Production of shiga toxin by Escherichia coli measured with reference to the membrane vesicle-associated toxins.

Production of Shiga toxin (Stx) 1 and 2 from Stx-producing Escherichia coli (STEC) was measured with reference to the membrane vesicle (MV)-associated toxins. An immunoblot analysis method using specific antibodies for Stx1 and Stx2 was developed for the detection of the extracellular toxins. All 46 STEC isolates, studied including 30 O157 and 16 other O-antigenic isolates, released Stx1 and Stx2 as MV-associated and MV-removed fractions under aerobic and anaerobic conditions. Treatment of vesicles with polymyxin B that disrupted MVs increased the release of Stx1 and Stx2. Therefore, delivery of Stx1 and Stx2 by MVs is a general mechanism in STEC. Stx1 remained within MVs rather than in the MV-removed fraction under an aerobic culture condition. On the other hand, a larger proportion of Stx2 was detected in the MV-removed fraction. The kinetic patterns of the release of the toxins from STEC strains showed that both Stx1 and Stx2 were released into the growth medium during the exponential growth phase. An rpoS-deficient mutation did not have altered levels of extracellular Stx1 and Stx2, supporting the idea that Stx1 and Stx2 are produced during exponential growth phase.