Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 1 http://www.agriculmag.uodiyala.edu.iq/ MOLECULAR DETECTION OF erm(A), mef(A) IN Staphylococcus spp AND Streptococcus spp RESISTANT TO MACROLIDE FROM DIFFERENT CLINICAL INFECTIONS Hadi R. Rasheed Al-Taai Dept. of Biology-College of Sciences-University of Diyala, Iraq (drhadialtaai@yahoo.com) ABSTRACT Isolates Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus albus, Streptococcus Pyogenes and Streptococcus agalactiae isolated from different clinical infections were activated. Results of susceptibility test showed that Staphylococcus aureus have resistance to antibiotics of erythromycin, azithromycin and clindomycin with percentage 45%, 45% 25% respectively, while Staphylococcus epidermidis resistant to erythromycin, azithromycin and clindomycin with ratio 66.6%, 44%, 22.2%, respectively. The resistance of erythromycin and azithromycin by Streptococcus pyogenes reached 25% and 37.5% respectively, whereas all isolates sensitive to clindomycin. The outcome disclosed that the values of minimum inhibitory concentrations of erythromycin ranged between 32 - ≥ 64 µg/ mL for all isolates. The results outcomes to 13(65%) isolates of Staphylococcus aureus produced Staphylokinase, 6(66.6%) isolates of Staphylococcus epidermidis able to produce the same enzyme. The results proved that 3(37.5%) isolates of Streptococcus pyogenes able to produce Streptokinase enzyme, but Streptococcus agalactiae could not produce streptokinase. The PCR-based assay, gene was detected in erm (A) gene was detected in 33.3% isolate of Staphylococcus spp. and 50% of Streptococcus spp, whereas mef (A) was detected in 46.6 % isolates of Staphylococcus spp. and 50% of Streptococcus spp .These findings demonstrated the variable phenotypic expression when compared to genotypic detection . Key words: Staphylococcus spp., Streptococcus spp, Macrolide, erm(A), mef(A). PCR assay. INTRODUCTION The Staphylococci are a diverse group of bacteria that cause diseases ranging from minor skin infections to life-threatening bacteraemia. In spite of large-scale efforts to control their spread, they persist as a major cause of both hospital and community acquired infections worldwide. The two major opportunistic pathogens of this genus are Staphylococcus aureus and Staphylococcus epidermidis (Fung-Tomc et al., 2002 ; Gill et al., 2005). Unlike coagulase-negative Staphylococci (CNS), Staphylococcus aureus strains are able Received: 29/9/2015 Accepted: 4/2/2016 http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 2 http://www.agriculmag.uodiyala.edu.iq/ to secrete free plasma coagulase, which is an important virulence factor for these bacteria. S. aureus is a common pathogen in nosocomial infections, so exact identification of S. aureus isolates is essential for microbiology laboratories (Emori & Gaynes, 1993). The proportion of clinical S. aureus isolates that are resistant to methicillin (MRSA) has increased over recent years (Tiemersma et al., 2004). Compared with methicillin-sensitive S. aureus (MSSA), infections by MRSA strains are associated with increased morbidity and mortality in affected patients (Cosgrove et al., 2003). Macrolide resistance in Streptococcus pyogenes results primarily from modification of the drug target site by methyl transferase encoded by erm genes, erm(A) and erm(B) or by active efflux mediated by a mef-encoded efflux pump. Of these, erm(A) is inducibly expressed (Malhotra- Kumar et al., 2009) and generally confers low-level resistance to macrolides, whereas lincosamides and streptogramins B (MLSB), which share overlapping binding sites, remain active against erm(A)-harboring S. pyogenes (Leclercq, 2002). erm(B)-mediated erythromycin resistance is the most common mechanism in many areas of the world, including some European and Asian countries (Monaco et al., 2005 ; Reinert et al., 2005), whereas mef, with its most common variants mef(A) and mef(E), is predominant in the United States (Farrell et al., 2007), Canada (Hoban et al.,2001 ), and the United Kingdom (Amezaga et al., 2002). That macrolide used is the main driver of macrolide resistance in streptococci has been well demonstrated at the population and individual levels (Goossens et al., 2005 ; Malhotra-Kumar et al., 2007). Because erm and mef are cocarried with tet genes on mobile elements, tetracycline also use affects macrolide resistance (Malhotra-Kumar et al., 2007). In Iraq such molecular studies about these important genes are rare so this study was done to focus on detection of erm A and mef A genes present in Staphylococcus spp. and Streptococcus spp. using phenotypic test followed by genotypic using specific primers for these target gene by PCR technique . MATERIAL AND METHODS Activation of isolates Forty isolates of Streptococcus spp and Staphylococcus spp were isolated from 200 samples clinical sources included (Urine, blood, middle ear, sputum, throat, wounds, and vaginal swabs) from hospitals (Baquba teaching hospital, Al-Batool hospital and Balad Ruz teaching hospital) in addition to some health centers for the period 1/9/2013 until 01/01/2014. Blood agar base medium, mannitol salts were used for activation isolates of Streptococcus spp and Staphylococcus spp., Nonobiocin disc for discrimination S. saprophyticus, while http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 3 http://www.agriculmag.uodiyala.edu.iq/ bacitracin disc and optochin disc were used for discrimination Streptococcus. Identification off all bacterial isolates was confirmed mediated Vitek2 system. Susceptibility Testing Susceptibility of all bacterial isolates to azithromycin, erythromycin and clindamycin antibiotics was determined by the disc diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2012). Each antibiotic concentration was applied on the surface of Muller-Hinton agar plates inoculated with S. epidermidis, S. aureus, Strp. pyogens, Strp. agalactiae, S. saprophyticus isolates and incubated at 37 ºC for 24 h. (Tankhiwale et al., 2004). Minimum inhibitory concentration (MIC) MIC was determined by agar dilution method in Muller Hinton agar (Oxoid/ UK). Serial two-fold dilution of erythromycin was incorporated in media plates from 0.25 to 1024 µg ml-1 as recommended by Clinical and Laboratory Standards Institute (CLSI, 2012). Staphylokinase and Streptokinase Production The Staphylokinase and Streptokinase Production of the different bacterial isolates were assayed as described by (Cruickshank et al., 1975; Rajamohan et al., 2000). PCR amplification Isolates with positive phenotypic tests were subjected to molecular screening study using PCR amplification technique according to following steps: Extraction of bacterial DNA Bacterial DNA was isolated using a DNA isolation kit (Geneaid Biotech. Ltd; Taiwan) PCR amplification procedure: Detection of erm A, mef A genes was performed by amplifying the genes by PCR. The primers sequences were previously reported by (Sutcliffe et al., 1996a ; Sutcliffe et al.,1996 b ; Lim, 2002) and obtained from Korea/Bioneer. Descriptions and sequences of the PCR primers used in this study are displayed in Table 1. Amplification was performed in a thermal cycler (Eppendorf, Germany) according to the methods described by (Sutcliffe et al., 1996 a ; Sutcliffe et al., 1996 b ; Lim 2002). For amplification of erm A, mef A genes, PCR mixture was composed from 2.5 microliters of template DNA and were http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 4 http://www.agriculmag.uodiyala.edu.iq/ mixed with PCR mixture that composed from 12.5 μl of GoTaq®Green Master Mix, 2.5 μl from each primers of erm A and mef A and 5 of D.D water to get final volume of 25 μl. The program, for mef A the reactions mixtures included an initial denaturation at 93 ºC for 3 min. consisted of 35 cycles of 93 ºC for one min, specific annealing temperature 52 °C for one min and a final extension at 72 ºC for 5 min in a Thermal Cycler , whereas for erm (A the reactions mixtures included an initial denaturation at 95 ºC for 5 min, specific annealing temperature 54 ºC for one min and a final cycle of primer extension at 72 ºC for 5 min. The thermocycler reaction conditions for each primer as fallow pair were calculated on the basis of the annealing temperature and the length of the product size. PCR amplified DNA was analyzed on 0.8 to 1% agarose gels by electrophoresis. Concentration and purity of DNA by Nano drop system. The phylogenetic group to which the S. epidermidis, S. aureus, Strp. pyogens strains belonged was determined by a PCR-based method. Table1. Primer sequences and predicted sizes of the amplified products of PCR Primer Oligonucleotide sequences (5`-3`) No. of base pair Size of amplicons erm A F 5- GTT CAA GAA CAA TCA ATA CAG AG – 3 23 421 R 5` - GGA TCA GGA AAA GGA CAT TTT AC – 3` 23 mef A F 5` - AGT ATC ATT AAT CAC TAG TGC – 3` 21 348 R 5` - TTC TTC TGG TAC TAA AAG TGG – 3` 21 RESULTS AND DISCUSSION Activation of Bacterial isolates Twenty Isolates of S. aureus, nine isolates of S. epidermidis, one isolate of S. saprophyticus, one isolate of S. albus, eight isolates of Strep. pyogenes, one isolate of Strep. agalactiae, isolated from urine, blood, sputum, wound, vagina, pharynx were activated. Diagnosis was confirmed mediated Vitek2 system. Susceptibility Testing The results showed the proportion of S.aureus resistance to antibiotics of erythromycin, azithromycin and clindomycin was 45%, 45% 25% respectively (Table 2). There are some studies have highlighted the high resistance to azithromycin where that Aktas et al., (2011) in Turkey found that S. aureus http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 5 http://www.agriculmag.uodiyala.edu.iq/ resistant to azithromycin with ratio amounted to 96%, while erythromycin 96.1% and for clindomycin 55.1%, which was also higher than the proportion of resistance to the compounds of Macrolide in the current study. 25.5 % of S.aureus was resistant to clindomycin, these results agreed with Al-Hasani (2011) pointed out ratio of resistance of clindomycin was 31.2%. As for Staphylococcus CO-agulase negative (CONS), S. epidermidis which constitute the bulk of them in the current study, the percentage of resistance to erythromycin was 66.6%, while for azithromycin was 44% and for clindomycin was 22.2%, the proportion of resistance of azithromycin was less than results of Hafeth (2010) demonstrated ratio was 87.2%. As for the results of clindomycin did not agree with Al-Hasani (2011) who found all isolates were sensitive to this antibiotic with 100%. The resistance of erythromycin and azithromycin by Strep. pyogenes, which constitute the bulk of the genus of Streptococcus reached 25 % and 37.5 % respectively, whereas all isolates sensitive to clindomycin as shown in (Table 2), these results of the current study agreed partly with Younge et al. (2004) who found resistance of erythromycin was 19%. As for clindomycin the ratio agreed with Moriangthem and Gurung (2013) found all isolates of Strep. pyogenes were sensitive to this antibiotic with 100%, in general that resistance of clindomycin is less than the resistance ratios in most countries of the world. Ciftci et al. (2003) found as no more than 3% in Turkey, and Grivea et al. (2012) point out that the resistance of clindomycin in Spain 0%. Results show that all bacterial isolates possess resistance to most antibiotics used in this study and varying rates, the results showed that the percentage of erythromycin, azithromycin and clindomycin by Staphylococcus spp were 51.6%, 45.1% and 22.5% respectively. As for Streptococcus spp. the proportion of resistance to erythromycin, azithromycin and clindomycin were 25.5%, 25.5% and 0.0% respectively. The reason of resistance of Staphylococcus spp. and Streptococcus spp. for erythromycin, azithromycin and clindomycin to the presence of genes located on plasmids or transposal elements which responsible for this resistance, there are two main ways to resist macrolide group during modulating ribosomal target site by producing 23 rRNA methylase encoding mediated by a gene erm including erm (A) and erm (B) and erm (C), which the resulting compounds macrolide resistance and lincosamides and Streptogramin B (MLSB Phenotype) or through acquisition mef (A) gene as the gene coding for the construction of efflux pump protein by the expulsion of the macrolide antibiotics outside bacterial cell ( Zmantar et al., 2011). http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 6 http://www.agriculmag.uodiyala.edu.iq/ Table 2. Percentage of the sensitivity of antibiotics by Staphylococcus spp. and Streptococcus spp Antibiotics Isolates Erythromycin Azithromycin Clindomycin S % I % R % S % I % R % S % I % R% Staphylococcu aureus 50 (10) 5 (1) 45% (9) 45 (9) 10 (2) 45 (9) 70 (14) 5 (1) 25 (5) Staphylococcus epidermidis 33.3 (3) 0 66.6% (6) 55.5 (5) 0 44.4 (4) 55.5 (5) 22.2 (2) 22.2 (2) Streptococcus pyogenes 75 (5) 12.5 (1) 25% (2) 62.5 (6) 0 37.5 (2) 62.5 (6) 28.5 (2) 0 Streptococcus agalactiae 100 (1) 0 0% 100 (1) 0 0 100 0 0 Staphylococcus albus 100 (1) 0 0% 100 (1) 0 0 100 (1) 0 0 Staphylococcussap rophyticus 100 (1) 0 0% 100 (1) 0 0 100 (1) 0 S: Sensitive - I: Immediate - R: Resist - NO. Above: Percentage –NO. Below: NO. of isolates Detection of isolates resistant to erythromycin Table 3 shows the existence of a high proportion of isolates resistance to erythromycin, as the 14(46.6%) isolates belonging to the genus Staphylococcus spp. can grow on erythromycin with concentration reach 100 ug ml-1, while erythromycin sensitive Staphylococcus spp. was 17 isolates with ratio 54.8%. Results indicated presence one isolate (11.11%) of total 9 isolates belonging to the genus Strep. pyogenes resistant to erythromycin. Despite the decline in the ratio, however, isolates of Staphylococcus spp., which are expected to form the strains MRSA large part may be the cause for many of the infections that require to treatment it processing specific varieties of antibiotics, as these resistant strains of several varieties of antibiotics including macrolide group (Duran et al., 2012). Table 3. Numbers and percentages of isolates resistant to erythromycin Isolates Sensitivity to erythromycin NO. Of isolates (%) Staphylococcus spp Resistant 14(46.66) Sensitive 16(53.33) Streptococcus spp Resistant 1(11.11) Sensitive 8(88.88) Minimal inhibitory concentration Depending on the results of Antimicrobial susceptibility test and growth at erythromycin medium, 17 isolates belonging to both Staphylococcus spp. and Streptococcus spp. was elected, isolated from different clinical sources, these http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 7 http://www.agriculmag.uodiyala.edu.iq/ isolates resistant to all antibiotics under study, so as to determine ability of these isolates to resistance of Macrolide antibiotics through the work of a series of concentrations different to determine the minimum inhibitory concentrations based on CLSI (2012). Results in Table 4 indicate that the values of minimum inhibitory concentrations of erythromycin ranged between 16-64 µg ml-1 for isolates of Staphylococcus spp., these results converged with the results of Richter et al., 2011 whom found MIC of erythromycin is ≥64 µg ml-1, and those results also partially converged with results Aktas et al., (2007) whom pointed out that values of MIC for erythromycin ≥128 µg ml-1. Table 4. Minimum inhibitory concentrations of erythromycin N o. o f Is ol at es Isolates M IC E ry th ro m yc in N o. o f Is ol at es Isolates M IC E ry th ro m yc in N o. o f Is ol at es Isolates M IC E ry th ro m yc in Break point ≥64 3 Streptococcus aureus ≥64 11 Streptococcus aureus ≥32 22 Streptococcus pyogenes ≥64 6 Staphylococcus aureus ≥64 12 Streptococcus epidermidis ≥64 25 Streptococcus epidermidis ≥64 7 Staphylococcus aureus ≥64 13 Staphylococcus aureus ≥32 26 Streptococcus epidermidis ≥32 8 Staphylococcus aureus ≥64 14 Staphylococcus aureus ≥64 27 Streptococcus epidermidis ≥64 9 Staphylococcus aureus ≥64 16 Streptococcus epidermidis ≥64 31 Streptococcus pyogenes ≥64 10 Staphylococcus aureus ≥64 21 Streptococcus epidermidis ≥64 Staphylokinase and Streptokinase Production The results (Table 5) indicate to 13(65%) isolates of S. aureus produced Staphylokinase, 6(66.6%) isolates of S. epidermidis able to produce the same enzyme. S. albus and S. saprophyticus produced Staphylokinase with ratio 100% Those results agreed with the results of the study conducted by Devi et al., (2012) whom found 7 out of 12(58%) isolates belong to the Staphylococcus spp produced this enzyme. While the results showed that 3 (37.5%) isolates able to produce Streptokinase enzyme, while Strp. agalactiae could not produce streptokinase (Table 5). The ratio was lower than of the results obtained by Razak and Al-Jebori (2012) found that 66.6% of its isolates able to produce this enzyme. http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 8 http://www.agriculmag.uodiyala.edu.iq/ Table 5. Percentage of Staphylococcus spp and Streptococcus spp. producing Staphylokinase and Streptokinase Staphylokinase production Isolates No. of Isolates No. of isolates producing enzyme (%) Staphylococcus aureus 20 13(65) S.epidermidis 9 6 (66.6) Staphylococcus albus 1 1(100) Staphylococcus saprophyticus 1 1(100) Streptokinase Production Streptococcus pyogenes 8 3(37.5) Streptococcus agalactiae 1 0(0) Detection of genes erm A and mef A using PCR technique The results showed that the purity ranged from 1.91 to 2, and the concentrations ranged between 5.993 ng µl-1 and 93.5 ng µl-1. The products of PCR technique were rolled on gel electrophorese with concentration of 1%, as observed one band on same level, after exposing the gel to ultraviolet light, these results induct the primer linkage with its sequence complementary, and notice do not appear any band for the second track which negative control sample comparing bands with DNA Ladder. The results showed the size of band similar to expected sizes, were a (412 bp) erm (A), (348 bp) for mef(A) gene and also agreed with result of Sutccliffe et al. (1996 a) and Lim (2002). The results of the current study showed the presence of a high percentage of isolates possess both or one of the genes, with 5 isolates have erm (A) gene 33.33% return of Staphylococcus spp, results inducted a high proportion of isolates of Staphylococcus spp possess the erm A gene, 5 out of 15 (33.33%), divided by 4 isolates 80% belong to S. aureus, one (20%) isolate belonging S. epidermidis Table 6 and Figure 1. The results also showed that 7(46.6 %) isolates belonging to Staphylococcus spp possess the mef A gene, divided by 5 isolates 71.4 % belong to S. aureus, 2 (28.5 %) isolate belonging to S. epidermidis. One out of two of Strep. pyogenes has been detected, results showed that one isolate possesses erm A gene, while another has mef A gene (Table 6) and (Figure 2, 3). http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 9 http://www.agriculmag.uodiyala.edu.iq/ Table 6. Numbers and percentages of isolates under study containing the genes erm A and mef A using the technique (PCR) Genes The number of isolates containing genes Staphylococcus spp Streptococcus spp erm A 5(33.33%) 1 (50%) S. aureus 80% S. epidermidis 20% Strep. Pyogenes 50% mef A 7(466.%) 1(50%) S.aureus 71.4% S.epidermidis 28.5% Strep. Pyogenes 50% Figure 1. Gel electrophoresis and Ethidium bromide staining to detect genes erm (A) and mef (A) by using PCR technique, concentration of agarose (1%) ,of 80 volts 45 minutes. Lanes 1, mef (A) of S. epidermidis ; 2,mef( A)of S.aureus;3, mef(A) of S.aureus;4, mef(A ) of S.pyogens;5, mef(A) of S.aureus; mef (A) of S.aureus 6, ; M, Leader DNA (1000 bp) 8, mef(A) of S.epidermidis ;9, mef(A) of S.epidermidis;10,erm (A) of S.aureus. The PCR was carried out in duplicate. http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 10 http://www.agriculmag.uodiyala.edu.iq/ Figure 2. Gel electrophoresis and Ethidium bromide staining to detect genes erm (A) and mef (A) by using PCR technique, concentration of agarose (1%), of 80 volts 45 minutes. Lanes 1, erm (A) of Strep. pyogenes ; 2, erm (A) of S.aureus ;3, erm (A) of S.aureus; 4, erm (A) S.aureus; M, Leader DNA (1000 bp 9, erm( A) of S.epidermidis; 10, mef (A) of S.aureus; mef (A) of S.aureus;11 mef (A )S.epidermidis. The PCR was carried out in duplicate. It was noted that some isolates in the current study did not show any band through using gel electrophorese technique, although that it resistant to erythromycin, it may come back to possess these isolates for resistance genes located on a small plasmid, sometimes may be lose this plasmid (Zmantar et al., 2011). The results partially converged with Aktas et al., (2011) in Turkey found that 50% of isolates of S. aureus containing the gene erm A, while in regards to S. epidermidis far less than reached his results of the current study where the ratio reached erm (A) 8.9%. Aktas (2011) pointed out that there are disparities in the distribution of resistance genes in the different countries of the world or the disparity of the hospital may be because randomly use of erythromycin in each country as well as to the person's age and the sample source. Figure 3. The percentages for the presence of genes in Streptococcus pyogenes http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(Special Issue): 1-15, 2017 Al-Taai 11 http://www.agriculmag.uodiyala.edu.iq/ The results agree with Duran et al., (2012) in a study by the Mustafa Kemal University in Turkey who pointed out that 33% of the total isolates of S.aureus possess the gene erm A, As for S.epidermidis results converged partly with the results with same researcher who found 11.4% of its Isolates possess the gene erm A, also converge with the results of Chaieb et al., (2007) in Tunisia since concluded that 12.5% of the isolates of S. epidermidis possess the gene erm A but differ with him in gene mef A as to reach all of its Isolates do not have this gene and 0%. As for Strep. pyogenes products have shown to detect the presence of a single gene 50% , these results Partially agree with López et al., (2012) who conducted studies (1994- 2006) in Spain since reached 35% of the Strep. pyogenes isolates possess the gene erm (A) while the results of the current study did not agree with López et al., (2012) for the mef (A) gene which found that 89% of its Isolates possess this gene. PCR technique was able to give a high- resolution results and the sensitivity of quality as one of the main diagnostic techniques. CONCLUSION This study evaluated that Staphylococcus spp and Streptococcus spp having resistance for Macrolide group and there is relationship between erm (A) and mef (A) genes and resistance of macrolide. REFERENCES Al-Hasani, H. M. H. 2011. Comparative Study between Methicillin-Resistant Coagulase Positive and Negative Staphylococci. M. Sc. thesis, College of Science, University of Baghdad Aktas, A., A. Aridogan, C. B. Kayacan, and B. 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Resea. 4(453): 2 9 ــ. .Staphylococcus sppفي mef(A)و erm(A)الكشف الجزيئي للجين والمعزولة من مصادر المقاومة لمضادات المايكرواليد Streptococcus sppو مختلفةسريرية هادي رحمن رشيد الطائي ( (drhadialtaai @yahoo.comالعراق ،ديالى –جامعة ديالى -كلية العلوم -قسم علوم الحياة المستخلص ،Staphylococcus aureus, Staphylococcus epidermidis نشطت العزالت البكتيرية Staphylococcus saprophyticus ,Staphylococcus albus،Streptococcus pyogenes ، Streptococcus agalactiae من مصادر سريرية مختلفة. اظهرت نتائج فحص الحساسية والمعزولة azithromycinو erythromycinقد قاومت مضادات Staphylococcus aureusان Staphylococcusعلى التوالي، بينما كانت مقاومة %25 و %45 و %45بنسب Clindomycinو epidermidis على التوالي. مقاومة المضادين %22.2و %44و %66.6لنفس المضادات بنسب erythromycin ،azithromycin من قبلStreptococcus pyogenes 25وصلت الى% ، . اوضحت النتائج ان التركيز %100ونسبة Clindomycinبينما جميع العزالت حساسة لـ 37.5% ( من عزالت % 65) 13. ان 1-مل مكغم 32- ≤ 64كان erythromycin لـ MICالمثبط االدنى Staphylococcus aureus منتجة النزيمStaphylokinase ( % 66) 6وانStaphylococcus epidermidis ( %37) 3منتجة لنفس االنزيم. بينت النتائج انStreptococcus pyogenes انتجت PCR. نتائج تقنية لم تستطع انتاج االنزيمStreptococcus agalactiae ولكنStreptokinase انزيم من عزالت %50و Staphylococcus sppمن عزالت %33.3 في erm (Aكشفت تواجد الجين ) Streptococcus spp. بينما بينت النتائج تواجد جينmef (A) من عزالت %46.6في Staphylococcus spp من عزالت %50وStreptococcus spp بينت النتائج هناك تنوع في النمط . المظهري مقارنة بالنمط الوراثي. ، جهاز erm(A)، mef(A)مضادات المايكرواليد، جين ،المسبحيات الكروية ،: المكورات العنقوديةالكلمات المفتاحية .بلمرة الدنا http://www.agriculmag.uodiyala.edu.iq/