Similar results were observed with the in vivo experiments as well. Although fewer pups died within 24 hrs post-infection in the GANT61 chemical structure groups infected with RS218cured as compared to the groups infected with wtRS218 and RS218compl, there was no statistically significant difference in mortality rates between the three groups (Figure 5B). No mortalities were detected in the negative control group treated with
PBS or E. coli DH5α. In groups infected with wtRS218 or RS218compl, 84-87% of rat this website pups that survived 24 hrs post-infection showed septicemia, whereas in groups treated with RS218cured strain, only 33% had septicemia. In all three groups the number of bacteria in the blood was too numerous to count (>1.5-2.8 *104 CFU/ml). Also, E. coli were re-isolated Ilomastat from CSF collected from 84-87% of rat pups infected with wtRS218 or RS218compl whereas only 29% CSF samples collected from rat pups infected with RS218cured strain contained E. coli suggesting a role of pRS218 in translocation of bacteria through the blood brain barrier (BBB) to cause meningitis. Similarly, histopathological
evaluation of brain tissue from the rat pups inoculated with wRS218 or RS218compl strains demonstrated lesions consistent with meningitis (Figure 6). The bacterial loads in CSF were 4.57 + 3.02*103 in rat pups infected with wtRS218 strain and 3.77 + 2.24*103 in rat pups infected with RS218cured strain. Figure 4 Confirmation of pRS218 curing. A, Plasmid profiles of wtRS218 and RS218cured. B, PCR amplification of selected pRS218 genes in wtRS218 and RS218cured. Lane 1,100 bp ladder; Lane 2, senB; Lane 3, scsD; Lane 4, transposase; Lane 5, traU; Lane 6, pRS218_113; Lane 7, ycfA; Lane 8, ompA. C, Adenosine triphosphate Growth of wtRS218 and RS218cured E. coli in LB broth, M9 medium containing 10 μg/ml niacin broth (M9),
and complete cell culture medium (CM). Figure 5 Evaluation of virulence potential of pRS218 in vitro and in vivo. A, Involvement of pRS218 in invasion of hCMEC cells. B, Comparison of mortality, septicemia and meningitis among the groups of rat pups infected with wtRS218, RS218cured, RS218compl. ** denotes statistical significance and * denotes no statistical significance. Figure 6 Histopathological evaluation of brain tissue from rat pups. Five-day-old rat pups were infected by the IP route with wtRS218, RS218compl, RS218cured, E. coli DH5α or PBS. Pups that survived were euthanized 24 hrs post-infection, and the brains were excised, embedded in formalin, sectioned in paraffin, and stained with haematoxylin and eosin. A-F: meningitic lesions observed in pups infected with wtRS218 (A and B) or RS218compl (C, D, E, and F). Arrows indicate rod-shaped bacteria in meninges and brain tissue (black), neutrophilic infiltration/neutrophilia (blue), and cerebral edema (orange). G to I: normal histology of brain tissue from pups inoculated with RS218cured (G), PBS (H) or DH5α (I).