Figure S1. Phage-based AST of MHB-suspended EV76 toward CL, GM, DC and CIP. EV76 was tested by phage-based AST with 7 h of exposure to CL, GM and DC, and 14 h of exposure to CIP as described in the methods. Blue bars represent RLU values of phage-based AST (MIC marked by a black frame around the relevant bar) and mustard curves represent OD630nm values of a standard microdilution test (MIC marked by a black dot) for comparison. Values are the mean of 3 wells in one experiment (error bars represent standard deviation). Figure S2. Detection of Y. pestis directly from positive human blood culture. A. Y. pestis strain Kimberley53D70D10 was inoculated (2 × 106 CFU/ml) into human blood cultures (10 ml human whole blood in Bactec aerobic plus/F bottle) and incubated at 180 × rpm, 37oC for 4 h (bacterial concentration at 4 h = 6.6 × 106 CFU/ml). Bacteria were separated from blood component by either centrifugation in serum separation tube (SST) or by differential centrifugation. SST separation was conducted as described at Methods section (bacterial concentration after SST = 6 × 106 CFU/ml). Differential centrifugation was done by centrifugation of 15 ml blood culture in a 50 ml tube at 300 × g, RT for 8 min, and the supernatant fraction containing the bacteria was transferred to a clean tube (bacterial concentration after differential centrifugation= 6.6 × 106 CFU/ml). For positive control, colonies of Kimberley53D70D10 grown on BHIA were resuspended to 2.2 × 107 CFU/ml. Phage-based ID assay was performed as described for Figure 1. B. For determining LOD of blood culture derived bacteria, 10-fold dilutions of Kimberley53D70D10 suspension were inoculated into three blood culture bottles and incubated for 4 h followed by SST purification and phage-based ID as described above. C. Human blood culture was spiked with either Kimberley53D70D10 (~104 CFU/ml) or EV76 (~104 CFU/ml) Y. pestis strains and incubated at the BACTEC FX40 devise until it became positive. Bacteria were purified by SST (bacterial concentration for Kimberley53D70D10 and EV76: 3 × 108 CFU/ml or 2.8 × 107 CFU/ml, respectively) and ID assay was performed as described for A. Results are the average of 3 wells and error bars are STDEV. Figure S3. Detection of Y. pestis directly from environmental asphalt samples. A. Y. pestis Kimberley53D70D10 were resuspended in MH or in PBS and diluted to 2 × 106 CFU/ml. Half of each sample was diluted by 3 in MHB (to 6.6 × 105 CFU/ml). All 4 samples were tested by phage-based ID as described in the methods. B. Environmental samples were spiked with Y. pestis Kimberley53D70D10 in low concentrations, diluted by 3 in MH and tested by the detection method as described in the Method section. Numbers in the legend represent the bacterial concentration of each sample prior to dilution. Values are the mean of 3 triplicated in the same experiment, and the error bars represent the STDEV. The red dashed line represents the LOD.