In Vivo Detection and Quantification of Tetracycline by Use of a Whole-Cell Biosensor in the Rat Intestine

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 tetracyclineregulated 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.Since their discovery in the 1940s, tetracyclines have been very useful antibiotics for the treatment of a whole range of bacterial infections in humans and animals and have also seen widespread use as growth promoters in animal husbandry (5). However, the emergence and characterization of some 36 different genes (17) conveying tetracycline or oxytetracycline resistance to the host bacteria by a number of different mechanisms have raised some concerns regarding the future usefulness of the tetracyclines. It is believed that subinhibitory concentrations of tetracycline have a positive selective effect on the dissemination and accumulation of tetracycline resistance genes in bacteria colonizing the gastrointestinal tract of mammals. This belief is based partly on molecular evidence, which indicates the tetracycline-induced transfer of mobile elements containing tetracycline resistance determinants (4), and partly on the observed increase in prevalence of various resistance determinants after therapeutic or prophylactic treatment with the drug (3, 9). To investigate this effect further it is important to be able to determine the actual in situ bioavailable concentration of the antibiotic in the microhabitats hosting bacterial activity. The traditional methods for measuring in situ antibiotic concentrations are based on an extraction of the compound from its natural matrix, followed by analysis by either chromatographic methods, such as high-pressure liquid chromatography (19), microbiological methods, such as bacterial inhibition assays (15), or in vitro biosensor ...

Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA , umuDC , or sulA Promoters

“…GFP is ideal for in vivo or in situ applications since it has a very stable signal, requires the presence of only oxygen to function, and needs no addition of substrates or cofactors. Studies in our laboratory have enabled in situ detection of specific compounds in a variety of complex environments by using a fluorescence-activated cell sorter-optimized version of GFP as reporter in combination with flow cytometry (4,7,19). In this study, we therefore decided to construct a genotoxicity biosensor based on GFP.…”

A novel hybrid dual-channel catalytic-biological sensor system for assessment of fruit quality

“…Synthetic-biology approaches for the design of cell-based biosensors have resulted in sensitive and economic methods for detecting and quantifying antibiotics (Bahl et al, 2004), heavy metals (Tibazarwa et al, 2001;Yoshida et al, 2008), metabolites (Burmolle et al, 2005;Prieto and Garcia, 1997), organic aromatic pollutants (Kim et al, 2005) and explosives (Radhika et al, 2007) as well as for discovering new drugs (Weber et al, 2008). Cellbased biosensors commonly rely on a receptor or a transcription factor, which, in the presence of the analyte, triggers de-repression or activation of cognate promoters, resulting in the expression of a quantifiable reporter gene.…”

Engineered cells as biosensing systems in biomedical analysis

“…For example, it has been shown that subinhibitory concentrations of tetracycline promote accumulation of tetracycline-resistance genes in bacteria colonizing the GI tract of mammals [53]. Bahl et al have investigated the above mentioned effect by determining, in vivo, bioavailable concentrations of the antibiotic in the intestines of tetracycline-treated rats, which continuously received drinking water containing the antibiotic [54]. This was achieved by using a whole-cell biosensing system consisting of E. coli MC4100 cells harboring the plasmid pTGFP2, which contains a gene fusion of the tetracycline inducible promoter P tet and gfp.…”