Physiological state of Escherichia coli BJ4 growing in the large intestines of streptomycin-treated mice

Abstract

Growth rates of Escherichia coli BJ4 colonizing the large intestine of streptomycin-treated mice were estimated by quantitative hybridization with rRNA target probes and by epifluorescence microscopy. The ribosomal contents in bacteria isolated from the cecal mucus, cecal contents, and feces were measured and correlated with the ribosomal contents of bacteria growing in vitro at defined rates. The data suggest that E. coli BJ4 grows at an overall high rate in the intestine. However, when taking into account the total intestinal volume and numbers of bacteria present in cecal mucus, cecal contents, and feces, we suggest that E. coli BJ4 in the intestine consists of two populations, one in the mucus which has an apparent generation time of 40 to 80 min and one in the luminal contents which is static.The animal intestinal tract harbors a vast number of bacteria representing a complex ecosystem in which the microorganisms are present without overgrowing the host but also without being flushed out by the host's intestinal activities, e.g., peristaltic movements and fluid flow. At least 400 to 500 different bacterial species are thought to be present at any time in the healthy human intestinal tract, and up to 10 12 bacteria are found per g of feces (3, 9). One question of interest is whether the intestinal flora grows as a homogeneous population at a low rate or as a heterogeneous population containing fast, slow, and nongrowing organisms.Accurate calculations of the rates of bacterial proliferation in the intestine have so far been represented only by average estimates at the level of populations. For example, the growth rate of Escherichia coli has been estimated in vivo, i.e., in the mouse intestine, by radioisotope techniques (8), by dilution by growth of a nonreplicating genetic marker (17,25), and simply by counting the number of viable cells (12,14). With these techniques, generation times from 30 min to 40 h have been estimated for E. coli (12,14,16,36). Also, continuous-flow cultures have been developed to mimic bacterial interactions in the gut and estimate overall growth rates (13, 27). These systems, however, do not reflect the physiological conditions in the gut, where entrapping of the bacteria in the mucus gel plays an important role. Furthermore, bacterial cell morphology, protein profiles, and growth physiology have been found to be distinct during growth in the intestine when compared with growth in laboratory media (22,33).We have previously found that in test tubes E. coli BJ4 is rod shaped, appears as large cells, and has a great potential for fast growth; however, soon after colonization of mice, E. coli BJ4 differentiates into a coccoid morphology, the so-called small variant, which grow more slowly than the rod-shaped E. coli cells in aerated test tubes (22). Here, we continue our investigation of the growth physiology of E. coli BJ4 present in its natural environment, the large intestine. Bacterial growth rates can be estimated from the cellular RNA and/or DNA contents, since the cellular RNA...

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