الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Dairy products are liable to contamination with different types of microorganisms from different sources during production, processing and handling, which lead them to be unfit for consumption and constitute a public health hazard. S. aureus remains the first causative agent involved in food-borne diseases in milk and milk products.
Staphylococcal enterotoxins (SEs) are resistant to inactivation by gastrointestinal proteases such as pepsin as well as by heat. Heat stability is one of the most important properties of SEs in terms of food safety. Although pasteurization kills S. aureus cells, thermostable SEs generally retain their biological activity. In addition, enterotoxins retain their biological activity even after pasteurization.
Raw milk and raw-milk cheese contaminated with MRSA have been incriminated in the transmission of the pathogen to humans. Handling or consumption of raw milk and dairy products may lead to the spread of antimicrobial resistance genes of S. aureus to humans.
The main objective of the present study was to assess prevalence of S. aureus, detect their enterotoxin productivity and anti-microbial resistance in raw milk and cheese products.
To fulfill this objective a total of 150 cheese samples of different types and 50 milk samples were taken during the study as follows:
• Thirty white soft cheese samples, 30 white hard cheese samples, 30 edge roamy cheese samples, 30 core roamy cheese and 30 cheddar samples collected from 9 supermarkets and 6 hypermarkets across East, Center and West districts.
• Thirty raw milk samples and 20 pasteurized milk samples collected from 9 supermarkets and 6 hypermarkets across East, Center and West districts.
Isolation and identification of coagulase negative staphylococci and S. aureus was done using ISO 6888-1:1999, performing the antimicrobial sensitivity test for the isolated S. aureus. Was done using Biomereux Vitek-2 compact system and detection of S. aureus enterotoxins genes was done using multiplex PCR.
The results of the present study revealed that:
Prevalence and count of coagulase negative staphylococci isolated from cheese and milk:
• As a total, 148 cheese samples out of 150 samples representing 98.6% were positive for coagulase negative staphylococci. with a count ranging from 2.7×104 to 4.6×107 CFU/g and a mean value of 6.8×106 CFU/g.
• 100.0% of white soft cheese, white hard cheese and roamy cheese collected from the three districts were positive for Staph. spp. While, 100.0% of cheddar cheese collected from East district, 90.0% of cheddar cheese collected from center and West districts were positive for coagulase negative staphylococci.
• As a total, 97.6 and 100.0% of different types of cheese sold in supermarkets and hypermarkets respectively were positive for coagulase negative staphylococci. The highest count was 1.5×107 CFU/g in white soft cheese samples sold in hypermarkets and the lowest count was 2.0×102 CFU/g in core roamy sold in supermarkets.
• 96.6%, 33.4%, 40.0%, 36.7% and 26.7% of soft white cheese samples, white hard cheese, edge roamy cheese, core roamy cheese and cheddar cheese had coagulase negative staphylococci counts of 105CFU/ml or more.
• As a total 100.0% of raw milk samples were positive for coagulase negativestaphylococciwith a mean value of 3.5×106 CFU/ml. While, 40.0% of pasteurized milk samples were positive for coagulase negativestaphylococci with a mean value of 2.6×103 CFU/ml.
• 100.0% of raw milk samples collected from East, Center and West districts was positive for coagulase negative staphylococciWhile, 28.5%, 71.4% and 16.7% of pasteurized milk samples collected from East, Center and West districts respectively were positive for coagulase negative staphylococci.
• 100.0% of raw and pasteurized milk samples sold in supermarkets were positive for coagulase negative staphylococciWhile, 33.3% of pasteurized milk sold in hypermarkets were positive for coagulase negative staphylococci.
• 46.7% of raw milk samples had coagulase negative staphylococcicounts of 105CFU/ml or more. All pasteurized milk samples had coagulase negative staphylococcicounts less than105CFU/ml.
Prevalence and count of S. aureus isolated from cheese and milk:
• As a total, 33 out of 150 cheese samples representing 22.0% were positive for
S. aureus with a count ranging from 1.2×105 CFU/g to 2.2×107 CFU/g with a mean value of 7.4×106 CFU/g.
• 46.7%, 30.0%, 20.0%, 13.3% and 0.0% of white soft cheese samples, white hard cheese, edge roamy cheese, core roamy and cheddar cheese samples, respectively, were positive for S. aureus.
o 40.0%, 60.0% and 40.0% of white soft cheese samples in East, Center and West districts, respectively, were positive for S. aureus.
o 10.0%, 50.0% and 30.0% of white hard cheese samples in East, Center and West districts, respectively, were positive for S. aureus.
o 0.0%, 40.0% and 20.0% of edge roamy cheese samples in East, Center and West districts, respectively, were positive for S. aureus.
o 10.0%, 20.0% and 10.0% of core roamy cheese samples in East, Center and West districts, respectively, were positive for S. aureus.
o S. aureus was not detected in cheddar cheese in the three districts.
• As a total samples 17.4% and 28.1% of the different types of cheese sold in supermarkets and hypermarkets respectively were positive to S. aureus. The highest count was 1.3×107 CFU/g in white soft cheese samples sold in supermarkets with a mean value of 7.4×106 CFU/g and the lowest count was 6.0×103 CFU/g in roamy edge sold in supermarkets with a mean value of 2.8×104 CFU/g.
• As a total 46.7% of raw milk samples sold in supermarkets were positive for
S. aureus, the highest count was 1.4×107 CFU/ml with a mean value of 4.0×106 CFU/ml.
• 40.0%, 30.0% and 70.0% of raw milk samples were positive for S. aureus in East, Center and West districts respectively.
• All pasteurized milk samples showed negative results for S. aureus in the three districts.
• 43.3%, 16.7%, 10.0%, 6.7% and 0.0% of soft white cheese samples, white hard cheese, edge roamy cheese, core roamy cheese and cheddar cheese had S. aureus counts of ≥105 CFU/ml.
• 36.7% of raw milk samples had S. aureus counts of ≥ 105 CFU/ml.
Prevalence of enterotoxigenic S. aureus isolated from cheese and milk:
• Out of 200 cheese and milk samples, 47 samples (23.5%) were positive for S. aureus and with further analysis, 34 samples (72.3% of S. aureus positive samples) had SEs genes. While, 13 (27.6%) had not SEs genes.
• Out of 150 different cheese samples collected from Alexandria market, 33 (22.0%) were positive S. aureus and with further analysis it was noted that 24 (16.0%) had SEs genes. While, 9 (6.0%) had not.
• Out of 24 cheese samples with SEs gene; 17 samples (70.8% of S. aureus positive samples) encoded see, 4 samples (16.7%) encoded multiple toxins genes sea+seb+see, one sample (4.2%) encoded seb, one sample (4.2%) encoded seb+see, and one sample (4.2%) encoded sea+seb.
• Out of 50 raw milk samples collected from Alexandria supermarkets, 14samples (28.0%) were positive for S. aureus and with further analysis 10 samples (20.0%) had SEs genes and 4 samples (8.0%) had no SEs genes.
• Out of 10 milk samples with SEs gene; 5 samples (50.0% of S. aureus positive samples) encoded see, 3 samples (30.0%) encoded seb, one sample (10.0%) encoded multiple toxins genes sea+seb+see, and one sample (10.0%) encoded seb+see.
• White soft cheese isolates were the most encoders for S. aureus enterotoxins genes representing 33.3%(23.3%, 6.7% and 3.3% for see, sea+seb+see and sea+see, respectively) followed by white hard cheese isolates representing 23.3% (20.0% and 3.3% for see, sea+seb+see respectively), followed by edge roamy cheese isolates representing 13% (10.0% and 3.3% for see, sea+seb+see respectively), then core roamy cheese isolates representing 1.0% (3.3% for seb, see or sea+seb+see).
Antimicrobial resistance and MRSA detection in S. aureus isolated from cheese and milk:
• In cheese, the highest antibiotics resistance rate belongs to benzylpenicillin (90.9%) followed by tetracycline (84.8%), cefoxitin (24.2%), oxacillin (24.2%), gentamycin (15.2%), ciprofloxacin (3.0%), erythromycin (3.0%), clindamycin (3.0%), trimethoprim/sulphamethoxazole (3.0%).
• In milk, the highest antibiotics resistance rate belongs to benzylpenicillin (64.3%) followed by tetracycline (57.1%), cefoxitin (14.3%), oxacillin (14.3%), gentamycin (14.3%), clindamycin (14.3%).
• Out of 47 S. aureus analyzed isolates, 10 isolates (21.3%) were identified as MRSA as a result of antimicrobial sensitivity test. Positive isolates were derived from different types of cheese and raw milk samples in particular four isolates from white soft cheese, two from raw milk, two from edge roamy cheese and one from white hard cheese and core roamy cheese respectively. Half (50.0%) of MRSA isolates encodedsee, 1 sample (10.0%) encodedseb and 40.0% non enterotoxigenic S. aureus. Half (50.0%) of MRSA isolates from Center district, 3 from West district and 2 samples from East. Six isolates belonged to samples collected from hypermarkets and 4 isolates belonged to samples collected from supermarkets.
• In MRSA, 100.0% of isolates were resistant to cefoxitin, benzylpenicillin and oxacillin, 70.0% of the isolates were resistant to gentamycin while, 10.0% were intermediately resistance to levofloxacin and 10.0% were resistant to ciprofloxacin, erythromycin, clindamycin, qunipristin/daphlopristin, tetracycline.
• Out of thirty three tested isolates fromcheese,seventeen (51.5%) were resistant to two antibiotics, five (15.2%) to one and five antibiotics respectively, three (9.1%) to three antibiotics, one (3.0%) to four and six antibiotics respectively. While, one (3.0%) had no resistance to any of the tested antibiotics.
• Out of fourteen tested isolates from raw milk, five (35.7%) were resistant to two antibiotics, three (21.4%) to one, 2 (14.3%) to five antibiotics and one (7.1%) to three antibiotics. While, three (21.4%) had no resistance to any of the tested antibiotics.