In vivo loops were prepared in the small intestine of rabbits and injected with mixtures of Vibrio cholerae and polystyrene spheres (1.1-tim diameter). The loops were removed and frozen after 15 min and then sectioned in a cryostat. The locations of particles and vibrios were determined microscopically. The vibrio/ particle ratio was unity in the lumen of the loops, but increased 10-fold in the deep intervillous spaces, indicating active invasion of the mucus gel by the chemotactic parent strain. Motile nonchemotactic mutants and nonmotile mutants of this strain invaded the mucus at the same rate as inert particles. Similar results were obtained with intestinal loops prepared in germfree mice. When germfree mice were diassociated with mixtures of chemotactic (parent or revertant) and nonchemotactic mutant vibrios in equal proportions, the chemotactic strain rapidly outgrew its nonchemotactic counterpart in the intestine. Nonchemotactic mutants introduced as monoassociates into germfree mice were rapidly overgrown by nonmotile mutants which apparently arose spontaneously in the gut. Motility was therefore beneficial to survival only when it was directed by chemotactic stimuli, whereas it was a liability in the absence of such stimuli. Growth of chemotactic vibrios in small intestinal loops of rabbits paralleled that of nonchemotactic mutants for the first 4 to 6 h. Thereafter, the growth rate of the chemotactic vibrios was significantly faster. This was correlated with a significantly higher degree of association with the mucosa on the part of the chemotactic vibrios.

Earlier reports from this laboratory have shown that chemotaxis is an important mechanism that expedites the in vitro association of cholera vibrios with intestinal slices and that affects the in vivo colonization and virulence of these bacteria to a significant degree. The data reported in the present communication indicate that there appears to be a chemotatic gradient attracting cholera vibrios not only to the surface of the mucus gel, but that this gradient continues for at least a considerable distance toward the base of the villi. It is shown further that a strain of Vibrio cholerae was attracted by all 20 amino acids tested, in contrast to Escherichia coli AW405 which is repelled by several of these. Finally, experiments are described that show that superior in vivo colonization of chemotatic vibrios (compared to nonchemotactic mutants) was correlated with a significantly higher degree of mucosal association. Such increased mucosal association of chemotatic vibrios has previously been shown only with mucosal slices in vitro.

Various Sephadex G-15 fractions of pepsin-digested mucosal extract inhibited the in vitro association of cholera vibrios with mucosal slices. Inhibitory activity paralleled the taxin activity of the fractions for these bacteria. This supports the theory that inhibition of mucosal association by pepsin-digested mucosal scrapings was due to the blocking of taxin receptors on the bacterial surface. Nonchemotactic mutants were significantly less efficient than the chemotactic parent or revertant strains in associating with mucosal slices in vitro. Control experiments in which filter paper disks replaced the mucosal slices showed a comparable extent of association of chemotactic and nonchemotactic vibrios with this material. Histological studies indicated that vibrios associated predominantly with the mucus gel of the intestinal slices rather than with the mucosal epithelium or the serosal surface. Intestinal slices attracted chemotactic vibrios even after prolonged washing, suggesting continuous production of the taxin by the tissue. Inert

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