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The Importance of Clindamycin Induction Test in Treating Patients Infected with Methicillin Resistance Staphylococcus aureus Iyad Kaddora Bioinformatics Introduction In recent years, strains of Staphylococcus aureus (S. aureus) resistance to erythromycin have been clinically isolated with increasing frequencies. S. aureus is a Gram positive cocci bacterium that belongs to the Micrococcaceae family(4). S. aureus can exist in air, dust, water, and can be normal flora of skin and respiratory tract of human However, the most common mode of transmission is by skin-to-skin contact from an infected host(4). This common bacterium is the number one cause of nosocomial infections, which are infections that are acquired while clients are receiving care in the hospital setting (8). S. aureus can live harmlessly on the skin surface, but when the surface is punctured or broken, it can cause serious infections that may ultimately lead to serious disease even death if untreated. Antibiotics have been successful in killing bacteria, but due to over use of medications and the incomplete drug course taken by infected individuals, the bacteria have started to develop different, mutated strains that raise the degrees of virulence these bacteria express to fight the effects of various antibiotics. In the past few years, Staphylococcus aureus has begun to show more resistant to commonly used antibiotics, which is expressed by developing mutated strains called Methicillin Resistant staphylococcus aureus (MRSA). This methicillin resistance now refers to multiantibiotic resistant groups (8). This evidence represents the growing resistant to β-lactam antibiotics such as penicillin and its offspring, as well as the cephalosporins drug. MRSA can cause a serious infection if it is not treated quickly and spreads very easily in the health care setting. Macrolides is a class of antibiotic that inhibits growth of bacteria by several different mechanisms (7). It is used clinically as a broad spectrum antibiotic against gram positive bacteria. It inhibits the bacterial growth by binding to specific sites in the bacteria’s ribosome to prevent the production of proteins. Resistance of the organisms to macrolides can occur in many ways; however, in mechanism of resistant to erythromycin occurs by one of these two mechanisms: efflux or ribosome alteration. Efflux mechanism is mediated by the macrolide streptogramin (type B) gene (mrsA), where the ribosome alteration is achieved by methylation of specific residues 23S rRNA and mediated by erythromycin ribosome methylase (erm) gene (2, 6, 7). Following exposure to macrolides the organism’s resistance can be either constantly presents (appear) where the rRNA methylase is always produced, or it can be inducible where methylase is produced only in the presence of inducing agent which is erythromycin in this case (1,6). These two types of resistance are called respectively constitutive and inducible resistance. Determinant (gene) Erythromycin Clindamycin Efflux mrsA R S Ribosome alteration erm R S* erm R R (constitutive) mrsA: macrolide streptogramin (type B) resistant. erm: erythromycin ribosome methylase; is a gene that encodes enzymes which confer inducible or constitutive resistance to macrolide lincosamide (clindamycin) streptogramin type B agents via methylation of the 23S rRNA. Clindamycin is a lincosamide class of antibiotics that has activity against different kinds of bacteria and possesses the ability to inhibit the synthesis of protein in bacterial organism. This activity prevents the bacteria from replicating by binding exclusively to the 50S ribosomal subunit of susceptible organisms (5). Clindamycin works very well in treating MRSA However, some strains have recently begun showing resistance to this drug, making treatment more difficult (2). The failure of Clindamycin therapy in the treatment of MRSA infected patients, those expressed sensitivity to the drug at the beginning of the course treatment, raises a big concern to health care professionals and attending physicians. In order to successfully treat individuals with MRSA, a new test has been developed to test the future resistance of bacteria to medicines that are currently being used to treat the infections. This test, clindamycin disk inductions test (D-Test) will aid in the future treatment for clients with MRSA (2). Many studies have been preformed regarding MRSA and the growing resistant strains that have begun to deter the normal treatment of this infectious disease process. The aim of this study is to determine whether Methicillin Resistant Staphylococcus aureus (MRSA) is showing more resistance to the clindamycin antibiotic drug during 2000/2001 year compared to 2004/2005 year. By using this new test, treatment for MRSA in the future will be able to be provided accurately and hopefully aid in the prolongation of the human race. Materials and Methodology The specimens were collected from patients presenting to clinical facilities in Huntington, WV. A loopful of the sample was streaked on a new Trypticase soy agar plate with 5% sheep’s blood (BAP) and incubated overnight at 37°C, with 5 % CO2 Another loopful of the sample was then combined with 5 ml of Trypticase soy broth (TSB) in a test tube and incubated overnight at 37°C with 5% CO2. Two test tubes were prepared from each sample. The test tubes were centrifuged at 1500 rpm (Beckman CS6R centrifuge with a GH-3.7 rotor) for 5 minutes to precipitate a pellet. After centrifugation 0.5 ml of TSB/ bacterial pellet was mixed with 0.5 ml of a sterile 24% glycerol solution and placed in a sterile 4 ml glass Wheaton vial. The vial was labeled with the sample number and frozen at -70 to -80°C. Two hundred and forty frozen samples of Methicillin Resistant Staphylococcus aureus (MRSA) were collected in year 2000/ 2001 in order to be evaluated for clindamycin resistance by performing the D-test. The frozen specimens were sub cultured on blood agar plates and the D test was performed on all specimens. Positive Dtest indicates the ability of MRSA strains to demonstrate resistance to the clindamycin during the antibiotic therapy. Negative D-test indicates the effectiveness of the clindamycin drug in treating those patients with MRSA (6). This test was performed on Mathecillin-resistant Staphylococcus aureus from sources other than urine, such as sputum, blood culture, wound, or body fluid. When the MRSA strains are resistant to erythromycin and sensitive to clindamycin, after using routine antimicrobial susceptibility test method, the drugs sensitivity is determined. Urine specimen is unacceptable because clindamycin is not used for treating urinary tract infection caused by MRSA strains. The principle of the D test is to make erythromycin induce production of the methylase, which will allow clindamycin resistant to be expressed and detected by forming a D shape letter on the blood agar plate (BAP). The first step in performing the D test is to make 0.5 McFarland suspension of the organism in 3ml sterile inoculum’s water. After the specimen is mixed in sterile water, a sterile swab is used to inoculate the organism to the blood agar plate, making sure the organism is covering the entire surface of the plate to ensure growth in the whole plate. An erythromycin disk is placed on the plate. This disk is used as an inducing agent when placed 15mm to 26 mm away from the clindamycin disk, on a (BAP). After the specimen is incubated for 18-24 hours at 35°C with 5% CO2, a flattening of the zone in the area between the two disks (letter D shape) will indicate the organism’s ability to induce clindamycin resistance in the future (6). References 1. Catchpole, I., and K. G. H. Dyke. 1990. A Staphylococcus aureus plasmid that specifies constitutive macrolide-lincosamide-streptogramin B resistance contains a novel deletion in the ermC attenuator. FEMS Microbiol. Lett. 69:43-48. 2. Drinkovic, D., E. R. Fuller, K. P. Shore, D. J. Holland, and R. Ellis-Pegler. 2001. Clindamycin treatment of Staphylococcus aureus expressing inducible clindamycin resistance. J. Antimicrob. Chemother. 48:315-329. 3. Fiebelkorn, K. R. S. A. Crawford, M. L. McElmeel, and J. H. Jorgensen. 2003. Practical Disk Diffusion Method for Detection of Inducible Clindamycin Resistance in Staphylococcus aureus and Coagulase-Negative Staphylococcus. J. Clinical Microbiology Oct. 2003; 41(10): 4740-4744. 4. Henry, John Bernard. 2001. Clinical Diagnosis and Management by Laboratory Methods. 20th Edition. W.B. Saunders Co., Philadelphia, USA. ……… 5. Paul C. Schreckenberger, E. Ilendo, and K. L. Ristow. 2004. Incidence of Constitutive and Inducible Clindamycin Resistance Staphylococcus aureus and Coagulase-Negative Staphylococci in Community and Tertiary Care Hospital. J. of Clinical Microbiology. 2004 June; 42(6): 2777-2779. 6. Steward, C., p. M. Raney, A. K. Morell, P. P. Williams, L. K. McDougal, L. Jevitt, J. E. McGowan, Jr., and F. C. Tenover. 2005. Testing for Induction of Clindamycin Resistance in Erythromycin-Resistant Isolates of Staphylococcus aureus. J. of Clinical Microbiology 1716-1721. 7. Weisblum, Bernard. 1995. Insights into Erythromycin Action from Studies of Its Activity as Inducer of Resistance. Antimicrobial Agents and Chemotherapy, Apr. 1995, p. 797-805. 8. http://www.link.med.ed.ac.uk/RIDU/Mrsa.htm