Phage Paride can kill dormant, antibiotic-tolerant cells of Pseudomonas aeruginosa by direct lytic replication

Bacteriophages are ubiquitous viral predators that have primarily been studied using fast-growing laboratory cultures of their bacterial hosts. However, microbial life in nature is mostly in a slow- or non-growing, dormant state. Here, we show that diverse phages can infect deep-dormant bacteria and suspend their replication until the host resuscitates (“hibernation”). However, a newly isolated Pseudomonas aeruginosa phage, named Paride, can directly replicate and induce the lysis of deep-dormant hosts. While non-growing bacteria are notoriously tolerant to antibiotic drugs, the combination with Paride enables the carbapenem meropenem to eradicate deep-dormant cultures in vitro and to reduce a resilient bacterial infection of a tissue cage implant in mice. Our work might inspire new treatments for persistent bacterial infections and, more broadly, highlights two viral strategies to infect dormant bacteria (hibernation and direct replication) that will guide future studies on phage-host interactions.

(a-c) E. coli K-12 MG1655 cultured for 8h or 12h after subculturing were challenged with antibiotics or phages (MOI ≈ 0.01) and viable cells (CFU/ml) as well as plaque-forming units (PFU/ml) of free virions and infected cells were recorded over time.Data points and error bars show the average of three biological replicates and standard error of the mean, with the exception of (c) where the individual replicates of the experiments performed with phage T7 are shown (matching Fig. 2a).Limits of detection are 2 log10 CFU/mL for viable cells, 3.6 log10 PFU/mL for free virions and 2.6 log10 PFU/mL for infected cells.Source data are provided as a Source Data file.(a) Deep-dormant cultures of P. aeruginosa Δpel Δpsl grown in M9Rich were treated with antibiotics or phages (MOI ≈ 0.01) and viable CFU/ml as well as free phages were recorded over time.Data points represent the average of three independent experiments and error bars show their standard error of the mean.Limits of detection are 2 log10 CFU/mL for viable cells, 3.6 log10 PFU/mL for free virions and 2.6 log10 PFU/mL for infected cells.(b, c) Free virions of one-step growth experiments with ancestral Paride and two lineages evolved on deep-dormant cultures for ca.600 generations were recorded over time in fast-growing cultures (b) and stationary phase cultures (c).Data points represent the average of six (regularly growing) and two (stationary phase) independent experiments, respectively.The dashed line represents the limit of detection (3.6 log10 PFU/mL).Source data are provided as a Source Data file.Top agars were set up with P. aeruginosa PAO1 Δpel Δpsl mutants lacking functional expression of one or more surface receptor genes before infection with serial dilutions of phage Paride and control phages E79 (targeting the LPS core; see Meadow & Wells, Microbiology (1978) 2 ), newly isolated phage Victoria (targeting the LPS O antigen), and DMS3vir (targeting type IV pili; see Budzik et al., J Bacteriol (2004) 3 ).Arrows highlight opaque plaque formation of phage Paride on several mutants.Strain EM-366 is is a spontaneously isolated mutant with a single nucleotide deletion which inactivates the ssg gene complemented with the same gene in cis.Strain EM-381 is a spontaneously isolated "brown mutant" as described previously with a large deletion around galU (Markwitz et   (a) Deep-dormant cultures of P. aeruginosa PAO1 Δpel Δpsl (wildtype) and its ΔrpoS derivative both grown in M9Glc were treated with antibiotics or phages (MOI ≈ 0.01) and viable CFU/ml as well as free phages were recorded over time.(b) Fast-growing cultures of P. aeruginosa PAO1 Δpel Δpsl (wildtype) and its ΔrpoS derivative both grown in M9Glc were treated with antibiotics or phages (MOI ≈ 0.001) and viable CFU/ml as well as free phages were recorded over time.(c,d) Fast-growing cultures of P. aeruginosa PAO1 Δpel Δpsl (wildtype) and its ΔrelA ΔspoT derivative both grown in M9Rich were treated with antibiotics or phages (MOI ≈ 0.001) and viable CFU/ml as well as free phages were recorded over time.(e) Growing cultures of P. aeruginosa were processed as described for the SHX treatment shown in Fig. 6c (as a control without SHX) and then challenged with antibiotics or phages (MOI ≈ 0.01).Viable CFU/ml as well as free phages were recorded over time.Data points and error bars show the average of three biological replicates and standard error of the mean.Limits of detection are 2 log10 CFU/mL for viable cells, 3.6 log10 PFU/mL for free phages.Source data are provided as a Source Data file.___________________________________

Figure S1 :
Figure S1: Whole proteomes of E. coli and P. aeruginosa at various growth stages.Heatmaps of the total proteomes of E. coli K-12 MG1655 (a) and P. aeruginosa Δpel Δpsl (b) sampled at 3, 12, 24, and 48h of growth in M9Glc.The dendrograms (top) indicate how the proteomes from different time points cluster together.Colors (blue to red) on the heatmaps indicate the log2 intensity of the mean-centered MS signals for each protein detected.The data indicate a clear progression of the proteomic profiles from the earliest (light brown, growing bacteria) to the latest time point (dark brown; deep dormancy 48h after subculturing).In this culture setup, the bacteria reach stationary phase (maximal optical density at 600nm wavelength) after ca.eight hours as shown in our previous work¹.All data are available in public repositories (see Data Availability).

Figure S2 :
Figure S2: Time-kill curves of early stationary phase E. coli.(a-c)E. coli K-12 MG1655 cultured for 8h or 12h after subculturing were challenged with antibiotics or phages (MOI ≈ 0.01) and viable cells (CFU/ml) as well as plaque-forming units (PFU/ml) of free virions and infected cells were recorded over time.Data points and error bars show the average of three biological replicates and standard error of the mean, with the exception of (c) where the individual replicates of the experiments performed with phage T7 are shown (matching Fig.2a).Limits of detection are 2 log10 CFU/mL for viable cells, 3.6 log10 PFU/mL for free virions and 2.6 log10 PFU/mL for infected cells.Source data are provided as a Source Data file.

Figure S3 :Figure S4 :
Figure S3: Time-kill curves of early stationary phase P. aeruginosa, infection of regularly growing P. aeruginosa with Paride and lysis of deep-dormant cultures by the same phage.(a-b) P. aeruginosa Δpel Δpsl cultured for 8 or 12h after subculturing were challenged with antibiotics or phages (MOI ≈ 0.01) and viable cells (CFU/ml) as well as plaque-forming units (PFU/ml) of free virions and infected cells were recorded over time.(c) Representative picture of a deep-dormant P. aeruginosa culture at 96h after infection with Paride in M9Glc (right) and untreated control (left).(d) Regularly growing cultures of P. aeruginosa were treated with antibiotics or phages (MOI ≈ 0.001) and viable CFU/ml as well as free phages were recorded over time.Data points represent the average of three independent experiments and error bars show their standard error of the mean.Limits of detection are 2 log10 CFU/mL for viable cells, 3.6 log10 PFU/mL for free phages and 2.6 log10 PFU/mL for infected cells.Source data are provided as a Source Data file.

Figure S5 :
Figure S5: Comparative genomics of evolved Paride lineages and mutation analysis.(a) Schematic genome alignment of Paride_1 and Paride_2 lineages from clones sampled at different time points (after ca.340, 470, and 600 generations of evolution).Grey bars highlight spots where the same or similar mutations have been detected.(b) Full list of mutations identified in the two evolved Paride lineages.Possible biological functions of mutated genes have been inferred from the genome annotation.The table also indicates the first detection of each mutation with respect to the three clones that have been sequenced sequentially for each lineage.All data are shown directly in this illustration.

Figure S6 :
Figure S6: Top agar assays with Paride and control phages on different surface receptor mutants of P. aeruginosa PAO1.Top agars were set up with P. aeruginosa PAO1 Δpel Δpsl mutants lacking functional expression of one or more surface receptor genes before infection with serial dilutions of phage Paride and control phages E79 (targeting the LPS core; seeMeadow & Wells, Microbiology (1978)  2 ), newly isolated phage Victoria (targeting the LPS O antigen), and DMS3vir (targeting type IV pili; seeBudzik et al., J Bacteriol (2004)  3 ).Arrows highlight opaque plaque formation of phage Paride on several mutants.Strain EM-366 is is a spontaneously isolated mutant with a single nucleotide deletion which inactivates the ssg gene complemented with the same gene in cis.Strain EM-381 is a spontaneously isolated "brown mutant" as described previously with a large deletion around galU(Markwitz et al., ISME J (2022)  4 ) complemented with galU in cis.The data are summarized in TableS2.Top agar plates are representatives of at least three independent replicates.___________________________________________

Figure S7 :
Figure S7: Additional data of phage-antibiotic combination treatments and tissue cage infections experiments.(a-c)Deep-dormant cultures of P. aeruginosa were treated with meropenem (12 μg/ml), tobramycin (40 μg/ml) or ciprofloxacin (10 μg/ml) in combination with phages (MOI ≈ 0.01) and viable CFU/ml as well as free phages were recorded over time.(d) P. aeruginosa was cultured for 48h after subculturing and spiked with 1% of either Paride-or meropenem resistant cells from analogous cultures before treatment with Paride (MOI ≈ 0.01), meropenem, or the combination thereof and viable CFU/ml (Fig.5) as well as free phages were recorded over time.Data points and error bars show the average of three independent experiments and their standard error of the mean.Limits of detection are 2 log10 CFU/ml for viable cells and 3.6 log10 PFU/ml for free phages.(e) The planktonic bacterial load of the tissue cage infection experiments was determined over time and plotted per mouse grouped by treatment condition (dashed line = treatment start).Each data point represents the CFU/ml recovered from tissue cage fluid of one mouse.(f) Boxplots analogous to Fig.5dshowing viable adherent bacteria recovered from tissue cages at the end of the experiment (see Methods).Each dot represents the surviving bacteria recovered from one mouse and the dashed line shows the median initial inoculum at the treatment start.The limit of detection is 1.6 log10 CFU/ml.Source data are provided as a Source Data file.______________Page 7

Table S1 -
Bacteriophages used in this study

Table S2 -
Phage susceptibility of P. aeruginosa PAO1 surface receptor mutants Phage E79 is known to depend on structures in the LPS core of P. aeruginosa PAO1(Meadow and  Wells, Microbiology (1978)  2 ) while phage DMS3vir targets type IV pili(Budzik et al., J Bacteriol

Table S3 -
Strains used in this study

Table S4 -
Oligonucleotide primers used in this study

Table S5 -
Plasmids used in this study

Table S6 -
Minimum inhibitory concentrations of antibiotics for clinical isolates

Table S6 . MIC values of tobramycin and ciprofloxacin for P. aeruginosa PAO1 Δpel Δpsl, CI249 and CI282 in M9Rich medium.
The values reported are the average of two independent experiments.

Table S7 -
Minimum inhibitory concentration of meropenem for a resistant strain

Table S7 . MIC values of P. aeruginosa PAO1 Δpel Δpsl and P. aeruginosa PAO1 Δpel Δpsl oprD_CI NDM in
M9Glucosemedium.The values are the average of two independent experiments.