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Alcohol tolerance in Enterococcus faecium

Primary Supervisor: Professor Willem van Schaik, Institute of Microbiology and Infection

Secondary supervisor: Professor Alan McNally

PhD project title: Alcohol tolerance in Enterococcus faecium

University of Registration: University of Birmingham

Project outline:

Alcohols have long been used in food production and healthcare as disinfectants. The most important alcohols used in disinfectants are ethanol and isopropanol. Indeed, these two alcohols are important ingredients in alcoholic handwash products that are widely used in clinical and food production settings, to minimise the spread of bacteria.

  1. faecium is a common commensal of the intestinal tract of humans and animals. A genetic sub-population, termed clade A-1, has recently emerged as a multi-drug resistant opportunistic pathogen. E. faecium has thus become a common cause of infections in immunocompromised patients. Other than antibiotic resistance genes, E. faecium clade A-1 strains have acquired genes that help it survive and spread in the hospital environment, by contributing to biofilm formation, high-level colonisation of the gut and attachment to epithelial cells [1].

Recently isolated clade A-1 strains are particularly tolerant to alcohols [2]. Recent work in the Van Schaik lab showed that E. faecium grown in a biofilm show particular tolerance to alcohols, with ~102 cells in the biofilm surviving after exposure to 70% ethanol for 5 minutes. The remarkable tolerance of E. faecium to alcohols may minimise the efficacy of disinfection products and alcoholic handwashes and could importantly contribute to the spread of this organism in hospitals.

In this project we will identify and characterise the determinants of alcohol tolerance of E. faecium and study the physiological mechanisms that contribute to this trait. We will use state-of-the-art functional and comparative genomics approaches in this PhD project, providing training opportunities in microbiology, and molecular biology (provided by Prof Van Schaik) and bioinformatics (provided by Prof McNally).

This project will encompass the following research lines

  1. In vitro evolution of alcohol tolerance. We will use multi-drug resistant faecium alcohol-susceptible strains and evolve them towards alcohol tolerance by repeated exposures to ethanol and culturing of surviving cells. We will then perform whole genome sequencing to identify mutations that emerge in the tolerant clones.
  2. Transposon mutant library screening for determinants involved in alcohol tolerance. We will generate high-density transposon mutant libraries in up to 5 alcohol-tolerant faecium strains and will then perform transposon-sequencing (Tn-seq) to identify genes that contribute to alcohol tolerance.
  3. Characterisation of mutations and genes involved in alcohol tolerance. The role of the mutations and genes identified by the in vitro evolution experiments and transposon mutant library screening will be confirmed using a recently developed CRISPR-Cas9 based method for the efficient generation of markerless, site-directed mutants in faecium. Mutants that are more alcohol-tolerant than their parental strain will be tested for their fitness in the absence of alcohols and their ability to withstand other stressors, including non-alcoholic disinfectants, like quaternary ammonium compounds and chlorhexidine, and antibiotics.

By the end of this project, the student will have elucidated the mechanism(s) by which E. faecium can gain tolerance to alcohols through a multi-disciplinary approach. Alcohol tolerance in opportunistic pathogens is a topic of considerable scientific and societal importance and the work in this PhD project is likely to lead to impactful publications.


  1. Pidot SJ et al. Increasing tolerance of hospital Enterococcus faecium to handwash alcohols. Sci Transl Med. 2018; 10:eaar6115.
  2. Lebreton et al. Emergence of epidemic multidrug-resistant Enterococcus faecium from animal and commensal strains. MBio. 2013; 4:e00534-13.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Microbiology

Techniques that will be undertaken during the project:

  • Microbiology: microbial culturing, in vitro evolution, susceptibility testing
  • Molecular microbiology: generation of targeted and random transposon mutants
  • Whole genome sequence analysis and bioinformatics

Contact: Professor Willem van Schaik, University of Birmingham