Experimental Animals

The mouse strain BALB/c (Jackson laboratories, Bar Harbor, ME) was used for Fig. 3 and Swiss Webster (Charles River Wilmington, MA) for Figs 4 and 5. All mice were 6 weeks of age and female. They received sterile food and water ad libitum and were housed individually in filtered cages post-infection or gavage. All methods performed on mice were approved in accordance with relevant guidelines and regulations from “The Guide and Care and Use of Laboratory Animals” (National Institute of Health) and approved by Baylor College of Medicine’s Institutional Animal Care and Use Committee (AN-5177).

Bacterial Strains and Growth Conditions

ExPEC strain CP936 and MG1655 K12 were used as phage isolation strains. E. coli MG1655 (λ 16831) was used for the lysogeny assays. Figure 1 lists other E. coli strains (kindly provided by Dr. James Johnson, University of Minnesota) that were permissive for phage infection. All strains were grown in Lysogeny Broth (LB) at 37 °C from individual colonies selected on plates after resuscitation from a frozen stock (−80 °C, 10% glycerol).

Phage Isolation and Plaque Assay

Geese, duck, and dog feces were collected from two parks (2 miles apart) in the Houston, Texas area. Chicken feces were collected from a private farm. Fecal samples were homogenized in phage buffer (1.0 M NaCl, 0.01 M Tris, pH 8.0, 0.001 M EDTA) by rocking gently using a benchtop rocker, separated by a Sorvall Lync 4000 Lynx centrifuge (Thermo Scientific, Waltham, Mass) at 13 K rpm (~29000 g, 50 min) and the supernatant filter sterilized (0.22 μm). The filtrate was added to a soft agar overlay assay containing the isolation bacterial strain. Plaques of different morphology were selected, grown to high titer in the isolation strain and the lysates used to generate the efficiency of plating (EOP’s) presented in Fig. 1.

Phage Purification

Small batch cultures of CP9 for ϕCF2 and JJ2547 for ϕHP3 were grown to an O.D. of 0.1 at 600 nm, infected with phage lysate (MOI 0.1) until complete lysis was achieved and used to infect a large batch culture prepared in 4 Liters of LB. The lysate was centrifuged at 10 K RPM (~17000 g, 4 °C, 30 min) and precipitated with 30 g NaCl/1000 mL and 300 g of Polyethylene Glycol 8000. After overnight precipitation at 4 °C, the phage was pelleted at 10 K RPM (17000 g, 4 °C, 30 min) and then resuspended in 40 mL of phage buffer. One volume of CHCl 3 was added to the resuspended phage, emulsified, and phases separated by centrifugation, 10 K RPM (17000 g, 10 min). Cesium Chloride (CsCl) was added to the aqueous phase, treated with 5 μg/ml DNAse/RNAse mix for 30 minutes at 37 °C, adjusted to a density of 1.5 and ultracentrifuged using SW41 rotor (20 K RPM, 10 °C, 18 hrs). The phage band was collected and dialyzed in phage buffer (2000X Vol, twice).

Lysogeny testing

A lysate consisting of ϕHP3 was added (MOI 10) to stationary phase E. coli JJ2528 in an LB overlay assay. The next day, bacterial colonies insensitive to ϕHP3 were isolated from these plates and were re-plated to remove residual phage. The induction of lysogens was tested using the method described for Zeng et al.30. Insensitive colonies from strains E. coli JJ2528 and E. coli MG1655 (λ 16831) were grown to log phase in LB. Cultures were incubated with the DNA damaging agent mitomycin C (10 μg/ml, Fisher BioReagents, Waltham, Mass.) for two hours. Chloroform (2%) was added to the flasks to lyse cells. Lysed cultures were pelleted via centrifugation (7,000 × G, 10 minutes) and an aliquot removed and serially plated on a lawn of the originator strain. Other ϕHP3 insensitive colonies, E. coli JJ2528 and E. coli MG1655 (λ 16831) were lysed after log phase growth and pelleted to plate for the spontaneous induction of phage.

Measurement of burst size and latent period

These procedures were based on the experiments of Ellis and Debruick71 with some modifications72. An overnight culture of E. coli JJ2528 was grown to mid-log phase and purified ϕHP3 was added at an MOI of 1. Phage was allowed to adsorb for 20 minutes with shaking (255 rpm, 37 °C). Following adsorption, the culture was pelleted via centrifugation (10,000 × G, 5 minutes). The pellet was washed and prewarmed LB media added to resuspend the pellet. The resuspended culture was grown as before (37 °C, 255 rpm). Samples (100 μL) were taken every 10 minutes after resuspension for 2 hours total. Samples were lysed (Chloroform 2–5%) and pelleted (8,000 × G, 5 minutes), supernatant aliquots were serially diluted and plated on a lawn of E. coli JJ2528. ϕHP3 burst size was calculated as the final titer of the plateau period to the initial titer of infected bacteria73. The latent period was determined to be the interval when there was no or little increase in the titer73.

Adsorption Assays

Adsorption was measured according to established methods72. Briefly, ϕHP3 (MOI 1) was added to a mid-log phase culture of E. coli JJ2528. Immediately after addition of ϕHP3, 100 μl of culture was removed (time point “0”). This sample was added to 900 μL of chilled LB media (choroform added) then vortexed and kept on ice. This was repeated with samples taken every minute for a total of 10 minutes. All samples were pelleted via centrifugation (8,000 × G, 5 minutes) and supernatant aliquots were serially diluted and plated on a lawn of JJ2528. An adsorption curve was generated using this data with the adsorption rate constant calculated as the natural log of the slope of the graph versus the bacterial titer72.

Bacteriophage sequencing and genome assembly

Phages HP3, EC1, and CF2 were grown to high titer and purified as described above. Phage DNA extraction, sequencing and assembly was done by the Center for Metagenomics and Microbiome Research (CMMR) at Baylor College of Medicine. Phage DNA was extracted using the Applied Biosystems MagMAX Viral DNA/RNA Isolation kit (Applied Biosystems, Foster City, CA) and eluted in 50 μl of Elution Buffer. Extracted DNA samples were constructed into Illumina paired-end libraries. DNA from phages HP3, EC1, and CF2 were sheared into fragments of approximately 500–600 base pairs using a Covaris E210 system (Covaris, Inc. Woburn, MA). Products were then amplified through Ligation Mediated-PCR (LM-PCR), which was performed using the KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc., Cat. no. KM2602). Purification was performed with Agencourt AMPure XP beads after each enzymatic reaction. Following the final XP bead purification, quantification and size distribution of the LM-PCR product was determined using the Agilent Bioanalyzer 7500 chip. The libraries had an average final size of 660 bp (including adapter and barcode) and were pooled in equimolar amounts to achieve a final concentration of 10 nM. The library templates were prepared for sequencing on an Illumina MiSeq. Briefly, this library was denatured with sodium hydroxide and diluted to 6 pM in hybridization buffer in order to achieve a load density of 670 K clusters/mm2. The library pool was loaded on a MiSeq flow cell which was spiked with 1% phiX control library for run quality control. The sample then underwent bridge amplification to form clonal clusters, followed by hybridization with the sequencing primer. Sequencing runs were performed in paired-end mode using the 600V3 kit. Sequencing-by-synthesis reactions were extended for 301 cycles from each end, with an additional 10 cycles for the index read. After sequencing, the.bcl files were processed through Illumina’s analysis software (CASAVA), which demultiplexes pooled samples and generates sequence reads and base-call confidence values (qualities). The average raw yield per sample was 3,064 Mbp. Raw data files were trimmed using Babraham Bioinformatics’ TrimGalore script74, and then had human and PhiX removed using a combination of Bowtie75 and PRINSEQ76 against the Genome Reference Consortium Human Build 38 (HG38) assembly. Phage genomes were assembled using SPAdes Genome Assembler77 and verified on a Platanus Assembler78 which generated 1 complete contig per genome. Genome annotation was done using DNA Master. This program concurrently runs a collection of programs, including GLIMMER79 V. 3.02 for the prediction of open reading frames or ORFs and GeneMarkS80, to verify the calling of ORFs. ARAGORN81 V. 1.1 was used to predict tRNAs and BLASTP searches were performed on predicted ORFs for a functional analysis. Bacterial virulence genes were searched using VirulenceFinder29 and genes encoding antibiotic resistance using ResFinder31.

Cryo-electron Microscopy

Quantifoil grids of mesh size 400 (Quantifoil Micro Tools GmbH, Großlöbichau, Germany) were rendered hydrophilic by 20–25 s glow discharge, or in the case of phage CF2, by 5 s plasma discharge in a Solarus Model 950 (Gatan, Pleasanton, CA). Next, 3 μL of phage were applied to the grid, blotted for 1–2.5 s, and vitrified in a Vitrobot Mk IV (FEI, Hillsboro, OR). Grids were imaged in JEOL 200 kV cryoelectron microscopes and images were recorded with a DE12 direct detection device (Direct Electron LP, San Diego, CA) or with a US4000 CCD (Gatan). To render figures, images were low-pass filtered to a resolution of 20 Å using EMAN282. Feature lengths were measured manually in EMAN2 based on calibrated detector pixel sizes.

Murine Infections

All methods described below were approved in accordance with relevant guidelines and regulations by the Institutional Animal Care and Use Committee.

Injectional bacteremia model

Overnight bacterial cultures were grown to log phase at 37 °C (250 rpm). Cultures were washed twice in 1XPBS. Colony forming units (CFU) were determined by plating on bacteriologic agar and a dose of 108 CFU injected intraperitonealy (IP)83. After 1 hour, infected mice were injected with purified ϕHP3, IP, with 109 plaque forming units - PFU in 100 μl of PBS.

Translocation bacteremia model

This model system is described in detail in reference36. Briefly, mice were gavaged with 109 CFU of ExPEC strain JJ2528 and animals administered cyclophosphamide (Cytoxan, Baxter Healthcare Corporation, Deerfield, IL, USA) IP for a total dose 450 mg/kg body weight on days 1, 3 and 5 as indicated in Fig. 3. Purified ϕHP3 was administered IP on the days 2, 4 and 6 as indicated in Fig. 3 and the mice necropsied on day 7 post infection.

Measurement of health scores

The scale for measuring murine health is based on four scored parameters, including activity (lethargy), grooming behavior (rough coat), body condition (hunched posture), and respiratory distress (hypernea or labored breathing), as outlined by the NIH Animal Research Advisory Committee Guidelines for the evaluation of rodent health84. Each observation of one of these criteria during a 20 minute period receives a score of 0, 0.5 or 1 depending on the severity. Mice with respiratory distress receive a score of 0 for normal breathing or a score of 1 for labored breathing (hyperpnea). All other parameters receive either a 0 (no disease), 0.5 (moderate) or 1 (severe). The scoring is performed by a blinded investigator that is trained to recognize these symptoms. When animals reach a score of 4, they are considered moribund and must be sacrificed according to our protocol approved by our institutional review board.

Bacterial and phage quantification of organ and fecal homogenates

Immediately following euthanization mice were necropsied under sterile conditions. Lungs, kidneys, liver and spleen were weighed and homogenized in 1XPBS with sterile blades. Organ homogenates were serially diluted and plated on LB plates and incubated at 37 °C overnight. Phage counts were quantified using a soft agar overlay assay. Fecal counts were determined on LB and Ampicillin, 100 μg/ml, plates to prevent growth of other commensal flora.

Statistics

Statistical significance values were determined using “R” version 3.2.4. For Fig. 3 panel B, and Fig. 4 panels A-i,B-i and C-i, a Wilcoxon rank sum test was used to assess significance. For Fig. 3 panel C, Fig. 4 panels A,ii–v,B,ii–v and C,ii–v and Fig. 5 panels B and C a two-tailed T-test on normalized data (log transformed) was used to test significance. For Figs 3D, 4D and 5D a One-Way ANOVA test was performed on normalized data and Tukey’s test for multiple comparison was used to determine significance. Unless otherwise noted, alpha values were set to 0.05 and statistical significance was determined if calculated P values were below 0.05. Figure “n” values are reported in the legend under each figure.