BACTERIOLOGICAL ASSESSMENT OF RAW MILK AND YOGHURT FOR PRESENCE OF STAPHYLOCOCCUS AUREUS WITH SPECIAL REFERENCE TO ITS ENTEROTOXINS

AssiutUniversity web-site: www.aun.edu.eg

BACTERIOLOGICAL ASSESSMENT OF RAW MILK AND YOGHURT FOR PRESENCE OF STAPHYLOCOCCUS AUREUS WITH SPECIAL REFERENCE TO ITS ENTEROTOXINS

M.F. HUSSIEN¹ and A.M. KOREIM²

¹ Food Hygiene Department, Animal Health Research Institute, Assiut Lab.

² Bacteriology Department, Animal Health Research Institute, Assiut Lab.

Received: 13 December 2015;     Accepted: 10 January 2016  

INTRODUCTION

Milk has long been referred to as the most perfect food for human consumption from birth to senility; it contains all the nutrients required for rapid growth and healthy development of the body. Milk and its products have been shown to be an ideal media for growth and multiplication of many microorganisms; including Staphylococcal food poisoning (Bone et al., 1989 and Wieneke et al., 1993). Staphylococcus aureus contamination can occur through its presence in raw milk itself or during its processing (Le Loir et al., 2003).

Yoghurt is the most popular type of fermented milk in Egypt, the nutritive value of yoghurt is attributed to the fat content, sugar and casein. Therefore, yoghurt is recommended for sick and convalescent people. It inhibits the bacterial flora of intestine which may lead to constipation, autointoxication and colitis as well as it helps in the absorbtion of calcium and phosphorous.

Staphylococcus aureus infection was estimated to be present in up to 90% of dairy farms and is responsible for 35% of the economic loss in the dairy industry(Bone et al., 1989). Moreover Staphylococcus aureus is facultative anaerobic Gram positive bacterium, the majority of Staph. aureus strains are catalse and coagulase positive which forms the basis of traditional identification methodology.

Corresponding author: Dr. M.F. HUSSIEN

E-mail address: Mahmoutfarghaly_1970@yahoo.com

Present address: Food Hygiene Department, Animal Health Research Institute, Assiut Lab.

Staphylococcus aureus is a leading cause of food poisoning resulting from the consumption of contaminated food with staphylococcal enterotoxins. Exactly, Staph. aureus is considered the world's third most important cause of food-born illnesses due to its ability to graw and produce enterotoxins (SEs) under a wide range of conditions.

The pathogenicity of Staph. aureus and its ability to cause diseases is attributed to a number of virulence factors such as the heat stable enterotoxins (Sande and Mckilli, 2004). Staph. aureus produces 15 enteotoxins (Atichou et al., 2004). The five classical enterotoxins (SEs type A, B, C, D and E) were known to be responsible for 95% of Staphylococcal food poisoning (SFP) cases, the rest of cases were due to the new types of SEs (SE G – SE O) (Wang et al., 2012). However, the role of newly identified enterotoxins in food poisoning is not fully clarified, and the development of methods for the detection of these novel SE genes is of critical importance for food poisoning investigations.

The staphylococcal food poisning (SEP) is a mild intoxication occurring after the ingestion of food containing from 20 ng up to1 ug of Staphylococal enterotoxins (SEs) which is enough to induce symptoms in human beings (Nermano et al., 2007). The SFP symptoms appear 1-6 h after ingestion of contaminated food, depending on individual and toxic dose ingested. They include nauseas, abdominal cramps, diarrhea, general malaise, weakness and characteristic projectile vomiting. Clinical signs of SFP generally disappear within 24-48 h. Deaths occur rarely and specifically in the very young or elderly (Jay et al., 2005).

Enterotoxins are highly thermostable, normal cooking and pasteurization cannot totally inactivate them leading to food poisoning (Nagarajappa et al., 2012). Pasteurization kills Staph. aureus cells, and fermentation or ripening of cheese may prevent growth of Staph. aureus in raw milk cheese. However, once formed the thermostable SEs generally retain their biological activity (Le Loir et al., 2003 and Becker et al., 2007). Although SEs a generally heat resistant and a heat denatured enterotoxin, it can be renatured by prolonged storage or in the presence of urea. The SEs also are resist most proteolytic enzymes such as pepsin or trypsin thus keep their activity in the digestive tract after ingestion and are capable of causing food poisoning (Lee Loir et al., 2003).

Because of the importance of these toxins in the public health and food sectors, an efficient screening methods to detect the prevalence of Staphylococcal enterotoxins (SEs) in food are required. The present work was planned to investigate the prevalence of Staph. aureus in raw milk and yoghurt samples and identification of enterotoxigenic Staph. aureus isolates using ELISA technique.

MATERIALS AND METHODS

A total of 150 random raw milk and yoghurt samples (75 of each) were collected from small dairies and supermarkets in Assiut city, Egypt. The samples were collected in clean, sterile plastic bags as marketed to the consumers. The samples were transferred in an ice box as rapidly as possible to the laboratory were subjected to microbilogical examination:-

I- Isolation of Staphylococcus aureus: accordingto (FDA, 2001).

A- Isolation.

Samples were inoculated on Baird-Parker agar plates and incubated at 35 ⁰C for 48h. The typical appearance of Staph. aureus colonies are circular, smooth, convex, moist, 2-3 mm in diameter, gray to jet-black, frequently with light-colored (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone. Inoculated typical colonies on TSA slants were used for maintenance and further identification.

B- Identification.

1- Microscopical appearance.

2- Catalase test.

Growth from TSA slant was used for catalase test on glass slide or spot plate, and illuminated properly to observe production of gas bubbles.

3- Coagulase test.

Suspected Staph. aureus colonies were inoculated into small tubes containing 0.2-0.3 ml BHI broth and emulsified thoroughly and incubated at 35 ⁰C for 18-24h then 0.5 ml of reconstituted coagulase plasma with EDTA was added to the BHI culture and mixed thoroughly. The tube was incubated at 35°C and examined periodically over 6 h period for clot formation.

4- Anaerobic utilization of glucose

Tube of carbohydrate fermentation medium containing glucose (0.5%) was inoculated with suspected organism. The tube surface covered with sterile paraffin oil at least 25 mm thick and incubated for 5 days at 37°C. Acid is produced anaerobically if indicator changes to yellow throughout tube, indicating presence of S. aureus.

5- Lysostaphin sensitivity.

Isolated colony transferred from agar plate with inoculating loop to 0.2 ml phosphate-saline buffer, and emulsified. Half of suspended cells was transferred to another tube (13 x 100 mm) and mixed with 0.1 ml phosphate saline buffer as control. Add 0.1 ml lysostaphin (dissolved in 0.02 M phosphate-saline buffer containing 1% NaCl) to original tube for concentration of 25 μg lysostaphin/ml. Both tubes were incubated at 35°C for not more than 2 h. If turbidity clears in test mixture, test is considered positive. If clearing has not occurred in 2 h, test is negative. Staph. aureus is generally positive.

II- Determination of Staphylococcus aureus enterotoxins: Staph. aureus isolates (39 strains) from raw milk samples were performed in Food Analysis Center, Faculty of Veterinary Medicine, Benha Univirsity, Egypt, by using ELISA technique according to (Ewald, 1988) as following:-

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

According to the test kit manual, a loopful of the culture 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 more 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 more 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 H2 SO4) 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 of Staphylococcus aureus in the examined raw milk and yoghurt samples.

Table 2: The distribution of Staphylococcus aureus enterotoxins recovered from raw milk samples.

DISCUSSION

The results illusterated in Table (1) revealed that, 52% of the examined raw milk samples were contaminated with Staphylococcus aureus. Nearly similar results 52 and 56.66 were reported by Pourhassan and Taravat, (2011) and EL-Jakee et al. (2013), respectively. However, higher results were recorded by Al-Tarazi et al. (2003); Ekici et al. (2004) and Oliveira et al. (2011) in percentages of 80, 75 and 58%, respectively. While, lower results were estimated by Yagoub et al. (2005); Singh et al. (2011); Khuder et al. (2012); Meshref (2013); Thaker et al. (2013) and Vahedi et al. (2013).

Presence of Staphylococus aureus in raw milk cold be attributed to many causes such as subclinical mastitis, where Staph. aureus was the most isolated bacteria or due to contamination of raw milk by milkers and milk handlers. Also, inadequate refrigeration and long storage of milk before use help growth of Staph. aureus (Asperger, 1994). In addition, negligence of hygienic condition such as improper cleaning of bulk tank, dirty udder, milk equipments, milk handling techniques and improper storage will increase the proportion of Gram-positive and Gram-negative bacteria in the bulk tank milk (Bonfoh et al., 2003).

Concerning examined yoghurt samples, Staph. aureus couldn't be detected in the present study. Similar results were obtained by Rodriguez et al. (1990); El-Bessery (2001) and Isam et al. (2011). The failure of Staph. aureus detection in this study may be due to pH of yoghurt, lactic acid and inhibitory substances produced by yoghurt culture which reduce or inhibit Staph. aureus growth in yoghurt (Pazakova et al., 1997). Moreover, such organisms was inhibited after few days during storage of fermented products (Estrada et al., 1999).

Table (2) illusterated the distribution of Staph. aureus enterotoxins recovered from raw milk samples by using ELISA technique, where the ability to synthesize enterotoxins was found in 12 (30.8%) out of 39 isolates of Staph. aureus.

Five isolate (41.7%) produce SEA, three isolate (25%) produce SEC, two isolates (16.7%) produce SED, one isolate produce SE A&C and another isolate produce SE C&D, both in percentage of 8.3%.

Of the twelve Enterotoxigenic positive isolates of Staph. aureus, two isolates only are able to produce more than one enterotoxins (one produce A & C and another produce C & D), while, all the remaining 10 isolates were able to produce one enterotoxin only.

These findings were nearly similar to that detected byNermano et al. (2007) and Rahimi et al. (2012)who detected similar findings of enterotoxin A. However, some of the present finding differ from those reported by other researchers in other countries, such studies have indicated that SEC was the most predominant type (Stephen et al., 2001; Jorgensen et al., 2001; Katsuda et al., 2005 and Loncarevic et al., 2005).

StaphylococcalenterotoxinsSEA and SEB are the most important gastroenteritis causing agents. In some areas, more than 50% of food poisoning is caused by SEA and SEB (> 60%) in USA and England (Kluytman and Werthein 2005). SEA is the most common enterotoxin recovered from food-poisoning outbreak in US (77.8%) followed by SED (37.5%) and SEB (10%) (Rall et al., 2008).

In conclusion Staph. aureus producing enterotoxins could be isolated from examind raw milk samples, so most official regulations should stricktly require the absence of Staph. aureus and its enterotoxins in milk and dairy products which could be highlighted Using ELISA technique.

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