Chemical and biological evaluation of antibiotic-based ionic liquids and GUMBOS against

234 Pages·2017·4.62 MB·English

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LSU Doctoral Dissertations Graduate School
2012
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Marsha Rae Cole
Louisiana State University and Agricultural and Mechanical College
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Cole, Marsha Rae, "Chemical and biological evaluation of antibiotic-based ionic liquids and GUMBOS
against pathogenic bacteria" (2012). LSU Doctoral Dissertations. 487.
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CHEMICAL AND BIOLOGICAL EVALUATION OF ANTIBIOTIC-BASED IONIC
LIQUIDS AND GUMBOS AGAINST PATHOGENIC BACTERIA

A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Chemistry

by
Marsha Cole
B.S., Grambling State University, 2007
August 2012
To the legacy of Williams and Coles…past, present, and future

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ACKNOWLEDGEMENTS

“Trust in the Lord with all thine heart and lean not unto thine own understanding, in all
thy ways acknowledge Him and He will make your paths straight.”
King James Bible, Proverbs 3:5-6
First and foremost, I would like to thank Jesus Christ, my Lord and Savior, for keeping me
while directing my path through the tumultuous journeys and painful experiences that I have tread
in my life. I understand that this accolade would be completely unattainable if it were not for Him.
So I humbly submit my gratitude for His continual direction from the start of this journey through
the beginning of my next. All glory is to Him.
Secondly, I would like to thank my advisor, Dr. Isiah M. Warner, for allowing me the
opportunity to conduct research in his laboratory. I am especially grateful to him for allowing me
to pursue my own ideas and experiments in the lab, thus molding me into the independent and
resourceful research chemist that I am. Although the numerous group meetings, journal revisions,
seminars, and research colloquia that we were required to attend each week sometimes seemed like
cruel and unusual punishment, I am appreciative for them since they have helped me to continue
learning even after my formal coursework was complete. I cannot thank both you and Mrs. Warner
enough for your support, encouragement, and kind gestures. I will miss the laughs and the hugs.
Thank you for being my extended family.
Thirdly, I would like to thank all of the graduate students, postdocs, and undergraduate
students that I have worked with over the years while matriculating as a Ph.D. student. I will miss
you all dearly. Many of you have grown to be more like brothers and sisters than friends and I
hope like family are bonds that will be forever. I thank you all for the laughter in times I wanted to

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cry, for reading scripture and fellowshipping when I’ve missed church, singing badly along to the
radio with, shopping and bargain hunting for clothes and shoes, for providing insight and comfort
during demanding and stressful times, falling victim to prank calls, and in general for making time
spent in and out of the lab much more enjoyable. All of you have contributed something so special
and endearing to me and have made this Ph.D. journey an invaluable experience. Likewise, I
appreciate you for always being there to help with experiments and presentations and never being
reluctant to help answer my thousands of questions.
I thank the members of my committee: Dr. Daniel Hayes, Dr. Robert Cook, Dr. Marlene
Janes, and Dr. James Moroney for taking the time out of each of their schedules to serve on my
committee and for always perpetuating my boundless curiosity with their insightful questions. I
also thank Dr. Jeffrey Hobden and Dr. George Stanley, although not formal committee members,
for being resourceful and honest when I sought guidance. Last but not least, I thank Dr. Saundra
McGuire, an angel in disguise, a wizard of academic success, and a doctor of educational crises.
You have selflessly listened and contributed encouragement while reinforcing confidence when I
thought the Ph.D. was too difficult to obtain. I thank you for getting me through many of the road
bumps that are evident in this journey, and I think God couldn’t have placed a woman any better
than you to serve as a mentor and comforter to me. Most importantly, I thank you for your
countless encouragement, recommendations, and support as I tried to collect my abstract approach
to science and mount it into something bigger than I could have ever imagined. Thank you for
understanding that honesty, hardwork, and serendipitous ambition are my best attributes.
As I conclude this chapter of my life, I cannot help but acknowledge the other people who
have also helped get me to this point. These people will always hold a special place in my heart
and will never be forgotten. Thank you to my undergraduate chemistry professors: Dr. Danny

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Hubbard, Dr. Allen Miles, Dr. Bobby Burkes, Dr. Frank Ohene amongst other “Grambling”
friends and family. You all have been instrumental in my future and I truly believe that each of you
have helped lay the foundation that was necessary for me to make it to this point. Special thanks to
my friends from home and at Grambling for the fun times that helped me relieve stress during
study hours, whether it was giggles over “Rotel Nachos”, free styling amateur rap lyrics, playing
beauty shop, running midnight “fat-attacks” to TCBY and Popeyes or simple sisterly relations
eating “skee-wee cakes” and taunting BB the cat, each moment shared is memorable and always
helped me enjoy my journey through each phase of this academic career.
Lastly, I give a very special acknowledgement to my own personal support system: Mark
and Patti Cole (my parents) and Marcus Cole (my brother) for without them I would have never
been encouraged to pursue something so daring, foreign, and pressing. I love you all with every
ounce of my breath and drop of my blood. I want you to know that I am proud to represent our
family and our name. I am eternally grateful for your love and support because this accolade was
not only for me, but it was for us. Thank you Daddy for teaching me the “6 P’s” and that the only
security in life is the one that I create for myself. Because of your wisdom, I have secured my
future as a trailblazer and not as a statistic. Papa Bear, consider yourself a success as a parent.
Mommy, I appreciate you for being my best friend during my loneliest times and my mother
during the hardest times. You are the hardest working woman that I have ever known, and I’m glad
to know that I have inherited such diligence and perseverance from you. It is because of your
strength that I have learned to push through the seeming insanities that accompany the Ph.D.
program. Most importantly, you have taught me to smile and stay kind through it all. Marcus,
thank you for keeping me focused on the meaningful things in life. You have been my silent knight
and I thank you for always protecting and encouraging me when others would belittle me because I

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was “smart”. Since you have stood guard over your kid sister, I have finally become who you
knew I could be. I also thank Aunt Paula Ross and Uncle Joseph Williams for always looking out
for me whenever my parents weren’t able. You two were always there for me both academically
and socially and I am grateful for the time that you have contributed to help make the final steps of
my education less intense. From teaching me how to read to building kinetic machines, you have
impacted me immensely and I will always cherish those memories. I will never forget how you
have nurtured my love for education and science. Because of our family, I have become a
trailblazer and no other first means more than logging this moment into our family history. Last
and definitely not least, I would like to thank Kevin Roberson for your inifinite encouragement,
continuous friendship, and enthusiasm during this journey which has given me the motivation to
realize this achievement. From the first week of school, we have grown closer and I am thankful to
God that he has placed such an awesome man by my side to travel this journey together with. Your
cupcakes, cards, and conversation has been a priceless investment to me accomplishing this
journey and I want express my gratitude for all you have done for me during this process. I love
you all more than time could measure and my gratitude to you extends infinitely beyond the
measures of this world.

Thank you….
XOXO
Rae

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TABLE OF CONTENTS
DEDICATION .................................................................................................................................. ii
ACKNOWLEDGEMENTS ........................................................................................................... ..iii
LIST OF TABLES ........................................................................................................................... xi
LIST OF FIGURES ....…………………………………………………………………………...xiii
ABSTRACT..…………………………………………………………………………………....xvii
CHAPTER 1 INTRODUCTION ...................................................................................................... 1
1.1 Bacteriology and Infectious Disease ......................................................................... 1
1.1.1 Bacteria ............................................................................................................... 1
1.1.2 Morphology and Cellular Structure .................................................................... 2
1.1.3 Growth and Reproduction .................................................................................. 3
1.1.4 Associations with Other Organisms ................................................................... 4
1.1.5 Pathogenic Bacteria and Infectious Disease ....................................................... 5
1.2 Introduction to Antibacterial Drugs .......................................................................... 7
1.2.1 History of Antibiotic Development .................................................................... 7
1.2.2 Types of Antibacterial Drugs by Class ............................................................. 10
1.2.3 Mechanisms of Antibiotic Action and Resistance ............................................ 17
1.3 Introduction to Antiseptics and Disinfectants ......................................................... 18
1.3.1 Quaternary Ammonium Compounds ................................................................ 21
1.3.2 Chlorhexidine and Bisbiguanidines .................................................................. 23
1.3.3 Structural Differences between QACs and Chlorhexidine and Their
Antibacterial Mechanism of Activity. .............................................................. 25
1.4 Combination Antibacterial Drug Therapy (CAT)................................................... 27
1.4.1 Hybrid Antibiotics ............................................................................................ 32
1.4.2 Loewe’s Additivity Model ................................................................................ 35
1.5 Ionic Liquids ........................................................................................................... 38
1.5.1 Groups of Uniform Materials Based on Organic Salts (GUMBOS) ................ 41
1.5.2 Antibacterial Ionic Liquids ............................................................................... 42
1.5.3 Active Pharmaceutical Ingredient Based Ionic Liquids ................................... 44
1.6 References ............................................................................................................... 47

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CHAPTER 2 ANALYTICAL TECHNIQUES, MECHANISM OF ACTION VALIDATION,
AND ANTIMICROBIAL CHARACTERIZATION ..................................................................... 54
2.1 Pharmacological Techniques and Characterization ................................................ 54
2.1.1 Rate of Dissolution ........................................................................................... 54
2.1.2 Predictive Intestinal Permeability ..................................................................... 55
2.2 Antimicrobial Testing and Preparation ................................................................... 57
2.2.1 Turbidity Standards for Inoculum Preparation ................................................. 57
2.2.2 Broth Dilution Tests ......................................................................................... 58
2.2.3 Kirby-Bauer Disk Diffusion Test ..................................................................... 60
2.2.4 Mechanism of Action Studies ........................................................................... 62
2.3 Absorbance-based Techniques................................................................................ 62
2.3.1 Michaelis-Menten Kinetics ............................................................................... 65
2.3.2 Mammalian Cell Cytotoxicity .......................................................................... 67
2.4 Fluorescent-based Techniques ................................................................................ 70
2.4.1 1-N-Phenylnaphthylamine (NPN) Permeability Assay .................................... 74
2.4.2 BacLight Live/Dead Assay ............................................................................... 75
2.4.3 BacLight Membrane Potential Assay ............................................................... 76
2.4.4 Lipopolysaccharide (LPS) Endotoxin Sequestration ........................................ 80
2.5 Scanning Electron Microscopy ............................................................................... 81
2.6 References ............................................................................................................... 83
CHAPTER 3 DESIGN, SYNTHESIS, AND BIOLOGICAL EVALUATION OF BETA (β)-
LACTAM ANTIBIOTIC-BASED IMIDAZOLIUM- AND PYRIDINIUM-TYPE IONIC
LIQUIDS ................................................................................................................................. 85
3.1 Introduction ............................................................................................................. 85
3.2 Experimental ........................................................................................................... 86
3.2.1 Materials and Methods ..................................................................................... 86
3.3 Results and Discussion ........................................................................................... 89
3.3.1 Physical Characterization of Amp-ILs ............................................................. 89
3.3.2 Antibacterial MIC of ILs .................................................................................. 97
3.3.3 Critical Micelle Concentration and Antibacterial Activity Relationship ....... 104
3.3.4 Effect of Ampicillin-content in ILs ................................................................ 107
3.3.5 Antibacterial Activity of Combinations ......................................................... 109
3.3.6 Loewe’s Additivity Model .............................................................................. 110
3.4 Conclusion ............................................................................................................ 112
3.5 References ............................................................................................................. 114

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CHAPTER 4 SYNTHESIS, CHARACTERIZATION, AND BIOLOGICAL EVALUATION OF
BETA (β)-LACTAM BASED CHLORHEXIDINE GUMBOS AGAINST
ENTEROHEMORRHAGIC ESCHERICHIA COLI ................................................................... 117
4.1 Introduction ........................................................................................................... 117
4.2 Materials and Methods .......................................................................................... 119
4.2.1 Synthesis of Chlorhexidine di-ampicillin GUMBOS ..................................... 119
4.2.2 Dissolution Profile Measurement ................................................................... 119
4.2.3 In Vivo Prediction of Intestinal Permeability and Absorption ........................ 120
4.2.4 Antimicrobial Activity .................................................................................... 120
4.2.5 GUMBOS Interaction Indices ........................................................................ 121
4.2.6 Time-Kill Kinetics of Chlorhexidine di-ampicillin ........................................ 122
4.2.7 Mechanism of Action Studies ......................................................................... 122
4.2.8 HeLa, NIH/3T3, and EOMA Cell Viability Tests .......................................... 124
4.3 Results ................................................................................................................... 125
4.3.1 Representative GUMBOS Structural Analysis:
Chlorhexidine di-Ampicillin ........................................................................... 125
4.3.2 Pharmacokinetic Properties and Bioavailability of β-lactam-based
Chlorhexidine GUMBOS ............................................................................... 131
4.3.3 Anti-EHEC Activity ....................................................................................... 139
4.3.4 Interaction Indices .......................................................................................... 146
4.3.5 Time-kill Activity of Chlorhexidine di-ampicillin on
E. coli O157:H7 43895 ................................................................................... 152
4.3.6 Mechanism of Action Studies ......................................................................... 152
4.3.7 Cytotoxicity .................................................................................................... 158
4.4 Discussion ............................................................................................................. 162
4.5 Conclusions ........................................................................................................... 166
4.6 Acknowledgements ............................................................................................... 167
4.7 References ............................................................................................................. 168
CHAPTER 5 ANTIBACTERIAL EFFECTS OF BETA (β)-LACTAM ANTIBIOTIC-
CHLORHEXIDINE HYBRID SALTS ON ANTIBIOTIC RESISTANT
MICROORGANISMS .................................................................................................................. 172
5.1 Introduction ........................................................................................................... 172
5.2 Materials and Methods .......................................................................................... 174
5.2.1 Antimicrobial Activity .................................................................................... 174
5.2.2 Lipopolysaccharide (LPS) Sequestration ....................................................... 176
5.3 Results and Discussion ......................................................................................... 176

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