Phages produce persisters

Abstract

Arguably, the greatest threat to bacteria is phages. It is often assumed that those bacteria that escape phage infection have mutated or utilized phage-defence systems; however, another possibility is that a subpopulation forms the dormant persister state in a manner similar to that demonstrated for bacterial cells undergoing nutritive, oxidative, and antibiotic stress. Persister cells do not undergo mutation and survive lethal conditions by ceasing growth transiently. Slower growth and dormancy play a key physiological role as they allow host phage defence systems more time to clear the phage infection. Here, we investigated how bacteria survive lytic phage infection by isolating surviving cells from the plaques of T2, T4, and lambda (cI mutant) virulent phages and sequencing their genomes. We found that bacteria in plaques can escape phage attack both by mutation (i.e. become resistant) and without mutation (i.e. become persistent). Specifically, whereas T4-resistant and lambda-resistant bacteria with over a 100,000-fold less sensitivity were isolated from plaques with obvious genetic mutations (e.g. causing mucoidy), cells were also found after T2 infection that undergo no significant mutation, retain wild-type phage sensitivity, and survive lethal doses of antibiotics. Corroborating this, adding T2 phage to persister cells resulted in 137,000-fold more survival compared to that of addition to exponentially growing cells. Furthermore, our results seem general in that phage treatments with Klebsiella pneumonia and Pseudomonas aeruginosa also generated persister cells. Hence, along with resistant strains, bacteria also form persister cells during phage infection.

FIGURE 1

Phages produce persisters. (A) Schematic of experiments used to demonstrate that phages produce persister cells. (B) Kill curves of exponentially growing Escherichia coli BW25113 treated with ampicillin (100 μg/mL, 10X MIC, blue) or T2 phage (MOI ≈ 0.1, orange). Note the increase in cell density with T2 phage indicates that persister cells are present and have revived (with a specific growth rate of 1.2 ± 0.1 h⁻¹). (C) Double‐layer TA plates, from left to right, of phages T2, T4, and lambda mutant cI infecting E. coli BW25113, showing surviving colonies inside the phage inhibition area (indicated with a black arrow). Colonies are visible after 1 day but allowed to grow for several days for the photo here. (D) Escherichia coli BW25113 persister cells were formed by rifampicin pre‐treatment (30 min, 100 μg/mL), and 0.1 MOI T2 phage was added for 3 h. Exponential cells (turbidity 0.5 at 600 nm) were treated with ampicillin (10X MIC) for 3 h. (E) First bar indicates initial cell density after phage attack (10⁸ E. coli cells/mL treated with 0.01 MOI T2 phage for 1 h, 10⁸  Klebsiella pneumoniae cells/mL treated with 0.01 MOI VAC25 phage for 1 h or 10⁸ Pseudomonas aeruginosa cells/mL treated with 1 MOI PaMx12 phage for 1 h), second bar indicates phage and antibiotic treatment (0.01 MOI T2 phage for 1 h followed by 10X MIC of ampicillin for 3 h for E. coli, 0.01 MOI VAC25 phage for 1 h followed by 10X MIC of colistin for 3 h for K. pneumoniae or 1 MOI PaMx12 phage for 1 h followed by 10X MIC of gentamicin for 3 h), and third bar indicates antibiotic treatment alone (10X MIC of ampicillin for 3 h added to 10⁴ cells/mL for E. coli,10X MIC of colistin for 3 h added to 10⁴ cells/mL for K. pneumoniae or 10X MIC of gentamicin for 3 h added to 10⁶ cells/mL for P. aeruginosa). One average deviation shown.

FIGURE 2

Schematic of model for cells in plaques.