The Lysis of Pathogenic Escherichia coli by Bacteriophages Releases Less Endotoxin Than by β-Lactams

Abstract

Background.: Other than numerous experimental data assessing phage therapy efficacy, questions regarding safety of this approach are not sufficiently addressed. In particular, as phages can kill bacterial cells within <10 minutes, the associated endotoxin release (ER) in severe infections caused by gram-negative bacteria could be a matter of concern.

Methods.: Two therapeutic virulent phages and 4 reference antibiotics were studied in vitro for their ability to kill 2 pathogenic strains of Escherichia coli and generate an ER. The early interaction (first 3 hours) between these actors was assessed over time by studying the instantaneous cell viability, the colony-forming unit count, the concentration of free endotoxin released, and the cell morphology under light microscope.

Results.: While β-lactams have a relatively slow effect, both tested phages, as well as amikacin, were able to rapidly abolish the bacterial growth. Even when considering the fastest phage (cell lysis in 9 minutes), the concentrations of phage-induced ER never reached the highest values, which were recorded with antibiotic treatments. Cumulative concentrations of endotoxin over time in phage-treated conditions were lower than those observed with β-lactams and close to those observed with amikacin. Whereas β-lactams were responsible for strong cell morphology changes (spheroplast with imipenem, filamentous cells with cefoxitin and ceftriaxone), amikacin and phages did not modify cell shape but produced intracellular inclusion bodies.

Conclusions.: This work provides important and comforting data regarding the safety of phage therapy. Therapeutically relevant phages, with their low endotoxin release profile and fast bactericidal effect, are not inferior to β-lactams.

Keywords: cell lysis; endotoxin; phage therapy; safety; β-lactams..

Figure 1.

Colony-forming unit (CFU) counts of strains LM33 and 536 according to time. Strain LM33 (A) and strain 536 (B) were cultured in absence (control) or presence of antibacterial agents: phages (LM33_P1 or 536_P1, multiplicity of infection of 5) or antibiotics (8-fold the minimum inhibitory concentration). Data are means with standard deviation, and the dotted line represents the detection threshold.

Figure 2.

Relative amount of metabolically active cells according to time in presence of different antibacterial agents. Strain LM33 (A) and strain 536 (B) were cultured in absence (control) or presence of antibacterial agents: phages (LM33_P1 or 536_P1, multiplicity of infection of 5) or antibiotics (8-fold the minimum inhibitory concentration). The viable cells were quantified using an ATP-driven luciferase assay (see Methods). Data are means with standard deviation and the y-axis is in log₂ scale. Abbreviation: RLU, relative luminescence unit.

Figure 3.

Morphology of Escherichia coli cells after a 3-hour exposition to different antibacterial agents. The liquid cultures of strains LM33 and 536 in absence (control) or presence of antibacterial agents (antibiotics with a concentration of 8-fold the minimum inhibitory concentration, phages LM33_P1 or 536_P1 with a multiplicity of infection of 5) were centrifuged to concentrate the residual biomass. Wet mount preparations were then observed under phase contrast (×100 lens).

Figure 4.

Concentration of free endotoxin released in the culture medium over time by cells exposed to different antibacterial agent. Strain LM33 (A) and strain 536 (B) were cultured in presence of phages (LM33_P1 or 536_P1, multiplicity of infection of 5) or antibiotics (8-fold the minimum inhibitory concentration). The symbols are the means of 2 independent experiments with standard deviation. The curves are nonlinear regressions from these points (see Methods). Abbreviation: EU, endotoxin unit.

Figure 5.

Areas under the concentration of free endotoxin vs time curve (0 to 180 minutes). Strain LM33 (left side) and strain 536 (right side) were cultured in presence of phages (respectively, LM33_P1 and 536_P1, multiplicity of infection of 5) or antibiotics (8-fold the minimum inhibitory concentration). Data are means with standard deviation. Abbreviations: AUC, area under the curve; EU, endotoxin unit.