Fig. 3

Internalization of S. aureus isolates and response to antibiotic treatment in Caco-2 cells. a Intracellular survivability of S. aureus isolates in Caco-2 cells. Approximately 2 × 10⁴ Caco-2 cells/well of a 96-well plate were exposed to the isolates maintained at 0.5 Macfarland standard (1.5 X 10⁸ cells/ mL). The cells were washed with PBS and subjected to gentamicin (10 µg/mL) to remove extracellular bacteria. The cells were washed further after 4 h of incubation and lysed using 0.5% (v/v) of Triton-X. Colony-forming units were determined using the drop culture method. b Epifluorescence microscopic images of non-infected control and Sa30-infected Caco-2 cells. After 4 h incubation of the Caco-2 cells exposed to bacteria, 30 µL of Hoechst-PI cocktail was added and incubated for 30 min in dark. These wells were imaged using an epifluorescence microscope with blue, green, and red filters. i-ii) Hoechst 33342 staining of non-infected and Sa30-infected Caco-2 cells. iii-iv) GFP-labeled Sa30 internalization in Caco-2 cells. Non-infected cells were considered as a control. v-vi) PI staining of infected and non-infected cells. vii-viii) Overlap of Hoechst and PI stained Sa30-infected and non-infected Caco-2 cells. The images confirmed Sa30 internalization and infection of the Caco-2 cells leading to cell death. c Antibiotic efficiency against intracellular Sa1158c, Sa30, and Sa3. The infected Caco-2 cells were exposed to ampicillin (AMP) (10 µg/mL), kanamycin (K) (30 µg/mL), streptomycin (S) (10 µg/mL), chloramphenicol (C) (30 µg/mL), tetracycline (TET) (30 µg/mL), and ceftiofur (CF) (30 µg/mL). The plates were incubated for 24 h, followed by washing, lysing of the cells, and drop-culture method to enumerate the cfu/well. Average values plotted in the graph with different alphabets indicate a significant difference (p < 0.05). ‘IF-control’ stands for infected Caco-2 cells without antibiotic treatment. The experiment was performed in quadruplicates and repeated thrice to ensure reproducibility