<i>S</i>. Typhi phage isolation from environmental samples collected in Nepal.

S. Typhi phage isolation from environmental samples collected in Nepal.</p

Host range activity of 10 <i>S</i>. Typhi phages isolated in our study.

S. Typhi phages (y-axis) were spotted on S. Typhi isolates (x-axis) belonging to different genotypes. Colored circles represent lytic activity, white circles represent no lytic activity.</p

Phylogenetic tree of 27 S. Typhi phages isolated from Nepal and other 27 phages based on two gene sequences (tail fiber and terminase).

Tail fiber and terminase large subunit sequences of related phages were downloaded from NCBI. Phages isolated in our study are annotated with a black circle. Pickard et al., 2010 [15]* represents a S. Typhi Vi phage collection obtained from Cambridge University, Cambridge, United Kingdom, and originally isolated between the 1930 and 1955. These historical phages are annotated with white circles.</p

The main characteristics of river and drinking water samples collected in the study area.

The main characteristics of river and drinking water samples collected in the study area.</p

Fig 1 -

(a) Sampling location and detection of Salmonella Typhi phages in Nepal. Environmental water sample collection locations and percentage of samples collected with phage detection performed in (b) Kathmandu Valley and neighboring Kavrepalanchok district, (c) rural Dolakha and (d) urban Biratnagar. Topographic maps are overlayed with population density by shades of red. The basemap layer is from Open Street Maps (https://download.geofabrik.de/asia/nepal.html) and was rendered using ArcGIS Pro (https://www.esri.com/en-us/arcgis/products/arcgis-pro/overview).</p

Genome characteristics of 27 <i>S</i>. Typhi phages sequenced in our study.

Genome characteristics of 27 S. Typhi phages sequenced in our study.</p

Bacterial strains used in this study.

BackgroundEnvironmental surveillance, using detection of Salmonella Typhi DNA, has emerged as a potentially useful tool to identify typhoid-endemic settings; however, it is relatively costly and requires molecular diagnostic capacity. We sought to determine whether S. Typhi bacteriophages are abundant in water sources in a typhoid-endemic setting, using low-cost assays.MethodologyWe collected drinking and surface water samples from urban, peri-urban and rural areas in 4 regions of Nepal. We performed a double agar overlay with S. Typhi to assess the presence of bacteriophages. We isolated and tested phages against multiple strains to assess their host range. We performed whole genome sequencing of isolated phages, and generated phylogenies using conserved genes.FindingsS. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river and 6.3% (1/16) drinking water samples from the Kathmandu Valley and Kavrepalanchok. Water samples collected within or downstream of population-dense areas were more likely to be positive (72.6%, 193/266) than those collected upstream from population centers (5.3%, 5/95) (p=0.005). In urban Biratnagar and rural Dolakha, where typhoid incidence is low, only 6.7% (1/15, Biratnagar) and 0% (0/16, Dolakha) river water samples contained phages. All S. Typhi phages were unable to infect other Salmonella and non-Salmonella strains, nor a Vi-knockout S. Typhi strain. Representative strains from S. Typhi lineages were variably susceptible to the isolated phages. Phylogenetic analysis showed that S. Typhi phages belonged to the class Caudoviricetes and clustered in three distinct groups.ConclusionsS. Typhi bacteriophages were highly abundant in surface waters of typhoid-endemic communities but rarely detected in low typhoid burden communities. Bacteriophages recovered were specific for S. Typhi and required Vi polysaccharide for infection. Screening small volumes of water with simple, low-cost (~$2) plaque assays enables detection of S. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance.</div

Phylogenetic tree of <i>S</i>. Typhi isolated from Nepal.

All pairwise comparisons of the nucleotide sequences were conducted using the Genome-BLAST Distance Phylogeny (GBDP) method under settings recommended for prokaryotic viruses using VICTOR software. The resulting intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME including SPR postprocessing. Branch support was inferred from 100 pseudo-bootstrap replicates each. Branches with bootstrap values below 50 were collapsed and the bootstrap values equal to or above 50 are shown on the remainder of the tree branches. (TIF)</p

Heatmap showing pairwise intergenomic similarities (%) of <i>S</i>. Typhi isolated from Nepal.

The numbers and colors indicate similarity between the phage genomes from none or lower (red) to high (dark red). (TIF)</p

Nucleotide-based intergenomic similarities of <i>S</i>. Typhi isolated from Nepal, using VIRIDIC.

A heatmap of hierarchical clustering of the intergenomic similarity values was generated and given as percentage values (right half, blue-green heatmap). Each genome pair is represented by three values (left half), where the top and bottom represent the aligned genome fraction for the genome in the row and column, respectively. The middle value represents the genome length ratio for each genome pair. (TIF)</p