Molecular identification of methicillin-resistance Staphylococcus aureus isolated from milk of breast infection woman in Diwanyia province.

Methicillin-resistance Staphylococcus aureus (MRSA) is the greatest important source of community-acquired infection in humans. The present study was carried out to use (PCR) assay as more specific molecular methods for recovering of methicillin resistant S.aureus (MRSA) isolates that isolated from milk of breast infection woman which collected from Diwanyia hospital . PCR assay was based on used specific primers that amplified of mecA gene in S.aureus isolates. The primers were designed in this study by using NCBI-Genbank data base (KM505042.1) and primer 3 plus primers design. The results of primary identification bacterial isolation were show 16 positive S.aureus isolates out of 50 milk samples. From those 5(31.25)were identification


Introduction
Staphylococcus-aureus is a main important bacterial pathogen in both hospitals acquired infection and the community acquired infection [1]. It is a member of family Micrococcacea, it be Gram positive-cocci, and itis occurs in either singly coccus or form pairs, or which show as clumps of cluster "grape-like" [2]. S. aureus is commonly colonized on external skin surfaces particularly the nasal passages in Human. This bacterium is opportunist pathogen, and can be cause more severe after burns woundbacteria infections, the bacterium which invaded the tissue and production of toxins e.g. toxic shock syndrome [3]. Methicillin resistance Staphylococcus aureus is clinical infection isolates which arise from the production of βlactamase enzyme that hydrolysis of methicillin [4]. The methicillin is member class of βlactamase antibiotic, that inhibiting cell wall formation which comprised in the peptidoglycan synthesis mechanism and composed mesh like polymer which protect and surrounds the bacterial cell [5]. Methicillin -resistant S. aureus (MRSA) are actuality recorded with high frequency in the public community and they have been named community-acquired-(MRSA), mostly associated with soft tissue and skin infection [6]. The presence of Methicillin resistance S.-aureus-(MRSA) in specimens of human-breast milk can be description for secondarycontamination from surrounded skin, breasts and the contamination nasal cavity of baby donor [7]. Many studies reported that the occurrenc of (MRSA) from human milk, and some of these strain capable to cause disease of mastitis by production exotoxins [8]. Other reports on (MRSA) especially in postpartum-mastitis mostly among young, while the other healthy milking women lack risk of factor for (MRSA) that arisen in the previous a small number of years [9][10]. Molecular techniques have the potential of offering highly sensitive and rapid in detection and identification of mecA gene in S. aureus with Methicillin-Resistans. Therefor our study was aimed to molecular detection of -(MRSA) which isolated from milk specimens of breast infection woman in Diwanyia province.

Materials and Methods
Milk specimens collection: 50 specimens of milk of breast infected milk woman were collected in Al-Diwanyia hospital. The milk specimens were collected in 25ml sterile containers after clean and washing the breast using disinfectant solution, then the milk specimens transported into laboratory and stored in a refrigerator until use for bacterial stady.
Bacterial Isolation: Staphylococcus aureus was isolated from milk specimens by inoculation on Brain-Heart-Infusion-Broth-(BHI) at 37°C for overnight for primary enrichment culture and the growth of bacteria were inoculated on mannitol-salt agar (MSA) at 37°C overnight for selective isolation of pure culture S. aureus isolates. After that, the positive growing S. aureus isolates were confirmative identification using (VITEK 2 Compact, Biomerieux).

Bacterial DNA extraction and PCR Method:
PCR technique was performed for detection Methicillin-Resistant Staphylococcus aureus basd mecA gene in S. aureus isolated from milk specimens by following steps:-

1-DNA extaraction:
The Staphylococcus aureus isolates were subjected to nucleic acid extraction using commercial total DNA extraction kit (Presto Mini-DNA Bacteria Kit. Geneaid Biotech Ltd.USA) The extraction method was don depend on the manufacturing instructions using gram positive bacteria DNA Protocol extraction method using (20 mg/ml) lysozyme buffer.

2-Nanodrop:
The extracted DNA was estimatd by nanodrop device at 260/280nm, and then kept at deep freezer until used in PCR technique.

3-Primers :
The primer of PCR that is useing in this study for deteced Methicillin-Resistant S. aureus based on mecA gene were designed in NCBI Gene sequnce data base (Genbank code: KM505042.1) and primer 3 plus design. These primers were provide from «Bioneer company, Korea» as following toble(1):-

Results :
the specimens were firstly inoculated on on Brain-Heart-Infusion-Broth-(BHI) and a total of 16(32%)the growth of bacteria were inoculated on mannitol-salt agar (MSA) The colonies appeared on the (MSA) pale yellow and gold color ,After that the positive growing S. aureus isolates were confirmative identification using (VITEK 2 Compact, Biomerieux).
The PCR was appeared as highly sensitive and more specific assay in direct detection of Methicillin-resistant S. aureus (MRSA) from S. aureus isolated milk infacted breast woman as following The PCR detection of mecA gene in5(31.25%) S. aureus isolates samples was shown good PCR product bands on examination by gel electrophoresis at 503bp product size. Figure (

Discussion :
In this investigation, a total number of 50 specimens were subjected to bacteriological examination for detection and isolation of S. aureus isolates. The Developing colonies appeared on the central Mannitol Salt Agar in yellow, pale yellow, golden, reaching diameters (1-2) milli meters, in addition to the center color of red was changed to yellow color due to the fermentation of mannitol and acid production These results were consistent with the mention [11], [12], [13]. The Incidence of S. aureus among the examined specimens was readed 16(8%) isolates and can be attributed of the high rates of S. aureus clinical samples to the fact that man is the center of a development rich in bacteria that you need and supplied to the temperature and humidity had the appropriate growth and reproduction and that an exceptionally versatile organism were adopt many ecological nichesand in spite of the presence of defensive means, immune to the human body but the Staphylococcus aureus You can find numerous citizen to breed long-term colonization [11].
The polymerase chain reaction (PCR) technique is found very specific and sensitive assay, and less time consumption, when using for direct detection of methicillin-resistant Staphylococcus aureus from breast milk, in contrast to other conventional diagnostic techniques.
Our results showed that PCR is very important tool for the identification of (MRSA) from milk by using universal primers for of mecA gene in methicillin-resistant Staphylococcus aureus , These results were in agreement with [14]who referred that 21 cases with methicillin resistance, 17 (81%) occurred in 2005and the increase is due tocommunity-acquired methicillin-resistant S.aureus ,in [15] MRSA has emerged in patients without established risk factors,that MRSA infections by (15%) were community-associated and (85%) were health care-associated.
Results indicated shown in table 1Samples of milk of breast infection woman This is consistent with a study [16],where he reached, 45.1% of S. aureus isolates were mecA-positive in the PCR assay. in another study, the researcher found from being isolated from human patients with mastitis with more reports indicate that between 30-50% of bacteria from human human mastitis is S. aureus [17]while (MRSA) is a relatively common result in human clinical mastitis rights this represents threat to for the patient. MRSA importance came through the resistance of many antibiotics, such as resistance to all types of anti-B-lactam antibiotics and may cause a problem for patients and their treatment and thus failed to control the infection occurs [18].
This case indicates the growing somewhat fear in bringing different injuries in the body, and difficult to treat, control and dissemination of the pathogen in the population and the environment. So as to anticipate the likely effects on further necessary public research to understand the dynamics of the diseas from S. aureus, especially in the city of Diwaniyah hospitals..