of (Nam et al., 2011) who detected resistance rate of 100%.

The observed resistance rate of Coagulase Positive Staphylococci against oxacillin was 86.7%.This result was similar to that of (Lee, 2003) who detected 100% resistance rate against oxacillin. While, this result disagreed with that of (Nam et al., 2011)  who detected resistance rate against oxacillin of(6.2%).

Control programs do not address laying hens whose eggs are produced for personal consumption or local sale, control measures should not forget home-produced eggs, as there is a risk of infection from their consumption, (Hardy et al., 2012).

The eaeA gene was tested to speculate the virulence of the isolated E. coli strains. There were 100% positive results for this gene. This result assured the virulence of these isolates because the eaeA gene encodes for intimin protein which is considered as a bacterial adhesion molecules that leads to the emersion of the A/E lesions (Kilici et al., 2007). The high incidence rate of eaeA gene detection was recorded by many authors as(El-Jakee et al., 2012) who detected eaeA gene in 95.9% of the tested E. coli O157:H7 isolates.

The iss gene had the 2nd incidence degree as 76.92% of the E. coli isolates showed positive amplification of the specific 266 bp of this gene. Iss (increased serum survival gene) is considered as a promising virulence gene that is usually associated with the APEC strains. Its rule was studied by(Lynne et al., 2007) when they observed a significant drop in E. coli resistance to serum with the iss- mutant. A bor- mutant also showed a drop in serum resistance but the drop in serum resistance was more violent in iss- mutant which indicates that iss contributes more to serum resistance than bor in the E. coli strains. This effect was assured when the level of serum resistance was restored after the iss was reintroduced into the iss- mutant.

The tsh (temperature-sensitive hemagglutinin) gene was detected in 4 isolates. This gene encodes for an autotransporter protein secreted by avian-pathogenic E. coli. This gene is rarely detected in commensal E. coli (Delicato et al., 2002) and is frequently found in highly pathogenic avian E. coli. The presence of this gene increases the danger of the harboring strains as it has a potential role as an adhesin (Provence & Curtiss, 1994) and it also has the ability to degrade haemoglobin (Otto et al., 1998).

None of the thirteen tested E. coli isolates showed positive PCR results for the stx1 gene. However, 5 isolates (38.46%) was recorded as positive for the stx2 gene. A close result was reported by (Zahraei    et al., 2007) who suggested that Stx2 may be widespread among APEC as they detected stx1 only in one isolate (8.33%) out of the 12 tested isolates, but stx2 was detected in 9 (75%) isolates. AL-Ashmawy, (2013) has much correlated results as she detected stx2 in 37/39 of the E. coli isolates from table eggs. The correlation was high also because none of the positive stx2 isolates showed positive results for stx1 gene. The heat stable toxin (Stx) causes disruption of chloride channels in the cell and secretion of fluid and electrolytes into the intestinal lumen causing diarrhea (Gaastra and Svennerholm, 1996). However the current results differed markedly from that obtained by many authors as (Wani et al., 2004; Zahraei et al., 2007 and AL-Ashmawy, 2013) who reported that all stx2 positive isolates were negative for eae genes, our results were supported by that obtained by (Dutta et al., 2011) who recorded eae and stx1 genes in 2 toxin producing Escherichia coli (STEC) isolates. The results of the current study also was supported by the study done in Egypt by (Galal et al., 2013) who detected both of the stx1 and stx2 genes with eaeA gene in 2/19 (10.52%) of the samples, and detected either stx1 or stx2 with eaeA gene in 3/19 (15.78%) of the samples.

The papC gene which encodes for the P fimbriae was detected in 3 isolates. Rocha et al. (2008) reported that papC operon is located in bacterial colonization in respiratory epithelium which directly affects the intensity of infection.

The gamma haemolysine (hlg) gene was positively amplified in 45.45% of the tested Coagulase Positive Staphylococci isolates. This gene is considered as one of the most important virulence genes of Staphylococci as it lead to generation of pores in the erythrocytes after initial binding of the two synergistically acting proteins hlgB and hlgA (Dickinson and Bisno, 1993).

The risk of Coagulase Positive Staphylococci isolates was accounted through the 81.81% positive percentage of the spa gene. This gene encodes for the protein A which antagonize the function of the immune system through hindering of the antibody mediated immune clearance of the organism through binding to the Fc receptor of IgG. Also, protein A interferes with the phagocytosis of opsonized bacteria via binding IgG (Murray et al., 2002).

The PCR result of the coa gene was enough to confirm the virulence of the Staphylococci isolates as it is considered as a marker for its virulence. The coagulase aids in the formation of a fibrin layer around a focal staphylococcal abscess, which leads to the localization of the infection and protecting the organism from phagocytosis (Sawai et al., 1997).

The results of the enterotoxin genes were so interesting, as 45.45% of the isolates were positive for Seb and Sec, while 27.27% of the isolates showed positive results for the See gene. The presence of these entrotoxins is so threatening as they resist the hydrolysis by gastric and jejunal enzymes and also they are heat stable at 100°C for 30 minutes which can explain why the staphylococcal food poisoning is the leading cause of food-borne microbial intoxication worldwide (Holmberg and Blake, 1984).

The blaTEM gene was tested for the 11 selected  E. coli isolates to assess its resistance to amoxicillin. Interestingly, the positive PCR percent (92.3%) was highly related to the phenotypic positive percent (92.3%) which confirmed the high degree of the resistance of these isolates to amoxicillin.

However, the PCR showed high positivity for the TetA(A) gene (100%) than the positive resistance percent obtained for the tetracycline by the antibiotic susceptibility test (92.3%), which may be related to the sensitivity of PCR itself. This also may be related to the antibiotic susceptibility test which may be influenced by several factors, some of which include the medium used for bacterial culture, type of drug tested, and the type of organism.

Conversely, the antibiotic susceptibility test showed higher positive percent for the trimethoprim-sulphamethazole (84.61%) than that showed for sul1 gene by PCR (61.53%). This was elucidated by (Gündoğdu et al., 2011) who recorded 10 strains of 96 carrying sul2 and intl1 were not positive for the presence of the sul1 gene. This was explained by (Grape et al., 2005) who referred to the sul1 gene as a semi-conserved segment.

Out of the 11 tested Staphylococci isolates, 4 were positive for mecAgene. This can increase the ferocity of this isolates. This was also reported by (Pyzik et al., 2014)who detected mecA gene in two S. aureus-like strains isolated from table eggs. To determine the ability of MRSA strains to infect human, (Lee, 2003) performed RAPD PCR and the results showed that their genome was very closely related to some human strains considering these isolates may be a possible source of food borne human infections.

However, the PCR positive percent for mecA gene (36.36%) was somewhat far from that obtained by antibiotic susceptibility test which showed 81.81% positive percent for the resistance to oxacillin. This was clarified by (Mathews et al., 2010) who reported two types of strains that show phenotypic resistance to oxacillin however they don't harbor the mecA gene. The 1st type of those two strains is called borderline oxacillin resistant S.aureus (BORSA) which hyper produces betalactamase and while they appear oxacillin resistant, do not possess the usual genetic mechanism for such resistance. They reported also that another type of strains known as modified S. aureus (MODSA) which possess a modification of existing penicillin binding proteins rather than the acquisition of a new PBP as is the mechanism for classical MRSA. 

The tetK gene was tested for the evaluation of the antibiotic resistance to tetracycline. The PCR result showed a 72.72% positive result which was lower than the resistance percent encountered by the antibiotic susceptibility test (90.9%). Schmitz et al. (2001) illustrated the mechanisms of tetracycline resistance for Staphylococcus species in two models. The 1st one is the active efflux resulting from the acquisition of the tetK and tetL genes located on a plasmid; and the 2nd one is the ribosomal protection mediated by tetMor tetOdeterminants located on either a transposon or the chromosome. And as recorded by (Duran et al., 2012), the tetM gene had higher positive percent (78%) than that recorded for tetK (43%) in the study performed to evaluate the association between the antibiotic susceptibility patterns and the antibiotic resistance genes in staphylococcal isolates.

The blaZ gene was positively amplified in 54.54% of the Staphylococci isolates. This result was dissimilar to the recorded penicillin resistance by the antibiotic susceptibility test (81.81%). The discrepancy between those results may also be related to the different mechanisms (other than blaZ) for the resistance of staphylococci to penicillins. Duran et al. (2012) mentioned those 2 mechanisms where the most important mechanism one is the production of beta-lactamase which inactivates penicillin by hydrolysis of its beta-lactam ring, another mechanism is associated with penicillin-binding protein 2a (PBP2a), encoded by mecA.

In conclusion, this study confirm that local house hold produced chicken eggs which are consumed as food can harbor resistant bacterial pathogen of zoonotic importance, those pathogens may impose public health hazard. The study recommends rising public awareness to the importance of proper thermal processing and cooking of eggs specially for immune-compromised group as pregnant women, children and old ages. The study also recommends regular monitoring and surveillance of house hold sector together with guidance programs to the public targeting rising awareness for safe house hold rearing procedures of chicken, storage and handling of eggs, in order to prevent dissemination of dangerous pathogens through environment and the transfer of infection to other animals and human.

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