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Isolation, Characterization and Possible Biocontrol Application of Bdellovibrionaceae (BD) Isolated From

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Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Isolation, characterization and possible biocontrol application of Bdellovibrionaceae (BD) isolated from NZ sources Muftikhar Ahmed A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) at Massey University 2008 Abstract Bdellovibrionaceae (BD) are unique, predatory, endoparasitic, Gram-negative bacteria. As the world's smalle t living hunter they prey on other Gram-negative bacteria giving them potential as biological control agents. Prior to this study, however, there were no reports of BD in New Zealand. The overall aim of thi research was to isolate BD from New Zealand ources, characterise them and investigate their potential role as a biological control agent. The history, characteristics, life cycle and mechanism of predation of this organism are reviewed and the possibil ity of the industrial applications of BD, are discussed. In this study, a halophilic specIes of BD was isolated from fo urteen coastal sea water sites around New Zealand. Thil1een isolates were characterised using proven characterisation techniques including general, microscopic and molecular techniques. It was fo und that the isolates were taxonomically identical or very closely related to each other and belong to the genus Bacteriovorax. The predation pattern of BD isolates was examined against a group of Gram negative bacteria in solid and liquid media. The predation patternsand efficiencies of the different BD isolates were similar, which confirms that the BD isolates are closely related, are selective in their predation, and prey on some Gram-negative bacteria but not all. The rapid loss of culture viabil ity of BD is well known, but no studies have been reported to date on the survival of pure cultures of BD at diffe rent temperatures. The survival rate of BD in dcnsc suspensions at diffe rent temperatures without host bacteria was investigated and it was observed that pure BD cultures can be stored with minimal reduction in numbers at temperatures ranging fr om 4°C to 20°C. However, significant reductions in numbers were observed at _1 8°C, 30°C and 37°C after 13 to 16 days. The effects of the 13 New Zealand BD isolates on the growth of a population of Photobacteriul11 phosphoreul11 were examined to elect the best isolate fo r in vitro application. All of the isolates tested had considerable reduction effect against P. phosphoreum. Some i olates were more 11 effective than others, despite their taxonomic similarity to each other. The isolate OT2 was selected for further studies based on these results. The in vi/ro efficacyof BD was assessed against late exponential cultures of a seafood spoilage bacterium, P. phosphorelll11, originally isolated from Cod fillets from Denmark. Log1o reductions of P. phosphoreum and ome other Gram-negative bacteria ranged from 4.5 to 4.8 after 9 h of incubation at 25°C. BD was effective in reducing the numbers of P. phosphoreul11 at pH 5.5 to 8.5 and salinity 0.9 to 4.5% (w/v). A significant interaction was observed between the prey and predator concentrations and nutrient concentration. Prey concentrations were ob erved to be the most vital fa ctor in predation and the most fa vourable predation conditions were at a prey concentration of �8 loglo colony fo nning units (CFU)/mL, together with a predator concentration of 3 - 7 loglo plaque fo mling unit ( PFU )/mL and a prey : predator ratio of >5.0. The thresholds of the prey and predator concentrations fo r predation were observed to be 3.7 loglo CFU/mL and 3.9 loglo PFU/mL, respectively. The trials carried out in this study focused on the efficiency of BD on a pure culture of one organism, P. phosphoreum and not on mixed cultures of Gram­ negative spoilage bacteria, the nomlal condition ob erved in saltwater fish. There has been very little research in this field and the results of these trial suggest fulther investigation into the effect of BD on mixed cultures of Gram-negative spoilage organisms is warranted. Since only one isolate of BD (OT2) was examined against only one spoilage bacteri um (P. phosphoreum) in liquid medium, the evidence of these fi ndings must be restricted to these particular conditions. Future studies, using a range of BD isolates against a mixture of spoilage and pathogenic organisms in solid medium are warranted. The biopreservation capability of BD in extending the helf life of king salmon was evaluated. A significant effect was observed at 20°C but not at 10° . At 20cC the shelf I ife was extended through extension of the lag phase of growth of the prey bacteria and a reduction in total numbers attained. Sensory evaluation of the salmon product being tested confirmed that the shelf life was extended. However, at 10cC there wa no reduction in prey organism , which ugge ted that the strain of BD used is ineffe ctive at refrigeration temperatures. III Acknowledgements I am particularly grateful to my chief supervisor, Or Quan Shu fo r his excellent guidance, support, encouragement and fo r organising the invaluable research training in the University of Maryland, Baltimore, USA. I would like to expre s my sincere gratitude to my supervisor Professor Jan S. Maddox of the Institute of Technology and Engineering, Massey University fo r his patience, encouragement, and invaluable contribution of ideas, advice and continuing support throughout the study period. I would also like to express my sincere appreciation to my supervisor and team leader Mr Graham C Fletcher of the New Zealand Institute fo r Crop & Food Research Limited fo r his total support, valuable guidance and direction, patience and invaluable advice throughout the course of the study and in the completion of my thesis. I also thank Or Lynn Mc1ntyre lately of the ESR, Christchurch for providing valuable supervision and encouragement throughout the period of my re earch. I am grateful to the New Zealand Institute fo r Crop & Food Research and my group leader Mr TC Chadderton fo r providing research and fe llowship funding, generous suppOl1, laboratory fa ci I ities and administrative support. I will always respect my great teacher Professor Henry N Williams of Florida A & M University and the University of Maryland, Baltimore, USA fo r his great guidance, invaluable advice, training, hospitality, confidence, and fo r preparing me fo r doing this study. I am particularly grateful to Or Silvia A Pii'ieiro of the Department of Medical and Research Technology, School of Medicine, University of Maryland, Baltimore, USA fo r her great teaching, advice and cientificcont ribution particularly in training me fo r the isolation and molecular characterisation of Bdellovibrionaceae. I would like to thank to the entire BD team of the University of Maryland, particularly Susan (Fluorescent microscope training), Tim (laboratory), Muhammad and all the others fo r their huge upport and encouragement. I gratefully acknowledge technical assistance from a number of staff and postgraduate students of Crop & Food Research particularly - Graeme Summers (sigma plot training), Joseph Youssef (microbiology laboratory), Michelle Miles (microbiology laboratory ), Simon Brown (sample collection), Salley Harrow (molecular biology laboratory), Jeanette McMonagle (administration) and fe llow postgraduate students Douglas Ro sendale, Lu Guangin and Sravani Gupta fo r their IV great help and valuable advice throughout the study period. I want to thank to Duncan Hedderley fo r his statistics advice. I gratefully acknowledge technical assistance from a number of staff of Hort Research and Massey University particularly - Paul Sutherland (electron microscope laboratory), Diane Barraclough (SDS-PAGE laboratory), Tim Holmes (photography unit), Sunita Bajaj (fluorescent microscope laboratory) and Rebecea Pattison (PFGE laboratory) of the Massey University. I would like to specially thank my entire fa mily, and my two lovely daughters (Nabila and Nazia) fo r their encouragement, patience, huge supp0l1 and sacrifices during the entire course of my studies. I want to dedicate this thesis to the sole memory of my fa ther, the late Mr Azim Uddin Ahmed and my mother Mrs Nurun Nahar fo r their excellent encouragement and total suppol1. v Table of Contents Abstract ........................................................................................................................................... i Ackno\vledgements ...................................................................................................................... iii Table of Contents .......................... .............. .................................................... .............................. v List of Tables ................................................................................................................................. x List of Figures .............................................................................................................................. xii Chapter I ................................................................................................................ .... ................. J General Introduction ........ ....................................................................................... ...... ..... ..... J I. I General review of literature ..................................... ........................................................
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