Metals Enhance the Killing of Bacteria by Bacteriophage in Human Blood

Li Ma,Barbara W Trautner,Robert F Ramig,Sabrina I Green,Anthony W Maresso

doi:10.1038/s41598-018-20698-2

Abstract

Multidrug-resistant bacterial pathogens are a major medical concern. E. coli, particularly the pathotype extraintestinal pathogenic E. coli (ExPEC), is a leading cause of bloodstream infections. As natural parasites of bacteria, bacteriophages are considered a possible solution to treat patients infected with antibiotic resistant strains of bacteria. However, the development of phage as an anti-infective therapeutic is hampered by limited knowledge of the physiologic factors that influence their properties in complex mammalian environments such as blood. To address this barrier, we tested the ability of phage to kill ExPEC in human blood. Phages are effective at killing ExPEC in conventional media but are substantially restricted in this ability in blood. This phage killing effect is dependent on the levels of free metals and is inhibited by the anticoagulant EDTA. The EDTA-dependent inhibition of ExPEC killing is overcome by exogenous iron, magnesium, and calcium. Metal-enhanced killing of ExPEC by phage was observed for several strains of ExPEC, suggesting a common mechanism. The addition of metals to a murine host infected with ExPEC stimulated a phage-dependent reduction in ExPEC levels. This work defines a role for circulating metals as a major factor that is essential for the phage-based killing of bacteria in blood.

Highlights

All three strains decreased in cell number by four hours, with the greatest effect observed for the laboratory bacterium E. coli K12
Hypothesizing that the negative effect on extraintestinal pathogenic E. coli (ExPEC) growth in blood was due to complement, the dominant innate antibacterial mechanism in serum[29], we sought to generate a blood formulation that preserves the cellular components of blood but eliminates the activity of complement
When ExPEC is grown in Heat-inactivated Plasma Blood (HIP-B) an increase in the growth is observed at nine hours post inoculation when compared to blood that was not heated and recombined (Fig. 2A,B, black versus grey bars)

Summary

Introduction

When bacterial numbers are assessed by plating a sample of the culture and counting the number of colonies (a more sensitive method than optical absorption), an approximately 3.5 logarithmic drop in CFUs is observed when ExPEC is incubated with phage in L broth (MOI of 1 for 4.5 hours - Fig. 3B). Unlike the very efficient 2.5 logarithmic reduction in ExPEC levels by φHP3 at an MOI of 0.1 in LB (Fig. 3), no decrease in bacterial cell numbers was observed under identical conditions in blood, regardless of the presence of active complement (Fig. 4A and B).

Results

Conclusion