Figure 1.
Tilivalline structure, chemical formula, and mass.
Table 1.
Sources and origin of Klebsiella oxytoca used in this study.
Table 2.
Antimicrobial resistance of K. oxytoca.
Figure 2.
Animal isolates of K. oxytoca produce cytotoxin.
HEp-2 cell culture inoculated with K. oxytoca supernatant and media control at 4, 10, and 30× magnifications. A) Media control showing normal morphology of cells stained with Diff-quick after 48 hours of incubation. B) K. oxytoca, ATCC 13182, isolate negative for cytotoxin production showing normal cell morphology. C) K. oxytoca isolate, 09-7231-1, positive for cytotoxin production showing abnormal cell morphology, decreased concentration of attached cells, small round cells, and multinucleated cells.
Table 3.
Cytotoxin of K. oxytoca varies with different growth conditions.
Figure 3.
Temporal pattern of cytotoxicity in various conventional bacterial growth media.
K. oxytoca, 09-7231-1, was cultured in A) LB; B) TSB; and C) HBI liquid media. At various time points, the supernatant was collected and used in the cytotoxicity assay using HEp-2 cells. TSB media has the strongest cytotoxin induction capability compared to LB and HBI media. The degree of cytoxicity is indirectly proportional to the level of monolayer confluency i.e., the higher % the confluency, the lower the cytotoxity and vice versa.
Figure 4.
Soy components in TSB induced HEp-2 cell detachment and death.
HEp-2 cells treated with A) media containing only casein extract (Tryptone; control); B) supernatant of K. oxytoca, 09-7231-1, grown in media containing only casein extract; C) media containing only soy extract (Soytone); D) supernatant of K. oxytoca, 09-7231-1, grown in the media containing only soy extract.
Figure 5.
Tilivalline induced HEp-2 cell detachment and death.
A) HEp-2 cells incubated with the control DMSO, which was used to dissolve tilivalline. The majority of cells adhered to the bottom of the cell culture well after 48 hours of culture; B) HEp-2 cells treated with purified tilivalline (1 µg/ml) were detached from the cell culture well after 48 hours of incubation.
Figure 6.
The reference proteins encoded by CTA, CFX B, and PAX B are displayed as colored bars.
The arrow beneath the bar shows the region in the reference protein to which the corresponding K. Oxytoca protein aligns from the BLAST analysis.