OCCURRENCE AND MOLECULAR DETECTION OF ENTEROTOXIGENIC STAPHYLOCOCCUS AUREUS IN SOME READY-TO-EAT MEAT SANDWICHES MARKETED IN ZAGAZIG CITY

OCCURRENCE AND MOLECULAR DETECTION OF ENTEROTOXIGENIC STAPHYLOCOCCUS AUREUS IN SOME READY-TO-EAT MEAT SANDWICHES MARKETED IN ZAGAZIG CITY

Abdallah F.A. Mahmoud* and Fardous M. Atta

Department of Food Control, Faculty of Veterinary Medicine,

Zagazig University, Egypt

*Correspondence to:          Abdallah Fikry A. Mahmoud

E-mail: afabdallah@zu.edu.eg (abdallah.fikry90@gmail.com)

Key Words:S. aureus, RTE meat sandwiches, Enterotoxins, MRSA, PCR.

ABSTRACT

Staphylococcus aureus in ready-to-eat (RTE) meat sandwiches is a consequence of insufficient hygienic handling and improper processing, posing a major health hazards. This study was conducted to assess RTE meat sandwiches as a potential source for Staphylococcus aureus (S. aureus). Furthermore, the identifiedS. aureus isolates were tested for detection of mecA virulence gene of MRSA (methicillin resistant Staphylococcus aureus) and enterotoxin encoding genes (sea, seb, sec, and sed). A total of 80 samples of ready-to-eat meat sandwiches represented by kofta, liver, shawarma and hawawshi (20 of each) were collected from various fast food restaurants of different sanitation levels at Zagazig city, Sharkia province, Egypt. The obtained results revealed that S.aureus were isolated from 4 (20%), 3 (15%), 4 (20%) and 5 (25%) of the examined kofta, liver, shawarma and hawawshi samples, meanwhile the average count of S. aureuswas4.21, 4.83, 4.79 and 4.32log10 cfu/g of the examined samples, respectively. Out of 16 identified S. aureusisolates,4 (25%) of MRSA strains were characterized by the presence of the mecA gene, comprising two isolates from kofta, one from liver and one from shawarma sandwiches. Enterotoxogenic virulence genes were evaluated by multiplex PCR, revealing that oneisolate from shawarma samples harbored the sea and sedgenes,whereas one isolate from kofta and hawawshi was positive for the sea gene, and only one isolate from examined liver sandwiches was positive for secgene. Thus, strict hygiene practices should be applied during preparation and processing of such meat sandwiches to ensure its safety before serving to consumers.

INTRODUCTION:

Ready-to-Eat foods are processed foodstuffs that can be consumed without further thermal treatments so in recent times there is increase in the demands of RTE fast foods because of a change in social patterns that characterized by increased mobility, large numbers of working woman and itinerary workers so they are dependent on fast served meals. Different terms can be used to describe RTE foods these include convenient, ready, and instant and fast foods.In Egypt, the most RTE meat products sold in street vendors and fast food restaurants are Kofta, liver, shawarma and hawawshi sandwiches.Despite the economic and nutritional benefits of RTE meat sandwiches, the consumption of these sandwiches has been suggested to potentially increase the risk of food borne diseases as they are readily contaminated from different sources (Tambekar et al., 2008).

Staphylococcal food poisoning (SFP) is one of the most prevalent food-borne illnessesin the world. It results from the ingestion of staphylococcal enterotoxins produced by enterotoxigenic types of coagulase-positive staphylococci in food, mainly S. aureus and usually occurs within 30minutes to 8 hours; resulting in several symptoms that includes vomiting, nausea, abdominal cramping, diarrhea, chills, and sweating.SFP is generally self-limiting and resolves typically within 24-48hours after beginningbased on the quantity of contaminated food consumed, the amount of the ingested toxin in food and the general health of the patients. Occasionally it can be serious enough to warrant hospitalization, especially when it comes to children, the elderly or debilitated people.

Lack of adequate hygienic measures during food preparation is one of the maincauses of contamination as the food-handlers themselves can carry the pathogenic bacterium. Besides that, S. aureus can withstand a broad variety of temperature, pH and salinity (Stewart et al., 2005).In addition, the most of the nosocomial S. aureus infections are triggered by methicillin-resistant S. aureus (MRSA) strains and have become a world-wide recognized cause of morbidity and mortality (Ho et al.,2008). MRSA strains have emerged resistant to quinolones or multiresistant to other antibiotics, leaving a restricted option for their control (Pesaventoet al., 2007).Therefore, the present work wasplanned out for detecting the incidence of enterotoxigenic S. aureus and methicillin-resistant S. aureus (MRSA) strains in different popular ready-to-eat sandwiches (Kofta, liver, shawarma and hawawshi sandwiches) in Zagazig city, Sharkia governorate, Egypt, as well as to determine the prevalence of the major SEs among the isolated S.aureus strains.

MATERIALS AND METHODS

1-Collection of samples:

Eighty random samples of ready- to-eat meat sandwiches (20 each of Kofta, liver, shawarma and hawawshi) were collected from different localities (fast food restaurants and street vendors) with varying sanitation levels in Zagazig City, Sharkia governorate, Egypt.  All collected samples were directly transferred without undue delay in an icebox under complete aseptic conditions to the laboratory for organoleptic and bacteriological examination.

2-Preparation of samples (APHA, 2001):

Twenty-five grams of each meat sandwiches cores only without bread were homogenized aseptically for 1 min with 225 ml of 0.1 % sterile buffered peptone water in stomacher to prepare a homogenate of 10^-1 initial dilution andallowed to stay for 5 minutes and then serially diluted 10-fold in the same diluent.

3-Determination of S. aureus count :( A.O.A.C., 2000)

0.1ml from each of the prepared dilutions was spread onto duplicate plates of Baird-Parker (BP) agar(Oxoid CM 275), supplemented with egg yolk tellurite emulsion (50ml/L, Oxoid SR54) and incubated at 37 °C for 24-48h. Colonies with typical S. aureus morphology (i.e. , circular, black, shiny with narrow white margins and surrounded by clear zones extending into the opaque medium were counted and recorded to obtain the total Staphylococcus aureus counts per gm. Each suspected colony was picked up and cultured on slope agar for further identification.Isolated purified strains were identified morphologically by Gram's stain and biochemically confirmed as S.aureus according to FDA (2001) by the conventional methods that included catalase, production of coagulaseand utilization of sugars.

4-Demonstration of enterotoxin and Methicillin Resistant S.aureus “MRSA” (mecA) virulence genesby PCR:

Accurately, variousspecific primers were used as shown in table (1) for demonstration of enterotoxin and Methicillin Resistant S.aureus “MRSA” (mecA) virulence genes by PCR.

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

S. aureus strains were incubated in 5 ml of brain heart infusion broth (Oxoid, CM509, Adelaide, Australia) and incubated aerobically at 37°C overnight. About 1.5 ml of the culture was centrifuged at 6000 rpm for 2 minutes and the supernatant was discarded. The DNA was extracted using QIA amp kit according to Shah et al. (2009).

6. DNA amplification:

Amplification reaction for enterotoxins genes ofS. aureus (Rall et al., 2008):

The DNA amplification was donein a Thermal Cycler (Master cycler, Eppendorf, Hamburg, Germany) using 25 μl of PCR mixture. The reaction mix invariably consisted of 5 µl of the bacterial lysate, 5 µl of 10x assay buffer for Taq polymerase containing 1.5 mM MgCl2, 2 µl of 10mM dNTP mix 1 µl each of forward and reverse primers (10 pmol) and 1.25 U of Taq DNA polymerase made upto50 µl using sterile distilled water. The following conditions: initial denaturation at 94°Cfor 5 min followed by 30 denaturation cycles (94°C for 2 min), annealing (50°C for 1 min), and extension (72°C for 1 min).After the completion of the cycles, a final extension step (72 °C for 5 min) was conducted. The PCR products were electrophoresed in 1% agarose gel and stained with ethidium bromide.                                                                                                                                                                      

7-Amplification reaction for MRSA gene(Jukes et al., 2010):

The bacterial genomic DNA samples were amplified by PCR in a reaction mixture (25 µl) 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 filling up to 25 µl PCR grade water. Amplification reaction was carried out in 25μl volumes, under the following conditions: initial denaturation at 92°C for 5min, accompanied by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds, followed by final extension at 72°C for 3min. The PCR products were electrophoresed in 1.5% of agarose gel electrophoresis stained with ethidium bromide and visualized on UV transilluminator.

Table (1):Primer sequences and expected product sizes of S. aureus virulence genes:

RESULTS

Table (2): Incidence and mean count (log cfu/g) of S. aureus in the examined samples (n=20)

n: number of examined samplesNo: number of positive samples

cfu/g: Colony Forming Unit per gram

Means are not significantly different at P> 0.05 (0.371)

Table (3): prevalence of virulence genes in the isolated S. aureus strains

n: Number of examined strains

SEA: SEA: S. aureus enterotoxin A; SEB: S. aureus enterotoxin B; SEC: S. aureus enterotoxin C; and SED: S. aureus enterotoxin D.MRSA: Methicillin Resistant S.aureus

Figure (1):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. Lanes 7& 15: Positive S. aureus strains for sea. Lanes 8: Positive S. aureus strain for sec gene. Lane 11: Positive S. aureus strain for sea and sed genes. Lanes 1, 2, 3, 4, 5, 6, 9, 10, 12, 13, 14 & 16: Negative S.aureus for enterotoxins.

Figure (2):Agarose gel electrophoresis of PCR amplification products of mecA gene for characterization of Methicillin Resistant S.aureus. Lane M: 100 bp ladder as molecular size DNA marker. Lane C+: Control positive S. aureus for mecA gene. Lane C-: Control negative. Lanes 5, 9, 11 and 16: Positive S. aureus strains for mecA gene. Lanes 1, 2, 3, 4, 6, 7, 8, 10, 12, 13, 14 & 15: Negative S. aureus strains for mecA gene.

DISCUSSION:

The results recordedin table (1&2) and figures (1&2) revealed that the prevalence of S. aureus was 20%, 15%, 20%, 25% in the examined kofta, liver, shawerma and hawawshi, respectively with mean value 4.21± 0.575 , 4.83± 0.423, 4.79±0.475,  4.32±0.512  log 10 cfu/g, respectively. With minimum count 3, 3, 3, 2.5 log 10 cfu/ g and maximum count 7, 7.3, 7.3, 7.7 log 10 cfu/g.

The four ready-to-eat products whose staphylococcal isolates were investigated were found earlier to be highly contaminated with staphylococci. Aycicek et al. (2005) reported that hawawshi and Liver samples contained S. aureus with counts ranging from 3.7-4.1 and 2.5-3.6 log cfu/g, respectively. In Egypt, El-Mossalami et al. (2008) mentioned that shawarma and liver sandwiches have S. aureus with counts rangedfrom 3.4×10² to 5.2×10⁴ and from 3.7×10² to 6×10⁴ cfu/g, respectively. While, Shalaby and Zaki (2008) could detect S.aureus in shawarma in numbers varied from 2×10² to 3×10³ with a mean value of 9.8×10² cfu/g.

   Regarding the incidence of S. aureus recorded in table (1), the obtained results of S. aureus in Kofta samples were similar tothe resultsobtained by Ibrahim (1991)and El-Shewehy (1994)(20%), lower thanthe results obtained by Morshdy et al. (2014) 65.62%, and higher than obtained by Sobieh(2014) 6.67%, while Masoad(2013)failed to detectedS. aureus in Kofta.

Concerning to the liver samples, the results were lower than the results recorded by El-Mossalami et al., (2008) 80%, El- Hanafy (2016) 56.67%.

   Moreover,the results of shawarma samples were lower than obtained by Ali and Abd El-Aziz (2011) 25% and higher than obtained by Odu and Akano (2012) 13.6% but the results of hawawshi were lower than obtained by Ghanem(2009) 40% and higher than obtained by Sobieh(2014) 20%.

   The obtained data in table (1&2) showed that the higher incidence in hawawshi followed by Kofta and liver then lower incidence in shawarma  while the higher mean count of S. aureus were liver followed by shawarma then hawawshi and finally Kofta . These results indicated that no significance difference between the four examined sandwiches in S. aureus counts (P> 0.05).

According to the US Food and Drug Administration (≥10⁵ CFU/g S. aureus)is capable of causing staphylococcal intoxication. SFP is one of the most common causes of food-borne illness due to the widespread occurrence of S. aureus and to the ability of many strains to synthesize one or more SEs.

In this study, the isolates of S. aureus were tested for demonstration of enterotoxins and mecAvirulence gene of MRSA (methicillin resistant Staphylococcus aureus) by multiplex PCR. The obtained data in table(3) showed that  only four strains out of  the 16 isolated  coagulase positive S. aureus strains were enterotoxigenic;  only one strains of S. aureus from the four tested Kofta isolates carry SEAgene with a percentage 25%, one strainfrom the three tested liver isolates carry SEC gene with a percentage 33.3 %, one from the four tested strains in shawarma carry more than one enterotoxin genes SEA (25%) and SED with percentage 25 % and one from the five tested strains in hawawshi carry SEA gene with a percentage 20%.

From these obtained results concluded that SEA is the most common enterotoxinproduced in the 16 tested isolates with a percentage 18.75% followed by SEC (6.25%) and SED (6.25%),While SEB failed to be detected in all strains isolated from the four tested RTE sandwiches. Pexara et al., (2010) concluded that SEA and SED are the most common enterotoxins isolated from SFP outbreaks.RTE meat sandwiches should be free from S. aureus enterotoxinsaccording to EOS (2005).

 The isolates were also tested for presence of mec A gene in this study, the obtained results in table (3) showed that mec A gene detected in only 4 strains out of the 16 isolates with a percentage 25% at 100 bp of coagulase positive S. aureus isolates; two strains isolated from Kofta (50%), one from the liver (33.3), one from shawarma (25%) but it couldn’t be detected in hawawshi. The results of the present study highlighted that; these foods may constitute a risk for consumers and especially for immunocompromised individuals. In immunocompromised persons, the specific and non-specific immune responses are not able to act as barriers to prevent colonization of the gastrointestinal tract and ingestion of food contaminated by MRSA may lead to sometimes lethal disease (Kluytmans et al., 1995).

Considering the importance and public health hazard of S. aureus organism recovered from fast food(ready-to-eat sandwiches), Longree and Blacker (1971) reported that preparing and serving food to the publicis a very important obligation that can only be fulfilled if every one in the establishment understand food hygiene, applying sanitary measures at every stage of the operation. Furthermore, ICMSF (1988) stated that cooked meat should not be touched by hands or by equipments that have come in contact with raw meat; raw products should be separated from cooked products to avoid cross-contamination.

To safe the ready -to-eat sandwiches sold in fast food services it must be focus on prevention of contamination and multiplication of microbes and production of toxins. Food should not be prepared long in advance of consumption (Bryan et al., 1992). Cooking usually give time temperature exposures that would have been lethal for vegetative form of food borne pathogens. On the other hands, holding of food provide time temperature exposures conductive to microbial growth, particularly in food holds overnight and large populations of aerobic organisms including S. aureus and others were recovered from these food. So, time temperature had variable effect of killing the microorganism but heat stable toxins still not affected (Jermini et al., 1997 and Pepe et al., 2006).

In conclusion, it can be achieved from the obtained data that fast meat products (sandwiches) have the potential to cause staphylococcal intoxication to consumers. Therefore, the rules of health agencies must reach to all workers in such field especially street vendors, and fast food takeaway restaurants besides safety programs for safe food preparation drawn by international organizations and national authoritiesshould be followed and effective preventive measures must be authorized and applied to safe the consumer health.

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مدى تواجد والکشف الجزیئی للمکورات العنقودیة الذهبیة المفرزة للسموم المعویة فی بعض ساندوتشات اللحوم الجاهزة للأکل المسوقةفی مدینة الزقازیق

عبدالله فکرى عبدالله محمود و فردوس محمد ربیع عطا

کلیة الطب البیطرى – جامعة الزقازیق

یعد انتشار المکورات العنقودیة الذهبیة فی ساندوتشاتاللحوم الجاهزة للأکلنتیجة لنقص التداول الصحی والمعالجة غیر السلیمة لها، مما یشکل مخاطر صحیة جسیمة على الصحة العامة. لذلک أجریت هذه الدراسة لتقییم ساندوتشات اللحومالجاهزة للأکلکونها مصدراً محتملاً للمکورات العنقودیة الذهبیة. علاوة على ذلک، تم اختبار عزلات المکورات العنقودیة الذهبیة المحددة للکشف عن وجود جین (mecA) الموجود بالمکورات العنقودیة الذهبیة المقاومة للمیثیسیلین (MRSA) وکذلک الجینات المسئوبه عن افراز السموم المعویة (sea, seb, sec, and sed). تم جمع 80 عینة من شطائر اللحوم الجاهزة للأکل ممثلة فی سندوتشات الکفتة والکبد والشاورما والحواشی (20 من کل نوع) تحت ظروف صحیة مختلفة من مطاعم الوجبات السریعة فی مدینة الزقازیق، محافظة الشرقیة، مصر. أظهرت النتائج التی تم الحصول علیها أنمیکروب المکور العنقودى الذهبىتم عزله من 4 (20٪)، 3 (15٪)، 4 (20٪) و 5 (25٪) من عینات الکفتة،الکبد، الشاورما و الحواشی على التوالى، فی الوقت نفسه کان متوسط ​​عدد المکورات العنقودیة الذهبیة 4.21 ، 4.83 ، 4.79 و 4.32لوغاریتم مستعمرة بکتیریة/جم فی العینات المفحوصة على التوالی. کما أوضحت النتائج أن من بین 16 عزلة من المکورات العنقودیة الذهبیة، تم تمییز 4 (25 ٪) من سلالاتبالمکورات العنقودیة الذهبیة المقاومة للمیثیسیلین (MRSA) بواقععزلتین من الکفتة، واحدة من الکبد وواحدة من ساندوتشات الشاورما. تم تقییم جینات الضراوة المسئولة عن افراز السموم المعویة عن طریق تفاعل البلمرة المتسلسل(PCR)، تبیناحتواء عزلة واحدة من الشاورما على جینات (sea and sed)، کما احتوت عزلتین من الکفتة والحواشی على الجین (sea)، بینما کانت عزلة واحدة فقط من ساندوتشات الکبد إیجابیة للجین (sec). وبالتالی یجب تطبیق الممارسات الصحیة الصارمة أثناء إعداد وتجهیزساندوتشات اللحوم لضمان سلامتها قبل تقدیمها للمستهلکین.