OCCURRENCE OF ENTEROTOXIGENIC STAPHYLOCOCCUS AUREUS IN SOME CHEESE VARIETIES IN ASWAN CITY – UPPER EGYPT

OCCURRENCE OF ENTEROTOXIGENIC STAPHYLOCOCCUS AUREUS IN SOME CHEESE VARIETIES IN ASWAN CITY – UPPER EGYPT                                                                 

LAILA M. EL MALT

Department of Food Hygiene, Faculty of Veterinary Medicine, South Valley University, Qena Upper Egypt.

Email: lailael.malt@ymail.com  

INTRODUCTION

Cheeses are ready to eat food products that do not undergo any further treatment to ensure their safety before consumption Although cheeses have been characterized as one of the safest food products by some authors (Little et al., 2008). In 2006 the consumption of contaminated cheese accounted for the 0.4% of the total food borne outbreaks in Europe (European Food Safety Authority "EFSA", 2008), furthermore, the scientific literature has reported severe food poisoning out breaks associated with various types of cheese. (Kongo et al., 2008).

Staphylococcus aureus is a leading cause of gastroenteritis resulting from the consumption of contaminated food. Staphylococcal food poisoning is due to the absorption of Staphylococcal enterotoxins preformed in the food (Loir et al., 2003)

Many contaminants find their way to raw milk, from which they gain access to dairy products (Al–khatib and Al-Mitwalli, 2009). Chapaval et al., (2010) found staphylococcalenterotoxins in milk when milk was stored at temperatures of 37 ºC to 42 ºC or when exposed to variations in temperature.

On heating at normal cooking temperature, the bacteria may be killed but the toxins remain active (Presscott et al., 2002). Staphylococcal enterotoxins are highly heat resistant and are thought to be more heat resistant in foodstuffs than in a laboratory culture medium (Bergdoll 1983).

Besides enterotoxins producing S. aureus are most dangerous and harmful for the human health about 50 % of this organism are able to produce enterotoxins associated with food poisoning (Payne, 1974). Illness  through S. aureus ranges from minor skin infection such as pimples, boils, cellulites, toxic shock syndrome impetigo, and abscesses to life threatening disease such as pneumonia, meningitis, endocarditis, and septicemia (Soomro et al., 2003).

SEA and SED are the most common Ses involved in food poisoning outbreaks (Genigeorgis, 1989). Growth of enteroxigenic strains of Staph. aureus to a population of 10⁶ or more cells/g of food is generally considered necessary for production of a sufficient amount of enterotoxins to cause intoxication if the food consumed (Newsome, 1988). The amount of SEA necessary to cause symptoms in humans is about 100ng (Balaban and Rasooly, 2001). The Ses functions as potent gastrointestinal toxins. After ingestion of contaminated food, the toxins are resorbed into the blood in the gastrointestinal tract, activate an emetic reflex and cause nausea, emesis, abdominal cramps an diarrhea (Tortora, 1995) after 2-6 hours of eating contaminated food and the recovery takes 1-3 days, while deaths occur rarely, specifically in very young or old age (Martin and Iandolo, 2000) Time of onset and severity of symptoms depend on the quantity of toxins consumed and the individuals susceptibility (Baird and Lee, 1995).

To detect the level of enterotoxin (100ng) several sensitive detection methods for SE detection have been developed in particular the rapid  methods based on specific antibodies such as a radioimmunoassay (RIA) an enzyme linked immunsorbent assay (ELISA) or enzyme immunoassay (EIA) and reversed passive latex agglutination assay (RPLA) are commonly used in staphylococcal intoxication the use of polymerase chain reaction (PCR) and DNA probes is limited in detection only the presence of Staph aureus genes, but not in detection the presence of preformed toxins  (Di Pinto et al., 2004).

Antimicrobial resistance is a major public health problem in many countries due to the persistent circulation of resistant strains of bacteria in the environment and the possible contamination of bacteria in the environment and the possible contamination of water and food. The development of resistance both in human and animal bacterial pathogens has been associated with the extensive therapeutic use of antimicrobials or with their administration as growth promoters in food animal production (Barber et al., 2003)

The present study was planned to deal with the following:

1-Enumeration, isolation and identification of staph. aureus in  some cheese varieties sold in Aswan city.

2- Identification of antibiotics resistant strains of staph. aureus.

3-Detection of enterotoxigencity of the isolated staph. aureus organisms                                                                                                                                                                                                                                                                                                                   

MATERIALS and METHODS

A) Collection of samples:                                                    

A total of 120 random samples of some cheese varieties were collected from various dairy shops, street vendors and supermarkets located at Aswan city. These samples included Fresh Kareish, pickled Kareish, Domiati and Processed cheese (30 samples each). Collected samples were transferred in an ice box directly to the laboratory with a minimum of delay to be examined.

B) Preparation of serial dilutions (ISO 8261. 2001):

C) Microbiological examination:

1- Enumeration and isolation of Staph. aureus (A.O.A.C., 2000):

Over duplicated plates of a dry surface of Baird Parker (B-P) agar, 0.1 ml from each prepared dilutions of examined samples were transferred and evenly spread using surface plating technique (Thatcher and Clark, 1988).

- The inoculated B-P agar plates were incubated at 37°C for 24-48 hrs. Suspected colonies are circular, smooth, convex, moist, 2-3 mm in diameter, gray to jet black, shiny, with light color (off-white) narrow margin, surrounded by hallow zones and had buttery to gummy consistency when touched with inoculating  needle were counted. The plates were then reincubated for additional 18-24 hrs before being counted for further growth, Staph. aureus count/ g  were recorded.

- Furthermore, an appropriate amount from each prepared sample was inoculated into sterile Nacl 10% broth. Inoculated Nacl 10% broths were inoculated at 37 °C for 24 hrs. A loopful of the incubated broth was streaked onto sterile plates of Mannitol Salt agar. (A.O.A.C., 2000)

Identification of Staph. aureus recovered from the examined samples:

A- Morphological characters for all isolates:

1-    Staining reaction (A.P.H.A., 2004):-

B- Biochemical reactions:- catalase activity (Koneman et al., 2005), anaerobic mannitol fermentation (Baird-Parker, 1962), coagulase test. according to (Cruickshank et al., 1973).

2- Isolation of antibiotics resistant strain of staph. aureus.

a- Identification of methicillin resistant Staph aureus (MRSA): (Simor et al., 2001).

The cultures of Staph aureus were subcultured on Oxacillin Resistance Screen Agar Base (ORSAB) (Oxoid Limited, Basingstoke, England) containing ORSAB Selective Supplement contained two antibiotics-oxacillin at 2mg/L and polymyxin B 50.000 IU/l. The plate was incubated at 37 ^OC for 24-48 h and examined for the presence of MRSA colonies, which were blue on ORSAB.

b- Identification of vancomycin resistant Staph aureus (VRSA) LTiwari and Sen, 2006).

3- Detection of the enterotoxigencity of Staph. aureus (Park et al., 1994)

Using RIDASCREE^®SET A, B, C, D, E (Art. No.:R4101) manufactured by R- Biopharm AG, Germany.

Selected strains of the isolated MRSA and VRSA from the examined samples were tested for –ability to produce entertoxins.

RESULTS

Table 1: Statistical analytical results of Staph.aureus count in the examined cheese samples.

  ^⃰E.S.: Egyptian Standard (2005).

Table 2: Frequency distribution of positive cheese samples based on their S.aureus count.

Table 3: Incidence of coagulase positive and coagulase negative strains of S. aureus in the examined cheese.

Table 4: Frequency distribution of MRSA and VRSA and recovered from the examined cheese samples.

Table 5: Incidence of MRSA and VRSA strains isolated from the examined cheese

Table 6: Enterotoxins produced by some strains of Staph. aureus isolated from the examined cheese samples.

Table 7: Entertoxins production in relation to coagulase producing Staph. aureus strains.

DISCUSSION

The results recorded in Table 1showed that 28(93%), 25(83%), 21(70%) and 6(20%) of the examined fresh Kareish, Pickled Kareish, Domiati and processed cheese samples were contaminated with Staph.aureus, respectively.

The S.aureus count ranged from 1 x10³ to 5.25 x10⁷ with a mean count of 3.08 X 10⁶ ± 1.79 x10⁶ in fresh Kareish cheese samples, from 4.17 x10² to 4.70 x10⁶ with a mean Count of 3.40 x 10⁵ ± 1.59x10⁵ in Pickled Kareish cheese samples, from 1 x10 to 1.27 x10⁷ with a mean count of 7.24 x10⁵± 4.10 x10⁵ and for Processed cheese the counts ranged from 1 x10 to 1.90 x10² with a mean count of 1.15 x10²±8.98 cfu/g.

Egyptian standards (2005) pointed out that the cheese must be free from Staph. aureus and its toxins It was evident that 28(93%), 25(83%), 21(70%) and 6(20%) of the examined fresh Kareish, Pickled Kareish Domiati and processed cheese samples failed to comply with the standerd, respectively.

The results in Table 2, revealed that the highest frequency distribution of fresh Kareish cheese samples was  8(28.5%) lied in the range of 10³ <10⁴ and 10⁶  <10⁷ cfu/g, while the rest of the positive samples were distributed  as 21,18 and 4% lied in between 10⁴ <10⁵, 10⁵ <10⁶ and 10⁷ <10⁸ cfu/g, respectively. In case of Pickled Kareish cheese samples the highest frequency distribution was (44%) lied in the range of 10³ <10⁴ cfu/g, while the rest of the positive samples were distributed as 36,8,8 and 4% lied in between 10⁵  <10⁶, 10² <10³ , 10⁴ - <10⁵ and 10⁷  <10⁸ cfu/g, respectively.

For Domiati cheese samples the highest frequency distribution was (43%) lied in the range of 10² <10³ cfu/g, while the rest of the positive samples were distributed as 33, 14 and 10 % lied between 10³ <10⁴, 10⁴ <105 and 10⁶ <10⁷, respectively. While the Processed cheese positive samples all of them lie in range10² <10³ fresh Kareish cheese samples ,comparatively higher counts were obtained by Tawfek et al. (1988) Relatively lower counts were detected by Shelaih (1979),Ahmed (1980). Ewida (2009),De Reu et al. (2002) and In case of the examined Pickled Kareish cheese samples comparatively lower counts were obtained by Kaldes (1997). While for Domiati cheese samples results, higher counts obtained by Sallam et al. (1985).Relatively lower counts obtained by El-Malt (1993)and Hassan (2009). Lower incidence of S.aureus in curd (6.66 %) was reported by Kumar and Prasad (2010) and  (3.33 %) Thaker et al. (2013). The difference in the prevalence of S.aureus between different types of cheese may originate from the method of manufacture, storage and handling. 

Turkoglu et al. (2001) and Soncini et al. (2002)failed to detect the S. aureus in the examined Processed cheese samples.

Fresh Kareish cheese is a popular Egyptian food. It is made at farmers houses from raw skim milk and the fresh product is either consumed fresh or consumed after pickling in its salted fresh whey with added spices and medical plants (mish cheese) so the source of Staph.aureus in Kareish cheese are the raw milk, during processing and distribution in village markets these sources lead to the expected high count of Staph. aureus. Domiati cheese is considered to be the main national popular pickled soft cheese produced in Egypt. It is traditionally prepared from raw milk without addition of lactic acid culture starters but by addition of appreciable amount of salt to milk before renneting. It can be consumed fresh, but usually it is consumed after pickling in its salted fresh whey for a period of not less than 8 weeks, the product may be soiled with pathogens which enter through different environmental sources that render it a source of infection by many diseases. Genigeorgis (1989) demonstrated that the higher concentration of competing microorganisms in milk, the lower the rate of S. aureus growth and SE production. Competition with lactic acid bacteria has been reported in other research on cheese (Otero et al., 1988).

Table 3 showed that 15(54%), 7(28%) and 13 (62 %) of the examined staphylococci strains recovered from fresh Kareish cheese, Pickled Kareish cheese and Domiati cheese, respectively. were coagulases positive. While, 13(46%), 18(72%), 8(38%) and 6(100%) of the isolated strains of staphylococci of the examined cheese, respectively. were coagulase negative. Concerning the result of coagulase positive S.aureus in the examined fresh Kareish cheese samples, higher results were obtained by Ahmed (1980), Al-Hawary et al. (2009) and Helmy et al. (2009). Lower results were detected by Aman (1994); Kaldes (1997) and Kolluman et al. (2011).

In case of coagulase positive S.aureus in the examined Domiati cheese samples, lower results were detected by Coveney et al. (1994), Kaldes (1997); Normanno   et al. (2005)  and Kolluman et al. (2011).

Various examples of staphylococcal food poisoning are described in the literature. In one case, cheese was involved in an outbreak because it had been made from unheat treated milk, milk contaminated after pasteurization and before inoculation with lactic starter culture or did not use culture starter. In this particular case, the starter culture did not grow properly, resulting in a fermentation accident that allowed the S. aureus strain to develop and produce SE (Vasavada, 1988) Although milk and milk products are frequently contaminated with S. aureus, dairy products are rarely involved in staphylococcal food poisoning because the critical cell density of >105 cfu/g-1 is usually not reached (Altekruse et al., 1994).

The result in Table 4 showed that 8(29%), 3(11%) and 3(11%) showed staph aureus strains isolated from fresh Kareish cheese were MRSA, VRSA, respectively. While 7(28%), 5(20%) and 4(16%) Staph aureus strains isolated from Pickled Kareish cheese were MRSA, VRSA, respectively. The frequency distribution of MRSA and VRSA in the Domiati cheese samples were 9(43%) and 8(38%) while 3(14%) can be resistant for both drugs. In Processed cheese there is no of isolated strains of MARSA, VRSA or resistant for both drugs.

In Table 5 the incidences of MRSA and VRSA from the examined samples were 26%, 3(10%), 7(23%), 5(17%), 9(30%) and 8(27%) of fresh Kareish cheese, Pickled Kareish cheese and Domiati cheese respectively.

Inspection of Table 6 revealed that out of 12 methicillin and vancomycin resistance Staph aureus strains tested for enterotoxins production 3 strains of fresh Kareish cheese, 4 strains of Pickled Kareish cheese and 5 strains of Domiati cheese. From fresh Kareish cheese 2 strains were synthesized enterotoxins A and SEB (one strains) in Pickled Kareish cheese there is no strains producing interotoxins. Domiati cheese 2 strains were isolated and produce enterotoxins A(2 strains), SEB (one strain), SEC (one strain) and SED (one strain).

This finding was in contrast to other studies from Spain, Kenya, Switzerland, Brazil, South Korea, the USA, Slovakia and Palestine, where most of the enterotoxigenic Staph aureus isolated usually synthesized the toxins SEA, SEC, or SED (Scherrer    et al., 2004).

The higher percentages of SEA among Staph aureus strains isolated from milk and milk products may be due to the fact that enterotoxins A are less common among the strains of animals origin than from human origin (Hajek and Marsalek, 1973). These strains of human origin contaminate milk and milk producte during different stages of production or at consumer outlet. On the other side the presence of SEC and SED can be attributed to the increased incidence of Sraphylococcal mastitis as enterotoxins C and D were found to be produced by Staph aureus strains isolated from bovine mastitis and were designated as ^“animal strains^” (Olson et al., 1970).

Although the selected 12 isolates were strongly producing coagulase, only 4 were enterotoxigenic which confirm what was stated by A.P.H.A. (1992) and Ryser (2001) Table (7) that attempts to associate enterotoxin production by Staph aureus with specific biochemical properties were generally failed. Consequently, confirmation of the toxin by serological or other means provide the only proof that the particular strain is enterotoxigenic.

Park et al. (1994) evaluated the RIDASCREEN SET kit for its efficiency. They concluded the major advantages of the kits were a high degree of specify (neither false –positive results due to the growth of nonstaphyloccal microorganisms nor cross –reaction among reagents of the kits was reported), excellent sensitivity, simplicity rapidity (results can obtained in 3h) and semi quantitative results.

CONCLUSION

The obtained results allow concluding that fresh Kareish cheese, Pickled Kareish cheese, Domiati cheese and Processed cheese samples sold in Aswan city markets were produced, handled, packed and distributed under neglected hygienic measures. The information given by the achieved results proved that most of the examined  fresh Kareish cheese, Pickled Kareish cheese, Domiati cheese and Processed cheese samples sold in Aswan city market were highly contaminated with high number of Staph aureus which may lead to undesirable changes of these products that render them unfit for consumption and indicate unpersonal hygiene and un- sanitary conditions during processing and handling of such product in compared with examined Processed cheese which show lower incidences or counts.

In the resent years, the extensive therapeutic use of antimicrobials or with their administration as growth promoters in food animal production lead to development of resistance both in human and animal's pathogens as occurred with Staph aureus. Nowdays, Staph aureus can resist many antibiotics as methicillin and vancomycin which lead to difficulty in their susceptibility of Staph aureus to these antibiotics and other group of antibiotics. In this study, methicillin resistance Staph aureus (MRSA) and vancomycin resistance Staph aureus (VRSA) could be isolated from examined samples in different percentages.

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