Pathogen-phage geomapping to overcome resistance
- Department of Molecular Virology and Microbiology, Baylor College of Medicine, United States
- TAILΦR LABS, Baylor College of Medicine, United States
- Department of Electrical & Computer Engineering, University of Houston, United States
- Section of Infection Diseases, Department of Medicine, Baylor College of Medicine, United States
Figures
Figure 1
Overview and schematic of ΦHD.
(A) Pie charts of patient isolate status and percentage of bacterial strain with no phages for in TAILΦR’s library. Unfortunately, 24% of isolates that TAILΦR receives have no phages; at 35%, most of the isolates are Pseudomonas aeruginosa. (B) Diagram from phage request to phage discovery and limitation. Clinicians and their patients can seek phage therapy for antibiotic-resistant infections when there are no other approved treatments available. After approval, clinicians send patient isolates to TAILΦR. TAILΦR’s phage library and wastewater concentrates are screened against the patient isolate. If phages are discovered, they are moved onto the next stage for preparation where phages are ultimately purified and tested for safety and efficacy before being handed to the clinician. When there are no phages for that isolate in the library, TAILΦR searches for phages through environmental sampling. Alternatively, they can train related phages to infect the isolate through directed phage evolution. (C) Diagram of strategies employed in our study. Geographical phage (Φ) mapping (geΦmapping) allowed us to pinpoint target sites rich in our pathogen of interest from our environment (left). Once a location is chosen, we used our capture device, the phage hunting device (ΦHD), to filter, concentrate, and enrich our sample with phage and bacteria (middle). Bar graph shown here was derived from Figure 2F as an example for phage concentration. With the samples, we further analyzed them with phage metagenomics (ΦMICS) and screened, isolated, and characterized phage present in the samples (right). This led to the curation of the resistant phage (Φ) library (RΦ-Library). (D) Schematic of ΦHD. The components on the right of the reservoir draw, filter, and concentrate water through ΦHD. On the left of the reservoir, the remaining component concentrates any material in the reservoir. Created with BioRender.com.
Figure 2 with 2 supplements
GEΦMAPPING highlights Bray’s Bayou as a Pseudomonas-rich target site, and performance of ΦHD sampling of several environmental sites.
(A) Geographical map of freshwater samples obtained in Houston, TX, USA. (B) Principal Coordinate Analysis (PCoA) analysis showing β-diversity differences between sewage, sea, and freshwater sources. The black arrow indicates the Vince Bayou sample. (C) PCoA analysis (Bray–Curtis dissimilarity) of environmental water bodies with Bray’s Bayou target sites highlighted (purple). (D) Heat map with z-score normalized by site shows distinct patterns of microbial prevalence at various sites. (E) Heat map, z-score normalized by pathogen, highlights the potential of Bray’s Bayou as a target site for Pseudomonas and enteric phages. (F) Quantification of anti-Pseudomonas (left) and anti-Vibrio (right) plaques (PFU/mL) from different stages of processing (input, first stage, and second stage processing). (G) Heatmap showing anti-pathogen phages (log10[PFU/mL]) from freshwater sites. We display the mean and SEM of three technical repeat measurements. (H) Plaque assay on P. aeruginosa PAO1 of unprocessed, first stage concentrate, and second stage concentrate of a freshwater sample. (I) TEM of unprocessed water from freshwater. (J) Plaque assay on Vibrio parahaemolyticus 17802 of unprocessed, first stage concentrate, and second stage concentrate seawater sample. (K) TEM of unprocessed water from seawater, respectively. Representative images shown (H–K). Statistical analysis. (F, left panel) Phage titers were compared using a two-way ANOVA with Dunnett’s multiple comparisons test (α=0.05; n=3 technical replicates per condition), revealing significant effects of sampling site (F(1,12) = 419.7, p<0.0001), processing stage (F(2,12) = 670.2, p<0.0001), and their interaction (F(2,12) = 338.3, p<0.0001), indicating that the effect of processing stage differed between sites. Second-stage processing significantly increased phage yield relative to unprocessed input at both Bray’s Bayou (mean difference = 25,744 PFU/mL, p<0.0001) and Clear Creek (mean difference = 4,325 PFU/mL, p<0.0001); first-stage processing did not reach significance at either site (p=0.075 and p=0.945, respectively). (F, right panel) Phage titers across processing stages were compared using a one-way ANOVA with Dunnett’s multiple comparisons test (α=0.05; n=3 technical replicates per condition), revealing a significant effect of processing stage (F(2,6) = 48.00, p=0.0002). Second-stage processing yielded significantly higher titers relative to unprocessed input (p=0.0003), while first-stage processing did not differ significantly from input (p>0.9999). We display the mean and SD of three replicates for both graphs.
Figure 2—figure supplement 1
16 S analysis shows adequate sampling (rarefaction curves) and high diversity (α-diversity indices) for sampling collection.
(A) Rarefaction curves for each site show no sites were under-sampled in environmental sites (top panel) or sewage sites (bottom panel). Curves represent the mean of triplicate biological samples for all sites except for West University WWTP. Rarefaction was performed in MOTHUR with the default 1000 randomizations. (B) α-diversity indices for sewage sites only (top panel) and environmental sites only (bottom panel). Left panels show the Inverse Simpson’s Index, middle panels show the Shannon index, and right panels show the Chao’s index. Averages represent biological triplicates, except for West University measurements where only single replicates were available, and error bars show SEM. α-diversity calculations were made using MOTHUR where we standardized the calculation to 50,000 random sequences for each group.
Figure 2—figure supplement 2
Spike-in and early on-field ΦHD sampling experiments.
(A) Schematic of ΦHD spike-in study where 60 L of pond water was spiked with 1x103 PFU/mL of Pseudomonas phage JB10. We used two set-ups with changes in the upstream filters: 100/25 µm or 25/5 µm set-up. (B) Bacterial counts (left) and phage titers (right) were measured for each upstream filtration (status post s/p 100/25 or s/p 25/5) and after concentration with ΦHD (retentates). Indicator strain used was P. aeruginosa PAO1. (C) Validation of cleaning in place (CIP) protocol on PAO1 after spike-in study. Mean and SEM from two to four technical replicates are shown. With ΦHD, we performed our field test at Santa Ana Capture Site (SACS). (D) Representative plates of unprocessed and first stage concentrated samples from the SACS, plated on LB agar or Pseudomonas PAO1 lawns. (E) Quantification of anti-Pseudomonas plaques and bacterial colonies from SACs from two separate runs. We incorporated an in-lab, benchtop concentration step to our ΦHD retentates. (F) Schematic of second concentration phase of the ΦHD system in-lab. Using ΦHD, we sampled freshwater from Pedernales Falls (PF), Hamilton Pool (HP), and SACS. (G) Quantification of anti-Pseudomonas phages from PF, HP, and SACS before processing and after second stage processing. (H) TEM imaging shows sparse virus-like particles (VLPs) in unprocessed SACS samples compared to concentrated VLPs in SACS retentate samples. Representative images shown. (I) Heat-map showing anti-pathogen phages from each sampled site. We display the mean and SEM of three technical repeat measurements.
Figure 3 with 1 supplement
GEΦMAPPING identified Enterococcus and Enterobacteriaceae was rich at West University WWTP, and performance of ΦHD sampling in wastewater.
(A) Schematic of geographical areas drained by WWTPs in Houston, TX, USA. (B) β-diversity analysis shows variation in OTUs present in WWTPs by PCoA analysis based on Bray–Curtis dissimilarity (AB, colors represent differing WWTPs). (C) Heatmap analysis of pathogenic taxa highlights (black box) location of enteric pathogens (z-score normalized by taxa). (D) 16 S analysis further localizes pathogenic taxa to wastewater influent (z-score normalized by taxa). (E) Quantitation of plaques on index strains confirms 16 S sequencing data. (F) Titration of VLPs from different WWTP processing areas on PAO1 (P. aeruginosa) and K-12 (Escherichia coli) index strains (representative data shown). (G) Outline of prefiltration and sedimentation step for wastewater sites. (H) Quantification of anti-Pseudomonas plaques and anti-Escherichia plaques from different stages of processing shows phage retention through the system and concentration of the end products. (I) Titration of VLPs from different WWTP processing areas on PAO1 (P. aeruginosa). (J) TEM of unprocessed ΦHD input from wastewater. (K) TEM of second-stage concentrated wastewater. Representative images shown (F, I–K). Statistical analysis. (E) Phage titers across wastewater treatment stages and indicator strains were compared using a two-way ANOVA with Tukey’s multiple comparisons test (α=0.05; n=3 technical replicates per condition), revealing significant effects of treatment stage (F(2,12) = 399.3, p<0.0001), indicator strain (F(1,12) = 35.31, p<0.0001), and their interaction (F(2,12) = 16.87, p=0.0003), indicating that the effect of treatment stage differed between indicator strains. Influent yielded significantly higher phage titers than both contact and aeration tanks for PAO1 (p<0.0001 for both) and K-12 (p<0.0001 for both), while no significant difference was detected between contact and aeration tanks for either strain (PAO1: p=0.093; K-12: p>0.9999). (H) Phage titers across wastewater processing stages and indicator strains were compared using a two-way ANOVA with Dunnett’s multiple comparisons test (α=0.05; n=3 technical replicates per condition). A significant effect of processing stage was detected (F(4,20) = 31.68, p<0.0001), while neither indicator strain (F(1,20) = 0.188, p=0.669) nor the interaction between strain and processing stage (F(4,20) = 0.262, p=0.899) reached significance, indicating that processing stage drove phage yield increases independent of indicator strain. Second-stage processing yielded significantly higher titers relative to unprocessed influent for both PAO1 (p<0.0001) and K-12 (p<0.0001), while sedimentation, pre-filtration, and first-stage processing did not differ significantly from unprocessed influent for either strain (all p>0.6). We display the mean and SD of three replicates for both graphs.
Figure 3—figure supplement 1
Field photos of ΦHD.
(A) Buffalo Bayou is accessible at Santa Ana Capture Site. It is relatively close to the Port of Houston where ships enter and exit the Gulf of Mexico. Generally, this location is polluted with trash and dead plant material. There is also a warning sign that cautions women and children to not eat any spotted seatrout, catfish, or blue crab caught in the Bayou due to toxic pollutants present. (B) West University WWTP (influent). Raw wastewater enters the plant through large pipes where they are subjected to a large filter (steel bars) to filter hygiene products and trash. In the picture, the influent collects in this container and is propelled upwards with a large Archimedean screw (toward the contact tank).
Figure 4 with 2 supplements
Taxonomic classification of viral contigs from metagenomic datasets reveals distinct viral signatures as well as biased presence of viruses of pathogenic hosts between different locations.
(A) vConTACT2 gene-sharing network of 16,198 viral operational taxonomic units (vOTUs; ≥5 kb) from West University WWTP, Brays Bayou, Clear Creek, and Galveston. Each dot (node) represents a vOTU, and each line (edge) represents the similarity between each genome. Additionally, 3508 reference genomes from the Prokaryotic Viral RefSeq v201 database are shown in grey. (B) Pie-chart of viral cluster status of vOTUs from vConTACT2. (C) Pie-chart of classified (family-level) vs unclassified vOTU from PhaGCN. (D) Bar graph of family-level classification (PhaGCN counts) of topmost abundant genera across the samples. Remaining genera are grouped together as ‘others’ in grey. Unclassified vOTUs are in black. (E) Heatmap with z-score normalized by viral taxa to show distinct viral signatures between sites (pheatmap separated each cluster). (F) Venn-diagram of shared vOTUs (cluster mode: amino acid identity [AAI] 45%, protein coverage [PC] 80%) depicts the number of unique vOTUs present at each site and shared between sites. (G) Heatmap with z-score normalized by host reveals the relevance of sampling specific sites for phage hunting.
Figure 4—figure supplement 1
ΦHD concentrate (processed 400 L) has a greater number of viruses present than unprocessed sample (5 L).
(A) Heatmap of viral population abundances, calculated using z-scores. The 21,918 vOTUs identified from the 400 L and 5 L freshwater samples (Supp. File 8) were used as the reference genomes for MetaPop’s macrodiversity pipeline. (B) Heatmap normalized by viral taxa (10 kb cut-off for vOTUs). We classified vOTUs with PhaGCN to the family level and generated heatmaps to visualize the concentration of taxa from a 5 L vs 400 L sample. (C) Heatmap normalized by predicted hosts (10 kb cut-off for vOTUs). We predicted hosts of each vOTU and generated heatmaps to visualize the concentration of pathogenic taxa from each sample. (D) Phylogenetic tree of all vOTUs predicted to target Pseudomonas hosts from 6667 X (100 vOTUs) and 5 L unprocessed sample (25 vOTUs). Greater than half of the vOTUs from the 5 L sample have some relatedness to the vOTUs from the 400 L ΦHD-concentrate. (E) vConTACT2 network analysis of clustered vOTUs infecting Pseudomonas and first neighbors, including reference genomes. (F) Venn-diagram of vOTUs-grouping (cluster mode: AAI 45%, 15 SP, PC 80%) shows shared vOTUs between samples.
Figure 4—figure supplement 2
Viral community patterns across biomes.
(A) Heatmap of z-scored viral population abundances reveals distinct feature-level patterns between biomes. Abundance Files were generated using the MetaPop macrodiversity pipeline with reference genome set as the collection of all vOTUs identified across the four biomes. (B) PCoA (based on Bray–Curtis dissimilarity) compares viral communities in seawater (blue), freshwater (green), brackish water (yellow), and wastewater (orange). For PCoA, 2000 features were randomly subsampled and analysis repeated across 1000 bootstrap iterations. Resulting ordinations were aligned to a reference with Procrustes alignment. Mean coordinates and standard deviations were calculated for each sample.
Figure 5
Relevance of multi-site sampling for increased diversity and collection of unique Pseudomonas and Escherichia phages.
(A) Phylogenetic tree of all vOTUs predicted to have Pseudomonas host from West University WWTP wastewater and Brays Bayou freshwater metagenomic dataset. Branch-length is non-scaled. (B) Venn-diagram of vOTUs-grouping (cluster mode: AAI 45%, 15 shared protein (SP), PC 80%) shows no common vOTUs shared between sites. (C) Phylogenetic tree of all vOTUs predicted to have Escherichia host from wastewater and Brays Bayou metagenomic dataset. Branch-length is non-scaled. (D) Venn-diagram of vOTUs-grouping (cluster mode: AAI 45%, 15 shared protein [SP], PC 80%) shows no common vOTUs shared between sites. Bar graphs (A, B) represent the genomic similarities between a contig and its closest neighbor based on genome-wide sequence similarities computed by tBLASTx from VipTree.
Figure 6 with 2 supplements
ΦHD increases search volumes and allowed efficient discovery of phages for phage-resistant bacterial pathogens.
(A) Schematic of ΦHD compared to standard identification and discovery of therapeutic phages. (B) Effective volumes that can be searched comparing standard spotting of concentrates with high-concentration, high-volume ΦHD retentates (n=2 retentates with differing final concentrations). We display the mean and SD of two replicates. (C) Heatmap showing success/failure of ΦHD compared to the TAILΦR library in finding therapeutic phage candidates. (D) Plaque assays (left column) on phage-resistant strains EUA02 (K. pneumoniae) and HPC3.1 (P. aeruginosa). Individual plaques (middle columns) were streaked and isolated from plaque assays. TEM images (right columns) of select isolated phages infecting EUA02 and HPC3.1. Representative images shown. (E) Dendrograms of all isolated phages for Pseudomonas, Escherichia, Klebsiella, Enterococcus. Phages from ΦHD are in red, and phages from the TAILΦR library in blue. (F) Diagram of the implementation of ΦHD and geΦmapping to the phage discovery pipeline. Created with BioRender.com.
Figure 6—figure supplement 1
Unique phages isolated from GEΦMAPPING and ΦHD sampling.
Proteomic clustering (VIPTree) of UCS29C1, MYC30C1_1, and MYC30C1_2 with their closest neighbor found through BLASTn to look at how distinct their genomes are (top panel). Aligned phages with their closest neighbor to depict their percent identity (bottom panel).
Figure 6—figure supplement 2
Annotated genomes of unique ΦHD-isolated phages UCS29C1, MYC30C1_1, and MYC30C1_2.
Organization of UCS29C1, MYC30C1_1, and MYC30C1_2 genomes. Circular genome map was displayed with CGView. Genomes were annotated using RAStk. Coding sequences (CDS, black) are depicted on the two outer rings. GC content is shown in black in the middle ring, while GC skew is represented on the two innermost rings, with positive skew in green and negative skew in purple.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Acinetobacter baumannii) | 17978 | ATCC 17978 Bouvet and Grimont, 1986 | Received from TAILФR LABS; bacterial strain originally isolated from fatal meningitis; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (Achromobacter xylosoxidans) | UTS1 | Gift-TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from cystic fibrosis (CF) lung infection; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (Enterobacter cloacae) | SLC1 | Gift-TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (Enterococcus faecalis) | VRE001 | Gift - TAILOR Labs. El Haddad et al., 2022 | Received from TAILФR LABS; Vancomycin-resistant isolate; bacterial strain originally isolated from patient stool; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (Escherichia coli) | K12 | Gift - TAILOR Labs. Blattner et al., 1997 | Received from TAILФR LABS; Wild-type, common laboratory strain; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G; also used as our phage indicator strain (Figure 3E, F, H and I) | |
| Strain, strain background (E. coli) | JJ2528 | Gift - TAILOR Labs. Price et al., 2013 | Received from TAILФR LABS; ExPEC ST131 H30-R; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (Klebsiella aerogenes) | UCF1 | Gift - TAILOR Labs. Terwilliger et al., 2021 | Received from TAILФR LABS; bacterial strain originally isolated from hip prosthesis wound; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (Klebsiella pneumoniae) | EUA2 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from urinary tract infection (UTI); Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (Pseudomonas aeruginosa) | DSA497 | Gift - TAILOR Labs | Received from TAILФR LABS; Ramig LAB; bacterial strain originally isolated from UTI; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (P. aeruginosa) | PA01 | Gift - TAILOR Labs. Holloway, 1955 | Received from TAILФR LABS; Common laboratory strain; bacterial strain originally isolated from wound; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G; also used as our indicator strain (Figure 3E, F and H) | |
| Strain, strain background (Staphylococcus aureus) | AZM28 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from dog skin swab #13–1 on MSA; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (Staphylococcus pseudintermedius) | AZM69 | Gift - TAILOR Labs | Received from TAILФR LABS; Coagulase positive; bacterial strain originally isolated from canine ear infection; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (P. aeruginosa) | AR0246 | CDC and FDA Antibiotic Resistance Isolate Bank; Pseudomonas aeruginosa (PSA) Panel | Received from TAILФR LABS; MDR strain; CDC and FDA Antibiotic Resistance Isolate Bank; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (P. aeruginosa) | AR0266 | CDC and FDA Antibiotic Resistance Isolate Bank; Pseudomonas aeruginosa (PSA) | Received from TAILФR LABS MDR strain; CDC & FDA Antibiotic Resistance Isolate Bank; Used in the multidrug-resistant isolate panel for phage screening - Figure 2G | |
| Strain, strain background (E. coli) | BSL02 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from UTI; Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (E. coli) | UCS29 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from UTI; Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (E. coli) | UCS37 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from urine (pyelonephritis, sepsis); Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (E. coli) | UCF06 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from bone (infected amputation); Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (K. aerogenes) | MYC16 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from shoulder prosthetic joint infection; Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (K. pneumoniae) | EUA02 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from UTI; Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (K. pneumoniae) | EUA03 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from UTI; Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (K. pneumoniae) | UCS26 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from UTI, bacteremia; Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (K. pneumoniae) | UCS26.1 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from UTI, bacteremia; Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (S. aureus) | MYC20 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from sputum (bronchiectasis); Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (E. coli) | UCS17.2 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from UTI; Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (P. aeruginosa) | CSM01 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from sputum (lung abscess, adjacent sternal osteomyelitis); Used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (P. aeruginosa) | UCS18 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from bile duct (biliary infection, sepsis); used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (P. aeruginosa) | UCS20 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from chest drain (lung, chest wall infection); used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (P. aeruginosa) | UCS23 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from UTI; used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (P. aeruginosa) | MGB1.4 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from sputum (CF lung); used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (P. aeruginosa) | HPC3.1 | Gift - TAILOR Labs | Received from TAILФR LABS; bacterial strain originally isolated from sputum (lung); used in the XΦRO screening panel for phage isolation - Figure 6C; | |
| Strain, strain background (Vibrio parahaemolyticus) | 17802 | ATCC 17802 Fujino et al., 1974 | Purchased from ATCC; isolated from Shirasu food poisoning; used to produce Figure 2F and J (right panel); used to isolate Vibrio phages | |
| Strain, strain background (Vibrio vulnificus) | 27562 | ATCC 27562 Reichelt et al., 1976 | Purchased from ATCC; isolated from human blood; used as part of our panel to screen concentrated seawater for Vibrio phages; did not result in phage | |
| Strain, strain background (Vibrio parahaemolytic phage) | VP1 | This paper | Novel bacteriophage isolated in this study; isolated from seawater concentrate; strain used to isolate was 17802 | |
| Strain, strain background (V. parahaemolytic phage) | VP2 | This paper | Novel bacteriophage isolated in this study; isolated from seawater concentrate; strain used to isolate was 17802 | |
| Strain, strain background (V. parahaemolytic phage) | VP3 | This paper | Novel bacteriophage isolated in this study; isolated from seawater concentrate; strain used to isolate was 17802 | |
| Strain, strain background (V. parahaemolytic phage) | VP4 | This paper | Novel bacteriophage isolated in this study; isolated from seawater concentrate; strain used to isolate was 17802 | |
| Strain, strain background (Escherichia phage) | UCS29C1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS29; used to produce Figure 6E | |
| Strain, strain background (E. phage) | UCS29C2 | This paper | Novel bacteriophage isolated in this study; isolated from UCS29; used to produce Figure 6E | |
| Strain, strain background (E. phage) | UCS37C1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS37; used to produce Figure 6E | |
| Strain, strain background (E. phage) | UCS37C2_1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS37; used to produce Figure 6E | |
| Strain, strain background (E. phage) | UCS37C2_2 | This paper | Novel bacteriophage isolated in this study; isolated from UCS37; used to produce Figure 6E | |
| Strain, strain background (E. phage) | BSL02C1_1 | This paper | Novel bacteriophage isolated in this study; isolated from BSL02; used to produce Figure 6E | |
| Strain, strain background (E. phage) | BSL02C1_2 | This paper | Novel bacteriophage isolated in this study; isolated from BSL02; used to produce Figure 6E | |
| Strain, strain background (E. phage) | BSL02C2 | This paper | Novel bacteriophage isolated in this study; isolated from BSL02; used to produce 6E | |
| Strain, strain background (Enterococcus phage) | MYC30C1_1 | This paper | Novel bacteriophage isolated in this study; isolated from MYC30; used to produce Figure 6E | |
| Strain, strain background (E. phage) | MYC30C1_2 | This paper | Novel bacteriophage isolated in this study; isolated from MYC30; used to produce Figure 6E | |
| Strain, strain background (E. phage) | UCS17X2C1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS17; used to produce Figure 6E | |
| Strain, strain background (E. phage) | UCS17X2C2 | This paper | Novel bacteriophage isolated in this study; isolated from UCS17; used to produce Figure 6E | |
| Strain, strain background (Klebsiella phage) | UCS26C1_1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26C1_2 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | EUA02C1 | This paper | Novel bacteriophage isolated in this study; isolated from EUA02; used to produce Figure 6E | |
| Strain, strain background (K. phage) | EUA3C1 | This paper | Novel bacteriophage isolated in this study; isolated from EUA03; used to produce Figure 6E | |
| Strain, strain background (K. phage) | MYC16C2 | This paper | Novel bacteriophage isolated in this study; isolated from MYC16; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26X1C1_1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26X1C2_2 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26X1C2_1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26X1C2_2 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26X1C2_3 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26X1C2_4 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26X1C2_5 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26X1C2_6 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26C2_1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26C2_2 | This paper | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | |
| Strain, strain background (K. phage) | UCS26C2_3 | Novel bacteriophage isolated in this study; isolated from UCS26; used to produce Figure 6E | ||
| Strain, strain background (K. phage) | MYC16C1 | This paper | Novel bacteriophage isolated in this study; isolated from MYC16; used to produce Figure 6E | |
| Strain, strain background (Pseudomonas phage) | CSM01C1 | This paper | Novel bacteriophage isolated in this study; isolated from CSM01; used to produce Figure 6E | |
| Strain, strain background (P. phage) | UCS20C3 | This paper | Novel bacteriophage isolated in this study; isolated from UCS20; used to produce Figure 6E | |
| Strain, strain background (P. phage) | UCS18C3_1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS18; used to produce Figure 6E | |
| Strain, strain background (P. phage) | UCS18C3_2 | This paper | Novel bacteriophage isolated in this study; isolated from UCS18; used to produce Figure 6E | |
| Strain, strain background (P. phage) | UCS20C5 | This paper | Novel bacteriophage isolated in this study; isolated from UCS20; used to produce Figure 6E | |
| Strain, strain background (P. phage) | UCS20C4 | This paper | Novel bacteriophage isolated in this study; isolated from UCS20; used to produce Figure 6E | |
| Strain, strain background (P. phage) | 6X1C4 | This paper | Novel bacteriophage isolated in this study; isolated from 6.1; used to produce Figure 6E | |
| Strain, strain background (P. phage) | UCS20C1 | This paper | Novel bacteriophage isolated in this study; isolated from UCS20; used to produce Figure 6E | |
| Strain, strain background (P. phage) | MGB1X4C4 | This paper | Novel bacteriophage isolated in this study; isolated from MGB1.4; used to produce Figure 6E | |
| Strain, strain background (P. phage) | MGB1X4C1 | This paper | Novel bacteriophage isolated in this study; isolated from MGB1.4; used to produce Figure 6E | |
| Strain, strain background (P. phage) | UCS18C4 | This paper | Novel bacteriophage isolated in this study; isolated from UCS18; used to produce Figure 6E | |
| Strain, strain background (P. phage) | MGB1X4C3 | This paper | Novel bacteriophage isolated in this study; isolated from MGB1.4; used to produce Figure 6E |
Additional files
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Supplementary file 1
TAILOR distinct case count and number of isolates in their bacterial library.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp1-v1.xlsx
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Supplementary file 2
TAILOR phage library.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp2-v1.xlsx
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Supplementary file 3
Number of isolates with NO phage in TAILOR’s library.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp3-v1.xlsx
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Supplementary file 4
Various viral metagenomic studies with approximate volume processed and reported viral contigs.
A total of 16,198 virus operational taxonomic units (vOTUs) were identified from four specific sites in this studies’ metagenomic datasets. 5095 vOTUs were ≥10 kb. Viral contigs were identified from trimmed and cleaned reads with VIBRANT. vOTUs were clustered with standard thresholds of 95% average nucleotide identity over 85% alignment fraction. Additional selected references are shown to depict total volume sampled, viral contigs/viral populations present, and sampling strategy used.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp4-v1.xlsx
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Supplementary file 5
Environmental sampling summary.
Water from various sites have been processed with ΦHD and further concentrated in-lab; volume processed and concentration after second stage processing are listed. Concentrates were titered on indicator strain P. aeruginosa PA01. DNA was extracted from 11.5 mL of each concentrate and sent for PCR-free shallow shotgun metagenomic sequencing. After metagenomic analysis, viral contigs were identified and clustered into vOTUs based on 5 kb and 10 kb cut-offs. Raw sequences were uploaded to SRA (BioProject #: PRJNA1308632).
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp5-v1.xlsx
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Supplementary file 6
α-diversity analysis for metagenomic datasets of unprocessed (5 L sample) and processed ϕHD concentrate (60 mL 6667 X concentrate) from freshwater.
We used MetaPop’s macrodiversity pipeline to calculate diversity.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp6-v1.xlsx
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Supplementary file 7
Viral counts of unprocessed vs. processed concentrates.
We compared the viromes of unprocessed and ΦHD processed freshwater samples. ΦHD was used to process 400 L of freshwater from Brays Bayou and further concentrated in-lab. Final concentration was 6667 X, and the total volume was used for DNA extraction. 5 L of unprocessed freshwater was concentrated down to 60 mL and used for DNA extraction. DNA was sent for PCR-free shallow shotgun metagenomic sequencing.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp7-v1.xlsx
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Supplementary file 8
α-diversity analysis for metagenomic datasets of brackish, sea, waste, and freshwater.
We used MetaPop’s macrodiversity pipeline to calculate diversity.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp8-v1.xlsx
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Supplementary file 9
Characteristics of sequences assembled from members of the R-Phage library.
Nearest neighbors determined by total score (BLASTn), coverage, and identity also from BLASTn output. Determination of assemblies representing the same phage by nucleotide alignment (BLASTn), proteomic clustering (VIPTree), plaque morphologies, and known origins of the host. Phages highlighted with the same color indicate they are identical. Submission of whole genome sequences to GenBank in progress.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp9-v1.xlsx
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Supplementary file 10
Samples collected for GEOMAPPING from environmental and wastewater.
Samples collected for 16 S sequencing were collected on the days recorded MM/DD/YYYY. Latitude/Longitude were recorded as well as temperature (High °F/ Low °F) and precipitation (inches). Raw sequences were uploaded to SRA (BioProject #: PRJNA1309115).
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp10-v1.xlsx
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Supplementary file 11
Strains used in the study.
We include short-hand names, full strain names, characteristics, and references for the strains.
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp11-v1.xlsx
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Supplementary file 12
Additional phages found from this study.
Nearest neighbors determined by total score (BLASTn), coverage, and identity also from BLASTn output. Determination of assemblies representing the same phage by nucleotide alignment (BLASTn), proteomic clustering (VIPTree), plaque morphologies, and known origins of the host. Phages highlighted with the same color indicate they are identical. Submission of whole genome sequences to GenBank in progress
- https://cdn.elifesciences.org/articles/109259/elife-109259-supp12-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/109259/elife-109259-mdarchecklist1-v1.docx
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Source data 1
Processed and raw outputs used to generate figures, graphs, and diagrams in this article.
- https://cdn.elifesciences.org/articles/109259/elife-109259-data1-v1.xlsx
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Pathogen-phage geomapping to overcome resistance
eLife 15:RP109259.
https://doi.org/10.7554/eLife.109259.3
