Contribution of Horizontal Gene Transfer to Virulence and Antibiotic Resistance in Pathogens ...

174 Pages·2011·1.53 MB·English

Contribution of Horizontal Gene Transfer to Virulence and Antibiotic
Resistance in Pathogens Transmitted in Healthcare Facilities

by
Erika Lea Flannery

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Epidemiological Science)
in the University of Michigan
2011

Doctoral Committee:
Professor Harry L.T. Mobley, Co-Chair
Professor Betsy Foxman, Co-Chair
Associate Professor Carl F. Marrs
Associate Professor Lona Mody
Assistant Professor Sebastian K. Zoellner

© Erika L. Flannery
All Rights Reserved
2011
DEDICATION

To my friends, family and Chris.
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ACKNOWLEDGEMENTS

I thank everyone in the Mobley lab, past and present members, for their scientific
guidance and unwavering support. Stef is an amazing lab manager and an amazing
scientist. She has been a pleasure to share a bay with the past 6 ½ years and will be
missed dearly as I move to a new laboratory. Dr. Chris Alteri has been instrumental in
my growth as a scientist. I appreciate his patience with questions and guidance on
experiments, and for spending many hours talking “science” with me. Kelsey, Greta and
Erin were excellent graduate students to learn from and great friends. Gregg Davis
played an important role in training me as a scientist when I first started in the Mobley
Lab and has remained a good friend. I thank Dan and Ariel for their support and
friendship and wish them the best of luck finishing their degrees.
I thank my committee for providing thoughtful guidance as my dissertation
developed and for allowing me to conduct research on, what may have seemed at times,
unrelated topics.
I would like to thank my parents Dave and Tracey Flannery for their support over
the past 6 years of graduate education and the many years before that. Without their
confidence in my ability, this work would not have been impossible. I would also like to
thank my siblings, Ryan and Lauren, for supporting my endeavors throughout the years.
My boyfriend Christoph, has been amazingly supportive through this process. I thank
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him for his understanding, guidance, and thoughtful scientific discussion as well as his
kind encouragement.
I thank Dr. Lona Mody for her guidance and use of data for Chapter IV. I was
able to observe the many steps involved with implementing a population-based study and
I am grateful for her clinical perspective on the project.
I thank Dr. Betsy Foxman for her mentorship during my 6 years at the School of
Public Health. Her expertise has been instrumental in my thorough learning of
Epidemiologic theory. I also thank Betsy for her genuine interest and guidance in my
endeavors beyond my time as a student.
Finally, I would like to thank my mentor, Dr. Harry Mobley, for his unconditional
support throughout my time here. Harry has taught me as much about science as he has
about life, all lessons that will be influential in my development as a scientist and a
person in years to come.

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TABLE OF CONTENTS
DEDICATION................................................................................................................ ii 
ACKNOWLEDGEMENTS ......................................................................................... iii 
LIST OF FIGURES .................................................................................................... viii 
LIST OF TABLES ..........................................................................................................x 
LIST OF APPENDICES .............................................................................................. xi 
ABSTRACT .................................................................................................................. xii 
Chapter 1 Introduction...................................................................................................1 
HEALTHCARE-ASSOCIATED INFECTIONS ..................................................................... 1 
Epidemiology ........................................................................................................................ 1 
Antibiotic Resistant Organisms ............................................................................................ 4 
Catheter-associated urinary tract infections ........................................................................ 8 
Vancomycin-resistant S. aureus (VRSA) ............................................................................ 13 
HORIZONTAL GENE TRANSFER AND INTEGRATIVE AND CONJUGATIVE
ELEMENTS ........................................................................................................................... 17 
Horizontal Gene Transfer (HGT) ....................................................................................... 17 
Integrative and Conjugative Elements (ICEs).................................................................... 25 
STATEMENT OF THE PROBLEM ...................................................................................... 31 
Chapter 2 Identification of a Modular Pathogenicity Island that is Widespread
among Urease-producing Uropathogens and Shares Features with a Diverse
Group of Mobile Elements ...........................................................................................34 
ABSTRACT ........................................................................................................................... 34 
INTRODUCTION .................................................................................................................. 36 
MATERIALS AND METHODS ........................................................................................... 39 
RESULTS ............................................................................................................................... 43 
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DISCUSSION ......................................................................................................................... 48 
ACKNOWLEDGEMENTS .................................................................................................... 53 
Chapter 3 Self-transmissibility of the Integrative and Conjugative Element
ICEPm1 Between Clinical Isolates Requires a Functional Integrase, Relaxase and
Type IV Secretion System ............................................................................................59 
ABSTRACT ........................................................................................................................... 59 
INTRODUCTION .................................................................................................................. 61 
MATERIALS AND METHODS ........................................................................................... 65 
Bacterial strains and growth conditions. ........................................................................... 65 
Molecular techniques. ........................................................................................................ 65 
Construction of mutants. .................................................................................................... 66 
RESULTS ............................................................................................................................... 68 
DISCUSSION ......................................................................................................................... 76 
ACKNOWLEDGEMENTS .................................................................................................... 81 
Chapter 4 Wounds and Functional Disability are Associated with Co-Colonization
by Methicillin-resistant Staphylococcus aureus and Vancomycin-resistant
Enterococci in Southeast Michigan .............................................................................93 
ABSTRACT ........................................................................................................................... 93 
INTRODUCTION .................................................................................................................. 95 
MATERIALS AND METHODS ........................................................................................... 97 
RESULTS ............................................................................................................................. 101 
DISCUSSION ....................................................................................................................... 106 
ACKNOWLEDGEMENTS .................................................................................................. 110 
Chapter 5 Conclusions and Future Directions .........................................................116 
SUMMARY OF RESULTS ................................................................................................. 116 
CONCLUSIONS AND PERSPECTIVES ........................................................................... 118 
Conservation of a genomic island in three uropathogens ................................................ 118 
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ICEPm1 is an active ICE that is self-transmissible. ........................................................ 122 
ICEPm1 excision is integrase dependent ......................................................................... 124 
ICEPm1 encodes a T4SS necessary for self-transmissibility ........................................... 125 
ICEPm1 can integrate into either phe tRNA gene ........................................................... 126 
Co-colonization with MRSA and VRE occurs most commonly in wound and rectal sites 126 
Functional disability is a risk-factor for MRSA/VRE co-colonization ............................. 127 
FUTURE DIRECTIONS ...................................................................................................... 128 
APPENDICES .............................................................................................................137 
REFERENCES ............................................................................................................144 
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LIST OF FIGURES
Figure 1-1. Depiction of genes necessary for transmissibility of plasmids. .................. 24 
Figure 1-2. Mobile elements contribute to bacterial genome diversity by created
genomic islands. .................................................................................................. 30 
Figure 2-1. Comparative genomic hybridization results. ............................................... 54 
Figure 2-2. Array results and characteristics of ICEPm1. ............................................. 55 
Figure 2-3. BlastP results of all 91ORFs of ICEPm1. ................................................... 56 
Figure 3-1. Location of primers used in this study to detect ICEPm1 integration and
excision. .............................................................................................................. 82 
Figure 3-2. ICEPm1 actively excises from the chromosome. ....................................... 83 
Figure 3-3. ICEPm1 precisely excises from the chromosome at the PheV-tRNA. ....... 84 
Figure 3-4. Confirmation of ICEPm1 transfer to a clinical P. mirabilis commensal
isolate and its retained function. ......................................................................... 85 
Figure 3-5. intP is necessary for ICEPm1 excision and transfer. .................................. 86 
Figure 3-6. ICEPm1- encoded T4SS genes are important for conjugative transfer. ..... 87 
Figure 3-7. Active excision of the SXT-like ICE from P. mirabilis HI4320. ............... 88 
Figure 3-8. Genetic organization of pheU and pheV sites in Escherichia coli. ............. 89 
Figure 3-9. Transfer of ICEPm1 to select recipients. .................................................... 90 
Figure 4-1. Colonization patterns of 14 residents co-colonized a minimum of one visit
during the study period. .................................................................................... 111 
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