COMPLEMENTARY AND CONVENTIONAL ANTIMICROBIALS FOR INTEGRATIVE TARGETED ...

181 Pages·2016·1.92 MB·English

COMPLEMENTARY AND CONVENTIONAL ANTIMICROBIALS FOR
INTEGRATIVE TARGETED CONTROL OF BACTERIAL VAGINOSIS
ASSOCIATED PATHOGENS
By
AMMAR R. Al-GBURI
A dissertation submitted to the
Graduate School-New Brunswick
Rutgers, The State University of New Jersey
In partial fulfillment of the requirements
For the degree of
Doctor of Philosophy
Graduate Program in Microbial Biology
Written under the direction of
Dr. Michael Chikindas
And approved by
___________________________________
___________________________________
___________________________________
___________________________________
New Brunswick, New Jersey
OCTOBER 2016
ABSTRACT OF THE DISSERTATION

Complementary and Conventional Antimicrobials For Intergrative Targeted Control of
Bacterial Vaginosis Associated Pthogens
By AMMAR R. Al-GBURI
Dissertation Director: Dr. Michael Chikindas
Bacterial vaginosis, the polymicrobial vaginal infection, occurs in women of an
adolescent and childbearing age and associated with numerous gynecological and
obstetric complications.This infection is characterized by the presence of thick-adherent
vaginal biofilms, composed mainly of Gardnerella vaginalis, Atopobium vaginae,
Mobiluncus curtisii, Prevotella bivia and Peptostreptococcus anaerobius. Gardnerella
vaginalis is thought to be one of the primary etiological agent of the infection paving the
way for various opportunists to colonize the ecological niche. The failure of conventional
treatment with synthetic antibiotics is largely due to antibiotic tolerance of biofilm-
associated cells and infection recurrence with antibiotic-resistant mutants. The
dissertation-related articles include four major components of the study. First one is a
review on importance of natural derived antimicrobials (alone and particularly in
combination with antibiotic) as an effective strategies for combating the tolerance of
biofilm-associated pathogens to antibiotic treatment. Second component is our study
aimed at evaluation of antimicrobial activity of natural derived substances, subtilosin, ε-
poly-L-lysine and lauramide arginine ethyl ester against established biofilms of G.
vaginalis, using three commonly utilized methods (plate counts, ATP viability and
resazurin assays) to assess cell viability in the antimicrobial-treated G. vaginalis biofilms.
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Subtilosin and lauramide arginine ethyl ester showed the strongest biofilm bactericidal

effect in comparison to the other tested antimicrobials. The plate count was reported as
the best method for estimating the bactericidal effect of the studied antimicrobials. The
study’s third component is a research article reporting on elucidation of antimicrobial
activity of subtilosin and lauramide arginine ethyl ester in combination with commonly
prescribed antibiotic clindamycin and metronidazole against bacterial vaginosis-
associated pathogens. All tested antimicrobial combinations were inhibitory for BV-
associated Mobiluncus curtisii and Peptostreptococcus anaerobius. LAE and subtilosin
synergized ith clindamycin and metronidazole against biofilms of G. vaginalis but not
biofilm-associated vaginal lactobacilli. Last but not least, the fourth component of our
study is a report on safety and putative probiotic properties of Bacillus amyloliquefaciens
B-1895 and subtilosin-producing Bacillus subtilis KATMIRA1933. The cell-free
supernatants of both Bacillus strains were non-mutagenic and having antimicrobial
properties against human pathogens Listeria monocytogenes, Streptococcus intermedius
and Porphyromonas gingivalis. The two strains were strongly co-aggregating with
pathogenic Escherichia coli and Pseudomonas aeruginosa. In addition, the endospores of
B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 were tolerant to 0.3% (w/v)
bile salts and survived when incubated for 4 h in MRS broth at pH 2.0 to 3.0. The
chapters are linked through the idea of using natural derived antimicrobial (particularly,
subtliosin and LAE) alone (II) and in combination with the commonly prescribed
antibiotics metronidazole and clindamycin (III) to counteract biofilm formation by BV-
associated pathogens. Since LAE is already certified as GRAS for certain applications,
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we evaluated safety of a putative probiotic Bacillus subtilis KATMIRA1933 (IV), the

producer of subtilosin which was used as anti-biofilm agent.

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Dedications

Endless appreciations to the Iraqi Army and Popular Mobilization
Forces, who are fighting bravely and shedding their blood to protect my
precious country so I was able to conduct my PhD research and reassuring
the safety of my family.
For Mom and Dad, I would not be able to survive and to be who I am today
without your love and supports.
For uncle Ali, and aunt Kawther, the biggest thanks for your fatherhood
love and emotional support.
For my wife, Halah, and my little princesses Ruqayah and Batool, You are
my heroines when I am a scared, my strength when I am a weak, my
sunshine when the life turns dark, and my unending lovers and supporters.

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Acknowledgements

First of all, I would like to thank Dr. Chikindas who was, and still, a father more than
being an advisor and mentor. I would like to thank him for his support, encouragement
and taking care of me at the personal and professional levels. I will always appreciate the
knowledge, experience, confidence, and the progress of writing I gained during the
period I spent in his lab. I am so grateful and feeling blessed to have myself closely
worked with him and got the benefit of his scientific and insightful advices. I am very
thankful and will never forget his thoughtful conversations and the kind manner of
dealing with all his laboratory members as one family. I am so glad and grateful to have
an opportunity to continue working with him in future.
Many thanks to Dr. Leon Dicks for sharing his ideas and for his willingness to read and
edit my scientific writing.
I would like to thank my committee members Dr. Zylstra, Dr. Schaffner, and Dr. Riazi
for their collaboration and agreement to serve in my Ph.D defense committee.
Big thanks to all instructors of Microbial Biology Department: for the challenges and
their support.
To all my friends and colleagues in the Food Science building, especially Jason, "my
brother,” as I call him, thanks for your constant support.
Finally, I would like to thank my lovely family, especially my wife, who I owe my whole
life, with all the great sacrifices she made to get my Ph.D degree done.

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Table of Contents

Pages
Abstract of The Dissertation ............................................................................................... ii
Dedications ......................................................................................................................... v
Acknowledgments.............................................................................................................. vi
Table of Contents .............................................................................................................. vii
Preface..................................................................................................................................x
Chapter I – Control of biofilm formation: antibiotics and beyond ......................................1
I.1. Abstract ..........................................................................................................................3
I.2. Introduction ...................................................................................................................4
I.3. Antibiotics combined with antimicrobial peptides ........................................................6
I.4. Antibiotics combined with biofilm-degrading enzymes .............................................10
I.5. Antibiotics and quorum sensing inhibitors ..................................................................12
I.6. Antibiotics and essential oils .......................................................................................16
I.7. Antibiotics and nanoparticles ......................................................................................18
I.8. Conclusions .................................................................................................................20
I.8. References ...................................................................................................................22
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I.9. Tables and Figures .......................................................................................................37

Chapter II – Susceptibility of Gardnerella vaginalis biofilms to natural antimicrobials
subtilosin, ε-poly-L-lysine and lauramide arginine ethyl ester ..........................................40
II.1. Abstract ......................................................................................................................42
II.2. Introduction ................................................................................................................43
II.3. Materials and Methods ...............................................................................................46
II.4. Results and Discussion ...............................................................................................51
II.5. Conclusion ..................................................................................................................56
II.6. Acknowledgments ......................................................................................................56
II.7. References ..................................................................................................................57
II.8. Figures Legends .........................................................................................................63
II.9. Tables and Figures .....................................................................................................65
Chapter III – Natural antimicrobials subtilosin and lauramide arginine ethyl ester (LAE)
synergize with conventional antibiotics clindamycin and metronidazole against biofilms
of Gardnerella vaginalis but not against biofilms of healthy vaginal lactobacilli ............72
III.1. Abstract .....................................................................................................................74
III.2. Introduction ...............................................................................................................75
III.3. Materials and Methods ..............................................................................................77
III.4. Results .......................................................................................................................84
III.5. Discussion .................................................................................................................87
III.6. Acknowledgments.....................................................................................................97
III.7. References .................................................................................................................98
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III.8. Tables and Figures ..................................................................................................107

Chapter IV – Safety properties and probiotic potential of Bacillus subtilis
KATMIRA1933 and Bacillus amyloliquefaciens B-189 .................................................115
IV.1. Abstract ...................................................................................................................117
IV.2. Introduction.............................................................................................................118
IV.3. Materials and Methods ...........................................................................................122
IV.4. Results.....................................................................................................................131
IV.5. Discussion ...............................................................................................................134
IV.6. Acknowledgments ..................................................................................................146
IV.7. References...............................................................................................................147
IV.8. Tables and Figures ..................................................................................................159
Chapter V: Suggestions for Future Studies ......................................................................167
Curriculum Vitae .............................................................................................................170

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PREFACE

Chapter 1 has been submitted for publication as “Control of biofilm formation:
antibiotics and beyond.”, Algburi, A., Comito, N., Kashtanov, D., Dicks, L.M.T.,
Chikindas, M.L. Applied and Environmental Microbiology, 2016. AEM01664-16. I was
the first author who wrote the first draft and took care of reviewers’ comments.
Chapter 2 has been published as “Susceptibility of Gardnerella vaginalis biofilms to
natural antimicrobials subtilosin, ε-poly-L-lysine, and lauramide arginine ethyl
ester.”, Turovskiy, Y., Cheryian, T., Algburi, A., Wirawan, R.E., Takhistov, P.,
Sinko, P.J. and Chikindas, M.L. 2012. Infectious diseases in obstetrics and gynecology,
2012, 284762. I was the second author who performed 25% of this manuscript.
Chapter 3 has been published as “Natural antimicrobials subtilosin and lauramide
arginine ethyl ester synergize with conventional antibiotics clindamycin and
metronidazole against biofilms of Gardnerella vaginalis but not against biofilms of
healthy vaginal lactobacilli.”, Algburi, A., Volski, A. and Chikindas, M.L. 2015.
FEMS Pathogens and disease, 73 (5), p.ftv018. I was the first author who did 100% of
bench work and wrote the first draft with taking care of the reviewers’ comments.
Chapter 4 has been published as “Safety properties and probiotic potential of Bacillus
subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895.”, Algburi, A.,
Volski, A., Cugini, C., Walsh, E.M., Chistyakov, V.A., Mazanko, M.S., Bren,
A.B., Dicks, L.M.T. and Chikindas, M.L. 2016. Advances in Microbiology, 6, 432-452.
I was the first author who did 100% of bench work and wrote the first draft with taking
care of the reviewers’ comments.
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