Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2022

Turning the spotlight on persisters: investigation of intracellular pH and redox activity of Escherichia coli antibiotic persisters with fluorescence-activated cell sorting (#164)

Joanna Urbaniec 1 2 , Simone Krings 1 , Alejandro Sanchez-Pedreno Jimenez 1 , Faisal Hai 2 , Johnjoe McFadden 1 , Martina Sanderson-Smith 3 , Suzanne Hingley-Wilson 1
  1. Microbial Sciences , University of Surrey, Guildford, United Kingdom
  2. Civil, Mining and Environmental Engineering, University of Wollongong , Wollongong, NSW, Australia
  3. Illawarra Health and Medical Research Institute, University of Wollongong , Wollongong, NSW, Australia

Antibiotic persistence is a phenomenon observed when a subpopulation of genetically susceptible cells survives a prolonged exposure to an otherwise bactericidal concentration of an antibiotic. These ‘persister cells’ or ‘persisters’ survive because their metabolic state renders the antibiotic temporarily ineffective, and later regrow into a susceptible population. Frequency of persisters varies depending on environmental factors and the antibiotic’s mechanism of action, however it is increased in ‘highly-persistent’ genetic mutants.

Antibiotic persistence is widely recognized as a public health concern as it prolongs treatment time and contributes to recurring infections. Recently, it has also been demonstrated that persistence can be a ‘stepping-stone’ to genetic resistance, further highlighting a need for the development of anti-persister agents (1).

Persister cells are notoriously difficult to investigate as they are a small subpopulation with unique physiology. In this study we employed flow cytometry and fluorescence-activated cell sorting, combined with the use of fluorescent intracellular pH (2) and NADH/NAD+ (3) biosensors to characterise the persister phenotype on a single-cell level prior to, during and post antibiotic exposure in the model organism Escherichia coli. Persisters have been previously shown to have a lower intracellular pH (4) and redox activity (5) than antibiotic-sensitive cells, however the mechanisms behind this remain unclear.  We focus on two highly-persistent E. coli strains, HipQ which contains a non-synonymous mutation in putative electron transfer protein ydiR and ΔydcI lacking pH stress response regulator ydcI (6). We aim to develop a universal framework for using biosensors to investigate persister physiology and to gain further understanding of how intracellular pH and redox activity affects antibiotic persistence.

  1. Windels EM, Michiels JE, Fauvart M, Wenseleers T, Van den Bergh B, Michiels J. Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates. ISME J. 2019 May;13(5):1239–51.
  2. 2. Arce‐Rodríguez A, Volke DC, Bense S, Häussler S, Nikel PI. Non‐invasive, ratiometric determination of intracellular PH in Pseudomonas species using a novel genetically encoded indicator. Microb Biotechnol. 2019 Jul;12(4):799–813.
  3. 3. Hung YP, Albeck JG, Tantama M, Yellen G. Imaging Cytosolic NADH-NAD+ Redox State with a Genetically Encoded Fluorescent Biosensor. Cell Metab. 2011 Oct;14(4):545–54
  4. 4. Goode O, Smith A, Zarkan A, Cama J, Invergo BM, Belgami D, et al. Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment. Balaban N, Levin BR, editors. mBio. 2021 Aug 31;12(4):e00909-21.
  5. 5. Mohiuddin SG, Hoang T, Saba A, Karki P, Orman MA. Identifying Metabolic Inhibitors to Reduce Bacterial Persistence. Front Microbiol. 2020 Mar 27;11:472.
  6. 6. Hingley-Wilson SM, Ma N, Hu Y, Casey R, Bramming A, Curry RJ, et al. Loss of phenotypic inheritance associated with ydcI mutation leads to increased frequency of small, slow persisters in Escherichia coli. Proc Natl Acad Sci. 2020 Feb 25;117(8):4152–7.