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Link to original content: http://pubmed.ncbi.nlm.nih.gov/15047509/
In vivo detection and quantification of tetracycline by use of a whole-cell biosensor in the rat intestine - PubMed Skip to main page content
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. 2004 Apr;48(4):1112-7.
doi: 10.1128/AAC.48.4.1112-1117.2004.

In vivo detection and quantification of tetracycline by use of a whole-cell biosensor in the rat intestine

Martin Iain Bahl  1 , Lars Hestbjerg Hansen, Tine Rask Licht, Søren J Sørensen
Affiliations

In vivo detection and quantification of tetracycline by use of a whole-cell biosensor in the rat intestine

Martin Iain Bahl et al. Antimicrob Agents Chemother. 2004 Apr.

Abstract

An Escherichia coli biosensor strain, harboring the plasmid pTGFP2, was introduced into the gastrointestinal tract of gnotobiotic rats that continuously received drinking water containing tetracycline. Plasmid pTGFP2 contains a transcriptional fusion between a green fluorescent protein (GFP) gene and a tetracycline-regulated promoter and was shown to produce a proportional GFP signal in response to exposure to various tetracycline concentrations when harbored by an E. coli strain. The plasmid was highly unstable in the host bacteria colonizing the intestinal system of the animals, and rapid plasmid loss was observed. Reintroduction of the E. coli MC4100/pTGFP2 strain into animals already colonized by the plasmid-free E. coli strain the day before euthanasia made it possible to extract and analyze the biosensors from intestinal samples. The induction of GFP in the biosensor cells extracted from the animals was estimated on a single-cell basis by use of flow cytometry, and the mean induction of GFP in the samples was compared to a standard curve prepared from known tetracycline concentrations. The results showed that the bioavailable tetracycline concentration within the bacterial growth habitat of the intestine was proportional to the concentration of tetracycline in drinking water but represented only approximately 0.4% of the intake concentration. This is a significant finding which will help to clarify antimicrobial therapy in the intestinal environment.

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Figures

FIG. 1.
FIG. 1.
FCM analysis of samples extracted on the day of euthanasia from the colon segments of animals receiving either 0.0 (a) or 50 (b) μg of tetracycline/ml of drinking water. Region R1 in the dot plots defines the population of GFP-expressing bacteria.
FIG. 2.
FIG. 2.
CFU of biosensor E. coli MC4100/pTGFP2 (closed squares) and E. coli MC4100 (open squares) in fecal samples from rats not receiving tetracycline in drinking water. Each point represents a geometric average of values obtained for three animals. Error bars designate standard errors of the means.
FIG. 3.
FIG. 3.
CFU of biosensor E. coli MC4100/pTGFP2 (closed squares) and E. coli MC4100 (open squares) in fecal samples from rats receiving either 0.0 (a), 5 (b), 10 (c), or 50 (d) μg of tetracycline/ml of drinking water. Samples were taken from five segments of the gastrointestinal tract immediately after euthanasia. Each point represents a geometric average of values obtained for three animals. Error bars designate standard errors of the means.
FIG. 4.
FIG. 4.
Relative mean fluorescence values estimated by FCM analysis of biosensor E. coli MC4100/pTGFP2 cells exposed to various concentrations of tetracycline in LB broth (a) and estimated bioavailable tetracycline concentrations in sterile diluted fecal samples supplemented with various tetracycline concentrations (b). Each point represents an average of three values. Error bars designate standard errors of the means. The line in panel b represents a linear regression of the obtained values (R2 = 0.979).
FIG. 5.
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 cecum and colon segments of three animals (two animals for the concentration of 10 μg/ml). Error bars designate standard errors of the means. The line represents a linear regression of the obtained values (R2 = 0.999).
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