Relation between tetR and tetA expression in tetracycline resistant Escherichia coli
© Møller et al. 2016
Received: 12 October 2015
Accepted: 29 February 2016
Published: 12 March 2016
Tetracyclines are among the most used antibiotics in livestock worldwide. Resistance is widely disseminated in Escherichia coli, where it is generally mediated by tetracycline efflux pumps, such as TetA. Expression of tetracycline efflux pumps is tightly controlled by the repressor TetR, which has been shown to be tetracycline-responsive at sub-MIC tetracycline concentrations. The objective of this study was to investigate the effects of increasing tetracycline concentrations on the growth of TetA-producing E. coli, and to determine how expression of tetA and tetR related to each other in different growth phases in the presence of tetracycline.
A tetracycline resistant E. coli strain containing tetA and tetR on the chromosome was constructed and cultured in the presence of increasing concentrations of tetracycline. Expression of tetR and tetA was measured at four time points in different growth phases by quantitative real-time PCR. The TetA-producing E. coli exhibited prolonged lag phase with increasing concentrations of tetracycline, while expression of tetA and tetR increased and decreased, respectively, with increasing tetracycline concentration. The levels of tetA and tetR mRNA varied depending on growth phase, resulting in a gradual decrease of the tetA/tetR ratio from approximately 4 in the lag phase to approximately 2 in the stationary phase.
This study shows that the expression of tetR and tetA is tetracycline concentration- and growth phase-dependent, contributing to improved understanding of the relationships between E. coli growth, tetracycline exposure and expression of tetracycline resistance.
Tetracyclines exert their bacteriostatic activity by binding to the bacterial ribosome and thereby interfering with protein translation . Over the years, the prevalence of tetracycline resistance has increased in bacteria isolated from both human patients [2, 3] and animals . In a study performed by Tadesse et al. it was shown that the prevalence of tetracycline resistance in Escherichia coli isolates from humans increased by 0.45 % per year from 1950 to 2001 . As a consequence of the spread of tetracycline resistance and the introduction of newer and more effective antimicrobial agents, the use of tetracylines in human medicine has gradually decreased. However, tetracyclines remain among the most used antibiotics in livestock production worldwide . In the European Union, they account for 37 % of the total sales of antimicrobial agents for livestock  and a significant association has been observed at the country level between tetracycline consumption and occurrence of tetracycline resistance among E. coli isolated from livestock .
More than 40 genes encoding tetracycline resistance (tet-genes) have been characterized to date and they are divided into 11 classes, with a majority of classes (60 %) encoding for membrane-associated efflux proteins [9–11]. These efflux pumps selectively transport tetracycline from the cytosol to the periplasm in exchange of a proton, thereby limiting the access of tetracycline to the ribosomes in the cell . They are proton motive force-dependent, single polypeptide, drug specific efflux pumps, which belong to the major facilitator superfamily [12, 13]. The flow of protons through the pump provides the required energy to pump the antibiotic to the periplasm [9, 14]. In E. coli, tet-resistance efflux pumps are among the best characterized transport systems . Studies from selected parts of the United States of America list TetA as the second most frequent tetracycline resistance efflux pump in both human and animal isolates . TetA is also the most common tetracycline efflux pump type found in clinical as well as commensal isolates of E. coli in animals from Denmark .
Bacterial strain construction
Bacterial strains and plasmids used in this study
Strain or plasmid
Reference or source
E. coli K-12 MG1655
E. coli MG1655 ∆YbeM::TetA (tetr)
tetR and tetA containing plasmid
Vector for Lambda Red mediated mutagenesis: λ-red expression from arabinose-inducible promoter; temperature sensitive (gmr)
E. coli ATCC 25922
Oligonucletide sequences for PCR based amplification and sequencing
for: 5’-ATGCTGGTGGCACTTCAGGCAGGAAACATCGTCGCCCGTTCAATCGTCACCCTTTCTCGG -3’
Proof of insertion
for: 5’-CGGTCTTCTTCATCATGCAAC -3’
Antimicrobial susceptibility testing
The broth microdilution method was used to determine the MIC of tetracycline following the CLSI guidelines . The tetracycline concentrations tested ranged from 0 to 1024 μg/mL by two-fold dilution increase. In addition, MIC determination was performed using serial increases of 2 μg/mL of tetracycline in the range close to the observed MIC to obtain a value as close to the real MIC as possible. Furthermore, MIC experiments with spent media containing tetracycline (MG1655/TetA incubated in MH-2 with 8 μg/mL tetracycline for 15 hours at 37 °C, centrifuged and filter sterilised) and media with tetracycline (MH-2 with 8 μg/mL tetracycline incubated for 15 hours at 37 °C) were performed using the same method to test for drug degradation and destruction.
A BioScreen CTM was used to perform growth experiments for 24 hours at 37 °C in biological triplicates. A volume of 200 μL Müller-Hinton-II (MH-2) broth (Sigma, Copenhagen, Denmark) was inoculated with cells from blood agar plates (blood agar base (Oxoid, Roskilde, Denmark) supplemented with 5 % blood from cattle) to a final cell density of 106 cfu/mL, using a Sensititre™ Nephelometer (Thermo Scientific™, Roskilde, Denmark) with a McFarland 0.5 standard (1–2 x 108 cfu/mL). The cultures were supplemented with tetracycline (ranging from 0 to 128 μg/mL by two-fold dilutions). Optical density (OD, recorded with 600 nm filter) was measured every 5 minutes keeping bacterial cultures under continuous shaking. The specific maximum growth rate at different tetracycline concentrations was calculated. For expression studies, the strain was grown in 100 mL of MH-2 broth in 250 mL flasks at 37 °C and 225 rpm. The medium was supplemented with three different concentrations of tetracycline representing 1/8 MIC (3.5 μg/mL), ¼ MIC (7 μg/mL) and ½ MIC (14 μg/mL) of the strain and inoculated with a preculture grown for 2 hours at 37 °C and 225 rpm using the method described above. The tetracycline concentrations represent therapeutic concentrations, according to published pharmacokinetics data . Although it has been shown that tetracycline in water solution is stable for 3 days at 37 °C , and approximately 5 % tetracycline is degraded after 2 days at 40 °C , we ensured that the late growth onset in our growth studies was not due to degradation of tetracycline by using an E. coli reference strain (ATCC 25922) with a MIC of tetracycline of 2 μg/mL . E. coli ATCC 25922 was grown with the same tetracycline concentrations as used for the tetracycline resistant strain and MIC values were determined using spent media.
Samples for RNA extraction were collected at four different time points during in vitro growth; the lag phase (OD600nm = 0.1-0.2), the logarithmic phase (OD600nm = 0.5-0.6), the late logarithmic phase (OD600nm = 1-1.3) and the stationary phase (OD600nm = 3.3-4.6). Sampling, RNA extraction, DNase treatment and reverse transcription were performed as previously described .
Quantitative real time polymerase chain reaction
Quantitative real time polymerase chain reaction (qPCR) was performed using a LightCycler 96 (Roche, Hvidovre, Denmark) as described previously . Primers are listed in Table 2. The genes gapA and nusG were used as reference genes according to previous validation experiments . Relative gene expression (fold change) was calculated compared to the lag phase sample of strain MG1655/TetA without antibiotics. Two independent biological replicates were performed using two technical replicates and the 2-∆∆Ct method, corrected by different primer efficiencies and multiple reference genes was used .
Statistical analysis was performed as previously described . Briefly, the differences in the normalised qPCR measurements between the tetracycline concentrations within each growth phase were compared by differences in least square means using analysis of variance. The Mixed procedure in SAS version 9.3 (SAS Institute, Cary, USA) was used and differences in least square means estimates were evaluated with an approximate t-test using the LSmeans-function. Differences between tetracycline concentrations and growth phases were evaluated by F-tests, and a P value < 0.05 was deemed statistically significant. To correct for multiple comparisons of the differences in the least-squares means, the Benjamini–Hockberg ‘false discovery rate’ (FDR) was used .
Results and Discussion
Lag phase duration increases in the presence of tetracycline in a concentration-dependent manner
The maximum specific growth rate of the strain decreased with increasing tetracycline concentrations (Additional file 1: Table S1), showing that increased tetracycline concentrations resulted in slower growth. However, this could not account for the increased lag phase. Since the concentrations we have investigated are within the range of those achieved during therapy , these observations indicate that even in tetracycline-resistant strains growth is delayed during therapy. A recent model study of growth kinetics of tetracycline-resistant strains during therapy in the gut of post weaning pigs showed that resistant strains having a 10 % reduction in the growth rate still could colonize and remain in the gut in a stable manner despite this disadvantage .
Expression of tetR mRNA is tetracycline concentration- and growth phase-dependent
Expression of tetR at a specific tetracycline concentration varied depending on growth phase (Fig. 3). The level of tetR mRNA in MG1655/TetA increased from lag to stationary phase at all concentrations tested. Statistically significant increases in the tetR mRNA level were observed from logarithmic to stationary phase when the strain was exposed to 7 μg/mL or 14 μg/mL of tetracycline (adjusted P-values: 0.026 and 0.025, respectively, see Additional file 1: Table S2).
Expression of tetA mRNA is also tetracycline- and growth phase-dependent
The tetA mRNA levels also varied according to growth phase with different patterns depending on the presence of tetracycline (Fig. 4). A decrease in tetA mRNA was observed from lag to stationary phase in the absence of tetracycline. In the presence of fixed concentrations of tetracycline, the level of tetA mRNA increased from lag to logarithmic phase (except in the logarithmic phase at 3.5 μg/mL tetracycline) and stabilised in the late logarithmic phase. In the stationary phase, the tetA mRNA level was approximately twice of that observed in the late logarithmic growth phase regardless of the drug concentration to which the strain was exposed. Significant P-values for growth phase dependency can be found in Additional file 1: Table S3.
tetA/tetR mRNA ratio
It is known that the TetR repressor protein has a 2-fold higher affinity for the tetA proximal operator O2 site compared to the tetR proximal operator O1 site . This can explain why much lower tetA/tetR mRNA ratios were observed in the absence of tetracycline induction. In the model, tetracycline binds to Mg2+, and these complexes bind to TetR dimers and cause a conformational change that makes the TetR protein unable to bind to the operator sites O1 and O2. This turns on the expression of both tetA and tetR in a tetracycline-dependent manner. In a previous study the tetA promoter was found to be about four times as strong as the two overlapping tetR promoters together . Another study performed by Daniels et al. showed that the tetA promoter was 7–11 fold more active than the tetR promoters combined . A stronger tetA promoter corresponds well with the rise in tetA/tetR mRNA ratios in our studies with increasing concentrations of tetracycline in the lag phase.
A tetracycline concentration-dependent change in tetA/tetR mRNA ratio was found between growth phases with a decrease from onset of growth to maximum growth rate. We speculate that once growth is in the exponential phase, each cell contains the maximum level of TetA pumps in the membrane. This facilitates that enough tetracycline is pumped out to reach a critical concentration below which TetR is again free to bind the operator sites. TetR preferably binds to O2, which results in blocking the transcription of tetA but not of tetR . This explain the increase in tetR mRNA from lag to logarithmic phase, the constant tetA mRNA level in lag, logarithmic and late logarithmic phase (Figs. 3 and 4, respectively), and the lowered tetA/tetR mRNA ratios in the logarithmic, late logarithmic and stationary growth phases compared to the lag phase (Fig. 5). The highest tetA and tetR mRNA levels were observed at stationary phase in the presence of tetracycline. However, interpretation of transcriptional regulation from stationary growth phase cultures should be done with caution due to the cell complexity with a mixture of cells in different physiological phases.
This study shows that a tetracycline resistant TetA-producing E. coli exhibited prolonged lag phase with increasing concentrations of tetracycline. This suggests that even tetracycline-resistant strains are growth-detained in the presence of tetracycline, likely due to the time needed to express the resistance genes and export tetracycline out of the cell. The expression of tetA and tetR increased and decreased, respectively, with increasing tetracycline concentration. Furthermore, expression of tetA and tetR was shown to be growth phase-dependent, and specifically increased from lag to stationary phase in the presence of tetracycline. However, such a growth-dependent increase was not proportional for the two genes, resulting in a gradual decrease of the tetA/tetR mRNA ratio from lag to stationary phases. The basic observations behind the tetR-tetA regulation model , the affinity studies and promoter strength studies [42, 44] previously used to explain regulation of TetA production at low concentrations of tetracycline also seem to be applicable to the data obtained in the current study, which shows that the tetracycline regulation model also applies at therapeutic tetracycline concentrations. Altogether these results contribute to improve understanding of the relationships between E. coli growth, exposure to tetracycline and expression of tetracycline resistance.
The work was funded by University of Copenhagen, Faculty of Health and Medical Sciences and the MINIRESIST project (10–093490). We acknowledge European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement N8289285.
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