MOLECULAR CHARACTERISATION OF MULTIDRUG-RESISTANT Pseudomonas aeruginosa IN SOUTHWESTERN NIGERIA. BY BAMIDELE TOLULOPE, ODUMOSU B.Sc Microbiology (OOU), M.Sc. Pharmaceutical Microbiology (Ibadan) A thesis in the Department of Pharmaceutical Microbiology Submitted to the Faculty of Pharmacy in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF IBADAN, IBADAN, NIGERIA. APRIL, 2013 i CERTIFICATION I certify that this project was carried out by Mr B.T. Odumosu under my supervision in the Department of Pharmaceutical Microbiology, University of Ibadan, Nigeria. ----------------------------------------------------------- Supervisor Bola A. Adeniyi, B.Sc, M.Sc (Ilorin), Ph.D. (Ibadan) Professor, Department of Pharmaceutical Microbiology University of Ibadan, Ibadan, Nigeria. ii DEDICATION This research work is dedicated to Almighty God, the God of Jacob who answers me in times of my distress and has been with me wherever I have gone. To you alone is glory. And to the loving memory of my beloved mother, Florence Titilayo Adunni Odumosu, continue to rest in the bosom of our dear Lord (amen). Mummy how I wish you were here! iii ACKNOWLEDGMENTS I acknowledge the love and support of Professor Bolanle Adeniyi who has been my supervisor since the days of my Master of Science (M.Sc) till present time. I enjoyed her commitment and dedication towards the successful completion of my postgraduate study, “Thank you ma”. I also want to acknowledge Professor Ram Chandra who was my host and supervisor in India and ensured I never lacked support as regarding my training and research in Lucknow India. My appreciation goes to the head of Department of Pharmaceutical Microbiology, Dr O. E. Adeleke. I will also like to appreciate other faculty members, the Dean of Faculty of Pharmacy Professor Moody, Professor Itiola, Professor Odeku, Dr Adegbolagun, Dr Odeniyi, Dr Lawal, Dr Idowu, Dr Ayeni, Mrs Coker, Mrs Oluremi and Mr Alabi. My gratitude goes to members of staff of the Department of Pharmaceutical Microbiology, Mr Odewale, Mr Sunday, Mrs Ekundayo, Mr Olajubutu, Mr Osho, and Miss Adejugba for their cooperation and assistance. My special appreciation goes to the University of Ibadan Postgraduate School for the award of Teaching and Research Assistantship given to me during my program. I thank the Dean of Postgraduate School Professor Olorunisola, the Sub-Deans, Dr Babalola , Dr Alarape and the former Sub-Dean Professor Aderinto for all their assistance and cooperation all along. My profound appreciation goes to The World Academy of Science, Italy (TWAS) and Council of Scientific and Industrial Research, India (CSIR) for providing financial support and travel expenses towards my research work in India. I cannot but specially acknowledge Dr Soge for his immense contributions, commitments and financial support towards the success of this research work. I also acknowledge and appreciate Professor Sanni of the Department of Microbiology, University of Ibadan for his fatherly support. My gratitude also goes to Dr Onasanwo of the Department of Physiology, University of Ibadan and Dr Adekunle of Federal University of Technology Akure for their helpful suggestions while we were in India. I say thank you to my friends and colleagues, Dr Okunye, Mr Onyenwe, Mrs Christiana, Mrs Ogunmola and Mrs Obisesan for all their wishes and goodwill. My special thanks go to Dr Bhargava, Dr Yadav, Mr Verma, Mr Jagdale, Mr Abishek and Miss Singh for all their cooperation while I was in Indian Institute of Toxicology research Gheru in Lucknow India. This acknowledgement would not complete without appreciating my father Mr. Olufemi Odumosu and my late mother Mrs. Titilayo Odumosu who both believed in me and supported iv me in prayers and in finances, and to my step mum Mrs Omowumi Odumosu for her continued support and prayers, I pray that God will continue to bless their efforts. I appreciate my siblings Mr Olumide Odumosu, Mr and Mrs Bukola Aluko-Olokun, Joke, Korede, Bayo and Dayo Odumosu as well as my cousins Dr Aina, Mrs Durojaiye, Mrs Alabi and Mrs Abiona for their prayers and support. I will not forget the love of my life Betty Ifeoluwa Odumosu and to my son Olusola Samuel Odumosu for all their support and understanding throughout my academic pursuit. Finally, unto the Lord Almighty the creator and giver of life be all glory for ever-Amen. v ABSTRACT Multidrug-Resistant (MDR) associated nosocomial infection is a global problem resulting in treatment failure particularly with respect to Pseudomonas aeruginosa. There is paucity of information on the molecular mechanisms of multidrug-resistant P. aeruginosa in Nigeria. Knowledge of the genetic basis of resistance of the organism to available antimicrobial agents will further improve empirical treatment. This study was undertaken to genetically characterise the multiple antibiotic resistance determinants in P. aeruginosa and describe the genetic locations of the resistance genes. Antimicrobial susceptibility of 54 clinical isolates of P. aeruginosa obtained from 5 hospitals in 3 southwestern states of Nigeria, to 21 antibiotics representing nine classes of antimicrobial agents was determined using the antibiotic disk-diffusion method. Minimum inhibitory concentrations were determined by Etest. Plasmid DNA for the isolates were extracted by alkaline lysis while plasmid curing was carried out using acridine orange, ethidium bromide and sodium dodecyl sulphate. The Extended-Spectrum Beta-Lactamase (ESBL) phenotypic detection was carried out using double-disk synergy method. Twenty clinical isolates with resistance to more than three “anti-pseudomonas” drugs were randomly selected for molecular studies. Genetic characterisation of ESBL and other drug resistant genes were achieved by polymerase chain reaction with specifically designed primers and direct sequencing of the amplicons. Significant trends in the association of plasmid counts and antimicrobial resistance among P. aeruginosa strains was achieved using Fisher’s Exact Test. All the strains of P. aeruginosa were found to be resistant to ampicillin, tetracycline and amoxicillin-clavulanic acid, while 53.7%, 63.0%, 79.6% and 87.0% were resistant to ceftriaxone, carbenicillin, kanamycin, and ticarcillin-clavulanic acid respectively. Plasmid profile of the 54 isolates revealed the presence of 1-4 resistance plasmids varying in sizes from 2.3 to 210.0 kb. Highest curing activity was achieved with ethidium bromide on 81% of the isolates at 40 µg/mL. The MDR P. aeruginosa strains harboured significantly more plasmids (≥3) compared to their non-MDR counterparts, which carried < 2 plasmids (p<0.01). Out of the 20 isolates randomly selected for molecular studies, 80% harboured blaOXA-10 that were plasmid encoded. Chromosomally encoded AmpC β-lactamase was found in 85%, while blaSHV and blaCTXM-1 were detected in one isolate each. Efflux pump regulators: mexR and nfxB were found in 45%, aac (6′) – I was detected in 50% and ant (2′′) – IV in 45% while both genes coding for aminoglycoside modifying enzymes were harboured vi in 35%. The class 1 integrons harbouring gene cassette array aaA6-orfD and aaA13, were also detected in the chromosomes of the isolates. The presence of resistance plasmids, class 1 integrons, extended-spectrum beta-lactamase, aminoglycoside modifying enzymes and efflux pump regulator genes among the population of P. aeruginosa tested indicated a high prevalence of multidrug resistance. Keywords: Multidrug-resistance Pseudomonas aeruginosa, Extended-spectrum beta-lactamase, Class 1 integrons. Word count: 433 vii LIST OF MAIN ABBREVIATIONS Abbreviation Full meaning AACs Aminoglycoside acetyltransferases AG Aminoglycosides AME Aminoglycoside modifying enzymes AmpC AmpC cephalosporinase beta lactamase ANTs Aminoglycoside nucleotidytransferases APHs Aminoglycoside phosphotransferases AST Antibiotic sensitivity test. ATCC American Type Culture Collection attI Receptor site BSA Bovine serum albumin CAUTI Catheter associated urinary tract infection CDC Centre for Disease Control and Prevention CFU Colony forming unit CLSI Clinical and laboratory Standards Institute CTX-M Cefotaximase CV-I Crystal violet complex dATP 2’-deoxyadenosine 5’-triphosphate DDH2O Double distilled water DDST Double disk synergy test dGTP 2’-deoxyguanosine 5’-triphosphate DNA Deoxyribonucleic acid dTTP 2’-deoxythymidine 5’-triphosphate E.coli Escherichia coli EDTA Ethylenediametetraacetic acid. ESBL Extended Spectrum β-lactamase Etbr Ethidium bromide FQ Fluoroquinolone H2O Water viii ICU Intensive care unit intI Integrase gene IS Insertion sequence Kb Kilo base KOH Potassium hydroxide LB Luria Bertani MDR multidrug resistance MDR Multiple drug resistant mexR Efflux pump regulator gene MgCl2 Magnessium hydroxide MHA Mueller Hinton agar MIC Minumum Inhibitory Concentration MLSK Macrolides-Lincosamides-Streptogramin- Ketolides MTCC Microbial type culture collection. NAOH Sodium hydroxide NDM New Delhi metallo-β-lactamase NER Non enzymatic resistance nfxB Efflux pump regulator gene Omp Outer membrane protein OprD Outer membrane porin ORF Open reading frame ori Origin OXA Oxacillinase OXA Oxacillinase P. aeruginosa Pseudomonas aeruginosa parC,ParE Topoisomerase genes PBP Penicillin binding protein PCR Polymerase chain reaction PEP Phosphoenolpyruvate. PER Pseudomonas extended resistance Qnr Plasmid-mediated quinolone resistance QRDR Quinolone resistance determining region QRDR Quinolone resistant determining region ix R Plasmid Resistance plasmid RFLP