MOLECULAR DETECTION OF ANTIBIOTIC RESISTANCE GENE (MECA) IN STAPHYLOCOCCUS EPIDERMIDIS LOCAL ISOLATES. Rusul Muzher Hussein and Marrib

Rusul Muzher Hussein and Marrib Nazih Rasheed. Ministry of Higher Education and Scientific ResearchUniversity of Baghdad Genetic Engineering and Biotechnology Institute for Postgraduate Studies. Iraq-Baghdad. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The mecAgene is a gene found in bacterial cells. The mecAgene allows a bacterium to be resistant to antibiotics such as methicillin, penicillin and other antibiotics. This study is based on the molecular analysis of the antibiotic resistance gene (mecA). The polymorphism of the mecAgene existing in different S. epidermidishave been analyzed in this study. Seventy five isolates (57) of S.epidermidiswere obtained from eighty four (84) which were collected three hospitals in Baghdad include Al-Yarmuk General Teaching Hospital , Al-Karama teaching hospital, Ibn-ALbalady teaching hospital. These isolates were collected from clinical sources the samples included, wound swab, burn swab, ear swab and urine. .DNA from each sample was isolated and the mecAgene was amplified with appropriate primers. The PCR results of 57 mecA gene positive S.epidermidisgave the band size of amplicons about 500pb.The presence of mecAgene seems to be enhanced in biofilm producers and enable S.epidermidis strains to show increasing resistance to different groups of antibiotics.
Copy Right, IJAR, 2016,. All rights reserved. …………………………………………………………………………………………………….... Introduction:-S. epidermidis which is known as a natural colonizer of healthy human skin and mucosa, is also a common nosocomial pathogen along with other Coagulase Negative Staphylococci (CONS) [1].S. epidermidis is the most important pathogen, responsible from many indwelling medical device related infections such as; catheter, prosthetic joint, vascular graft, surgical site, central nervous system shunt and cardiac device related ones. Therefore, S. epidermidis strains especially emerge as life-threatening pathogens triggering septicemia, meningitis and other serious conditions in medical device using and immunocomprised patient [2,3,4,5]. One of the most important mechanisms making these commensal inhabitants dangerous for medical device using and immunocomprised patients is known as biofilm formation. By means of having ability to adhere to various surfaces and form slimy layer known as biofilm on them [6,7,8,9]. Biofilm is believed to make clinical S.epidermidis strains more resistant to administered antibiotics and to host defense mechanisms and highly contributed to cause nosocomial infections(NI) in patients [10].
In clinical practice, S. epidermidishas become one of the most significant species among methicillin-resistant coagulase negative staphylococci (CONS). There are various scientific world reports that stated that approximately between 80% and 90% of S. epidermidis strains isolated from patients with nosocomial infections(NI )carried the ISSN: 2320-5407 Int. J. Adv. Res. 4(9), 506-510 507 mecAgene [ 11 , 12]. The presence of mecA gene seems to be enhanced in biofilm producers and enable S. epidermidis strains to show increasing resistance to different groups of antibiotics [13,14].
The mecAgene is a component of a large DNA fragment designated as mecDNA, Theacquisition of mecDNA is considered to be the first genetic requisite for methicillin resistance in staphylococci [15].The aim of this study was to detect the presence ofmecA gene in S. epidermidis from clinical sources by PCR amplification.

Material and method:-
Sample collection:-Fifty seven isolates were obtained from 84 samples taken from clinical sources which were collected from three hospitals in Baghdad include Al-Yarmuk General Teaching Hospital , Al-Karama teaching hospital, Ibn-ALbalady teaching hospital. These sample include (wound swab,ear swab,burn swab and urine). All samples were cultured on blood agar and incubated at 37ºC for 24 hours. And then on differential media manittol salt agar S.epidermidis is not color change.
All the isolates were identified on the basis of colony morphology, biochemical test catalaseand coagulase test. Then identifiedusing a commercial biotyping system (api STAPH, bioMerieux, Inc., Hazelwood, MO) [16,17,18]. The strains were stored in brain heart Infusion (BHI) broth with 20% of glycerol at-20°C. The working cultures of the isolates were prepared in BHI broth at 37°C for 18 h. Chromosomal DNA from the S. epidermidisstrains isolated from different samples was extracted

Antibiotic Susceptibility Test:-
The Kirby-Bauer"s disc diffusion method using methicillin 5 μg disc on Muller-Hinton agar incubated overnight at37ºC was done for the detection of methicillin-resistant strains of S. epidermidis according to the zone that form around antibiotic disc.
DNA extraction:-DNA extraction using Genomic DNA extraction Kit, concentration and purity were determined using Nano drop 1000 spectrophotometer at 260/280nm. mecAgene Detection:-Staphylococcus epidermidisisolates were investigated for the presence of antibiotic resistance gene (mecA). The PCR for amplification of mecAgene was performed in a total reaction volume of 20 μl for one sample. The sequence of primer used for amplification of mecAgene forward 5-AAAATCGATGGTAAAGGTTGGC-3, Reverse 5-CGTGTTTTCAACATTTAATGCAA-3 the band size (500pb). The PCR cycling protocol was applied as following: initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 second, annealing at 55°C for 30 second and extension at 72°C for 30 second, followed by a final extension at 72°C for 10 min. Agarose Gel Electrophoresis and Visualization of PCR Products: 5 μof each amplicon was electrophoresed in 2 % agarose gel.

Results and Discussion:-
A total number of 57 isolates were obtained from 84 samples collected from clinical sources included wound swab, burn swab, ear swab and urine. All samples were cultured on blood agar and incubated at 37ºC for 24 hours. According to the preliminary results, table [1] showed the ratio of sample contain mecA gene of S.epidermidis from clinical sources.

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The results it is reported that from (105) clinical sample isolates (84) isolates were Staphylococcus epidermidis. Several biochemical tests were carried out to identify the S. epidermidis.All Gram-positive isolates gave positive results in the Catalase tests. The positive reaction indicated the liberation of free oxygen as gas bubbles after mixing of hydrogen peroxide solution with a little amount of bacterial growth [19]. In order to support the previous biochemical test, coagulase-negativeand detect the ability produce of biofilm by growth on Congo-red agarbiofilm producers form black colonies on CRA, whereas non-producers form red colonies [20]. The results revealed that from all 84 isolates, it was obtained 62 (73.8%) showed resistant to the Methicillin while 22 (26.2%) were sensitive to the Methicillin (inhibition zone of 23mm or less by Kirby-Bauer"s disc diffusion method using 5μg methicillin disc on Mueller-Hinton agar).This result agreed with the study by (Martinset al., 2013) which found 73.8% of S. epidermidis isolates were resistant to methicillin by disc diffusion method [21]. PCR results indicated that seventy five 57(61.2%) isolates were positive for mecAgenes as indicated by 500 bp Whereas 27 (38.8%)mecAgene negative. The result that obtain by PCR of the of S. epidermidis isolate agree with our study by (Contreras et al., 2013) it was found 64% of the S. epidermidis isolated resistance to methicillin and carried themecAgene [22].Genotypic methods are more accurate in detecting methicillin resistant Staphylococci as compared to conventional susceptibility methods. In addition, several culture conditions can also influence methicillin resistance such as the temperature, pH and concentration of NaCl in the medium [23]. Strains with low resistance level are usually complicated by these factors and impair the process of detection. The greatest disadvantage of phenotypic methods is that it is time-consuming [24]. And sometimes difficult to detect some strains; PCR can produce results within 24 hours as compared to the conventional methods which requires at least 48 hours there for the molecular detection methods are more preferred, favorable and accurate than the phenotypic method [25]. The study results indicate that 61.2% of the 57methicillin-resistant strains of S. epidermidis samples were mecAgene positive. Whereas 27(38.8) % samples as mecA negative by PCR method. However, the reason for the of the mecA negativity can be attributed to one of the following: 1. Differing levels of mecA gene expression of methicillin resistance, occurring every 104 or 106 cells [26].