Drug accumulation in gram-negative bacteria is growth phase dependent

Whittle, Emily (2021). Drug accumulation in gram-negative bacteria is growth phase dependent. University of Birmingham. Ph.D.

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Abstract

For antibiotic classes with intracellular targets, effective treatment of bacterial infections requires the drug to accumulate to a high concentration inside cells. Decreased permeability of the cell envelope and increased efflux pump activity causes low intracellular accumulation of antibiotics, contributing to the development of antimicrobial resistance. Here, the aim was to identify whether the balance between influx and efflux differs depending on bacterial growth phase in Salmonella enterica serovar Typhimurium. A novel flow cytometry assay was developed and used to measure accumulation of ethidium bromide (EtBr) in S. Typhimurium SL1344 and ∆acrB strains during growth. In SL1344, EtBr accumulation remained low, regardless of growth phase and did not correlate with acrAB transcription. EtBr accumulation in ∆acrB was high in exponential phase but dropped sharply later in growth and by stationary phase, EtBr accumulation was not significantly different from SL1344. Low EtBr accumulation in both strains was shown not to be due to the upregulation of other efflux pumps, but instead, due to decreased permeability of the membrane in stationary phase. RNAseq identified changes in expression of several pathways that are associated with decreased membrane permeability in stationary phase. This study shows that efflux is only important for maintaining low drug accumulation in actively growing cells, and that membrane permeability is the predominant factor in stationary phase. This conclusion means that (i) more consideration may be required when prescribing existing antibiotics that have intracellular targets in complex non growing or slow growing bacterial infections where accumulation concentration may be low, and (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):