Exploring the Mechanistic Implications of Pseudomonas aeruginosa Antimicrobial Resistance Open Access

Abstract

The antibiotic resistance crisis causes nearly 5 million deaths globally every year. Pseudomonas aeruginosa is a highly virulent Gram-negative bacteria that is resistant to commonly used antibiotics. Novel quaternary phosphonium compounds were highly effective against clinical isolates of P. aeruginosa compared to the widely-used disinfectant, benzalkonium chloride (BAC), marking the extent of the defensive strategy implemented by P. aeruginosa clinical isolates. The offensive strategy of  P. aeruginosa of pyocyanin production showed that BAC-resistant strains of  P. aeruginosa increased in pyocyanin production compared to their parent strains. Previous studies have demonstrated P. aeruginosa sensitization to Gram-positive antibiotics, which has piqued interest in the mechanism behind this phenomenon. Using a dilute growth media, P. aeruginosa was tested against a panel of Gram-positive antibiotics, with vancomycin yielding the most promising results. A significantly lower minimal inhibitory concentration of vancomycin was needed to prevent sensitized bacterial growth (16 μg/mL) than unmodified bacterial growth (>256 μg/mL). The structure of the outer membrane was investigated with N-Phenylnaphthalen-1-amine and nitrocefin. Both outer-membrane assays suggest that sensitization is not the result of a permeabilized outer membrane. In addition, divalent cation supplementation in the minimal media was done to further affirm that sensitization is not due to a permeabilized outer membrane. Additional assays such as growth inhibition over time, swimming motility, and carbon source supplementation were monitored to further explore the vancomycin mechanism of action on sensitized P. aeruginosa. Therefore, P. aeruginosa is sensitized to vancomycin through an alternate mechanism. Further experimentation with multidrug-resistant clinical isolates was conducted to explore the implications of P. aeruginosa sensitization. Future directions include seeking transient changes through RNA sequencing of the sensitized P. aeruginosa.

Table of Contents

1. Introduction

a. Antibiotic Resistance Crisis                                          1

b. Pseudomonas aeruginosa Infections and Resistance Mechanisms           1

c. Sensitization of Pseudomonas aeruginosa to Vancomycin                  3

d. Cationic Biocides                                                  5

2. Studying Pseudomonas aeruginosa Antimicrobial Resistance Behaviors     6

a. Defensive Strategy                                                 6

b. Offensive Strategy                                                 8

c. Conclusions                                                       9

3. Sensitization of Pseudomonas aeruginosa to Gram-positive antibiotics           10

a. Analysis of Gram-positive Antibiotic Sensitization and Efflux Pump System   10

b. Vancomycin Sensitization is not due to Outer Membrane Permeability        13

c. Exploring the Mechanism of Action of Vancomycin in Sensitized PA14       16

d. Clinical Isolates Respond to Sensitization                               21

e. Conclusions                                                      24

4. References                                                            25

5. Supplemental Information                                                35

a. Figures and Tables                                                 35

b. Methods                                                         37

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Biology, Microbiology
Keyword	Pseudomonas aeruginosa Antimicrobial Resistance
Committee Chair / Thesis Advisor	Dr. William Wuest, Emory University
Committee Members	Dr. David Lynn, Emory University Dr. Sheila Cavanagh, Emory University

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