STRAIN CHARACTERIZATION, ANTIMICROBIAL SUSCEPTIBILITY AND COLD-INDUCED GENES OF FLAVOBACTERIUMPSYCHROPHILUM A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by SHOHREH HESAMI In partial fulfillment of requirements for the degree of Doctor of Philosophy December, 2009 © Shohreh Hesami, 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-58289-3 Our file Notre reference ISBN: 978-0-494-58289-3 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 14-1 Canada ABSTRACT STRAIN CHARACTERIZATION, ANTIMICROBIAL SUSCEPTIBILITY AND COLD-INDUCED GENES OF FLA VOBACTERIUMPSYCHROPHILUM Shohreh Hesami, Advisors: University of Guelph, 2009 John S. Lumsden, DVM, PhD Janet I. Machines, PhD Seventy-five isolates of Flavobacterium psychrophilum collected from salmonids with clinical signs of bacterial cold water disease (BCWD) were characterized morphologically, biochemically, serologically, and genotypically. Although the isolates were morphologically and serologically homogeneous, API-ZYM testing revealed two distinct biovars. Four restriction pattern types were detected by 16S rRNA PCR-RFLP analysis. There were significant correlations between biovar I and digestion with Maelll (p<0.00\) and between biovar II and digestion with Mnll (p<0.05). Detection of nine genotypes within a 194 bp region of 16S sequence type revealed further heterogeneity of which type "a" was the predominant genotype. More than one biovar and genotype was identified among the strains recovered from a single BCWD outbreak, however, no association between genotype or biotype and clinical disease presentation was found. Management of outbreaks of BCWD often requires the use of antibiotics. The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents were determined by adapting a broth microdilution method, established by the Clinical and Laboratory Standards Institute for aquatic bacteria with optimal growth temperature below 35 °C. For most F. psychrophilum isolates there was a very high MIC for two of the four antibiotics licensed for use in Ontario (i.e., ormetoprim/sulfadimethoxine and trimethoprim/sulfamethoxazole). High MICs of florfenicol and oxytetracycline were obtained for 53% and 61% of isolates, respectively. For the majority of strains, the MICs for ampicillin, oxolinic acid and gentamicin were high, while for 83% of the strains tested the MICs for erythromycin were medium and low. cDNA suppression subtractive hybridization (SSH) was used to identify cold- induced genes in a ulcerative dermatitis and necrotizing stomatitis isolate F. psychrophilum B382-90-4. Genes predicted to encode a two-component system sensor histidine kinase LytS, an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein and four housekeeping genes, were up-regulated at 8 °C versus 20 °C. Since no reference gene of F. psychrophilum was available as an internal standard for use in quantitative real-time PCR (qPCR), the expression stability of 9 commonly used reference genes was evaluated at 8 °C and 20 °C. Expression of thel6S rRNA gene was equivalent at both temperatures and this gene was used in qPCR experiments to verify the SSH findings. DECLARATION OF WORK DONE I hereby declare that all work presented in this thesis was performed or directed by myself except for the statistical analysis, which was done by William Sears (Chapter 2) and Dr. John Lumsden (Chapter 3). Jing Zhang provided advice regarding the experimental design of the quantitative real time PCR performed at the Genomics Facility at University of Guelph. i ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my advisor Dr. John Lumsden for his dedication to assisting me in my graduate work and for providing valuable guidance and support throughout the duration of these studies. I would also like to thank my other advisor, Dr. Janet Maclnnes for her support and encouragement throughout my research. I was fortunate enough to have a great advisory committee members Dr. Carlton Gyles and Dr. Laura Brown who provided me with many helpful suggestions and sound advice. A particular thanks goes to Devon Metcalf a PhD student of Dr. Weese lab for her guidance and her friendship during this sometimes overwhelming experience. I would also like to thank the members of the fish Pathology lab department of Pathobiology for their technical assistance, friendship and making my time spend during this work enjoyable. I wish to give a special thank you to my family for their support and for being so patient and understanding. Financial support for this project was provided by the OMAFRA, ACRDP and NSERC. I was supported in part by Ministry of Science, Research & Technology of Iran. ii TABLE OF CONTENTS DECLARATION OF WORK DONE i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVATIONS ix CHAPTER 1. REVIEW OF THE LITERATURE 1.1. Introduction 1 1.2. The microbiology of Flavobacterium psychrophilum 1 1.2.1. Taxonomy and isolation 1 1.2.2. Identification : 3 1.3. Diseases caused by Flavobacterium psychrophilum 4 1.3.1. Clinical signs and pathology 4 1.4. Transmission 7 1.5. Putative virulence factors 8 1.5.1. Extracellular protease 8 1.5.2. Bacterial adhesions 10 1.5.3. Cell envelope 11 1.5.3.1. Lipopolysaccharide and outer membrane proteins 11 1.5.3.2. Glycocalyx 13 1.5.3.3. Biofilm formation 14 1.6. Typing schemes 15 1.6.1. Serotyping 15 1.6.2. Molecular typing 16 1.7. Control and treatment 18 iii 1.7.1 Antimicrobial therapy 19 1.7.2.Vaccination 21 1.8. Research proposal 23 CHAPTER 2. PHENOTYPIC AND GENOTYPIC ANALYSIS OF Flavobacterium psychrophilum ISOLATES FROM ONTARIO SALMONIDS WITH COLDWATER DISEASE 2.1. Introduction 27 2.2. Materials and Methods 29 2.2.1. Bacterial strains and growth conditions 29 2.2.2. Biochemical testing 30 2.2.3. API-ZYM profiles 31 2.2.4. Slide agglutination tests 32 2.2.5. DNA extraction and PCR amplification 32 2.2.5. PCR-restriction fragment length polymorphism 33 2.2.6. Sequencing of 16S rRNA gene PCR products 34 2.2.7. Statistical analysis 35 2.3. Results 35 2.3.1. Strain characterization 35 2.3.2. API-ZYM profiles 36 2.3.3. Slide agglutination tests 36 2.3.4. PCR amplification 36 2.3.5. PCR-RFLP 37 2.3.6. Sequencing of 16S rRNA gene PCR products 37 2.3.7. Association between biovars, sequence types and mortality events.. .* 38 2.4. Discussion 46 iv CHAPTER 3. ANTIMICROBIAL SUSCEPTIBILITY OF ONTARIO FLAVOBACTERIUM PSYCHROPHILUM 3.1. Introduction 50 3.2. Materials and Methods 53 3.2.1. Bacterial strains and growth conditions 53 3.2.2. Antimicrobial susceptibility testing 54 3.2.3. Statistical methods 56 3.3. Results and Discussion 56 3.3.1. Strain characterization 56 3.3.2. Antimicrobial susceptibility testing 57 3.3.3. Ormetoprim/sulfadimethoxine and trimethoprim/ sulfamethoxazole 69 3.3.4. Oxytetracycline 69 3.3.5. Florfenicol 70 3.3.6. Erythromycin 71 3.3.7. Oxolinic acid, flumequine and enrofloxacin 71 3.3.8. Gentamicin 72 3.3.9. Ampicillin 73 CHAPTER 4. IDENTIFICATION OF COLD TEMPERATURE REGULATED GENES IN FLA VOBACTERIUMPSYCHROPHILUM 4.1. Introduction 78 4.2. Materials and Methods 80 4.2.1. Bacterial strain and growth conditions 80 4.2.2. RNA isolation 80 4.2.3. mRNA isolation 80 4.2.4. Suppression subtractive hybridization 81 4.2.5. Cloning of PCR products 83 4.2.6. Rapid screening of clones 83 4.2.7. Sequencing 84 4.2.8. Expression analysis of reference gene candidates 84 v 4.2.9. Quantitative real-time PCR (qPCR) amplification of cold-induced genes 85 4.3. Results 86 4.3.1. Suppression subtractive hybridization 86 4.3.2. Quantitative real-time PCR of differentially expressed gene 87 4.4. Discussion 93 CHAPTER 5. GENERAL DISCUSSION 103 REFERENCES 114 APPENDIX 141 A. Nucleotide sequences of the longest insert sequence obtained from subtracted library for each identified gene 141 vi LIST OF TABLES 2.1 Flavobacterium psychrophilum strains used in this study 40 2.2 Sequence of 16S rRNA, gyrase A and gyrase B primers 44 2.3 16S rRNA PCR-RFLP cut-site and sequence type of F.