Models to Elucidate Intraspecies Interactions Between Staphylococcus aureus and Pseudomonas aeruginosa Restricted; Files Only
Luu, Justin (Fall 2025)
Abstract
Pseudomonas aeruginosa and Staphylococcus aureus are pathogenic bacteria of serious public
health concern. These bacteria can cause various infections in immunocompromised people, including
chronic respiratory infections in patients with cystic fibrosis (CF). In CF patients, bacterial respiratory
infections remain a leading cause of morbidity. P. aeruginosa and S. aureus are the two most frequently
isolated bacteria from CF respiratory samples. In addition, these bacteria can often be concurrently isolated
(co-isolated) together. Co-infection with these P. aeruginosa and S. aureus has been associated with worse
clinical outcomes compared to infections with a single species. Therefore, understanding the dynamics
between P. aeruginosa and S. aureus during co-infection is paramount in elucidating its impact on CF
patients. The work presented in this dissertation characterizes the interactions between a P. aeruginosa and
S. aureus co-isolated pair derived from a CF respiratory sample. We found that this P. aeruginosa isolate
was hyper aggressive towards S. aureus compared to our laboratory P. aeruginosa when competed in
environmental conditions designed to mimic CF sputum. This aggressiveness towards S. aureus was also
observed in the secreted products from this P. aeruginosa isolate. In addition, we identified that the S.
aureus co-isolate can increase its tolerance to the co-isolated P. aeruginosa by acquiring mutations in the
genes for the aspartate transporter gltT and the regulator of a fatty efflux pump farR. Our findings identified
potentially new mechanisms of antagonism and tolerance between co-isolated P. aeruginosa and S. aureus
strains. We also began to develop an in vivo model that recapitulates how these bacteria are acquired in CF
patients, where S. aureus is acquired in earlier stages of life and P. aeruginosa is acquired in later stages.
We found that culturing S. aureus in environmental conditions that mimic CF sputum prior to infection can
increase lung colonization in both acute and long-term infections. Finally, we developed and validated an
efficient protocol that gauges antagonistic bacterial interactions between species using fluorescence.
Collectively, these studies have expanded our knowledge on how P. aeruginosa and S. aureus interact and
developed tools to address promising questions in future studies.
Table of Contents
Table of Contents
Chapter 1. Introduction: Models to elucidate intraspecies interactions between
Staphylococcus aureus and Pseudomonas aeruginosa .................................................................1
The importance of Pseudomonas aeruginosa and Staphylococcus aureus ............................................... 2
P. aeruginosa and S. aureus Infections in Cystic Fibrosis Patients .......................................................... 3
Interactions Between P. aeruginosa and S. aureus ................................................................................... 4
Current Approaches to Characterize Interactions between these Species ................................................. 9
Outstanding questions on the interactions between P. aeruginosa and S. aureus .................................. 12
References ................................................................................................................................. 15
Chapter 2. Growing Staphylococcus aureus in Synthetic Cystic Fibrosis Medium Promotes
Colonization in a Murine Pneumonia Model ............................................................................ 27
Abstract ................................................................................................................................................... 28
Importance ............................................................................................................................................... 28
Introduction ............................................................................................................................................. 29
Results ..................................................................................................................................................... 32
Discussion ............................................................................................................................................... 50
Materials and Methods ............................................................................................................................ 54
Acknowledgements ................................................................................................................................. 59
Supplemental Materials ........................................................................................................................... 60
References ............................................................................................................................................... 64
Chapter 3. Mutations in genes encoding the amino acid transporter pump gltT and fatty
efflux pump regulator farR increase Staphylococcus aureus survival from Pseudomonas
aeruginosa antagonism in Synthetic Cystic Fibrosis Medium ................................................. 74
Introduction ............................................................................................................................................. 75
Results ..................................................................................................................................................... 77
Discussion ............................................................................................................................................... 93
Material and Methods .............................................................................................................................. 96
References ............................................................................................................................................. 104
Chapter 4. A Fluorescence-Based Protocol to Monitor Bacterial Antagonism ................... 112
Abstract ................................................................................................................................................. 113
Importance ............................................................................................................................................. 113
Introduction ........................................................................................................................................... 114
Results ................................................................................................................................................... 119
Discussion ............................................................................................................................................. 127
Material and Methods ............................................................................................................................ 129
Acknowledgments ................................................................................................................................. 132
Supplemental Materials ......................................................................................................................... 133
References ............................................................................................................................................. 137
Chapter 5. Discussion and Future Directions.......................................................................... 143
Interactions between Pseudomonas aeruginosa and Staphylococcus aureus during murine pneumonia
infection. ................................................................................................................................................ 144
Interactions between concurrently isolated (co-isolated) Pseudomonas aeruginosa and Staphylococcus
aureus in vitro ....................................................................................................................................... 151
Methods to measure bacterial competition in vitro ............................................................................... 154
Concluding remarks .............................................................................................................................. 156
References ............................................................................................................................................. 158
Appendix 1. Host-Microbe transcriptomic analysis of Staphylococcus aureus in a murine
model of acute pneumonia......................................................................................................... 164
Introduction ........................................................................................................................................... 165
Results and Discussion .......................................................................................................................... 166
Materials and Methods .......................................................................................................................... 171
References ............................................................................................................................................. 174
Appendix 2. Transcriptomics of Staphylococcus aureus and Pseudomonas aeruginosa after
co-culture on agar plates ........................................................................................................... 177
Introduction ........................................................................................................................................... 178
Results and Discussion .......................................................................................................................... 179
Material and Methods ............................................................................................................................ 190
References ............................................................................................................................................. 192
Tables .................................................................................................................................................... 195
List of Figures
Chapter 2.
Figure 1. Strains used in this study exhibit genetic diversity. ..................................................................... 34
Figure 2. Culturing S. aureus in SCFM2 led to similar growth kinetics compared to growth in LB for
most strains. ................................................................................................................................................. 36
Figure 3. Schematic of the S. aureus preparation workflows used in this study. ........................................ 39
Figure 4. Culturing S. aureus in SCFM2 generally increases lung colonization in mice during acute
pneumonia independent of mouse strain. .................................................................................................... 41
Figure 5. Compared to agar plate preparation, culturing WU1 in SCFM2 improves colonization for the
first 2 days post-infection in the throat and for 4 days post-infection in the lungs. .................................... 43
Figure 6. Infections with WU1 cultured in SCFM2 leads to increased inflammation and neutrophil
recruitment in both lung lobes compared to agar plate preparation. ........................................................... 46
Figure 7. Cytokine profile from lungs infected with WU1 cultured on agar plates and SCFM2. ............... 49
Supplemental Figure 1. Impact of different culture conditions on nasal cavity colonization in mice during
acute pneumonia. ......................................................................................................................................... 61
Supplemental Figure 2. Workflow for lung lobe separation for cytokine analysis and histology. ............. 62
Chapter 3.
Figure 1. Pa32 is hyper aggressive towards S. aureus when competed in SCFM2 compared to PAO1. .... 80
Figure 2. Sa46 is more sensitive to secreted products from Pa32 than PAO1 secreted products when
cultured in SCFM2. ..................................................................................................................................... 84
Figure 3. Mutation in both gltT and farR are required in Sa46 to increase tolerance to Pa32. ................... 90
Figure 4. Complementation of either gltT or farR into ECR restores sensitivity towards Pa32 ................. 91
Chapter 4.
Figure 1. Schematic representation of the fluorescence-based protocol. .................................................. 117
Figure 2. Reporter strains show significantly higher fluorescence than competitor bacteria. ................... 120
Figure 3. Reporter strain growth corresponds to red fluorescent protein (RFP) production. .................... 122
Figure 4. Co-culturing with competitor strain results in reduction in both CFUs and RFUs. ................... 125
Figure 5: SA RFP signal is reduced when co-cultured with PA in liquid. ................................................ 126
Supplemental figure 1. A reduction in RFUs in SA corresponds to a reduction in CFUs when co-cultured
the competitor strains under various conditions. ....................................................................................... 133
Supplemental figure 2. A reduction in RFUs in EC corresponds to a reduction in CFUs when co-cultured
with competitor strains under various conditions. ..................................................................................... 135
Appendix 1.
Figure 1. JE2 recovered after a 24-hour acute pneumonia murine infection. ............................................ 169
Appendix 2.
Figure 1. Bacteria recovery after co-culture. ............................................................................................. 183
Figure 2. RNA-seq visualizations of the co-culture between Pa24 and JE2. ............................................ 186
Figure 3. RNA-seq visualizations of the co-culture between PAO1 and JE2. .......................................... 189
List of Tables
Chapter 2.
Table S1. Average nucleotide identity values..................................................................................... 60
Chapter 3.
Table 1. Protein hits identified through mass spectrometry on the ~40 kDA band of Pa32..................... 86
Table 2. Mutations Identified in Evolved Colony Sensitive (ECS) and Evolved Colony Resistant (ECR)
relative to Sa46. ........................................................................................................................................... 92
Table 3. Mutations Identified in Evolved Population 2 at Passage 14 relative to Sa46. ............................. 92
Table 4. Bacterial strains utilized in this study. ........................................................................................... 97
Table 5. Plasmids utilized in this study. ...................................................................................................... 97
Table 6. Primers utilized in this study. ........................................................................................................ 97
Chapter 4.
Table 1. Strains used in this study. ............................................................................................................ 118
Appendix 1.
Table 1. RNA-sequencing Reads and Mapping Results to JE2 ................................................................. 170
Appendix 2.
Table 1. Differentially expressed genes in Pa24 when co-cultured with JE2............................................ 195
Table 2. Differentially expressed genes in JE2 when co-cultured with Pa24. .......................................... 202
Table 3. Differentially expressed genes in PAO1 when co-cultured with JE2.......................................... 210
Table 4. Differentially expressed genes in JE2 when co-cultured with PAO1. ........................................ 211
About this Dissertation
| School | |
|---|---|
| Department | |
| Subfield / Discipline | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Keyword | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
File download under embargo until 12 January 2032 | 2025-12-10 20:03:21 -0500 | File download under embargo until 12 January 2032 |
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|