Multiplexed PCR to measure in situ growth rates of uropathogenic E. coli during experimental urinary tract infection.

Abstract

Measuring bacterial growth rates in vitro is routine; however, determining growth rates during infection within a host has been more challenging. Peak-to-trough ratio (PTR) is a technique for studying microbial growth dynamics, calculated using the ratio of replication origin (ori) copies to those of the terminus (ter), as originally defined by whole-genome sequencing (WGS). WGS presents significant challenges in terms of expense and data analysis complexity due to the presence of host DNA in the samples. Here, we used multiplexed PCR with fluorescent probes to estimate bacterial growth rates based on the abundance of ori- and ter-adjacent loci, without the need for WGS. We establish the utility of this approach by comparing growth rates of the uropathogenic Escherichia coli (UPEC) strain HM86 by WGS (PTR) and qPCR to measure the equivalent ori:ter (O:T^PCR). We found that PTR and O:T^PCR were highly correlated and that O:T^PCR reliably predicted growth rates calculated by conventional methods. O:T^PCR was then used to calculate the in situ E. coli growth rates in urine, bladder, and kidneys collected over the course of a week from a murine model of urinary tract infection (UTI). These analyses revealed that the growth rate of UPEC strains gradually increased during the early stages of infection (0-6 h), followed by a slow decrease in growth rates during later time points (1-7 days). This rapid and convenient method provides valuable insights into bacterial growth dynamics during infection and can be applied to other bacterial species in both animal models and clinical infections.

Importance: Accurately measuring bacterial growth rates in the host, which plays a crucial role in determining the success of pathogens in establishing infections, poses significant challenges. To address this, bacterial replication rate has been measured as a proxy for the growth rate estimation. While whole-genome sequencing (WGS) has been used for this purpose, it comes with drawbacks such as high costs and difficulties in analyzing bacterial sequences due to the overwhelming presence of host DNA. In this study, we validate a more accessible PCR-based approach compared to the established WGS method and confirmed the reliability of our PCR-based technique. We then applied it to measure the growth rate of Escherichia coli during experimental urinary tract infection in a mouse model. This study provides a cost-effective and efficient alternative to WGS for studying bacterial replication dynamics during infection, potentially offering new insights into pathogen behavior and host-microbe interactions.