Phage-Antibiotic Synergy via Delayed Lysis

Abstract

When phages infect bacteria cultured in the presence of sublethal doses of antibiotics, the sizes of the phage plaques are significantly increased. This phenomenon is known as phage-antibiotic synergy (PAS). In this study, the observation of PAS was extended to a wide variety of bacterium-phage pairs using different classes of antibiotics. PAS was shown in both Gram-positive and Gram-negative bacteria. Cells stressed with β-lactam antibiotics filamented or swelled extensively, resulting in an increase in phage production. PAS was also sometimes observed in the presence of other classes of antibiotics with or without bacterial filamentation. The addition of antibiotics induced recA expression in various bacteria, but a recA deletion mutant strain of Escherichia coli also showed filamentation and PAS in the presence of quinolone antibiotics. The phage adsorption efficiency did not change in the presence of the antibiotics when the cell surfaces were enlarged as they filamented. Increases in the production of phage DNA and mRNAs encoding phage proteins were observed in these cells, with only a limited increase in protein production. The data suggest that PAS is the product of a prolonged period of particle assembly due to delayed lysis. The increase in the cell surface area far exceeded the increase in phage holin production in the filamented host cells, leading to a relatively limited availability of intracellular holins for aggregating and forming holes in the host membrane. Reactive oxygen species (ROS) stress also led to an increased production of phages, while heat stress resulted in only a limited increase in phage production.IMPORTANCE Phage-antibiotic synergy (PAS) has been reported for a decade, but the underlying mechanism has never been vigorously investigated. This study shows the presence of PAS from a variety of phage-bacterium-antibiotic pairings. We show that increased phage production resulted directly from a lysis delay caused by the relative shortage of holin in filamented bacterial hosts in the presence of sublethal concentrations of stress-inducing substances, such as antibiotics and reactive oxygen species (ROS).

Keywords: filamentation; holin; lysis delay; phage-antibiotic synergy; recA; stress.

FIG 1

Relationship between recA expression and phage production. (A) Determination of recA expression level in different bacteria in the presence of a sublethal dose of ciprofloxacin using real-time RT-PCR. Negative, without antibiotic treatment and given a value of 1 for comparison purposes; *, P < 0.05. (B) Bacterial morphological change was observed under a light microscope. E. coli K-12 strain or its recA deletion mutant (ΔrecA K-12, see Materials and Methods) was grown in the presence or absence of the sublethal dose of ciprofloxacin; (left) Western blot analysis showing a lack of RecA protein expression. Scale bar, 10 mm. (C) Observation of plaque sizes when phage T4 infected E. coli K-12 strain or its recA deletion mutant (ΔrecA K-12) in the presence of different doses of ciprofloxacin. Scale bar, 10 mm. (D) Burst sizes measured when phage T4 infected wild-type or recA deletion mutant strain (ΔrecA K-12) of E. coli K-12 in the presence or absence of the sublethal dose of ciprofloxacin. **, P < 0.01.

FIG 2

Adsorption of phage T4 to E. coli K-12 strain (MOI = 0.01) in the presence of sublethal concentrations of various antibiotics. After phages were added to a fresh culture of E. coli, the mixture was incubated at 37°C. Aliquots of the culture were taken at the indicated time points and the titer of free phages was measured. Remaining phages were counted every minute for 5 min postinfection. CON, control. (A) CPFX, ciprofloxacin; (B) CTX, cefotaxime; (C) KM, kanamycin; (D) MitC, mitomycin C.

FIG 3

Changes in transcription of phage T4 mRNA and replication of phage DNA in the presence of a sublethal concentration of ciprofloxacin (CPFX) as measured by real-time RT-PCR or real-time PCR. Total RNA or total DNA was isolated from T4-infected E. coli 13 min postinfection and analyzed. (A) mRNA encoding T4 DNA polymerase; (B) phage T4 genomic DNA; (C) mRNA encoding T4 major capsid protein; (D) Western blot analysis of bacterial RecA protein and phage T4 major capsid protein. GAPDH was used as an internal control. *, P < 0.05; **, P < 0.01.

FIG 4

Delayed lysis of T4-infected E. coli K-12 strain in the presence of sublethal doses of different antibiotics. (A) One-step multiplication curve of phage T4 with or without the antibiotics inducing PAS. CPFX, ciprofloxacin; CTX, cefotaxime. An aliquot of the culture was taken, and the titers of free phages were measured at each time point postinfection (MOI = 0.001). (B) One-step multiplication curve of phage T4 with or without the antibiotics not inducing PAS. CHL, chloramphenicol; ERY, erythromycin. MICs were 16 mg/ml and 256 mg/ml for CHL and ERY, respectively. One-half MIC was used as the sublethal dose for this experiment. (C) Burst sizes measured in the presence of antibiotics inducing PAS. *, P < 0.05; **, P < 0.01. (D) Phage titers after a forced lysis at 17 min postinfection with the addition of chloroform (5% [vol/vol]) to the culture.

FIG 5

Underlying mechanism of the delayed lysis in the presence of sublethal doses of CPFX. (A) Observation of T4 holin (t) fused to enhanced green fluorescent protein (EGFP) expressed in bacterial host under a fluorescence microscope at 0, 15, and 23 min postinfection. T4_t_EGFP, engineered phage T4 expressing EGFP-fused holin (see Materials and Methods). Scale bar, 10 mm. (B) Increase in bacterial length due to filamentation and increase in T4 holin production in the presence of the sublethal dose of ciprofloxacin. (C) Real-time RT-PCR measurement of phage T4 holin and antiholins. T, T4 holin; rI and rIII, T4 antiholins. Total RNA was isolated from T4-infected E. coli grown in the presence of ciprofloxacin at 20 min postinfection and subjected to a real-time RT-PCR. *, P < 0.05. (D) One-step multiplication curve of phage T4 in bacterial host expressing the nonsense mutant t (holin) gene (tamA3) from a plasmid. The host is either the suppressor strain (DH5α) or the wild type (K-12). MOI, 0.001.

FIG 6

Changes in phage T4 production in the presence of other stresses. (A) Bacterial filamentation in the presence of the sublethal dose of hydrogen peroxide (4.5 mM) or elevated temperature (45°C) was observed under a light microscope. (B) One-step multiplication curve of phage T4 in the presence of stresses other than antibiotics. After phages were added to a fresh culture of host bacteria, an aliquot of the culture was taken and the titers of free phages were measured at each time point postinfection (MOI = 0.001).