DETECTION OF ENTEROTOXIGENIC STAPHYLOCOCCUS AUREUS IN SOME MEAT PRODUCTS

DETECTION OF ENTEROTOXIGENIC STAPHYLOCOCCUS AUREUS IN SOME MEAT PRODUCTS

ALAA EL-DEEIN M. MORSHDY; ADEL I. EL-ATABANY; MOHAMED A. HUSSEIN and MOHAMMAD AL-AMEN SH. NASR

Faculty of Vet. Med. - Food Hygiene Department (Meat Hygiene) Zagazig University.

INTRODUCTION

Meat and meat products are considered the most important sources of food for people in every where as they supply the consumers with the required nutritive elements such as high quality proteins, essential amino acids, B-complex vitamins and certain minerals especially iron and phosphorous and beside that the meat has high calories value.

In comparison to fresh meat, meat products retain more consumers' palatability and easily preparation characteristics. Food safety is a global health goal and the food borne diseases take a major crisis on health. Therefore, detection of microbial pathogens in food is the solution to the prevention and recognition of problems related to health and safety (Velusamy        et al., 2010). Staphylococcus aureus (S. aureus) is a major cause of food borne intoxication and its presence in food constitutes an important problem for food processors, food service workers and consumers.  The main reservoirs of S. aureus are humans and animals. Healthy people carry the organism in their nose and throat (50 %), on their hands (5-30 %), and in wounds. S. aureus can also colonize food contact surfaces, and it can become a persistent organism in slaughter houses. S. aureus can contaminate foods through contact with contaminated hands, materials and surfaces, but also via the air coughing (Rao et al., 1980). Considering this hazard, meat and meat products should not be subjected to unnecessary contamination and they should be free from such serious pathogen to ensure a maximum margin of consumer safety.

S. aureus, if posses Staphylococcal enterotoxins genes may produce several enterotoxins (SEA to SEJ) (Monday and Bohach, 1999). Staphylococcal food poisoning (SFP) is one of the most prevalent causes of gastroenteritis worldwide. Symptoms of SFP have a rapid onset characterized by abdominal cramps, nausea, and vomiting, sometimes followed by diarrhea. Patients usually suffer from symptoms within 2-4 hours after ingestion of thermostable Staphylococcal enterotoxins (SEs). The approximate dose of SEs ranged from 0.1 to 1.0 mg/kg of body weight (Jorgensen et al., 2003 and Stewart et al., 2005). Since SEs are more stable than S. aureus bacteria, it is possible to test food product and obtain negative S. aureus culture results and positive SE tests. Today, up to 15 SEs are known; the last one discovered was the recently identified SEU (Letertre et al., 2003a). Multiplex PCR assay for detection of Staphylococcal enterotoxins genes (SEA, SEB, SEC, SED and SEE) was developed and proved to be specific, sensitive, and rapid method. (Omoe et al., 2002 and Zschock et al., 2005). There fore the current study was planed to through light on the incidence and count of S. aureus followed by PCR identification of enterotoxigenic strains.

MATERIALS and METHODS

Sampling:

Eighty four meat product samples (minced meats, sausages and burgers) 28 of each were collected randomly from supermarkets and butchery shops at Sharkia province, Egypt, at different levels of sanitations. The collected samples were identified, packed in sterile polyethylene bags and transferred to the laboratory as soon as possible.

Methodology:

The collected samples were examined for detection of S. aureus by ordinary culture method followed by biochemical identification and examined for coagulase enzyme production and then the detected strains were subjected to DNA extraction which was used as template of PCR amplification for detection of S. aureus enterotoxins genes.

Preparation of samples, (ICMSF, 1978):

Twenty five grams of each examined samples were transferred to a sterile polyethylene bags, and 225 ml of 0.1% of sterile buffered peptone water was aseptically added to the content of the bag. Each sample was homogenized in a blender at 2500 rpm for 1-2 minute to provide homogenate of 1/10 dilution. One ml from the original dilution was transferred to another sterile tube containing 9ml sterile buffered peptone water and mixed well to make next dilution, from which further decimal serial dilutions were prepared. Then, dilutions were subjected to the following examinations.

Count and isolation of Staphylococcus aureus, (APHA, 1992):

From the previously prepared decimal dilutions of the examined samples 0.1 ml was transferred and evenly spread on dry surface of Baired-Parker agar medium plates (Oxoid, 1998). Inoculated and control plates were incubated at 37º C for 48 hours and Staphylococci count was calculated and recorded. Each suspected colony (black shiny with narrow white margin and surrounded by clear hallow-zone extended into the opaque medium) was picked up and cultured on slope agar for further biochemical identification according to (APHA, 1992).

Identification of isolated Staphylococci organisms:

- Morphological characters:

Staining (microscopical examination):

Films were made from the pure culture of isolated organisms stained by Gram's stain and examined microscopically. Gram +ve, grapes shape, cocci were considered as positive Staphylococci.

- Biochemical reactions:

1- Catalase test.

2- Oxidation-fermentation test.

3- Coagulase test.

4- Mannitol test.

PCR detection of Staphylococcal enterotoxins genes:

Extraction of the DNA from Bacterial culture:

Bacterial DNA was extracted using GeneJET Genomic DNA Purification kit (Fermentase, USA # k0722) following the manufacture's protocols.

PCR assays:

Oligonucleotide sequences of SE genes' were listed in table (1).  PCR amplification was performed in a thermal cycler (Thermal cycler: PTC-100 ^TM programmable thermal controller, Peltier-Effect cycling, MJ, RESEARCH, INC.) with  an initial denaturation of 94° C for 5 minutes; 35 cycles of amplification (denaturation at 94° C for 2 minutes, annealing at  57° C  for  2 minutes, and extension at  72° C  for  1 minute), with a  final  extension  at 72° C  for 7 minutes. Electrophoration  was  done  using  1.5% agarose  gel  in  0.5%  Trisacetate- EDTA  (TEA)  buffer,  stained with  ethidium  bromide. The amplified products then were visualized using ultra violet lamp wherein, the images were taken.

Multiplex PCR for detection of selected Staphylococcal genes:

The reaction conditions for the multiplex PCR assaywere optimized to ensure that all of the target gene sequenceswere satisfactorily amplified. The primers were designed totarget the coding regions of the genes; care was taken to avoidareas of homology within the structural genes for the enterotoxins.The primers used in each set had almost equal annealingtemperatures, which reduced the possibility of occurrenceof unwanted bands originating from nonspecific amplification.

Table 1: Nucleotide sequences, gene locations, and anticipated sizes of PCR products for the S. aureus gene-specific oligonucleotide primers used in this study (Mehrotra et al., 2000).

RESULTS

Table 2: Incidence and count of S. aureus in examined meat product samples and its percentage (n = 28).

  ׳Min: minimum.

״ Max: maximum.    

**Means carrying the different letter were significant; (P < 0.05).

* S.E: Standard error of mean.

Table 3: Incidence of enterotoxins of coagulase positive S. aureus detected in examined meat product samples and its percentage.

(1)                                                                                            (2)

                                         (3)                                                                                               (4)     

                                          (5)                                                                                            (6)

Figureure: Agarose gel electrophoretic pattern of:

- 1, 2 minced meat samples; 3, 4, 5 sausage samples.

- 3 minced meat samples; 1, 2,4,5,6 sausage samples.

- 1, 2 sausage samples; 3,4,5,6 beef burger samples.

- 6 sausage samples; 1, 2,3,4,5 beef burger samples.

- 1, 2, 4 minced meat samples; 3, 5 beef burger samples.

- 1, 2, 3, 4, 5, 6 beef burger samples.

DISCUSSION

Staphylococcus aureus count:

Staphylococcal food-poisoning (SFP) is caused by Staphylococcal enterotoxins (SEs) produced during massive growth of S. aureus in food. SFP is a prevalent cause of food-borne disease worldwide, (Jablonski and Bohach, 2001).

The results showed in table (2) revealed that, the incidence of S. aureus in minced meat was 6 (21.4 %) similar result was recorded byVorster et al. (1994) while lower incidence was detected byEl-Said (2005).Higher incidences were recorded byDepourcq and Poucke, (1991); El-Gohary (1993); Ramasastry (1999); Salek (2000); Huffman (2002) and Tavakoli and Riazipour (2008). The mean count was 4.3x10² ± 3.6x10² cfu / gm and the count ranged from <10² to 10x10³ cfu / gm. Such results were lower than that recorded byTharwat (2008)andNossier (2010).

In sausage samples, the incidence was 11 (39.3 %). This incidence was similar to that obtained byEL-Sherbeeny et al. (1990) who detect S. aureus in 43.1% of examined sausage samples. On contrary lower incidences were reported by Abd El-Aziz et al. (1996); Soultos et al. (2003); El-Said (2005)andGehad et al. (2006). Higher incidences were recorded byRashad (1990), El-Gohary (1993) in the examined sausage samples.

The mean value was 5.1x10³ ±  2.1x10³ cfu / gm with a minimum  of <10² and a maximum of 40x10³ cfu / gm. Lower results were obtained by El-Mossalami, (2002) who found that S. aureus count was <50 cfu/gmand Gehad et al. (2006) whom failed to detect S. aureus in the examined sausage samples.

Burger samples showed a high incidence of 17 (60.7 %), while lower incidences were reported by Ranucci et al. (2004) they isolated S. aureus from 21.1% of the examined hamburgers also El-Said (2005) recorded that S. aureus was isolated from frozen beef burger with an incidence of 2 (4%) and Shahraz et al. (2012) isolated 64 (24%) S. aureus strains from 256 samples of packaged hamburger. The mean value was 8.7x10³ ±1.8x10³ organisms/ gm with a minimum of <10² and a maximum of 30x10³ organisms/ gm.Lower count was recorded by Essa and Makar, (2003).

Comparatively, the obtained results declared that burger samples gave a higher incidence of S. aureus followed by sausage then minced meat. This indicates that the unsanitary handling of the prepared meat products during processing, transportation and marketing. Also, was due to using a low quality meat and meat additives. The shape of burger may explain the higher incidence than that of sausage. Statistical analysis revealed that both sausage and burger were significantly higher than minced meat at p value < 0.05.

The United Kingdom Public Health Laboratory Service in 2000 classified meat products to four categories satisfactory, acceptable, unsatisfactory and unacceptable according to number of S. aureus as < 20, 20 to <100, 100 to <10 ⁴  and  ≥ 10 ⁴ CFU/g, respectively. We found that, all examined minced meat samples were within the permissible limit and considered acceptable except only one sample that show unsatisfactory level of contamination (10x10³ organisms/ gm).The examined samples of sausage and burger showed unsatisfactory level of contamination and were 11(39.3 %) and 17 (60.7%) for sausage and burger.

PCR assays used to identify the pathogen and its enterotoxin genes in food samples. The PCR assays could be made in hours rather than days, with a high sensitivity and method accuracy, allowing for the detection of very low concentrations of micro-organisms(Najera-Sanchez et al., 2003).

Data in table (3) revealed that the incidence of   identified enterotoxigenic S. aureus isolated from meat product samples by PCR technique was 9 (32.1 %) from sausage and 8 (28.6 %) from burger, while it couldn't be detected from minced meat. Nearly similar result was detected by Pinto et al. (2005). They found that forty out of 131 isolates (31%) tested were positive for enterotoxins production. Lower incidence of enterotoxins was detected by Kevin       et al. (2009). They examined 155 samples of fermented pork product, and found that 39.35% of the samples were positive for S .aureus, but none were positive for the Staphylococcal enterotoxins. Higher incidence of enterotoxigenic S. aureus was detected by Oh et al. (2007) they found that 47% of the isolated S. aureus strains from the contaminated meat product were enterotoxigenic S. aureus. Also,El-Shater (2010) detect enterotoxigenic S. aureus with an incidence of (55.6 %) in sausage samples and Aydin et al. (2011) found that out of 147 S. aureus isolated from different foods including sausage, 92(62.6%) were enterotoxigenic S. aureus.

Enterotoxins are a group of serologically distinct proteins (A, B, C1–3, D, E and F) that are the causative agents of Staphylococcal food poisoning. Although their exact mode of action has yet to be fully elucidated, they are believed to stimulate an enteric-vagus nerve reflex triggering the vomiting centers of the brain (Sears and Kaper, 1996; Arbuthnott et al., 1990). The enterotoxins can also act as super antigens, stimulating T lymphocytes to release cytokines and T-cell proliferation (Balaban and Rasooly, 2000; Krakauer, 1999).

The predominant enterotoxins isolated from examined sausage samples were enterotoxin A 8 (88.9 %) figures (1, 2, 3 and 4) detected at 102 bp then enterotoxins B 1 (11.1 %) figure (2) detected at 164 bp.Application of PCR assays for detection of enterotoxin production from coagulase positive S. aureus isolated from beef burger samples revealed that 6 (75 %) enterotoxin A detected at 102 bp figures (3, 4,5 and 6), followed by enterotoxin B figure (5) detected at 164 bp. With a percentage of 1 (12.5%) and enterotoxins A and B figure (3) detected in one sample at 102 and 164 bp. With a percentage of (12.5%). Such results substantiate what have been reported by Hwang et al. (2007); Ruzickova et al. (2008); El-Shater (2010) and Aydin et al. (2011).

Finally we conclude that:

Not all coagulase positive S. aureus are enterotoxigenic, since minced meat samples were negative, only nine sausage samples from eleven coagulase positive were enterotoxigenic (82 %) and only eight burger samples from seventeen coagulase positive were enterotoxigenic (47 %).

REFERENCES

Abd El-Aziz, A.S.; El-Neklawy, E.; Hussien-Azza and Niazi- Zeineb (1996): Food poisoning Microorganisms in Some Local Meat Products. Vet. Med. J., Giza. 44 (4), 691-698.

APHA (1992): Compendium of methods for the microbiological examination of food. 3rd Ed., Washington, D. C., USA.

Arbuthnott, J.P.; Coleman, D.C. and De Azavedo, J.S. (1990): Staphylococcal toxin in human disease. Soc Appl. Bacteriol. Symp Ser69, 101S–107S.

Aydin, A.; Sudagidan, M. and Muratoglu, K. (2011): Prevalence of Staphylococcal enterotoxins, toxin genes and genetic-relatedness of foodborne Staphylococcus aureus strains isolated in the Marmara Region of Turkey. Int.J. Food Microbiol., 148 :99–106.

Balaban, N. and Rasooly, A. (2000): Staphylococcal enterotoxins. Int J Food Microbiol., 61: 1–10.

Beuchat, T.J. (2001): Montville, Editors, Food Microbiology Fundamentals and Frontiers (2nd ed.), ASM Press, Washington, D.C., USA, pp. 411–434.

Depourcq, G. and Poucke, L.V. (1991): Evaluation of the microbiological quality of minced meat. Lab. For pharmaceutical microbial. Hygiene Univ. of Gent. Harelbekes traat 72, B. 9000 Ghent, Belgium.

Doyle, L.R. Letertre, C.; Perelle, S.; Dilasser, F. and Fach, P. (2003b): Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus. J. Appl. Microbiol., 95, 38–43.

El-Gohary, A.H. (1993): Sausage and minced meat as a source of food poisoning microorganisms to man. Assuit Vet. Med. J., 30 (59): 146-153.

El-Mossalami, M.K. (2002): Bacterial behaviour of some meat products stored in home fridge. Assiut Vet. Med. J. Vol., 47:94.

El-Said, SH. A.A. (2005): Psychrophilic microorganisms in frozen meat products. M.V.Sc. Thesis, Meat Hygiene, Fac. Vet., Med., Zag. Uni. Egypt.

El-Shater, N.S.L. (2010): Evaluation of immunological and bacteriological patterns of some food poisoning micro-organisms (Staphylococcus aureus and Clostridium perfringens), Ph. D. Thesis, Microbiol. Dept., Fac. Vet. Med. Zag. Uni. Egypt.

EL-Sherbeeny, M.; Saddik, M.; El-Hossany, M. and Abd-el-Kadir, M. (1990): Staphylococcus aureus associated with meat and meat products Bull. Nut. Instit. A.R.E., 9 (1) 27.

Erlich, H.A.; Gelfand, D. and Sninisky, J.J. (1991): Recent advances in polymerase chain reaction. Science, 252: 1643.

Essa, H.H. and Makar, N.H. (2003): Bacteriological quality of beef burgers in Assiut City, Assiut Vet. Med. J., 49:81-88.

Gehad, F.A.; Eman, M.S. and Takwa, H.I. (2006): Bacteriological and chemical studies of frozen sausage in Giza province. Zag. Vet. J., 34 (1): 156-163.

Guven, K.; Mutlu, B.M.; Gulbandilar, A. and Cakir, P. (2010): Occurrence and characterization of Staphylococcus aureus isolated from meat and dairy products consumed in Turkey. J. Food Safety 30: 196–212.

Hoda, A.M.A. and Afaf, A.Y. (2005): Effect of Nisin and Nitrite on some food poisoning microorganisms and their toxins production in frozen sausage.  J. Egypt. Vet. Med. Assoc., 65 (3): 113-122.

Huffman, R.D. (2002): Current and future technologies for the decontamination of carcasses and fresh meat. Meat Sci., 62: 285-294.

Hwang, S.Y.; Kim, S.H.; Jang, E.J.; Kwon, N.H.; Park, Y.K.; Koo, H.C.; Jung, W.K.; Jun Man Kim, J.M. and Park, Y.H. (2007): Novel multiplex PCR for the detection of the Staphylococcus aureus super antigen and its application to raw meat isolates in Korea. Int. J. Food Microbiol., 117:99–105.

I.C.M.S.F (1978): International Commission on Microbiological Specifications for Foods. Using of several media for identification of Clostridium spices. Univ. of Toronto Press. Toronto, Canada.

Jablonski, L.M. and Bohach, G.A.  (2001): Staphylococcus aureus. In: M.P.

Jorgensen, H.; Mork, T.; Caugant, D.A.; Kearns, A. and Rørvik, L.M. (2003): Genetic Variation among Staphylococcus aureus strains from Norwegian Bulk Milk. Appl. Environ. microbiol. p. 835.

Kevin, E.; Juan, C. and Hans, R. (2009): Incidence of Staphylococcus aureus and associated risk factors in Nham, a Thai fermented pork product. Food Microbiol., 26 (5): 547.

Krakauer, T. (1999): Immune response to Staphylococcal super antigens. Immunol Res 20, 163–173.

Letertre, C.; Perelle, S.; Dilasser, F. and Fach, P. (2003a): Detection and genotyping by real-time PCR of the staphylococcal enterotoxin genes sea to sej. Mol Cell Probes. 17(4):139.

Mehrotra, M.; Wang, G. and Johnson, W.M. (2000): Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance. J. Clinic. Microbiol.; 38:1032–5.

Monday, S.R. and Bohach, G.A. (1999): Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in Staphylococcal isolates. J. Clinic. Microbiol.; 37:3411–4.

Najera-Sanchez, G.; Maldonado-Rodriguez, R.; Olvera, P.R and de la GarzaL, M. (2003): Development of two multiplex polymerase chain reaction for the detection of enterotoxigenic strains of Staphylococcus aureus isolated from foods. J Food Prot; 66:1055–62.

Nossier, A.M.M. (2010): Control of microbiological hazard of some meat products preserved at refrigeration, M.V.Sc. Thesis (Meat Hygiene), Fac. Vet. Med. Zag. Univ. Egypt.

Oh, S.K.; Lee, N.; Cho, Y.S.; Shin, D.B.; Choi, S.Y. and Koo, M. (2007): Occurrence of toxigenic Staphylococcus aureus in ready-to-eat food in Korea. J. Food Prot. 70(5):1153.

Omoe, K.; Ishikawa, M.; Shimoda, Y.; Hu, D.L.; Ueda, S. and Shinagawa, K. (2002): Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates Harboring seg, seh, or sei genes. J. Clin Microbiol. 40 (3):857. 

Orwin, P.M.; Fitzgerald, J.R.; Leung, D.Y.; Gutierrez, J.A.; Bohach, G.A. and Schlievert, P.M. (2003): Characterization of Staphylococcus aureus enterotoxin L. Infect Immun 71, 2916–2919.

Oxoid (1998): The oxoid manual 8th edition. Oxoid, Basingstoke, England.

Pinto, B.; Chenoll, E. and Aznar, R. (2005): Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques. Sys. Appl. Microbiol., 28: 340–352.

Ramasastry, P.; Rao, M.R. and Mrunalini, N. (1999): Bacterial profiles of frozen meat. Indian Vet. J., 5409-411.

Ranucci, D.; Miraglia, D.; Branciari, R.; D'Ovidio, V. and Severini, M. (2004): Microbiological characteristics of hamburgers and raw pork sausages, and antibiotic-resistance of isolated bacteria. Vet. Res. Communic., 28(1), 269–272.

Rao, K.S.; Patish, V.K. and Ghodeker, D.R. (1980): Screening kulfi for staphylococcal enterotoxins with the thermonuclease test. J. Food Prot., 43 (1): 49.

Rashad, F.M. (1990): Microbiological studies on Egyption Fresh sausage. Arch. Fur lebensmitt. Hyg. 41 (1):11.

Ruzickova, V.; Karpiskova, R.; Pantucek, R.; Pospisilova, M.; Cernikova, P. and Doskar, J. (2008): Genotype analysis of enterotoxin H-positive Staphylococcus aureus strains isolated from food samples in the Czech Republic.Int. J. Food Microbiol., 121 :60–65.

Salek, M. (2000): Microbial control of cooked meat foods and lettuces served in Beheshti Medical Sciences University restaurants. Ph. D.  Thesis; (154): 63-9.

Sears, C.L. and Kaper, J.B. (1996): Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev 60, 167–215.

Shahraz, F.; Dadkhah, H.; Khaksar, R.; Mahmoudzadeh, M.; Hosseini, H.; Kamran, M. and Bourke, P. (2012): Analysis of antibiotic resistance patterns and detection of mecA gene in Staphylococcus aureus isolated from packaged hamburger. Meat Sci., 90: 759–763.

Soultos, N.; Abrahim, A. and Abrosiadis, I. (2003): Incidence of some food born bacterial pathogens in traditional sausages produced in northen Greece. Archiv. Furer. Lebensmittel. Hygiene, 54(3):55-57.

Stewart, C.M.; Cole, M.B. and Schaffner, D.W. (2005): Managing the Risk of Staphylococcal Food Poisoning from Cream-Filled Baked Goods to Meet a Food Safety Objective.  J. Food Prot. (66) 7: 1310.

Tavakoli, H.R. and Riazipour, M. (2008): Microbial quality of cooked meat foods in Tehran university’s restaurants. Pak. J. Med. Sci., 24 (4): 595-599.

Tharwat, A.E. (2008): Microbial aspects of some meat products with a trial to improve their quality and shelf life at refrigeration, M.V.Sc. Thesis (Meat Hygiene), Fac. Vet. Med. Zag. Univ. Egypt.

Velusamy, V.; Arshak, KH.; Korostynska, O.; Oliwa, K. and Adley, C. (2010): An overview of food borne pathogen detection: In the perspective of biosensors Biotechnology Advances,28(2):232-254.

Vorster, S.M.; Greebe, R.P. and Nortje, G.L. (1994): Incidence of Staphylococcus aureus and Escherichia coli in ground beef, broilers and processed meat in Pretoria, South Africa. J. Food Prot., 57(4):305-310.

Zschock, M.; Kloppert, B.; Wolter, W.; Hamann, H.P. and Lammler, C.H. (2005): Pattern of enterotoxin genes seg ,seh,sei and sej positive Staphylococcus aureus isolated from bovine mastitis. Vet. Microbiol., 108(3-4): 243.