After the initial colonization of the E. coli
biosensor strain MC4100/pTGFP2 in the intestinal systems of the rats, high plasmid instability was observed for all four groups of animals (Fig. and data not shown). This observation is in line with the general belief that high-copy-number cloned plasmids, such as pTGFP2, convey a selective disadvantage to the host cell when no selection for the plasmid is present (6
). In this case, tetracycline increases the selective disadvantage by inducing GFP synthesis, which requires increased energy. If all plasmids are lost from one E. coli
cell through segregational and structural instability, or if the inoculum contains plasmid-free MC4100 cells, these cells will inevitably be in close proximity to other isogenic, plasmid-harboring cells in the dynamic gastrointestinal environment. The in situ growth rates of the competing cell lines in the same microhabitat will determine the ratio between the two cell types and ultimately lead to loss of the type with the slowest in situ growth rate, i.e., the plasmid-harboring bacteria. The observed rapid plasmid loss from the intestinal E. coli
population indicated a highly competitive intestinal environment.
The day after the reinoculation of the biosensor strain E. coli MC4100/pTGFP2 into the E. coli MC4100-colonized animals, the CFU counts for samples obtained from five segments of the gastrointestinal tract showed an approximately constant ratio between the two E. coli types throughout the gastrointestinal system (Fig. ). In samples from the cecum and colon segments, between 2 and 8% of the E. coli MC4100 population carried the pTGFP2 plasmid. These findings indicate the ability of the biosensor strain to coexist with an isogenic strain in the intestine.
Increasing concentrations of tetracycline in drinking water caused an increase in the mean fluorescence of the E. coli
MC4100/pTGFP2 biosensor bacteria extracted from the cecum and colon segments of the gut. This finding showed that the concentration of tetracycline encountered by active biosensor bacteria in these segments was proportional to the concentration of tetracycline in the drinking water. Pilot experiments indicated that drinking water containing 1 μg of tetracycline/ml or less did not induce a significant biosensor response in the rats and consequently did not affect the intestinal microbial population (data not shown). This finding is in line with that of other investigations showing that the minimum selective concentration of tetracycline in drinking water is between 1 and 10 μg/ml (8
). However, it is still possible that tetracycline concentrations below 1 μg/ml could have a long-term effect on the microbial population.
In order to correlate the observed induction of the biosensor with the actual bioavailable concentration of tetracycline in the bacterial growth habitat, a number of issues must be addressed. It has been suggested (16
) that there is a lateral movement of intestinal bacteria, produced by growth in the mucus layer, into the luminal contents followed by excretion in feces. In connection with this lateral movement, it is plausible that the biosensor bacteria encounter various tetracycline concentrations; however, this is only translated into a GFP response if the cells are active and if protein synthesis occurs. Two factors point towards low bacterial activity in the luminal contents: first, previous studies of E. coli
bacteria have shown that the major bacterial growth compartment in the gut is within the mucus layer and that little growth takes place in the luminal contents (14
). Second, in a parallel study, inhibitory bioavailable tetracycline concentrations were measured in extracts from the cecum and colon segments and in fecal samples from groups B, C, and D (2
). The measured concentrations represented between 6 and 10% of the administered drug concentration in drinking water in the cecum and colon segments and between 13 and 34% in the fecal samples. These ranges are similar to those measured in other investigations of fecal samples from tetracycline-treated mice (8
) and cause inhibition of growth and of protein synthesis in the E. coli
biosensor cells, especially in group D. Furthermore, the FCM data showed a well-defined biosensor population with respect to green fluorescence, indicating that the cells did not respond to a different concentration of tetracycline when shed to the luminal contents (Fig. ). This finding suggests a fast transition from the growth habitat in the mucus layer to the luminal contents. We propose that (i) the biosensor bacteria respond to the relatively low concentration of bioavailable tetracycline within the mucus layer, which constitutes a partially tetracycline-protective environment, and (ii) the biosensor bacteria do not respond to higher concentrations when shed into the luminal contents, due to the lack of protein synthesis. Consequently, it was possible to estimate the actual bioavailable tetracycline concentrations in the fraction of the gastrointestinal tract which hosted the growth of the biosensor bacteria by comparing the relative mean fluorescence values obtained from the animal experiment with the standard curve (Fig. ). The bioavailable tetracycline concentration within the bacterial growth habitat of the intestine was proportional to the concentration of tetracycline in the drinking water and represented approximately 0.4% of the intake concentration of the drug (Fig. ). This is significantly less than the proportion of bioavailable tetracycline in sterile diluted fecal samples supplemented with tetracycline (Fig. ) and explains the ability of the sensitive E. coli
cells to proliferate in the intestinal environment, even when tetracycline concentrations in drinking water, as well as in luminal contents, far exceeded inhibitory concentrations. The rapid plasmid loss observed in the intestinal system (Fig. ) could reduce the plasmid copy number in some biosensor bacteria and cause a slight underestimation of the tetracycline concentration due to lower gfp
expression. However, the experience gained in our laboratories with the biosensor strain shows that gfp
expression is not very sensitive to the plasmid copy number. This view is supported by a recent study of the effect of plasmid copy number on protein expression levels (20
). Additionally, the well-defined population of biosensor bacteria exposed to tetracycline (Fig. ) indicates a homogeneous expression of gfp
in all E. coli
MC4100/pTGFP2 cells. Simultaneous colonization studies of two isogenic E. faecalis
OG1 strains, one of which was tetracycline resistant, were performed with the animals used in the present study and were reported previously (2
). The results of that study showed that the two isogenic E. faecalis
strains were able to coexist in the intestines of rats receiving up to 50 μg of tetracycline/ml of drinking water, which is well above the inhibitory concentration for the sensitive strain and suggests the existence of tetracycline-depleted microhabitats in the intestinal environment. These findings further indicate that the in situ bioavailable tetracycline concentrations were much lower than the intake concentration.
FIG. 5. Estimated bioavailable tetracycline concentrations, within the bacterial growth habitat of the intestine, in animals receiving various tetracycline concentrations in drinking water. Each point represents an average value for samples extracted from the (more ...)
This is, to our knowledge, the first time that a bacterial biosensor strain has been used to quantify in vivo tetracycline concentrations directly in the bacterial growth compartments of the gastrointestinal tract. Because the induction levels of the biosensor bacteria were almost identical in intestinal samples from the cecum and colon segments, and no bacterial activity was present in the luminal contents, it seems probable that analysis of tetracycline in excreted feces would reveal similar results. This would further allow real-time detection and quantification of the in situ bioavailable tetracycline contents in the intestinal environment. The results obtained from this study and in future work will contribute importantly to our understanding of antimicrobial therapy in the intestinal environment.