MOLECULAR CHARACTERIZATION OF CARBAPENEMASE PRODUCING BACTERIAL STRAIN OF ENTEROBACTERIACEAE FAMILY THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF Doctor of Philosophy IN BIOTECHNOLOGY BY NAYEEM AHMAD UNDER THE SUPERVISION OF PROF. ASAD ULLAH KHAN MaulanaINTERDISCIPLINARY Azad Library, BIAligarhOTECHNOLOGY Muslim UNITUniversity ALIGARH MUSLIM UNIVERSITY ALIGARH - 202002 (INDIA) 2019 Certificate This is to certify that the thesis entitled “Molecular characterization of carbapenemase producing bacterial strain of enterobacteriaceae family” herewith submitted by Nayeem Ahmad, in fulfilment of the requirement for the degree of Doctor of Philosophy in Biotechnology of the Aligarh Muslim University, Aligarh, is an authentic record of the research work carried out by him under my supervision and guidance and that no part, thereof, has been presented before for any other degree. He has fulfilled all the prerequisites necessary for the submission of the Ph.D. thesis according to 2009 regulation of University Grants Commission, New Delhi. Date: (Prof. Asad U Khan) Maulana Azad Library, Aligarh Muslim (Ph.D. University Supervisor) Candidate DECLARATION I, hereby declare that thesis entitled “Molecular characterization of carbapenemase producing bacterial strain of enterobacteriaceae family” embodies the work carried out by me. Dated: (Nayeem Ahmad) Maulana Azad Library, Aligarh Muslim University COURSE WORK/COMPREHENSIVE EXAMINATION/PRE-SUBMISSION SEMINAR COMPLETION CERTIFICATE This is to certify that Mr. Nayeem Ahmad, Interdisciplinary Biotechnology Unit, has satisfactorily completed the Coursework/Comprehensive examination/Pre-submission seminar requirement which is part of his Ph.D. programme. MaulanaDate:…….. Azad Library, Aligarh Muslim (Signature ofUniversity Chairman) Contents LIST OF CONTENTS Page No. Acknowledgement i-iii List of Abbreviation iv-v List of Figures vi-viii List of Tables ix Abstract x-xiii 1-33 Chapter 1: Review of Literature 1.1. Antimicrobial resistance (AMR) 1 1.2. General concepts 1 1.3. Carbapenems 3 1.3.1. Evolution of carbapenems 3 1.3.2. Classification 5 1.3.3. Mechanism of action 6 1.3.4. Activity of carbapenems 7 1.3.5. Pharmacology and clinical use 7 1.3.6. Differences among Individual Carbapenems 7 1.3.6.1. Imipenem 7 1.3.6.2. Meropenem 8 1.3.6.3. Ertapenem 8 1.3.6.4. Doripenem 8 1.3.7. Mechanism of carbapenem resistance 8 1.3.7.1. Carbapenemases 8 1.3.7.2. Efflux pumps 9 1.3.7.3. Loss of porins 10 1.3.7.4. Penicillin-binding proteins (PBP) 10 Maulana1.3.8. Azad Carbapenem Library, resistance in AligarhEnterobacteriaceae Muslim University10 1.3.9. Molecular class A carbapenemases 12 1.3.9.1. The KPC family 12 1.3.10. Class D serine-carbapenemases: the OXA β-lactamases 13 1.3.10.1. The OXA-48 family 1.3.10.1 13 Contents 1.3.11. Molecular class B carbapenemases: the Metallo-β-lactamases 14 1.3.11.1. The IMP family 14 1.3.11.2. The VIM family 15 1.3.11.3. New Delhi Metallo-β-lactamase (NDM) 15 1.3.11.3.1. Multi-drug resistance by NDM-1 producing 15 bacteria 1.3.11.3.2. Major healthcare risk of NDM producers 16 1.3.11.3.3. Global distribution of NDM variants 16 1.3.11.3.4. NDM and clinical infections 26 1.4. Microbiology of neonatal infections 26 1.4.1. Major pathogens in neonatal septicemia 28 1.4.1.1. Klebsiellas spp. 28 1.4.1.2. Infections by Klebsiella pneumoniae 28 1.4.1.3. Infections by Escherichia coli 29 1.4.1.4. Clinical categories of E. coli 29 1.4.1.5. Extra-intestinal infections due to E. coli 30 1.4.1.6. ExPEC and neonatal sepsis 31 1.4.1.7. Infections by Enterobacter species 31 1.5 Aims and objective of the study 33 34-45 Chapter 2: Material and Methods 2.1. Antibiotic disc 34 2.2. Antibiotic powders 35 2.3. Culture media 35 2.4. Some of the important reagent, chemical and kits 36 2.5. Collection of bacterial strains and hospital setting 37 2.6. Ethical approval 38 2.7. Identification of isolates 38 2.8. Antimicrobial susceptibility testing and MICs 38 Maulana Azad Library, Aligarh Muslim University 2.9. Detection of Metallo-β-Lactamase 39 2.10. Carba NP test for detection of carbapenemase 39 2.11. Amplification of resistance genes by Polymerase chain reaction 39 2.12. DNA sequencing 40 Contents 2.13. Conjugation experiment 40 2.14. Molecular characterization of plasmid 40 2.15. Genetic environment analysis 41 2.16. Integron analysis 41 2.17. Molecular genotyping of isolates 41 2.18. Generate dendrogram by using PyElph version 1.4 Software 42 2.19. Multi-locus sequence typing (MLST) 42 Chapter 3: Occurrence of bla variants among NDM 46-68 Enterobacteriaceae from a Neonatal Intensive Care Unit in a northern India Hospital 3.1. Introduction 46 3.2. Experimental outline 47 3.3. Results 48 3.3.1. Antimicrobial susceptibility, Metallo-β-lactamase (MBL) and 48 MICs 3.3.2. Isolate identification 53 3.3.3. Carbapenemase production 53 3.3.4. Detection of antibiotic resistance markers 53 3.3.5. Conjugation 60 3.3.6. Replicon typing 60 3.3.7. Integron analysis 60 3.3.8. Genetic relatedness of the carbapenem-resistant NDM 62 producing Enterobacteriaceae isolates 3.3.9. Genetic environment of the blaNDM gene 62 3.4. Discussion 64 3.5. Conclusions 67 Chapter 4: Isolation and characterization of NDM producing bacterial (E. aerogenes, C. lepagi, and M. wisconsensis) 69-80 in the NICU of Indian Hospital 4.1. Objective: To detect NDM variants among Enterobacter Maulana Azad Library, Aligarh Muslim University69 aerogenes from NICU of Indian Hospital. 4.1.1. Introduction 69 4.1.2. Experimental outline 70 4.1.3. Results 70 Contents 4.1.3.1. Isolate identification, antimicrobial susceptibility test and 70 MICs 4.1.3.2 Metallo-β-lactamase (MBL) detection 73 4.1.3.3. Carba NP test 74 4.1.3.4 Identification and Genetic context of bla bla NDM-4, NDM-5 74 and blaNDM-7 4.1.3.5 Genetic environment analysis 75 4.1.3.6 Molecular typing 75 4.1.3.7 Replicon typing 75 4.1.4. Discussion 79 4.1.5. Nucleotide sequence Accession numbers 79 4.1.6. Conclusion 80 4.2: Objective: To characterize NDM-1 among rare species of 81 Enterobacteriaceae family 4.2.1. Introduction 81 4.2.2. Case Presentation 81 4.2.3. Nucleotide sequence accession number 84 4.2.4. Conclusion 84 4.3. Objective: To characterize carbapenem-resistant genes in the north Indian pediatrics patients to know if blaNDM-1 and blaVIM-1 are 85 disseminating through any rare species. 4.3.1. Introduction 85 4.3.2. Case Presentation 86 4.3.3. Discussion 90 4.3.4. Nucleotide sequence accession number: 91 4.3.5. Conclusion 91 Chapter 5: Molecular characterization of novel sequence type of 92-109 carbapenem-resistant NDM-1 producing Klebsiella pneumoniae in NICU of Indian Hospital. 5.1. Introduction 92 5.2. Experimental outline 93 Maulana5.3. Results Azad Library, Aligarh Muslim University93 5.3.1. Clinical characteristics of the carbapenem-resistant K. 93 pneumoniae isolates 5.3.2. Antibacterial susceptibility, MICs and Metallo-β-Lactamase 94 (MBL) production 5.3.3. Carbapenemase Production 94 Contents 5.3.4. Detection of antibiotic resistance genes 94 5.3.5. Genetic environment of the blaNDM 94 5.3.6. Plasmid analysis 101 5.3.7. Molecular typing 101 5.4. Discussion 106 5.5. Conclusion 109 Chapter 6: Conclusion 110 BIBLIOGRAPHY 111-130 LIST OF PUBLICATIONS CONFERENCES ATTENDED Maulana Azad Library, Aligarh Muslim University DEDICATED TO MY BELOVED PARENTS AND FAMILY Maulana Azad Library, Aligarh Muslim University Acknowledgement Acknowledgment Beginning with the name of Allah (SWT), the most Gracious and the most Merciful. Praise is to Allah (SWT), Lord of the Universe, who bestowed upon me the strength, capability, and perseverance to complete Ph.D. work. Peace and prayers to be on His last Prophet and Messenger Mohammad (SAW), the ideal role model for all Makhluqaat. Among the mortal beings, the courage and motivation to pursue and complete this research work came from my supervisor and esteemed teacher, Prof. Asad Ullah Khan, Coordinator, Interdisciplinary Biotechnology Unit, AMU, Aligarh. From his rich fund of knowledge and immaculate conceptual clarity, he regularly rendered brief, precise and to the point comments and suggestions. His positive attitude, wholehearted cooperation, and constructive criticism kept me moving forward towards my goal. His very presence in mind sustained my motivation. I consider myself fortunate to be his student. I shall always remain in debt of gratitude to my supervisor with respect and humbleness. I take this opportunity to express my sincere thanks to Prof. Rizwan Hasan Khan, Prof. M. Owais, Dr. Waseem Ahmad Siddiqui, Dr. Hina Yunus and Dr. Shaper Nazeer Khan of Interdisciplinary Biotechnology Unit for their valuable cooperation and encouragement. I very special thank goes to Prof. Syed Munazir Ali, Department of pediatrics JNMCH for providing clinical sample from neonates admitted in NICU. I appreciate the support and cooperation of my seniors Dr. Sadaf Hasan, Dr. Shakir Khan, Dr. Danishuddin, Dr. Shariq Qayyum, Dr. Lama Misbah, Dr. Divakar Sharma and Dr. Sneha Lata. I also extend my sincere thanks to my colleague Dr. Azna Zuberi, Mr. Abid Ali, Mr. Mohd Waqar Azam and Dr. Lubna Maryam for being such a wonderful friend. Their advice, guidance, and endless support kept me focused and helped me during difficult phases of my journey. They were always there whenever I am in need of a help and Maulanacooperated Azad with me in Library, every possible way.