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Octot of $!)Ilos(Opl)P Tilfe^^iJ BIOCHEMICAL STUDIES ON ANTIBIOTIC RESISTANCE AMONG STAPHYLOCOCCI FROM EYES AND OTHER CLINICAL SOURCES IN HEALTH AND DISEASE ABSTRACT THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF ©octot of $!)ilos(opl)p Piocfjemisitrp BY JAVID AHMAD DAR DEPARTMENT OF BIOCHEMISTRY & MICROBIOLOGY SECTION, INSTITUTE OF OPHTHALMOLOGY JAWAHARLAL NEHRU MEDICAL COLLEGE FACULTY OF MEDICINE ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA) 2003 •XBESlS Oor.-.V^'*^ Vsd •'« '4 .'-LGrcOd THESIS Biochemical Studies on Antibiotic Resistance among Staphylococci from Eyes and other Clinical Sources in Health and Disease Abstract The emergence of multidrug-resistant bacteria is a phenomenon of concem to the clinician and the pharmaceutical industry, as it is the major cause of failure in the treatment of infectious diseases. Staphylococci have a record of developing resistance quickly and successfully to antibiotics. This defensive response is a consequence of the acquisition and transfer of antibiotic resistance plasmids and the possession of intrinsic resistance mechanisms. The acquired defense systems by staphylococci may have originated from antibiotic-producing organisms, where they may have been developed and then passed on to other genera. There are reports of emergence and high occurrence of staphylococcus strains resistant to methicillin, especially MRSA, from various parts of the World. These organisms are mostly resistant to multiple antibiotics; therefore the treatment of infections due to these organisms and their eradication is very difficult. The present study was undertaken with the aim of determining epidemiology of clinical and carrier staphylococci and biochemical studies of their acquisition and dissemination of resistance. The prevalence of methicillin-resistant Staphylococci was determined in 750 subjects infected/colonized with staphylococci providing 850 isolates. Of 850. strains 575 were isolated from clinical specimens, 100 from nasal cultures of hospitalized patients, 125 from nasal and 50 strains were isolated from ocular swabs of hospital workers. It was shown that 35.1% (180/513) of Staphylococcus aureus and 22.5% (76/337) of coagulase-negative staphylococcal isolates were resistant to methicillin. Highest percentage of MRSA (35.5%) was found in pus specimens (n=151) while as urine samples (n=153) contributed 25.0% of methicillin-resistant coagulase-negative staphylococci. 29.7% of methicillin resistant Staphylococcus aureus isolates were found in nasal swabs (n=175) while as 10.0% and 16.5% of methicillin- resistant coagulase-negative staphylococci were observed in nasal swabs (n=50) and ocular cultures (n=50) respectively. The results indicated that major reservoir of methicillin resistant staphylococci in hospitals are colonized/infected inpatients and colonized hospital workers, with carriers at risk for developing endogenous infection or transmitting infection to health care workers and patients. The results were confirmed by restriction mapping and SDS-PAGE protein profiles of clinical and carrier methicillin-resistant staphylococcus aureus isolates. The multiple drug resistance of all MRSA (n=180) and MRCNS (n=76) isolates was detected. In case of both methicillin- resistant as well as methicillin-sensitive staphylococcal isolates zero resistance was found to fusidic acid and vancomycin while as highest resistance was found to penicillin G followed by ampicillin. Selective methicillin-resistant staphylococcal isolates were subjected to plasmid isolation and curing treatments to study their mode of resistance. The piasmids were transformed into antibiotic sensitive Escherichia coli Strain DH5-a. In one of the experiments plasmid pJMRIO from Staphylococcus aureus coding for ampicillin (A'), gentamicin (G') and amikacin (Ak^) resistance was transformed into Escherichia Coli. Transformation efficiency was about 2 x 10^ transformants/ng of plasmid DNA. The minimal inhibitory concentrations (MICs) for A and G were lower in E. coli than in S. aureus. However, the MIC for AK was higher in E. coli transformants than in S. aureus. In one of Conjugation Studies transfer of erythromycin resistance was observed between clinical and carrier strains of Staphylococcus aureus. Furthermore Enzyme mediated resistance particularly through y5-lactamase and chloramphenicol acetyl transferase was studied in selected strains. The substrate profile of /?- lactamase so extracted was studied against different classes of pencillins and cephalosporins. The results indicated that Methicillin remains the most stable pencillin to the staphylococcal y9-lactamase. The anti-MRSA activities of a synthesized compound from an Inorganic Lab of A.M.U; was determined by in vitro testing. The results indicated that the Nickle- metalo, complex of 2-mercaptobenzimidazole has good activity against MRSA (MIC > 64 |jg/ml) and demands to be analyzed for clinical applications in future. The widespread occurrence and dissemination of /^-lactamase and PBP mediated resistance leading to multiple antibiotics ineffective, thus increasing the cost of health care, needs to be tackled logistically by wise and judicious use of existing antibiotics and by developing ideal and cost effective antibiotics having least chances of acquiring resistance. Furthermore the hospital acquired MRSA infections through colonization of patients and hospital workers demands appropriate and timely measure to counteract this health problem. BIOCHEMICAL STUDIES ON ANTIBIOTIC RESISTANCE AMONG STAPHYLOCOCCI FROM EYES AND OTHER CLINICAL SOURCES IN HEALTH AND DISEASE THG5I5 . SUBMITTED FOR THE AWARD OF THE DEGREE OF / / ©ottor of 5f)ilo2iopi)p an iSiocfjemigtrp / BY V JAVID AHMAD DAR DEPARTMENT OF BIOCHEMISTRY & MICROBIOLOGY SECTION, INSTITUTE OF OPHTHALMOLOGY JAWAHARLAL NEHRU MEDICAL COLLEGE FACULTY OF MEDICINE ALIGARH MUSLIM UNIVERSITY ALIGARH-202002 (INDIA) 2003 THESIS P!^ I 4 AUb 2-'^ T6158 rii£sis A^ OF % ALIGARH MUSLIM UNIVERSITY u: ALIGARH, INDIA. c?giim33f3C(iAmjg This is to certify that the thesis entitled "BIOCHEMICAL STUDIES ON ANTIBIOTIC RESISTANCE AMONG STAPHYLOCOCCI FROM EYES AND OTHER CLINICAL SOURCES IN HEALTH AND DISEASE" herewith submitted by Mr. Javid Ahmad Dar in fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry of the Aligarh Muslim University, is an authentic record of research work carried out under our supervision and guidance. The work embodied in this thesis has not been presented in part or fiill to any other university or institute for the award of any degree. (Dr. Shaitiim Ahmad) (Di< A^ Ali) Supervisor Co-Supervis'or "DAAD" Fellow (W. Germany), Reader, "JSPS" Fellow (Japan), Department of Biochemistry, Senior Reader and Officer Incharge, J. N. Medical College, Microbiology Section, Aligarh Muslim University, histitute of Ophthalmology, Aligarh-202002 (U.P.) India. J. N. Medical College, E. mail: [email protected] Aligarh Muslim University, Aligarh-202002 (U.P.) India. E. mail: shamimshamim@redif&nail.com Research is to see what everybody else has seen and thinking what nobody has thought. Albert Src>it-Gv^or5Vf PUSMOOOMOR- HASMO /MWtTAHT GENf •V r RESISTANT ^ OEM < PLASMA) TRANSFU TMHSfU x.. 'Of FfitE ONA VWUS Of to MAO {hronvmomt ' /b KACnWUM "^/ TRANSFER -^>l@ BYVWAL BACURIUM RESISTANT W:Ctrv»i& GENI REMSTAMCE -^ GENU BACTERIUM' MFECTIO av VWUS To get profit without risk, experience without danger, and reward without hard work, is as impossible as it is to live like without being born. A. ?. Oo\iXhc\\ ACKNOWLEDGEMENT In the name of Allah, Most Gracious Most Merciful First of all I would like to thank my supervisor Dr. Shamim Ahmad and co-supervisor, Prof. Asif All, at the moment of my great pleasure. I am indeed indebted to them for helping me to accomplish my Ph.D. They have been very solicitous all through these years and made themselves available whenever approached, even at the time of their leisure. I owe my humble gratefulness for their indispensable guidance, constructive criticism and unceasing encouragement at every step, which enabled me to make my research work a humble success. I feel short of words to acknowledge Dr. Manzoor Ahmad Thoker, Department of Bacteriology, S.K. Institute of Sciences, Kashmir; Dr. Jamal A. Khan, Department of Microbiology, J.N. Medical College A.M.U., Aligarh; Dr. M Owais, Interdisciplinary Biotechnology Unit, A.M.U., Aligarh, and Dr. Niyaz Ahmad and Dr. Prashant Kenchapa, Lab of Molecular and Cell Biology (LMCB), CDFD, Hyderabad; for their valuable help they provided me in accomplishment of different experiments of my Ph.D. I am really thankful to all of them for their magnanimous generosity and unflagging interest they showed for my Ph.D. work, that helped me in all my endeavors. I pa^ lot of thanks to Prof. Raghavan Varadarajan, Dr. Sidartha P. Sharma and all lab mates especially (Deepall'iiumar of the Molecular Biophysics Unit, lISc, Bangalore. Prof. RV's scientific temperament, dedication to science and management of research are really guiding lights, and the time I spend in his lab is a big milestone to my research dream. I also wish to offer my thanks to Prof. Rashid All, Chairman Department of Biochemistry and Prof. Leela Ahuja, Director, Institute of Ophthalmology, J.N. Medical College, A.M.U., Aligarh, for providing me the necessary facilities to carry out this research. My special thanks are due to technical staff of all the departments mentioned above. I treasure my friendship most and want to share the moment of my gusto with all my friends, especially, (Dr. J^isar
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