Document Type

Dissertation - Open Access

Award Date

2024

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Nicholas Butzin

Abstract

This research undertook an interdisciplinary approach, integrating bioengineering, microbiology, molecular biology, and systems biology to investigate bacterial dynamics behavior. Specifically, it delved into the development of microfluidic devices for biological applications such as bacterial cell counts, real-time observation of plant roots (here, specialized lectin-coated microbeads are used that mimic root characteristics), and soil microbe interactions. Furthermore, Next-Generation Sequencing and systems biology methodologies were employed to explore the intricate, multifaceted survival mechanisms of Escherichia coli persister population. Studying and quantifying persisters or testing for the existence of VBNC (viable but nonculturable) is challenging. These experiments require precise counts. It has been predicted that VBNCs cannot grow on Petri plates but can only grow in liquid, and that VBNCs may be related to antibiotic-resistant development. However, the VBNCs count showed a high variation (>2 fold) between CFU/ml and hemocytometer (20-30% count discrepancy is expected). It was hypothesized that VBNC cells might account for the higher counts observed under the microscope. However, attempts to revive VBNCs were unsuccessful, indicating no revival of VBNCs in our laboratory strains. This discrepancy raised a pertinent question: how can we reconcile cell numbers for comparison when potential counting errors exist? As VBNC is a small fraction of cells, variations in counts could obscure the detection of these cells, resulting in either overestimation or underestimation of total cell numbers. The difficulties of Z-stacks imaging (we often wrongly estimate the highest and lowest point and cannot identify cells, as this range varies from chamber-to-chamber); merging and counting them may also cause counting discrepancy; thus, VBNC remains undetected. Also, the available live/dead staining kits sometimes give us false-positive results and cannot detect VBNC. Even after killing the cells, some cells do not show any fluorescence using propidium iodide (PI) dyes, which bind to dead cell DNA. Similar results were observed for viable cells using CFDA dye (stain only live cells). Thus, a convenient and timely counting approach may be a helpful tool to investigate the intricate bacterial intelligence. To address this, I constructed microfabricated cell counters called “counter-on-chip”, coupled with high-precision 3D printing and an advanced inverted microscope system to enable quick, precise quantification of small bacteria (Chapter 2:2.1.). This study also explored providing a quantitative and qualitative analysis of the competitive binding ability of Bradyrhizobium strains to selective lectin-coated microbeads using a microfluidic chip (Chapter 2:2.2). However, currently, there is no established method for real-time monitoring of plant root-microbes interaction. The major limitation is studying real plants is challenging, especially in the case of soybeans, which have larger roots. So, I choose to use lectin-coated microbeads instead of real plants. Moreover, different Bradyrhizobium strains' binding efficiency varies, so it is challenging to analyze which strains are ahead in the competition qualitatively. To keep that in mind, I developed an innovative microfluidic device to observe the Bradyrhizobium’s binding to lectin-coated microbeads in real-time. This device will allow us to evaluate the binding efficiency of Bradyrhizobium strains within a shorter period of time. Employing this system, we can examine the binding process through an inverted microscope without growing real plants. I implemented my knowledge of microfluidic lab-on-a-chip technology and conducted a review work related to human pulmonary arterial hypertension (PAH). PAH is a fatal and enigmatic disease of the pulmonary circulatory system for which there is currently no cure. As no animal models can fully recapitulate human PAH features, the review highlights the emerging lab-on-a-chip technology as an excellent model for the disease and testing therapeutics (Chapter 2:2.3). Another exciting study I have done to challenge the conventional wisdom of one persister population and show strong evidence that multiple persister subpopulations are within an isogenic Escherichia coli population, allowing them to survive lethal antibiotic stress (Chapter 3). Bacterial persisters, a subpopulation known for their multidrug tolerance and ability to survive lethal antibiotic treatments, have long posed challenges in understanding their formation and long-term survival. They are a driving force of antibiotic resistance, so it is paramount that we learn more about them as the antibiotic resistance problem continues to grow. This study challenges the long-held consensus that persisters are completely dormant and are of one single population. Through wet lab (gene knockout, persister assay, RNA isolation) and dry lab (network biology, bioinformatics, mathematical modeling) approaches it has clearly shown that persisters are not as dormant as once thought, and multiple populations of persisters form during lethal antibiotic treatment despite the cells being genetically identical. These results challenge the idea of complete dormancy, suggest the presence of intricate, multifaceted survival mechanisms, and indicate that the persister population itself is heterogeneous.

Publisher

South Dakota State University

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Rights Statement

In Copyright