Abstract:The phylogenetic interrelationships of saccharolytic C. botulinum types B, E and F together with eleven other saccharolytic clostridia were examined by 16S rRNA gene sequencing. Comparative analysis of the sequence data revealed that the saccharolytic C. botulinum types B, E and F were highly related and represent a single genetic group. Strains of C. barati and C. butyricum that produce botulinal neurotoxin revealed almost 100% 16S rRNA sequence identity with their respective non-toxigenic counterparts and were phylogenetically distinct from saccharolytic C. botulinum (types B, E and F). Proteolytic C. botulinum type F was shown to be phylogenetically remote from the saccharolytic C. botulinum group. The implications of the sequence data for the taxonomy of the C. botulinum complex are discussed.

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phasedependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.In recent years there has been growing interest in the use of high hydrostatic pressure (HHP) as a means of food preservation. The attraction of HHP lies in the production of microbiologically safe foodstuffs with minimal use of chemical additives and without adversely affecting the organoleptic qualities of the food (11). HHP processing could replace traditional thermal pasteurization or be used in conjunction with existing techniques (3,10,14,22). However, exploitation of this potential requires a better understanding of the effects of HHP on microorganisms. HHP resistance varies among genera and species and is dependent on the physiological state of the organisms at the time of pressurization (6, 18). It is of concern that certain E. coli O157:H7 strains are among the most pressureresistant vegetative bacteria known (2, 18). It is therefore critically important to characterize the innate HHP resistance in these strains.We demonstrated recently that the pressure resistance of certain natural isolates of E. coli O157:H7 varied greatly (2, 17). Strains C9490 and 30-2C4 were the most pressure resistant and were able to withstand 500 MPa for 5 min with little viability loss; strains NCTC 12079 and W2-2 were of intermediate pressure resistance (ca. 3 to 4 log units decrease under the same conditions), whereas H1071 and an O124 strain, NCTC 8003, were the least pressure resistant (5 to 6 log decrease). However, this variation in pressure resistance was stationary-phase dependent, with the strains exhibiting similar pressure resistance in exponential phase (2). This led us to speculate that the differences in pressure resistance among the isolates were related to differences in RpoS. This sigma factor changes the specificity of RNA polymerase, allowing it to activate more than 30 genes, some of which are involved in stationary-phase stress survival (8). Stationary-phase bacteria are generally more resistant to other stresses, such as oxidative and osmotic stress (21). It is thus possible that resistance to inactivation by HHP is also controlled by mechanisms used to survive stationary-phase stress. These studies attempted to identify the genetic basis, and specifically the role of RpoS, in the wide variation observed in HHP resistance of natural isolates of E. coli O157:H7 stra...