Analysis of microbial properties using flow cytometry

Abstract

The widespread and non-regulated use of antibiotics has resulted in pathogens developing resistance against them. Mis-prescribing of antibiotics is partly responsible for the current crisis of antimicrobial resistance, and it would be ideal for a patient to know the correct antibiotic against the causative organism before leaving a GP’s surgery. To enable this strategy, a rapid antimicrobial susceptibility test needs to be performed with clinical samples. Flow cytometry was used to detect changes in bacterial cell size, viability, membrane energisation and DNA distribution. 3,3'-dipropylthiadicarbocyanine iodide (di-S-C3(5)), a red fluorescent membrane energisation dye, enabled assessment of bacterial cell numbers, and could be used to assess those capable of proliferating. E. coli were inoculated in ‘terrific broth (TB)’ at a concentration of 105 cells.mL-1 (representing bacteriuria), and bacterial proliferation was observed within 20 minutes by counting the live stained E. coli cells using a flow cytometer. Initially, this assay was used to see the effects of antibiotics (commonly used in UTIs), on lab-grown sensitive strains. The time point for the earliest detection of antimicrobial susceptibility ranged between 20-40 minutes, depending on the antibiotic used. A new growth medium was developed, from TB, to improve the period of the assay to a maximum of 30 minutes. The newly developed assay was cross-validated with real, UTIsuspected clinical samples. The results from the above assay could help in the development of a single-laser-based benchtop flow cytometer to be placed in a GP’s surgery. While developing the rapid antimicrobial susceptibility assay, heterogeneous uptake of the cationic dye (di-S-C3(5)) was observed by the individual cells in a bacterial population. It was intriguing to understand how the dyes entered into the cells. The assumption still persists that most xenobiotics cross phospholipid bilayer by diffusion. Alternatively, different transporters are involved in the uptake of xenobiotics and the directed unfacilitated transport through phospholipid bilayers is negligible. High-throughput flow cytometry was used in this study to measure the uptake of xenobiotics (the fluorescent dyes di-S-C3(5) and SYBR Green I were used here) in individual cells. A subset of the single-gene knockout E. coli Keio collection was used to analyse the contribution of each transporter to the dye uptake. With di-S-C3(5), the range of steady-state uptake was observed to be 36-fold. Many knockouts with lower uptake than the wild-type consisted of y-genes (unknown function). Four of the five most fluorescent strains were knockouts of known major efflux transporters. Knockout of ATP synthase subunits a- and b- decreased the ATP production and also the dye uptake signifying that the uptake was ATP-driven. About half of the knockout strains analysed showed more than a 50% change (up and down) in the uptake of the dye as compared to the wild-type. Overexpression of some of these genes (ASKA collection) showed opposite effects as compared to their knockout counterparts. The uptake range with SYBR Green I was even higher than di-S-C3(5) at 69-fold. There was no correlation found between the uptake distributions of SYBR Green I and the DNA content of the cells in different strains. The results strengthen the belief that multiple transporters could be responsible for the uptake of these dyes. The broad range of uptake also signifies that diffusion through phospholipid bilayer is negligible, and emphasises the role of transporters in the dye-based staining of the intact bacteria.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Philip Day (Supervisor)