# A Ratiometric pH Sensor for Gram-Positive and Gram-Negative Bacteria

## TL;DR

A new pH sensor helps track how individual bacteria respond to environmental changes, including during interactions with immune cells.

## Contribution

A ratiometric fluorescent probe for real-time cytoplasmic pH sensing in both Gram-positive and Gram-negative bacteria.

## Key findings

- The probe is selective and works in a wide range of biologically relevant conditions.
- It successfully monitored pH fluctuations in E. coli, S. epidermidis, and MRSA.
- The probe identified pH-sensitive phenotypes and tracked phagocytosis of virulent strains.

## Abstract

Fluctuating environments can lead to phenotypic heterogeneity
within
a monoclonal bacterial population, especially in response to antibiotics
or the human immune system. Methods are required to analyze the physiology
of single cells to understand how individual cells interact with their
environment and adapt to pH stress. We report a ratiometric, fluorescent
probe to sense cytoplasmic pH in bacteria. Our probes are based on
hemicyanine dyes and are taken up into both Gram-positive and Gram-negative
bacteria. The probes are selective under a broad range of biologically
relevant conditions. The response to pH changes is reversible and
rapid, allowing for the real-time tracking of pH fluctuations. The
sensing of these probes was tuned to allow for monitoring fluctuations
around neutrality and biologically relevant acidifications. These
probes were validated for cytoplasmic pH sensing in Escherichia coli, Staphylococcus epidermidis, and a clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) strain. Furthermore,
the probes enabled the identification of pH-sensitive phenotypes and
monitored phagocytosis of virulent clinical strains in immune cells.
Our probes are a promising tool for detecting phenotypic heterogeneity
within bacterial populations and may help unravel the physiological
state of resistant or persistent strains of clinical relevance.