title: Towards the Study of Bacterial Communities at Single-Cell Level Using Vibrational Spectroscopy
Creators: name: Richardson, Paul
Publisher: name: University of Liverpool Repository
Year: 2024

description: Bacteria can be found in virtually all environments on earth, from soil and the ocean to toxic sludge, and are find on and inside all animal and plant bodies. Within these environments, they form complex ecosystems and interact with each other to competitive and cooperative ends through biochemical means. These interactions are vital towards our understanding of the role that bacteria play within these different niches; however, their small size places stringent limitations on our ability to study them in isolation or their interactions with other bacteria or their eukaryotic hosts. Most common methods involve biological testing, either through the probing of specific metabolisms or growth rates or genomic research. These methods, while powerful, are also often laborious and only provide a part of the overall picture.
Vibrational spectroscopy is a family of physicochemical methods probing the presence of various functional groups through bond excitation. These provide a holistic image of the bacterial phenotype without necessitating external labelling, making them a relatively quick and simple option. Furthermore, recent developments in optical photothermal infrared (O-PTIR) spectroscopy and the first commercial instrument utilising this method, presents the opportunity to bypass the spatial resolution limitations of infrared spectroscopy engendered by the diffraction limit to study single bacterial cells.
In this work, the information obtainable from individual bacteria via O-PTIR was studied using the Staphylococcus genus as a case study. First, intergenetic, interspecific and intraspecific discrimination were tested using two strains of S. capitis, two strains of S. epidermidis and one strain of Micrococcus luteus and benchmarked against bulk measurements of millions of cells from the already established Fourier transform infrared (FTIR) spectroscopy. Alongside successful discrimination, small spectral differences putatively assigned to protein secondary structure were described. Then, 22 strains of S. capitis comprising both subspecies capitis and urealyticus were studied, aiming not only to find the limit of bacterial discrimination of O-PTIR, but also to compare phenotypic classification obtained from O-PTIR and FTIR with genotypic classification obtained from whole-genome sequencing. FTIR and O-PTIR classifications did not match genotypic classification or each other demonstrating that, given the experimental conditions, the portion of the phenotype probed via infrared spectroscopy is unable to fully differentiate between these two subspecies. Third, a method was developed to probe differences in the phenotypes of S. capitis ssp. capitis and ssp. urealyticus when cultured in consortia and axenic conditions using FTIR spectroscopy and the Transwell® co-culture insert system. While these cultures could be discriminated, significant differences were also noticed based on growth conditions, notably between cells cultured in the insert and the well.
Additionally, this thesis contains an aside involving a study started during the COVID pandemic that successfully quantified the propylene glycol: glycerol ratio and nicotine content of e-cigarette liquids using portable Raman spectroscopy.