**Classification of cefazolin as ‘active’ or ‘less active’: When difference in cleavage rates (fluorescence change) in the absence and presence of cefazolin was minimal, antibiotic predicted to be ‘active’. Drastically lowered cleavage rate in presence of cefazolin compared to when probe assayed alone led to prediction of cefazolin as ‘less active’ respectively (also see Figure 2). Details of Disk Diffusion results are presented in Table 3. Bacteria-free controls (PBS only) were included in each assay-set to account for non-specific probe cleavage that may occur. As expected, a negligible fluorescence change over time was observed. Comparison of cleavage rates (mRFU/min) for
#1, #2 and the PBS only control are shown in Additional file 1: Figure S1. Nitrocefin test for detection of β-lactamase validates results from β-LEAF Defactinib assay In order to validate the β-lactamase phenotypes determined by the β-LEAF assay, a CLSI recommended β-lactamase screening method, the chromogenic nitrocefin test, was utilized [41]. All bacterial isolates that were strongly positive by the β-LEAF assay were also found to be positive by nitrocefin conversion with the nitrocefin disks, showing a change in colour from yellow to deep orange in a positive reaction for β-lactamase (Table 1, right-most
column). Comparison of conventional disk diffusion and β-LEAF assay results In order to compare predictions of cefazolin activity by the β-LEAF assay to a conventional AST method, we performed cefazolin disk diffusion check details assays with the S. aureus isolates. Based on respective zone of inhibition diameters, each isolate was classified as susceptible, intermediate or resistant using the CLSI zone interpretive criteria (Table 3, Additional file 2: Figure S2). Interestingly, all the isolates
fell in the cefazolin ‘susceptible’ range with this methodology (Table 3). Table 3 Cefazolin disk diffusion results S. aureus isolate # Zone of inhibition diameter (mm) AS* Zone edge Interpretation as per zone edge test criteria& 1 21.5 ± 1.0 S Sharp β 2 31.0 ± 1.0 S Fuzzy 3 33.5 ± 0.5 S Fuzzy 4 33.0 ± 2.0 S Fuzzy 5 32.5 ± 0.5 S Fuzzy 6 36.5 ± 0.5 Mannose-binding protein-associated serine protease S Sharp β 7 32.0 ± 0.5 S Fuzzy 8 39.5 ± 1.5 S Fuzzy 9 29.5 ± 1.5 S Fuzzy 10 41.5 ± 0.5 S Fuzzy 11 34.5 ± 2.5 S Little fuzzy Weak β? 12 41.0 ± 1.6 S Fuzzy 13 32.5 ± 0.5 S Fuzzy 14 33.0 ± 0.0 S Fuzzy 15 35.5 ± 2.5 S Fuzzy 16 36.5 ± 0.5 S Fuzzy 17 36.5 ± 0.5 S Fuzzy 18 33.5 ± 0.5 S Sharp β 19 31.0 ± 0.0 S Sharp β 20 20.5 ± 0.3 S Sharp β 21 38.0 ± 1.0 S Fuzzy 22 34.0 ± 1.1 S Little fuzzy Weak β? 23 33.5 ± 1.5 S Fuzzy 24 34.5 ± 1.5 S Fuzzy 25 30.5 ± 0.5 S Fuzzy 26 34.0 ± 0.0 S Fuzzy 27 36.0 ± 2.0 S Little fuzzy/sharpish Weak β? *The Antibiotic Susceptibility (AS) was determined using the CLSI Zone Diameter Interpretive Criteria for Cefazolin Disk Diffusion [41].