The Biology of Colistin Resistance: A Heteroresistance Mechanism and Inhibition of a Resistance Pathway Open Access

Abstract

Antibiotic resistant infections are a significant and increasing cause of morbidity and mortality worldwide. In particular, infections caused by multi-drug resistant Gram negative bacteria recalcitrant to commonly used classes of antibiotics are of particular concern, as the antibiotic colistin is often the only remaining treatment option. Given the importance of this last-resort antibiotic, it is imperative that we fully understand the scope of colistin resistance mechanisms so that novel treatment strategies can be identified. In this work, we utilized an Enterobacter cloacae model of colistin heteroresistance to gain insight into this elusive resistance mechanism. The heteroresistant strain harbored both colistin resistant and susceptible subpopulations that were genetically indistinguishable yet transcriptionally distinct. The resistant subpopulation increased during antibiotic treatment, receded to baseline after subculture without drug, and was distinct from persisters. Presence of the resistant subpopulation and modification of colistin drug target lipid A was dependent on the histidine kinase gene phoQ. Colistin therapy failed to rescue mice infected with the heteroresistant strain, however treatment of mice infected with the phoQ mutant was successful, indicating that resistant bacterial subpopulations can cause antibiotic treatment failures. As a further approach to reduce the impacts of colistin resistance, we utilized a model of colistin resistant Acinetobacter baumannii to develop a resistance inhibition strategy. We identified a small molecule inhibitor of the naxD-controlled colistin resistance lipid A modification pathway via tandem in vitro and in silico screens. The inhibitor restored colistin susceptibility in a naxD-dependent manner and blocked the addition of galactosamine onto lipid A. Finally, the inhibitor reduced colistin resistance in a panel of clinical A. baumannii isolates, highlighting the potential for modulation of resistance expression to restore colistin efficacy for diverse strains. Altogether, these results provide fundamental insights into the biology of colistin resistance, and set the stage for continued development of novel therapeutics to combat antibiotic resistance in the clinic. 

Table of Contents

CHAPTER 1: Introduction.......................................................................................................... 1

Overview of the Antibiotic Resistance Problem......................................................................... 2

Evolution of Antibiotic Resistance ............................................................................................. 3

Colistin as a last-line therapy ...................................................................................................... 6

Colistin Resistance in the Clinic ................................................................................................. 8 References................................................................................................................................. 11

CHAPTER 2: Antibiotic Failure Mediated by a Resistant Subpopulation in Enterobacter cloacae .... 28

Abstract ..................................................................................................................................... 31 Introduction............................................................................................................................... 32 Results....................................................................................................................................... 33 Figures....................................................................................................................................... 44 Methods..................................................................................................................................... 54 References................................................................................................................................. 61

Supplementary Figures ............................................................................................................. 67

Supplementary Tables............................................................................................................... 97

CHAPTER 3: Resistance Inhibitor Suppresses Lipid A Modifications and Reverses Colistin Resistance in Acinetobacter baumannii ...122

Abstract ................................................................................................................................... 124 Introduction............................................................................................................................. 125 Results..................................................................................................................................... 127

Discussion ............................................................................................................................... 133 Figures..................................................................................................................................... 135 Methods................................................................................................................................... 141 References:.............................................................................................................................. 143

CHAPTER 4: Discussion and Conclusion .............................................................................. 147

References:.............................................................................................................................. 153

APPENDIX I: Antimicrobial Peptide Resistance Mechanisms of Gram-Positive Bacteria ... 156

Abstract ................................................................................................................................... 158

1. Introduction......................................................................................................................... 159

2. Extracellular Mechanisms of Resistance: Enzymatic Degradation and AMP Blocking .... 160

3. Membrane and Cell Wall Modifications............................................................................. 165

4. AMP Efflux Mechanisms ................................................................................................... 171

5. Conclusions......................................................................................................................... 176 References............................................................................................................................... 183 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Microbiology
Keyword	heteroresistance, colistin, naxD, phoQ
Committee Chair / Thesis Advisor	Weiss, David, Emory University
Committee Members	Moran, Charles , Emory University Shafer, William, Emory University Rather, Philip, Emory University Kim, Minsu, Emory University

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