ENTEROTOXIGENIC STAPHYLOCOCCUS AUREUS IN RAW AND PASTEURIZED MILK AND SOME MILK PRODUCTS

Assiut University web-site: www.aun.edu.eg

 ENTEROTOXIGENIC STAPHYLOCOCCUS AUREUS IN RAW AND PASTEURIZED MILK AND SOME MILK PRODUCTS

SANYA, T. EL-GHAMRY and EL-DOSOKY H.F.A

Food Hygiene Dept. Mansoura Provential Lab., Animal Health Research Institute, Egypt

Received: 31 March 2018;     Accepted: 29 April 2018

INTRODUCTION

Milk and Milk products are highly nutritious products specially for young and old aged due to its contents of proteins, fats, sugars, minerals and vitamins hence, they may exposed to be contaminated with bacteria through animals or its contact environment or handling and distribution. Staph. aureus was one of the dominant bacteria associated with raw milk. This might be due to the fact that milk is a good nutritive medium for microorganisms growth especially in poor sanitary conditions and lack of cooling facilities. Sattar et al. (2001) and Mubarack et al. (2010) added that Staph. aureus introduced into the milk also by droplet infection or from udder surface and milker’s hands.

Normanno et al. (2005); Bhatia and Zahoor (2007) and Rabello et al. (2007) mentioned that Staph. aureus commonly causes gastroenteritis resulting from consumption of contaminated food in which enterotoxigenic   staphylococci    have     grown    and

Corresponding author: Dr. EL-DOSOKY H.F.A

E-mail address: rafat552008@yahoo.com

Present address: Food Hygiene Dept. Mansoura Provential Lab, Animal Health Research Institute, Egypt

produced toxins. As these toxins are excreted from the organism, they are referred to as exotoxins. Staphylococcal enterotoxins are considered a potential biological threat because of their stability at 100°C for 1 hour.

Zhang et al. (1998); Atanassova et al. (2001); Loir et al. (2003) and Alegro et al. (2007) assured that Staphylococcal enterotoxicosis has a very rapid onset and course characterized by vomiting, headache, abdominal pain, and diarrhea develop as early as one to six hours after consumption of contaminated food. The symptoms resolve spontaneously within 24–48 hours. Meanwhile, Lina et al. (2004) added that Staph. aureus enterotoxicosis are due  to the classical enterotoxins (SEA, SEB, SEC, SED, SEE) and  several new variants of SEs.

Bergdoll (1983) and Letertre et al. (2003) concluded that the first five (A to E) classical enterotoxins are known to cause 95% of the food poisoning globally and Argudin et al. (2010) isolate 22 types of SEs designated with letters A-V are currently known. While, Bennett, (2005) demonstrated that there is a strong association between the ability of Staph. aureus strains to produce one or more of the SEs and the occurrence of staphylococcal food poisoning. Weronika and Jacek (2014) found that 11.9% of the isolated strains were positive for one or more classical SE markers. The aim of this study was to detect enterotoxigenic Staph. aureus prevalence which is a potential source of food poisoning

MATERIALS AND METHODS

Two hundred and fifty samples of milk and milk products (50 samples each) of raw market milk, pasteurized milk, soft white cheese, table butter and small scale ice cream were collected from different shops at Mansoura city and sent to the laboratory in icebox for examination without delay.

Enumeration and Isolation of Coagulase positive Staph. aureus according to (APHA 2001) 10 ml or g each of examined milk and milk product samples were taken aseptically and homogenized with 90 ml 0.1% peptone water in a stomacher for 3 minutes at 3000 rpm and filtered through a sterile cheese cloth filter, followed by six fold serial dilutions in 0.1% peptone water then 0.1 ml were taken from each dilution aseptically and inoculated onto Baird-Parker medium, the plates were incubated for 24-48 hours at 37°C. The plates containing 20-200 colonies were selected. Typical colonies of Staph. aureus were circular, smooth, convex, moist 2-3mm in diameter, grey to black (potassium tellurite reaction) with white margin and surrounded by outer clear zone (egg yolk reaction) the suspected colonies were streaked onto agar slant of nutrient agar medium and incubated at 37°C for 24 hours for further purification and identification by microscopical and biochemical examination by catalase, coagulase, thermostable nuclease and Voges-Proskauer tests.

Staph. aureus culture supernatant were collected  by Sac cultural method (Donnelly et al., 1967) and tested serologically by reversed passive latex agglutination technique using Oxoid SET-RPLA kits for the presence of SEA, SEB, SEC, SED and SEE.

Extraction of Staph. aureus enterotoxins from the examined samples were completed by blending of 10 ml of milk or milk product samples with 10 ml of sodium chloride solution (0.85%) and centrifuged. The supernatant was retained for toxin detection using Oxoid SET-RPLA kits Shingaki et al. (1981).

Detection of virulence genes in Staph. aureus using PCR (Reference Lab for Quality Control on Poultry Production, Animal Health Research Institute, Dokki -Egypt)

1-DNA extraction:

DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56^OC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

2- Oligonucleotide Primer. Primers used were supplied from Metabion (Germany) are listed in Table (1).

3- For multiplex PCR of enterotoxins, Primers were utilized in a 50-µl reaction containing 25 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 8 µl of water, and 7 µl of DNA template. The reaction was performed in an Applied biosystem 2720 thermal cycler.

4- Analysis of the PCR Products.

The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1xTBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 30 µl of the multiplex PCR products were loaded in each gel slot. Gelpilot 100 bp DNA ladder (Qiagen, Germany, GmbH) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

Table 1: Primers sequences, target genes, amplicon sizes and cycling conditions of Staphylococcus aureus enterotoxins.

Statistical analysis:

The results are expressed as log mean ± standard error (SE). Data were statistically analyzed using statistical analysis systems.

RESULTS

Table 2: Mean counts of Staph. aureus in the examined samples expressed as log₁₀cfu/g or ml (n=50).

NB: n= number of the examined samples

Table 3: Incidence and Disribution of enterotoxins produced by Staph. aureus strains isolated from the examined samples by SET-RPLA kits and PCR (n=50).

Results of Polymerase chain reaction:

Multiplex PCR for enterotoxiginic Staph. aureus genes:

Results of the isolated Staph. aureus from the examined raw milk by using multiplex PCR using sets of primers for enterotoxins (A,B,C,D and E) showed that

Fig (1): Agarose gel electrophoresis of Staph. aureus PCR products using enterotoxins Staph. aureus primer

Pos=positive control, Neg=negative control, L=100 bp DNA ladder

Lane "1": positive amplification of 102 bp for enterotoxin A

Lane "2 "and Lane "4" were negative 

Lane "3": positive amplification of 164 bp for enterotoxin B

Lane "5": positive amplification of 102 bp for enterotoxin A, 209 bp for enterotoxin E

Results of the isolated Staph. aureus from examined white soft cheese samples (Lane 1,2&3) and butter samples (Lane 4&5) by using multiplex PCR using sets of primers producing enterotoxins (A,B,C,D and E) showed that

Fig (2): Agarose gel electrophoresis of Staph. aureus PCR products using enterotoxins Staph. aureus primer

Pos=positive control, Neg=negative control, L=100 bp DNA ladder

Lane "1": positive amplification of 102 bp for enterotoxin A and 278 bp for enterotoxin D

Lane "2" positive amplification of 102 bp for enterotoxin A and 209 bp for enterotoxin E

Lane "3" positive amplification of 278 bp for enterotoxin D

Lane "4": positive amplification of 164 bp for enterotoxin B

Lane "5": positive amplification of 102 bp for enterotoxin A and164 bp for enterotoxin B

DISCUSSION

Presence of lactic acid bacteria lowering the pH in raw milk that may prevent Staph. aureus growth and enterotoxin production (Alomar et al., 2008 and  Janstova et al., 2012). Pinchuk et al. (2010) mentioned that bacterial counts of Staph. aureus need to reach 10⁵-10⁸ cfu/mL before sufficient amount of toxin to cause illness is produced while, Evenson et al. (1988) showed that growth of enterotoxigenic Staph. aureus up to 10⁶ or more/g of food enables them to produce a sufficient amount of enterotoxins to cause intoxication. As little as 20ng of SE can induce nausea, violent vomiting, abdominal cramps, and diarrhea between 1 to 8 h after food consumption. The achieved results in Tables 2,3 and Fig. 1 declared that the highest contamination of Staph. aureus were found in raw market milk with mean counts of 3.8 ±1.4 log₁₀cfu/ml with incidence percent of 36% mean while, 3 out of the examined 18 isolates by PCR and SET-RPLA kits were enterotoxigenic. The enterotoxigenic strains have sea, seb and see virulent genes, these results were nearly in accordance with Manfreda et al. (2005) who found 34.6% of milk samples were contaminated with Staph. aureus, 6.6% of which were enterotoxin producers. Enterotoxigenic strains were most frequently detected in milk with Staph. aureus count 4.47 log₁₀cfu/ml; Bianchi et al. (2013) declared that 53% of raw milk were positive for one or more SE genes and Staph. aureus count were 3.5 ±2.38 log₁₀cfu/ml with incidence percent of 32%. Also, Thabet et al. (2014) revealed that Staph. aureus was isolated with a percentage of 26.6% from raw milk; Hu Shou Kui et al. (2013) found  Staph. aureus in 30.0% of examined raw milk and 43.7% of the isolated Staph. aureus produced enterotoxins. These results were lower than that reported by Weronika and Jacek (2014) and Gundogan and Avc (2014) who found the incidence percent in raw milk were 56% and higher than Rajeev and Amit (2010) who could isolate Staphylococcus from milk by 10%.

The obtained results of Staph. aureus count and its incidence in Pasteurized milk in Tables 2 and 3 declared that the mean counts were 1.78±0.48 log₁₀cfu/ml with incidence percent of 4% mean while, the enterotoxigenic strains of Staph. aureus could not be detected by PCR and SET-RPLA kits. These results were in accordance with those obtained by Gad EL-Said et al. (2013) who reported that no enterotoxigenic Staph. aureus were detected in pasteurized milk and Asao et al. (2003) who  added that pasteurizing raw milk would eliminate Staph. aureus from raw milk, however once the pathogens have produced enterotoxins the toxins will remain stable even after pasteurization. Also, Jicinska and Havlova (1995) concluded that because of its heat resistance, Staph. aureus can be detected even in pasteurized milk in addition to Anderson et al. (1996) shown that Staph. aureus enterotoxins are highly resistant to heat treatment, a good example is sea, which retained its biological activity even after exposure to 121°C for 28 minutes.

Jablonski and Bohach (2001) reported that 10³ and 10⁵cfu/g Staph. aureus is able to produce enterotoxin in amounts that can pose a health risk to the consumers.

The achieved results of white soft cheese in Tables 2,3 and Fig 2 declared that the mean counts of Staph. aureus were 3.4±1.25 log₁₀cfu/g with incidence percent 24% mean while, 3 out of the examined 12 isolates were enterotoxigenic detected in examined samples by PCR and SET-RPLA kits have the enterotoxigenic virulent genes sea, sed and see. These results were lower than Gundogan and Avc (2014) who found that 48% of white cheese were contaminated with Staph. aureus. While, Thabet et al. (2014) revealed that Staph. aureus was isolated with a percentage of 6.6% in Damietta cheese samples and  Hu Shoukui et al. (2013) the positive rate of Staph. aureus in milk products including cheese were 7.5% and 43.7% of the isolated Staph. aureus produced enterotoxins. Gucukoglu et al. (2012) investigated that the enterotoxigenic Staph. aureus was detected in white cheese by 19%, two isolates from cheese samples 50% were found to be enterotoxigenic. Rahimi (2013) reported that 11.1% of examined cheese were found to be contaminated with Staph. aureus and the ability to synthesize classical staphylococcal enterotoxins (SEA-E) was determined in 7 of 20 (35%) isolates.

Bianchi et al. (2013) found that milk and dairy products account for 5% of all the incriminated foods poisoning.

Theresults of Staph. aureus incidence in Tables 2, 3 and Fig 2 of the examined table butter samples were 8% with mean count of 2.39±0.95log₁₀cfu/g and the enterotoxigenic virulent genes of Staph. aureus was detected in 2out of 4 isolates from the examined table butter samples. The isolated enterotoxigenic strains of Staph. aureus by PCR and SET-RPLA kits have sea and sebvirulent genes. These results were nearly in accordance with Rahimi, (2013) who found 5.3% of butter samples contaminated with Staph. aureus and 35% of the isolated Staph. aureus were able to synthesize the classical staphylococcal enterotoxins (SEA-E). While, Gucukoglu et al. (2012) investigated that the enterotoxigenic Staph. aureus was detected in 30% of the examined butter samples and 25% of them showed enterotoxigenic character (SEB 100%).

The results in Tables 2 and 3 indicated that the incidence percent of Staph. aureus in ice cream samples were 4%, with mean counts 2.14±0.78 log₁₀cfu/ml. The enterotoxigenic strains could not be detected in the examined samples either by PCR or by SET-RPLA kits. Bostan and Akn (2002); Sagdc et al. (2003) and Hu ShouKui et al. (2013) could not found Staph. aureus in ice creams samples while, higher percentage were reported by Gunsen (2002) who found Staph. aureus in 5% of lemon ice cream samples; Rahimi, (2013) found 5.9% ice-cream contaminated with Staph. aureus and Gundogan and Avc (2014) found Staph. aureus in 36% of the examined ice cream samples. Nazem et al. (2010) isolate Staph. aureus from 5% of ice cream collected from supermarkets; lower percentage were reported by Rajeev and Amit (2010) who isolated Staphylococcus from Ice cream by 1%; Yucel and Ctak (2002) found Staph. aureus count 1.0x10²-3.0x10³cfu/ml in ice cream samples. Guner et al. (2004) added that counts of Staph. aureus in ice cream were 1.2-1.7x10³cfu/g and El-Ansary (2015) found Staph. aureus count was 1.10x10³± 2.45x10²cfu/ml in Vanilla ice cream samples, which could be associated with potential food poisoning hazards. On the other side, Gucukoglu et al. (2012) investigated that the enterotoxigenic Staph. aureus was detected in 10% of ice cream samples.

Asao et al. (2003) mentioned that Staph. aureus were frequently contaminator for ice cream. Hence, improvement of the hygienic practice in processing, preparing and storage should be stressed and Schmitt et al. (1990) declared that the causes of staphylococcal enterotoxicosis are classical SEs. SEA, SEB, SEC1, SEC2, SEC3, SED, and SEE and the production of SEs is unlikely at temperatures below 10⁰C.

Bergdoll (1989) concluded that a very small amount of Staph. aureus enterotoxins ranging from 20 ng to < 1 μg is needed to cause a typical symptoms of staphylococcal food poisoning. An outbreak in Japan caused by low-fat milk contaminated with SEA showed that the total intake of SEA per individual was estimated to be 20–100 ng, More recently, Ostyn et al. (2010) in France found an outbreak caused by contaminated cheese, doses of SEE ingested by symptomatic persons were estimated to be about 90 ng, based on the mean weight of the cheese portion (about 200 g) and the total amount of SEE in food samples were 0.45ng/g.

CONCLUSION

The presence of enterotoxigenic Staph. aureus in raw milk and milk products poses a potential health hazard to the consumers. However, not only identification of such strains but also appropriate conditions for Staph. aureus enterotoxin genes during production and storage of milk and milk products should be taken into account in hazard risk analysis.

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