Detection of enterotoxins genes in staphylococci isolated from milk and cheese

Other Title(s)

التحري عن جينات السموم المعوية في المكورات العنقودية المعزولة من الحليب و الجبن

Dissertant

al-Khafaji, Marwah Hamid Muṭashshar

Thesis advisor

Fulayyih, Mayy Talib

Comitee Members

al-Hassani, Hayfa Hadi
Kazim, Shruq R.
Jumah, Intisar M.
Fadil, Iyad Muhammad Ali
Muslih, Rashid Mahjub

University

University of Baghdad

Faculty

College of Science

Department

Department of Biology

University Country

Iraq

Degree

Ph.D.

Degree Date

2013

English Abstract

During 2011; 300 milk and cheese samples were collected from Baghdad markets.

Two hundred staphylococcal isolates were isolated from milk and cheese samples, the predominant species was Staphylococcus aureus, 97 isolates (48%), followed by S.chromogenes 82 isolates (41%) and 21 (11%) S.epidermidis isolates.

The pattern of antibiotic susceptibility of Coagulase Positive Staphylococci (COPS) and Coagulase Negative Staphylococci (CONS) isolates to 3 antibiotics (Methicillin, Tetracycline and Vancomycin) was determined using disc diffusion method; the results revealed that 80 S.

aureus isolates (82.47%) found to be methicillin resistant (MRSA) and 18 S.

aureus isolates (19%) resist tetracycline while 8 isolates (8.24%) were vancomycin resistant (VRSA).

Sixty four CONS isolates (62.13%) were methicillin resistant, 28 CONS isolates (27.18%) resist tetracycline, and 5 CONS isolates (4.85%) were vancomycin resistant.

Deoxyribo nucleic acid (DNA) extraction from staphylococcal isolates and directly from milk and cheese samples was done manually.

The isolates were subjected to Polymerase chain reaction (PCR) technique in a monoplex pattern to amplify coagulase encoding gene: the coa gene; results by this study showed that 76 (78.35%) S.

aureus isolates gave the amplicon size 730 base pair of the coa gene.

The genetic determinants of methicillin resistance femA and mecA genes were amplified using monoplex PCR technique in order to identify methicillin resistant (mecA+) and susceptible (lacking mecA) staphylococci and to differentiate S.

aureus (femA+) from coagulase negative staphylococci (lacking femA).

Ninety six S.

aureus isolates (98.96%) were found to harbour femA gene, it is species specific marker for S.

aureus.

The mecA gene was detected in 91 (93.81%) MRSA isolates, while it was detected in 70 (67%) CONS isolates.

The detection of staphylococcal enterotoxigenicity according to four classical enterotoxins genes which are sea, seb, sec, and sed was performed simultaneously using multiplex PCR assay.

One hundred fifty three staphylococcal isolates (76.5%) found to be enterotoxigenic, 95 S.aureus isolate (62.09%) found to harbor one or more enterotoxin gene.

CONS isolates showed to be enterotoxigenic in this research, they accomplished 37.9% of the total enterotoxigenic strains (58 strains).

The sea gene was the most frequent enterotoxin coding gene among the others tested; sea accomplished 51.85% of the detected enterotoxin genes followed by the sed gene, which constituted 34.73%, and then the seb coding gene by 12.5% while the sec gene was very rare as 0.92%.

The results obtained by this study showed that 102 (66.66%) staphylococcal isolates harboured only one enterotoxin coding gene, while 39 (25.5%) of the isolates possessed two toxin genes, which were mostly sea and sed genes and 12 staphylococcal isolates (7.8%) harboured three toxin coding gene: sea, seb and sed genes.

Suckling mouse bioassay was tested to investigate the staphylococcal enterotoxin biological activity.

Results showed that 131 isolates which constitutes 65.5% of the examined isolates, gave a positive result.

Both COPS and CONS isolates were shown to be enterotoxigenic, COPS represented by S.

aureus species occupied the higher ratio of the enterotoxigenic staphylococci, 86 S.aureus isolates (65.64% of the enterotoxigenic staphylococci) gave the positive ratio of the intestine weight to the body weight which was ≥ 0.083 while 45 CONS isolates (34.35%) were enterotoxin producers.

These toxins were thermostable staphylococcal enterotoxins which gave the same toxic effect after heating to 100°C for 30 minutes.

Comparing the results obtained by multiplex PCR assay detecting four classical enterotoxin coding genes, with those obtained by suckling mouse bioassay concerning the phenotypic expression of enterotoxin coding genes, 153 (76.5%) staphylococcal isolates harboured one or more enterotoxin coding genes, while the suckling mouse bioassay revealed that 131 (65.5%) of those isolates produced detectable amounts of enterotoxins.

Main Subjects

Biology

Topics

• Microbiology
• Milk
• Iraq
• Cheese

No. of Pages

163

Table of Contents

• APA
• MLA
• AMA

Language

English

Data Type

Arab Theses

Record ID

BIM-598836