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Characterization and Colistin Susceptibility Of CHARACTERIZATION AND COLISTIN SUSCEPTIBILITY OF CARBAPENEM RESISTANT ISOLATES OF PSEUDOMONAS AERUGINOSA AND ACINETOBACTER BAUMANNII IN A TERTIARY CARE HOSPITAL Dissertation submitted for M.D. MICROBIOLOGY BRANCH – 1V DEGREE EXAMINATION THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI – 600 032 TAMILNADU MAY 2018 CERTIFICATE This is to certify that this dissertation titled “CHARACTERIZATION AND COLISTIN SUSCEPTIBILITY OF CARBAPENEM RESISTANT ISOLATES OF PSEUDOMONAS AERUGINOSA AND ACINETOBACTER BAUMANNII IN A TERTIARY CARE HOSPITAL” is a bonafide record of work done by Dr.M.SOWNDARYA, during the period of April 2016 to March 2017 under the guidance of Prof.Dr.THASNEEM BANU.S. M.D., Professor of Microbiology, Institute of Microbiology , Madras Medical College and Rajiv Gandhi Government General Hospital, Chennai - 600003, in partial fulfillment of the requirement of M.D. MICROBIOLOGY Degree Examination of The Tamilnadu Dr.M.G.R. Medical University to be held in May 2018. Dr.R.NARAYANA BABU, MD.,DCH Dr.ROSY VENNILA., M.D., Dean, Director, Madras Medical College & Institute of Microbiology, Rajiv Gandhi Government Madras Medical College & General Hospital, Rajiv Gandhi Government Chennai – 600003 General Hospital, Chennai – 600003 DECLARATION I, Dr.M.SOWNDARYA, Post Graduate , Institute of Microbiology, Madras Medical College, solemnly declare that the dissertation titled “CHARACTERIZATION AND COLISTIN SUSCEPTIBILITY OF CARBAPENEM RESISTANT ISOLATES OF PSEUDOMONAS AERUGINOSA AND ACINETOBACTER BAUMANNII IN A TERTIARY CARE HOSPITAL”is the bonafide work done by me at Institute of Microbiology, Madras Medical College under the expert guidance and supervision of Prof.Dr. THASNEEM BANU.S M.D., Professor, Institute of Microbiology, Madras Medical College. The dissertation is submitted to the Tamil Nadu Dr.M.G.R Medical University towards partial fulfillment of requirement for the award of M.D., Degree (Branch IV) in Microbiology. Place: Chennai Date: Dr.M.SOWNDARYA Signature of the Guide Prof. Dr.THASNEEM BANU.S, MD., Professor, Institute of Microbiology Madras Medical College, Chennai-600 003. ACKNOWLEDGEMENTS I wish to express my sincere thanks to Dr. R.Narayana Babu, MD, DCH, Dean, Rajiv Gandhi Government General Hospital & Madras Medical College, Chennai-3 for permitting me to use the resources of this institution for my study. I express my thanks to Dr.Rosy Vennila, M.D., Director, Institute of Microbiology for her guidance and support. Sincere thanks to Former Professor Dr.Mangala Adisesh M.D., Institute of Microbiology for her constant encouragement and support during this work. I owe my heartfelt gratitude and sincere thanks to my guide Dr.Thasneem Banu.S, M.D., Professor, Institute of Microbiology for her valuable suggestions, guidance, constant support, motivation and encouragement throughout this study. I would like to thank all my Professors Dr.U.Umadevi M.D., Dr.Vanaja.R M.D., and Dr.C.P.Ramani M.D., for their support during this study. I extend my gratitude to my co-guide Dr.K.Usha Krishnan, M.D., Senior Assistant Professor, Institute of Microbiology for her valuable guidance and constant support in this study. I wish to extend my thanks to our Assistant Professors Dr.Deepa.R M.D., Dr.Rathnapriya.N M.D., Dr.K.G.Venkatesh M.D., Dr.Sripriya.C.S M.D., Dr.Lakshmi priya.N M.D.DCH., Dr. David Agatha M.D., Dr. B.Natesan M.D.DLO., for their support. I would like to extend my thanks to all my postgraduate colleagues and technicians for their constant support and help in this study. I am thankful to my dear parents, beloved sisters and mother in-law for their unconditioned love, sacrifice and constant emotional support. I thank my husband Dr.K.Vijay Prakash M.D., for his constant motivation, emotional support and help in completing the dissertation work. Last but not least, I would like to thank the patients participated in this study for their co-operation and support. LIST OF ABBREVIATIONS ACT – AmpC Type AmpC – AmpC β-lactamases ATCC – American Type Culture Collection bla – β-lactamases CAUTI – Catheter Associated Urinary Tract Infection CDC – Centre for Disease Control and prevention CLABSI – Central Line Associated Blood Stream Infection CLSI – Clinical Laboratory Standard Institute CMY – Cephamycin hydrolysing CNS – Central Nervous System CRAB – Carbapenem Resistant Acinetobacter baumannii CRPA – Carbapenem Resistant Pseudomonas aeruginosa CTX-M – Cefotaxime hydrolyzing enzyme CV – Clavulanic acid DHP – Dehydropeptidase DNA – Deoxy ribonucleic acid DPA – Di picolinic Acid EDTA – Ethylene Diamine Tetra Acetate ESBL – Extended Spectrum β-lactamases E-test – Epsilometer test FOX – Cefoxitin GIM – German Imipenemase GNB – Gram-negative bacilli ICU – Intensive Care Unit IMP – Imipenemase IRMR – Imipenem Resistant Meropenem Resistant IRMS – Imipenem Resistant Meropenem Sensitive IS – Insertion Sequence kDA – Kilo Dalton KPC – Klebsiella pneumoniae carbapenemase LPS – Lipopolysaccharide MBL – Metallo β-lactamases mcr – mobilizable colisin resistance MDR – Multidrug Resistant MDRO – Multidrug Resistant Organism MHT – Modified Hodge Test MIC – Minimal Inhibitory Concentration MIR – Miriam Hospital MRIS – Meropenem Resistant Imipenem Sensitive MRSA – Methicillin Resistant Staphylococcus aureus NDM – New Delhi Metallo β-lactamase NFGNB – Non-fermenting Gram-negative Bacilli NHSN – National Healthcare Safety Network OF – Oxidative/ Fermentative OMP – Outer Membrane Protein OXA – Oxacillinase PBA – Phenyl Boronic Acid PBP – Penicillin Binding Protein PCR – Polymerase Chain Reaction PER-1 – Pseudomonas Extended Resistant β-lactamase PTZ – Piperacillin-Tazobactam RND – Resistant Nodulation Division rRNA – Ribosomal Ribonucleic Acid SENTRY – Antimicrobial Surveillance Program SHV – Sulfhydryl variable SIM – Seoul imipenemase SME – Serratia marcescens enzyme SPM – Sao Paulo metallo-β-lactamase SSI – Surgical Site Infections TEM – Temoneira VAP – Ventilator Associated Pneumonia VEB – Vietnam extended spectrum β-lactamase VIM – Verona Integron encoded Metallo β-lactamase WHO – World Health Organization TABLE OF CONTENTS Sl.No. TITLE Page No. 1 INTRODUCTION 1 2 AIMS & OBJECTIVES 4 3 REVIEW OF LITERATURE 5 4 MATERIALS & METHODS 42 5 RESULTS 56 6 DISCUSSION 73 7 LIMITATIONS OF THE STUDY 81 8 SUMMARY 82 9 CONCLUSION 83 9 COLOUR PLATES 84 10 BIBLIOGRAPHY 89 11 ANNEXURES (i) PROFORMA (ii) CONSENT FORM (iii) INFORMATION SHEET (iv) MASTER CHART (v) LEGENDS FOR MASTER CHART (vi) ETHICS COMMITTEE RECEIPT LIST OF TABLES Sl.No TITLE Page No. 1 Classification of medically important pseudomonads 6 2 Classification of beta-lactamases 16 3 Classification of Acinetobacter species 19 4 Putative genes for hospital adaptiveness 21 5 Biochemical reactions of Pseudomonas aeruginosa 45 6 Biochemical reactions of Acinetobacter baumannii 46 Panel of Antibiotics for Pseudomonas aeruginosa isolates and 7 47 interpretative criteria Panel of Antibiotics for Acinetobacter baumannii isolates and 8 48 interpretative criteria 9 MIC interpretative criteria for Imipenem 49 10 MIC interpretive criteria for Colistin according to CLSI 52 Distribution of Pseudomonas aeruginosa isolates among 11 56 various clinical samples Distribution of Acinetobacter baumannii isolates among 12 57 various clinical samples Distribution of Pseudomonas aeruginosa isolates among 13 58 various Clinical settings Distribution of Acinetobacter baumannii isolates among 14 59 various Clinical settings Antimicrobial Susceptibility pattern of Pseudomonas 15 60 aeruginosa Antimicrobial Susceptibility pattern of Acinetobacter 16 61 baumannii Distribution of Multidrug Resistance and Carbapenem 17 62 resistance among P.aeruginosa and A.baumannii isolates Distribution of Multidrug Resistant P.aeruginosa and 18 62 A.baumannii isolates among various samples Phenotypic characterization of resistance among Pseudomonas 19 64 aeruginosa isolates Phenotypic characterization of resistance among 20 65 Acinetobacter baumannii isolates Distribution of Carbapenem resistant P.aeruginosa and 21 66 A.baumannii isolates among various samples Distribution of Carbapenem resistant isolates among various 22 67 Clinical settings MIC of Imipenem for the Imipenem resistant isolates by disc 23 68 diffusion method 24 Risk factors associated with carbapenem resistance 68 Antimicrobial resistance pattern among Carbapenem 25 69 susceptible and Carbapenem resistant P.aeruginosa isolates Antimicrobial resistance pattern among Carbapenem 26 70 susceptible and Carbapenem resistant A.baumannii isolates Molecular characterization of MBL positive P.aeruginosa 27 71 isolates Molecular characterization of MHT positive A.baumannii 28 71 isolates 29 MIC of Colistin for the Carbapenem resistant isolates 72 LIST OF FIGURES Sl.No. TITLE Page No. 1 P.aeruginosa on MacConkey agar 9 2 P.aeruginosa on Blood agar 10 3 Colonies of A.baumannii on MacConkey agar 20 Sample wise distribution of P.aeruginosa and 4 57 A.baumannii Distribution of P.aeruginosa among various clinical 5 58 settings Distribution of A.baumannii among various clinical 6 59 settings 7 Sample wise distribution of MDR isolates 63 Distribution of Carbapenem resistant isolates among 8 66 clinical samples Distribution of Carbapenem resistant isolates among 9 67 clinical settings 10 Nitrate reduction test 84 11 AmpC betalactamase detection by combined disc method 85 MRIS – Meropenem Resistant Imipenem Sensitive 12 85 phenotype 13 Modified Hodge Test (MHT) 86 14 Imipenem MIC detection by E-test 86 Metallo Beta-Lactamase detection -Combined disc 15 87 method 16 Colistin MIC by E-test 87 Polymerase
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