Fig 4. Extreme acid survival of S. Typhi strains depends upon the pH of the pre-growth media.

A. Tested bacteria were pre-grown in either TSB-MES pH 4.5, TSB-MES pH 5.5, TSB-MOPS pH 6.5, commercial TSB (unbuffered pH 7.2), or TSB-MOPS pH 7.5 for 18 hours prior to the acid challenge, and survival at pH 2.5 for 3 hours was determined. Ty21a with pGad showed good survival when bacteria were pre-grown in mildly acidic pH media and in commercial unbuffered TSB, with the p value significance between Ty21apGad and Ty21a EV at pH 4.5, 5.5, 6.5 and 7.2 (unbuffered) are 0.0236, 0.0251, 0.0230 and 0.0468, respectively; however following pre-growth at pH 7.5, acid survival was decreased by ~ 3 logs (non-significant p value 0.1827). Our negative control Ty21a EV did not show any survival after pre-growth under all pH conditions. Although Ty2 showed good low pH-survival when pregrown at slightly acidic pH, it showed very poor survival when pre-grown at pH 7.5 or in commercial TSB at pH 7.2. Sf showed good survival under all pre-growth conditions, including pre-growth in TSB-MOS pH 7.5. B. The Gad protein expression of selected bacteria after 18 hours of growth in TSB-MES pH 4.5, TSB-MES pH 5.5, TSB-MOPS pH 6.5, unbuffered pH 7.2 TSB, and TSB-MOPS pH 7.5 was determined by Western blot with anti-Gad A/B antibody [35]. Gad protein is expressed under all the pH conditions in Ty21a pGad, where Gad protein expression is under the control of an arabinose inducible promoter. However, in Sf where Gad protein expression is tightly regulated, Gad protein expression is only seen in TSB-MES pH 5.5, TSB-MOPS pH 6.5 and unbuffered pH 7.2 TSB. As expected, the negative control Ty2 grown in TSB-MES pH 5.5 did not express any Gad proteins. An antibody-binding, non-specific band was used as a loading control; qualitative differences in GadA/B expression were monitored.