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Polyamines Mitigate Antibiotic Inhibition of A.Actinomycetemcomitans Growth

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Polyamines Mitigate Antibiotic Inhibition of A.Actinomycetemcomitans Growth Polyamines Mitigate Antibiotic Inhibition of A.actinomycetemcomitans Growth THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Allan Wattimena Graduate Program in Dentistry The Ohio State University 2017 Master's Examination Committee: Dr John Walters, Advisor Dr Purnima Kumar Dr Sara Palmer Dr Shareef Dabdoub Copyright by Allan Wattimena 2017 Abstract Polyamines are ubiquitous polycationic molecules that are present in all prokaryotic and eukaryotic cells. They are the breakdown products of amino acids and are important modulators of cell growth, stress and cell proliferation. Polyamines are present in higher concentrations in the periodontal pocket and may affect antibiotic resistance of bacterial biofilms. The effect of polyamines was investigated with amoxicillin (AMX), azithromycin (AZM) and doxycycline (DOX) on the growth of Aggregatibacter actinomycetemcomitans (A.a.) Y4 strain. Bacteria were grown in brain heart infusion broth under the following conditions: 1) A.a. only, 2) A.a. + antibiotic, 3) A.a. + antibiotic + polyamine mix (1.4mM putrescine, 0.4mM spermidine, 0.4mM spermine). Growth curve analysis, MIC determination and metatranscriptomic analysis were carried out. The presence of exogenous polyamines produced a small, but significant increase in growth of A.a. Polyamines mitigated the inhibitory effect of AMX, AZM and DOX on A.a. growth. Metatranscriptomic analysis revealed differing transcriptomic profiles when comparing AMX and AZM in the presence of polyamines. Polyamines produced a transient mitigation of AMX inhibition, but did not have a significant effect on gene transcription. Many gene transcription changes were seen when polyamines were in the presence of AZM. Polyamines in a periodontal pocket may help protect A.a. and contribute to antimicrobial resistance. ii Dedication This document is dedicated to my family. iii Acknowledgments Thanks to Dr. Walters, Dr. Kumar, Dr. Dabdoub, Dr. Palmer and Dr. Sukirth Ganesan for guidance throughout the project. iv Vita 2004................................................................St Joan of Arc CHS 2008................................................................B.S. Human Biology, University of Toronto 2014................................................................D.D.S., University of Minnesota 2017................................................................M.S., Dentistry, The Ohio State University Fields of Study Major Field: Dentistry v Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Introduction ......................................................................................................................... 1 Aim of study .................................................................................................................... 8 Hypotheses ...................................................................................................................... 8 Materials and Methods ........................................................................................................ 8 Materials .......................................................................................................................... 8 Bacterial culture .............................................................................................................. 9 Growth Curves ................................................................................................................ 9 Minimal Inhibitory Concentration (MIC) determinations ............................................ 10 Bacterial viability determinations ................................................................................. 11 vi Metatranscriptomic analysis .......................................................................................... 11 Results ............................................................................................................................... 13 Effect of polyamines on inhibition of A.a. growth by AMX, AZM and DOX ............. 13 Effect of polyamines on MIC for A. actinomycetemcomitans ..................................... 14 Bacterial viability determinations ................................................................................. 15 Transcriptome analysis .................................................................................................. 15 Effect of polyamines on gene expression in control cultures........................................ 15 Effect of polyamines on cultures inhibited by AMX and AZM ................................... 16 Discussion ......................................................................................................................... 17 Conclusion ........................................................................................................................ 21 References ......................................................................................................................... 22 Appendix A: Tables and Figures ...................................................................................... 27 Appendix B: Relative abundances of SEED transcripts in A.a. ....................................... 45 vii List of Tables Table 1: Description of antibiotics used ........................................................................... 27 Table 2: The effect of polyamines on the MIC of AMX, AZM and DOX. ...................... 28 Table 3: Summary of A.a. cDNA samples ........................................................................ 29 Table 4: Number of genes upregulated/downregulated >2 fold ....................................... 30 Table 5: Effect of polyamine mixture on gene transcription in A.a. ................................. 31 Table 6: Effect of polyamines and AMX on gene transcription in A.a. ........................... 32 Table 7: Effect of polyamines and AZM on gene transcription in A.a. ............................ 33 viii List of Figures Figure 1: Polyamine synthesis pathway ............................................................................ 35 Figure 2: Growth curve: AMX ......................................................................................... 36 Figure 3: Growth curve: AZM .......................................................................................... 37 Figure 4: Growth curve: DOX .......................................................................................... 38 Figure 5: Circle Plot: Polyamines vs Ctrl ......................................................................... 39 Figure 6: Circle Plot: AMX + Polyamines vs AMX......................................................... 40 Figure 7: Circle Plot: AZM + Polyamines vs AZM ......................................................... 41 Figure 8: KEGG Pathway Map: Polyamines vs Ctrl. ....................................................... 42 Figure 9: KEGG Pathway Map: AMX + Polyamines vs AMX ....................................... 43 Figure 10: KEGG Pathway Map: AZM + Polyamines vs AZM ...................................... 44 ix Introduction Periodontitis is characterized by the inflammatory destruction of tissues surrounding the tooth. Its cause is complex and thought to be due to host interactions with bacteria present in the oral cavity. The host response plays a significant role in determining the rate of destruction and attachment loss that occurs1. About 0.5% of individuals are susceptible to a form of the disease called aggressive periodontitis2. Aggressive periodontitis has a rapid rate of disease progression and occurs in individuals who are otherwise healthy. The disease has a tendency for familial aggregation and typically affects those under 35 years of age3. The amount of periodontal tissue destruction is not consistent with the amount of local factors, as patients usually present with low plaque and calculus levels. There are two forms of the disease: a generalized and a localized form. The localized form affects at least two permanent teeth that are first molars and/or incisors and no more than two teeth other than first molars and incisors. The generalized form is defined as attachment loss affecting at least three teeth other than first molars and incisors. Approximately 35% of individuals with localized aggressive periodontitis progress into generalized aggressive periodontitis. These individuals may exhibit a poor serum antibody response to infecting agents4. The localized form appears to be self-limiting, whereas the generalized form can recur various times in a patient’s life. The bacterial
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