Serum Killing Assay

Human blood was obtained from 10 healthy individuals. Pooled serum was separated and stored in small volumes at −80°C until use. Serum killing assay was performed as previously described. An inoculums of 25 μL (adjusted to 10⁶ cfu/ml) prepared from the mid-log phase was diluted by 0.9% saline, and was added to 75 μL of pooled human sera contained in a 10 × 75 mm Falcon polypropylene tube (BD Biosciences, Franklin Lakes, NJ, USA). Serial dilutions were plated on MHA for 0, 1, 2, and 3 h to obtain colony counts.

Each strain was tested at least three times, and the mean results were expressed as percent inoculums. The results were expressed as percentage of inoculation and the responses in terms of viable counts were graded from 1 to 6, as previously described (Abate et al., 2012). A strain was defined as serum sensitive at grades of 1–2, intermediately sensitive at grades of 3–4, and resistant at grades of 5–6. K. pneumoniae ATCC 700603 and 19432 (NDM-1 positive) strains are used for comparison in the serum killing assay.