Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Objective: To develop attenuated strains of Salmonella enterica serovar Typhi (S. typhi) for the
candidate vaccine by osmolar stress. Methods: S. typhi SS3 and SS5 strains were isolated from
asymptomatic typhoid carriers in Namakkal, Tamil Nadu, India. Both strains were grown in LB
(Luria Bertani) medium supplemented with various concentration of NaCl (0.1- 0.7M) respectively.
The effect of osmolar stress was determined at molecular level by PCR using MGR 06 and MGR
07 primers corresponding to ompR with chromosomal DNA of S. typhi SS3 and SS5 strains.
Attenuation by osmolar stress results in deletion mutation of the S. typhi strains was determined
by agglutination assays, precipitation method, SDS PAGE analysis and by animal models.
Results: The 799 bp amplified ompR gene product from wild type S. typhi SS3 and SS5 illustrate
the presence of virulent gene. Interestingly, there was only a 282 bp amplified product from S.
typhi SS3 and SS5 grown in the presence of 0.5, 0.6 and 0.7 M NaCl. This illustrates the occurrence
of deletion mutation in ompR gene at high concentration of NaCl. Furthermore, both the wildtype and mutant S. typhi outer membrane SDS-PAGE profile reveals the differences in the
expression of ompF, ompC and ompA proteins. In mice, wild type and mutant strains lethal dose
(LD50) were determined. The mice died within 72 h when both the wild type strains were injected
intraperitoneally with 3 log CFU.mL-1
. When the mice were injected with the mutants in same
dosage, no clinical symptoms were observed; whereas the serum antibody titre was elicited within
two weeks indicated that the mutants have the ability to induce protective humoral immune
response. These results suggest that S. typhi SS3 and SS5 may be used as good candidate strains
for the development of live attenuated vaccine against salmonellosis. Conclusions: This study
demonstrates that the S. typhi strains were attenuated and could be good vaccine candidates in