A novel method to determine antibiotic sensitivity in Bdellovibrio bacteriovorus reveals a DHFR-dependent natural trimethoprim resistance

Emanuele Marine,Klaas Martinus Pos,David Stephen Milner,Renee Elizabeth Sockett,Carey Lambert

doi:10.1038/s41598-020-62014-x

Abstract

Bdellovibrio bacteriovorus is a small Gram-negative bacterium and an obligate predator of other Gram-negative bacteria. Prey resistance to B. bacteriovorus attack is rare and transient. This consideration together with its safety and low immunogenicity makes B. bacteriovorus a valid alternative to antibiotics, especially in the treatment of multidrug resistant pathogens. In this study we developed a novel technique to estimate B. bacteriovorus sensitivity against antibiotics in order to make feasible the development and testing of co-therapies with antibiotics that would increase its antimicrobial efficacy and at the same time reduce the development of drug resistance. Results from tests performed with this technique show that among all tested antibiotics, trimethoprim has the lowest antimicrobial effect on B. bacteriovorus. Additional experiments revealed that the mechanism of trimethoprim resistance in B. bacteriovorus depends on the low affinity of this compound for the B. bacteriovorus dihydrofolate reductase (Bd DHFR).

Highlights

The increasing development and spread of antibiotic resistant bacteria is an ever-rising problem worldwide with an alarming rise of cases involving Gram-negative bacteria[1]
Direct cell growth measurements of B. bacteriovorus upon prey bacteria can be estimated by reduction of the optical density at 600 nm (OD600)
B. bacteriovorus HD100 cells were added to an E. coli culture and the reduction of OD600 measured in absence and presence of different concentrations of antibiotics

Summary

Introduction

The increasing development and spread of antibiotic resistant bacteria is an ever-rising problem worldwide with an alarming rise of cases involving Gram-negative bacteria[1]. We developed a novel assay in liquid medium, which allows testing of B. bacteriovorus sensitivity against antibiotics For this purpose, we tested antibiotics belonging to different classes of pharmaceuticals with different specificities for Gram-positive and -negative bacteria. In order to ascertain whether this mechanism is the cause of resistance in B. bacteriovorus, we cloned three B. bacteriovorus genes (bd3231, bd0323, bd1356) with homologies to the E. coli folA chromosomal gene (b0048, encoding the DHFR enzyme) and analyzed their sensitivity against TMP by plate dilution assays. By this screening we identified the B. bacteriovorus DHFR (Bd DHFR) as the protein encoded by the bd3231 gene. The kinetic properties and TMP inhibition of activities are studied on the purified protein and its substituted variants

Methods

Results

Conclusion