Detection of Salmonella Typhi bacteriophages in surface waters as a scalable approach to environmental surveillance

Environmental 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. We 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. S. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river water samples 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) 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 two different viral families (Autographiviridae and Siphoviridae) and clustered in three distinct groups. S. 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 plaque assays enables detection of S. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance. HighlightsO_LITyphoid phages are detectable in surface water using simple assays, in communities with high typhoid burden. C_LIO_LIBacteriophages are highly specific for S. Typhi and required Vi polysaccharide for infection. C_LIO_LIS. Typhi phages have a broad lytic activity against the S. Typhi strains circulating in Nepal. C_LIO_LIPhage plaque assay can be used as a low-cost tool to identify communities where typhoid is endemic. C_LIO_LIThe high abundance of phages in river water suggest that this could be an alternative to molecular methods for environmental surveillance for typhoid. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/23285806v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1da6b3dorg.highwire.dtl.DTLVardef@b6a91corg.highwire.dtl.DTLVardef@1896aaorg.highwire.dtl.DTLVardef@4b770d_HPS_FORMAT_FIGEXP M_FIG C_FIG