OCCURRENCE OF BACILLUS CEREUS AND STAPHYLOCOCCUS AUREUS ORGANISMS IN SOME DAIRY DESSERTS

OCCURRENCE OF BACILLUS CEREUS AND STAPHYLOCOCCUS AUREUS ORGANISMS IN SOME DAIRY DESSERTS

M.F. HUSSEIN; O.A. SADEK and EL TAHER, S.G.

Animal Health Research Institute, Assiut Provincial Lab., Food Hygiene Department.

Email: onsi_2000@yahoo.com                                                                              Assiut University Email: www.aun.edu.eg

INTRODUCTION

Dairy desserts as ice cream, mehallabia (a traditional dessert in Egypt) and rice with milk are popular frozen and refrigerated foods consumed particularly in summer, as well as, throughout all the year. They continue to present a dominant interest for a large segment of population.

The ingredients of dairy desserts may be various combinations of milk, cream, evaporated or condensed milk, dried milk, coloring materials, flavors, fruits, nuts, rice, starch, sweetening agents, eggs, eggs products, and stabilizers. One or more of these components may contribute microorganisms and affect the quality of the product as judged by its bacterial load or its content of various specific species of bacteria especially food poisoning ones.

Bacillus cereus has raised concern in dairy industry by its deteriorating effect, this because B. cereus is a psychrotrophic microorganism able to grow under chilling temperature and produce lipases and proteases enzymes that change the components of dairy products (Zhou et al., 2010). Several B. cereus strains have been identified as the causative agent of two different types of food poisoning: The emetic type (Ehling-Schulz et al., 2004) and the diarrheal type, and both type can occasionally be fatal (Dierick et al., 2005).

Staphylococcal intoxication, which is due to the consumption of food containing one or more preformed staphylococcal enterotoxins (SE), is one of the leading food-borne diseases worldwide (Balaban and Rasooly, 2000). Staph. aureus enterotoxins exhibit two separate biological activities; they cause gastroenteritis in the gastrointestinal tract and act as a superantigen on the immune system (Shinefield and Ruff, 2009).

As most of the dairy desserts consumers are children of vulnerable age groups, it is required to be microbiologically safe (Warke et al., 2000 and Caglayanlar et al., 2009). Therefore, the objective of this study was to isolate, quantify and identify Bacillus cereus and Staph. aureus organisms from some dairy desserts (ice cream, mehallabia and rice with milk) and detection of enterotoxins of Staph. aureus in these desserts consumed in Assiut city, Egypt.

MATERIALS and METHODS

A total of 60 dairy desserts samples comprising ice cream, mehallabia and rice with milk (20 samples of each) were collected from small dairies in Assiut city, Egypt. The samples were collected in its container as sold to the public and transported as soon as possible to the laboratory and subjected to the following microbiological examination:-  

1-Enumeration and isolation of Bacillus cereus according to Holbrook and Anderson (1980).

2-Enumeration and isolation of Staphylococcus aureus according to FDA (2001).

3-Demonstration of Staph. aureus enterotoxins in dairy desserts samples (which was performed in Food Analysis Center, Faculty of Veterinary Medicine, Benha University, Egypt) by ELISA according to Ewald (1988):

Accurately, RIDASCREEN set C (Art No.: R4101, R-Biopharm AG, Darmstadt, Germany) is an enzyme immunoassay for the determination of S. aureus enterotoxins by using their definite kits.

According to the test kit manual, food sample was mixed in sterile buffer saline and then shaken for 15 minutes. After centrifugation for 10 minutes at 3500 r.p.m, sterile filtration of the supernatant was applied. An aliquot (100 µl per kit well) of this solution was used in the test. Further, the last well was represented as positive control. They were mixed gently and incubated for one hour at room temperature (20-25°C) in the dark.

The liquid was dumped out of the wells into a sink to remove all of the remaining liquid from the wells. Therefore, the wells were then filled with 250 µl of washing buffer and the liquid was poured out again. The washing step was repeated 3 times to remove the unbound conjugate.

Subsequently, 100 µl of enzyme conjugate were added to each well and incubated for one hour at room temperature in the dark after mixing gently. The liquid was dumped out of the wells into a sink and the wells were each filled with 250 µl of the washing buffer. The liquid was poured out again and the wells were emptied to remove all of the remaining liquid.  The washing step was repeated 3 times again.

Afterwards, 50 µl of substrate and 250 µl of chromogen solutions were added to each well. The solutions were mixed gently and incubated for 30 minutes at room temperature in the dark. Finally, 100 µl of the stop solution (1M H₂SO₄) were added to each well with gentler mixing.

By using ELISA, the absorbance was measured at 450 nm in an ELISA plate reader (ELX800, BioTek Instruments, Bad Friedrichshall, Germany). The results were calculated from standard curve.

RESULTS

Table 1: Incidence and counts of Bacillus cereus in some dairy desserts.

Table 2: Incidence and counts of Staphylococcus aureus in some dairy desserts.

Table 3: Types of enterotoxins detected in Staph. aureus positive dairy desserts samples.

DISCUSSION

Results reported in Table 1 revealed that, B. cereus was isolated from 55% of ice cream samples in counts ranging from 2 X 10⁶ to 3 X 10¹⁰ with an average count of 3.1 X 10⁹ cfu/ml. Nearly similar result (52%) was estimated by Wong et al. (1988). Lower results (4, 44, 38, 40, 19, 48, 16 and 4%) were found by Masud (1989); Saleh et al. (1993); Al-Ashmawy et al. (1996); Warke et al. (2000); Yaman et al. (2006); Hassan et al. (2010); Altaf et al. (2012) and Maryam et al. (2013), respectively. In contrary, higher result (62.7%) was obtained by Messelhäusser et al. (2010).

The high average count of B. cereus obtained in this study may be attributed to fraud of the product with starch which is a favorable medium for B. cereus organisms or due to insufficient heating during the product manufacturing.

Concerning mehallabia samples, 60% of tested samples were contained B. cereus with counts ranged from 6 X 10⁶ to 1.2 X 10¹¹ with an average count of 1.07 X 10¹⁰ cfu/g (Table 1). Lower result (44%) was revealed by Al-Ashmawy et al. (1996). As no available literature could be traced dealing with the incidence of B. cereus in mehallabia, therefore, it was hard to discuss the aforementioned result.

With regard to rice with milk, 15% of samples were contaminated with B. cereus in counts ranged from 6 X 10⁶ to 5 X 10⁹ with an average value of 1.7 X 10⁹ cells/g (Table 1). Lower result (5.8%) was estimated by Çadirci et al. (2013). On the other hand, higher results (64, 60 and 35%) were noticed, respectively, by Al-Ashmawy et al. (1996); Sadek et al. (2006) and Reyes et al. (2007).

From Table 1, one can easily conclude that, mehallabia samples had higher B. cereus incidence and counts than ice cream and rice with milk. This could be attributed to that main constituent of mehallabia is starch which is the main medium for B. cereus growth. Furthermore, rice with milk had the lowest B. cereus incidence and counts in this study and this could be referred to over heating during rice with milk manufacturing or due to other factors which need further investigation.  

From estimated results in Table 1, it is obvious that, the B. cereus containing samples were having B. cereus counts that sufficient enough to cause food poisoning as the infective dose of the microorganism ranged from 10³ to 10¹⁰ cfu/g (Notermans and Batt, 1998).

The results recorded in Table 2 revealed that, Staph. aureus was detected in 15% of ice cream samples in counts ranging from 1.9 X 10³ to 2 X 10⁶ with an average count of 6.7 X 10⁵ cfu/ml. Relatively similar result (18.7%) was observed by Mathews et al. (2013). Lower results (4.3, 4.4, 10 and 5.6%) were reported by Kocak et al. (1998); Anuranjini et al. (2008); Gücükoğlu et al. (2012) and Rahimi (2013), respectively. While, higher results (20, 26, 40, 50, 50, 26, 28 and 23 %) were estimated by Ahmed et al. (1988); Masud (1989); Abdel-Hameed and El-Malt (2009); Fadel and Ismail (2009); Zakary et al. (2011); Mirzaei et al. (2012); Maryam et al. (2013) and Abou-Elkhair et al. (2014), respectively.

It worth to mention that, the sources of Staph. aureus in ice cream may be due to use of infected milk or from infected handlers during preparation and retailing the product.

All the mehallabia samples in this study were negative to Staph. aureus isolation (Table 2). The failure of Staph. aureus detection in mehallabia samples could be attributed to use of starch during product manufacturing instead of milk or other factors which need further investigation.

Concerning rice with milk, 15% of tested samples gave Staph. aureus with counts ranged from 8 X 10⁴ to 8 X 10⁷ with an average value of 2.7 X 10⁷ cells/g. Lower result (4%) was reported by Alisarli et al. (2003). Also, Staph. aureus was detected in 6.9% of rice cake samples tested by Cho et al. (2013). However, higher results (35 and 66.7%) were estimated by Sina et al. (2011) and Tang et al. (2015) from rice with sauce and rice products, respectively.

Due to paucity of available literature could be traced dealing with the incidence of Staph. aureus in rice with milk at Egypt, therefore, it was hard to discuss the aforementioned result.

Examination of ice cream samples for detection of staphylococcal enterotoxins (SE) revealed that, only one sample (sample No. 19) was positive for enterotoxin type A (Table 3). This result coincided with Rivas et al. (1983) as the authors reported that, in ice cream, SEA was the most commonly produced enterotoxin by enterotoxigenic staphylococci. Moreover, several authors (Balaban and Rasooly, 2000; Rall et al., 2008; Imani et al., 2010; Rahimi    et al., 2012 and Cho et al., 2013) estimated that, SE type A gene was the predominant gene in enterotoxigenic Staph. aureus isolated from various food samples. Furthermore, SE type A was detected in ice cream sample harbouring Staph. aureus count exceeded 10⁵ cfu/ml, while, the counts in the two SEA negative ice cream samples were less than 10⁵ cfu/ml (Table 3). This observation upheld what obtained by Tranter (1996) who reported that, Staph. aureus intoxication occurred when the count exceeded 10⁵ cfu/g in food.  

From the three positive Staph. aureus rice with milk samples, two samples (Samples No. 28 and 35) were containing SE type A & C and Type A, respectively (Table 3). It worth to mention that, rice with milk sample No. 28 was containing Staph. aureus count of 8 x 10⁴ cfu/g and this observation indicated that, even when the staphylococci count less than 10⁵ cfu/g food, the organism was capable to produce enterotoxins in food.

Rice with milk sample No. 33 was negative for SE, while the count was 8 x 10⁵ cfu/g (Table 3). The failure of SE detection in that samples could be attributed to, the expression of enterotoxin genes depends on some factors such as the origin and identity of the bacterial isolate and the host environment of bacteria.

Staphylococcal toxins are fast acting, sometimes causing illness in as little as 30 minutes after eating contaminated foods, but symptoms usually develop within one to six hours. Patients typically experience several of the following: nausea, retching, vomiting, stomach cramps and diarrhea. In more severe cases, dehydration, headache, muscle cramping, and changes in blood pressure and pulse rate may occur (CDC, 2015).

The aforementioned results revealed that, the staphylococcal positive dairy desserts samples represented the risk of food poisoning to the consumers.

CONCLUSION

This study revealed that, some dairy desserts sold in Assiut city, Egypt were contaminated with B. cereus in a count that sufficient to produce food poisoning. Likewise, Staph. aureus enterotoxins were detected in some food samples which representing potential risk of intoxication by consumption of such food. Thorough food inspection and frequent bacteriological surveillance by food control agencies is highly recommended to control the incidence of B. cereus and Staph. aureus in dairy products to safeguard the consumers from risks of food poisoning.

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