# Cell-to-cell heterogeneity and the emergence of antibiotic resistance

> **NIH NIH R01** · BOSTON UNIVERSITY (CHARLES RIVER CAMPUS) · 2020 · $412,196

## Abstract

Project Summary / Abstract
Antimicrobial drug resistance is a major clinical problem, with resistant strains of bacteria emerging at a rate
that dramatically outpaces development of new drugs. Traditionally, studies on antibiotic resistance have
focused on genetic changes that confer resistance, such as those encoding mechanisms that block the drug
target or modify the drug itself. However, bacteria can also evade antibiotics through expression of transient
resistance mechanisms, such as multi-drug efflux pumps that turn on either stochastically or in response to
antibiotic stress. Studies have implicated these transient resistance mechanisms in chronic, recalcitrant
infections, however, recent research has revealed examples where they also play a critical role in increasing
mutation propensity. It is unclear how heterogeneity and temporal variability in expression of resistance genes
leads to mutations and what the ultimate implications are for the evolution of drug resistance at the population-
level. This proposal addresses this gap directly by measuring expression of resistance genes over time
alongside reporters for mutation. These single-cell level studies are joined by population-level experiments that
modulate expression of the transient resistance genes while measuring growth under antibiotic stress. A
complementary modeling approach uses stochastic models to describe heterogeneity in gene expression,
mutation rate, and growth. Our central hypothesis is that heterogeneity in expression of transient resistance
genes can lead to single-cell-level differences in mutation rate, both via inducing spontaneous mutations due to
elevated endogenous stress in the absence of antibiotics and by extending survival times in the presence of
antibiotics. We will test this hypothesis using a quantitative approach that integrates single-cell time-lapse
microscopy, stochastic modeling, whole genome sequencing, parallelized continuous culture methods, and
optogenetic control. The project is organized around three Aims: (1) Measure expression history of transient
resistance genes in cells prior to spontaneous mutation. (2) Quantify time to death of single cells and the
evolution of resistance under antibiotic treatment. (3) Control temporal variation of AcrAB efflux pump
expression to determine frequency-dependent resistance levels and mutation rate. This research is significant
because it links dynamic, single-cell-level effects due to heterogeneity in expression of transient resistance
genes to the emergence of population-level increases in resistance. Identifying and eliminating nucleation
points for the emergence of drug resistance can inform assessment and treatment approaches.

## Key facts

- **NIH application ID:** 9942378
- **Project number:** 5R01AI102922-07
- **Recipient organization:** BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
- **Principal Investigator:** Mary J. Dunlop
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $412,196
- **Award type:** 5
- **Project period:** 2014-02-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9942378

## Citation

> US National Institutes of Health, RePORTER application 9942378, Cell-to-cell heterogeneity and the emergence of antibiotic resistance (5R01AI102922-07). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9942378. Licensed CC0.

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