Despite the widespread use of tetracycline antibiotics since the late 1940s, tetracycline hypersensitivity reactions have rarely been described in the literature. A comprehensive PubMed search was performed, including allergic and serious adverse reactions attributed to the tetracyclines class of antibiotics. Of the evaluated tetracycline analogs, minocycline was attributed to the greatest overall number and severity of serious adverse events reported in the literature, with notable reactions primarily reported as respiratory and dermatologic in nature. Reactions to tetracycline have also been well described in the literature, and although dermatologic reactions are typically less severe in comparison with minocycline and doxycycline, various reports of anaphylactic reactions exist. Although doxycycline has been noted to have had the fewest reports of severe allergic reactions, rare descriptions of life-threatening reactions are still reported in the literature. Allergic reactions regarding tetracyclines are rare; however, adverse reaction type, severity, and frequency among different tetracycline analogs is somewhat variable. A consideration of hypersensitivity and adverse reaction incidence should be performed prior to the selection of individual tetracycline entities.

The tetracycline operon is an important gene network component, commonly used in synthetic biology applications because of its switch-like character. At the heart of this system is the highly specific interaction of the tet repressor protein (TetR) with its cognate DNA sequence (tetO). TetR binding on tetO practically stops expression of genes downstream of tetO by excluding RNA polymerase from binding the promoter and initiating transcription. Mutating the tetO sequence alters the strength of TetR-tetO binding and thus provides a tool to synthetic biologists to manipulate gene expression levels. We employ molecular dynamics (MD) simulations coupled with the free energy perturbation method to investigate the binding affinity of TetR to different tetO mutants. We also carry out in vivo tests in Escherichia coli for a series of promoters based on these mutants. We obtain reasonable agreement between experimental green fluorescent protein (GFP) repression levels and binding free energy differences computed from molecular simulations. In all cases, the wild-type tetO sequence yields the strongest TetR binding, which is observed both experimentally, in terms of GFP levels, and in simulation, in terms of free energy changes. Two of the four tetO mutants we tested yield relatively strong binding, whereas the other two mutants tend to be significantly weaker. The clustering and relative ranking of this subset of tetO mutants is generally consistent between our own experimental data, previous experiments with different systems and the free energy changes computed from our simulations. Overall, this work offers insights into an important synthetic biological system and demonstrates the potential, as well as limitations of molecular simulations to quantitatively explain biologically relevant behavior.

Tetracycline resistance by antibiotic inactivation was first identified in commensal organisms but has since been reported in environmental and pathogenic microbes. Here, we identify and characterize an expanded pool of tet(X)-like genes in environmental and human commensal metagenomes via inactivation by antibiotic selection of metagenomic libraries. These genes formed two distinct clades according to habitat of origin, and resistance phenotypes were similarly correlated. Each gene isolated from the human gut encodes resistance to all tetracyclines tested, including eravacycline and omadacycline. We report a biochemical and structural characterization of one enzyme, Tet(X7). Further, we identify Tet(X7) in a clinical Pseudomonas aeruginosa isolate and demonstrate its contribution to tetracycline resistance. Lastly, we show anhydrotetracycline and semi-synthetic analogues inhibit Tet(X7) to prevent enzymatic tetracycline degradation and increase tetracycline efficacy against strains expressing tet(X7). This work improves our understanding of resistance by tetracycline-inactivation and provides the foundation for an inhibition-based strategy for countering resistance.

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.

The resistance of the emerging human pathogen Stenotrophomonas maltophilia to tetracycline antibiotics mainly depends on multidrug efflux pumps and ribosomal protection enzymes. However, the genomes of several strains of this Gram-negative bacterium code for a FAD-dependent monooxygenase (SmTetX) homologous to tetracycline destructases. This protein was recombinantly produced and its structure and function were investigated. Activity assays using SmTetX showed its ability to modify oxytetracycline with a catalytic rate comparable to those of other destructases. SmTetX shares its fold with the tetracycline destructase TetX from Bacteroides thetaiotaomicron; however, its active site possesses an aromatic region that is unique in this enzyme family. A docking study confirmed tetracycline and its analogues to be the preferred binders amongst various classes of antibiotics.

Manure compost has been thought of as a potential important route of transmission of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) from livestock to humans. To clarify the abundance of ARB and ARGs, ARB and ARGs were quantitatively determined in tetracycline-resistant Escherichia coli (harboring the tetA gene)-spiked feces in simulated composts. In the simulated composts, the concentration of spiked E. coli decreased below the detection limit at day 7. The tetA gene remained in manure compost for 20 days, although the levels of the gene decreased. Next, to clarify the field conditions of manure compost in Japan, the quantities of tetracycline-resistant bacteria, tetracycline resistance genes, and residual tetracyclines were determined using field-manure-matured composts in livestock farms. Tetracycline-resistant bacteria were detected in 54.5% of tested matured compost (6/11 farms). The copy number of the tetA gene and the concentrations of residual tetracyclines in field manure compost were significantly correlated. These results suggest that the use of antimicrobials in livestock constitutes a selective pressure, not only in livestock feces but also in manure compost. The appropriate use of antimicrobials in livestock and treatment of manure compost are important for avoiding the spread of ARB and ARGs.

The negligible water solubility of tetracycline (TC), a well-known antibiotic of clinical use, is the major disadvantage for its oral administration. With the aim to improve the water solubility of TC, the micelles of formulae SLS@TC and CTAB@TC (SLS = sodium lauryl sulphate and CTAB = cetrimonium bromide) were synthesized. The micelles SLS@TC and CTAB@TC were characterized by melting point (m.p.), thermogravimetric differential thermal analysis (TG-DTA), differential scanning calorimetry (DTG/DSC), attenuated total reflection spectroscopy (FT-IR-ATR), ultra-violet visible (UV/vis) spectroscopy, proton nucleus magnetic resonance (1H-NMR) spectroscopy, and the ultrasonically-induced biregringence technique. The antimicrobial activity of SLS@TC and CTAB@TC was evaluated, by means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and inhibition zone (IZ), against the Gram negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) and the Gram positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus). Generally, both micelles show better activity than that of TC against the microbial strains tested. Thus, the MIC value of CTAB@TC is 550-fold higher than that of free TC against S. epidermidis. Despite the stronger activity of CTAB@TC than SLS@TC against both Gram negative and Gram positive microbes, SLS@TC is classified as a bactericidal agent (in that it eliminates 99.9% of the microbes), in contrast to CTAB@TC, which is bacteriostatic one (inhibits, but does not kill the organisms). The toxicity of SLS@TC and CTAB@TC was evaluated against human corneal eukaryotic cells (HCECs). Moreover, SLS@TC and CTAB@TC exhibit low in vivo toxicity against Artemia salina, even at concentrations up to threefold higher than those of their MICmax. Therefore, SLS@TC and CTAB@TC can be candidates for the development of new antibiotics.

Sertraline, an antidepressive drug, has been reported to inhibit general bacterial efflux pumps. In the present study, we report for the first time a synergistic effect of sertraline and tetracycline in a TetA-encoded tetracycline-resistant strain of Escherichia coli. Synergy between sertraline and tetracycline in an E. coli strain with TetA-mediated tetracycline resistance (E. coli APEC_O2) was assessed by the MIC and checkerboard assays. The global transcriptome of E. coli APEC_O2 exposed to ½ MIC concentrations of sertraline and/or tetracycline was analyzed to elucidate the interaction mechanism between sertraline and tetracycline. The fractional inhibitory concentration index for tetracycline and sertraline in E. coli APEC_O2 was 0.5. In addition, in the presence of ½ MIC of sertraline, the sensitivity of E. coli APEC_O2 to tetracycline could be restored according to clinical standards (from 64 to 4 mg l-1). RNA data suggest changes in respiration that is likely to decrease intracellular pH and thereby the proton-motive force, which provides the energy for the tetracycline efflux pump. Furthermore, sertraline and tetracycline may induce a change from oxidation to fermentation in the E.coli, which further decreases pH, resulting in cell death. This study shows that sertraline interacts with tetracycline in a synergistic and AcrAB-TolC pump-independent manner. The combinational treatment was further shown to induce many changes in the global transcriptome, including altered tetA and tetR expression. The results indicate that sertraline may be used as a helper compound with the aim to reverse tetracycline resistance encoded by tetA.

Antimicrobial helper-compounds may reverse antimicrobial resistance. Sertraline, a antidepressant drug, has been suggested as a tetracycline helper-compound. Tetracycline is the preferred antimicrobial for treatment of enteric diseases in pigs. This study is the first to evaluate the potency of sertraline as a tetracycline adjuvant in pigs.

The aims of the present study were to assess the prevalence of Chlamydia suis in an Italian pig herd, determine the tetracycline susceptibility of C. suis isolates, and evaluate tet(C) and tetR(C) gene expression. Conjunctival swabs from 20 pigs were tested for C. suis by real-time polymerase chain reaction, and 55% (11) were positive. C. suis was then isolated from 11 conjunctival swabs resampled from the same herd. All positive samples and isolates were positive for the tet(C) resistance gene. The in vitro susceptibility to tetracycline of the C. suis isolates showed MIC values ranging from 0.5 to 4 μg/mL. Tet(C) and tetR(C) transcripts were found in all the isolates, cultured both in the absence and presence of tetracycline. This contrasts with other Gram-negative bacteria in which both genes are repressed in the absence of the drug. Further investigation into tet gene regulation in C. suis is needed.

The combination of nicotinamide and tetracycline has been anecdotally reported to be effective in the treatment of bullous pemphigoid. We conducted a randomized, open-labeled trial comparing the combination of 500 mg of nicotinamide, three times daily, and 500 mg of tetracycline four times daily, with prednisone therapy in 20 patients with bullous pemphigoid. The study was divided between an 8-week acute phase with fixed drug dosages and a 10-month follow-up phase in which study medications were tapered based on patient response.

Mitohormesis defines the increase in fitness mediated by adaptive responses to mild mitochondrial stress. Tetracyclines inhibit not only bacterial but also mitochondrial translation, thus imposing a low level of mitochondrial stress on eukaryotic cells. We demonstrate in cell and germ-free mouse models that tetracyclines induce a mild adaptive mitochondrial stress response (MSR), involving both the ATF4-mediated integrative stress response and type I interferon (IFN) signaling. To overcome the interferences of tetracyclines with the host microbiome, we identify tetracycline derivatives that have minimal antimicrobial activity, yet retain full capacity to induce the MSR, such as the lead compound, 9-tert-butyl doxycycline (9-TB). The MSR induced by doxycycline (Dox) and 9-TB improves survival and disease tolerance against lethal influenza virus (IFV) infection when given preventively. 9-TB, unlike Dox, did not affect the gut microbiome and also showed encouraging results against IFV when given in a therapeutic setting. Tolerance to IFV infection is associated with the induction of genes involved in lung epithelial cell and cilia function, and with downregulation of inflammatory and immune gene sets in lungs, liver, and kidneys. Mitohormesis induced by non-antimicrobial tetracyclines and the ensuing IFN response may dampen excessive inflammation and tissue damage during viral infections, opening innovative therapeutic avenues.

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics, since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring; however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TET-resistance genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50% resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate. In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates' BOX-PCR profiles, suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding, intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and expression merit further research.

Although tetracyclines are an important class of antibiotics for use in agriculture and the clinic, their efficacy is threatened by increasing resistance. Resistance to tetracyclines can occur through efflux, ribosomal protection, or enzymatic inactivation. Surprisingly, tetracycline enzymatic inactivation has remained largely unexplored, despite providing the distinct advantage of antibiotic clearance. The tetracycline destructases are a recently discovered family of tetracycline-inactivating flavoenzymes from pathogens and soil metagenomes that have a high potential for broad dissemination. Here, we show that tetracycline destructases accommodate tetracycline-class antibiotics in diverse and novel orientations for catalysis, and antibiotic binding drives unprecedented structural dynamics facilitating tetracycline inactivation. We identify a key inhibitor binding mode that locks the flavin adenine dinucleotide cofactor in an inactive state, functionally rescuing tetracycline activity. Our results reveal the potential of a new tetracycline and tetracycline destructase inhibitor combination therapy strategy to overcome resistance by enzymatic inactivation and restore the use of an important class of antibiotics.

The specificity and selectivity of protein-protein interactions are of central importance for many biological processes, including signal transduction and transcription control. We used the in-house side-chain packing program MUMBO to computationally design a chain-specific heterodimeric variant of the bacterial transcription regulator tetracycline repressor (TetR), called T-A(A)B. Our goal was to engineer two different TetR chain variants, A and B, that no longer interact as AA or BB homodimers but selectively recombine to form heterodimers. Although 56 residues from each chain contribute to a dimer interface as large as 2200 Å(2) in wild-type TetR, the substitution of only three residues in one chain and two residues in a second chain sufficed for generating specificity in a T-A(A)B heterodimer variant. The design was corroborated in vivo by a cell-based transcription assay, and in vitro by CD spectroscopy and X-ray crystallography. Crystal structure analyses showed that while selectivity in the B chain is achieved entirely through van der Waals repulsion, the best selectivity in the A chain is obtained for the variant with the lowest number of atoms in the interface, thus possibly leading to underpacking of the dimer interface. This results in a marked decrease in thermal stability and a drastic reduction in the solubility of the T-A(A)A(A) homodimer in comparison to the designed T-A(A)B heterodimer variant.

Plasma levels of tetracycline in chickens were determined after intravenous (iv) administration of a 65-mg/kg dose. The disposition kinetics of tetracycline in chickens were fitted to a two-compartment open model. Pharmacokinetic parameters were found to be: A (microgram/ml) = 2000 +/- 450, alpha (hr-1) = 4.3 +/- .5, B (microgram/ml) = 82 +/- 6, beta (hr-1) = .252 +/- .009, K12 (hr-1) = 1.515, K21 (hr-1) = .049, and K10 (hr-1) = 2.652. Biliary excretion of tetracycline was also studied in chickens fitted with cannulae inserted into both bile ducts. The maximum values for tetracycline biliary excretion rate (407 and 606 micrograms/hr) were reached at about 1 hr after iv administration of 10- and 15-mg/kg doses. First-order rate constants for the biliary excretion, Kbi (hr-1), were .834 and .665, respectively. The cumulative biliary excretion study showed that about 7% of both administered doses was recovered from the bile within the first 6 hr. In contrast, there was a low recovery of antibiotic in the bile after oral administration of 100 and 200 mg/kg doses.

We developed a competition-based screening strategy to identify compounds that invert the selective advantage of antibiotic resistance. Using our assay, we screened over 19,000 compounds for the ability to select against the TetA tetracycline-resistance efflux pump in Escherichia coli and identified two hits, β-thujaplicin and disulfiram. Treating a tetracycline-resistant population with β-thujaplicin selects for loss of the resistance gene, enabling an effective second-phase treatment with doxycycline.

Antibiotics have become a new kind of organic pollutant as they are widely used in the water environment of China. Tetracycline (TC) is a class of broad-spectrum antibiotics produced or semi-synthesized by actinomycetes. Metronidazole (MTZ) is the first generation of typical nitroimidazoles. The content of nitroimidazoles is relatively high in medical wastewater, and their ecotoxicity is worthy of attention because they are difficult to completely eliminate. In this paper, the effects of TC and MTZ on the growth, cell morphology, extracellular polymer and oxidative stress of Chlorella pyrenoidosa (C. pyrenoidosa) were studied, and the toxic interactions between TC and MTZ mixture components were analyzed. The results showed that the 96h-EC50 of TC and MTZ was 8.72 mg/L and 45.125 mg/L, respectively. The toxicity of TC to C. pyrenoidosa was higher than that of MTZ, and the combined toxicity effect of TC and MTZ was synergistic after the combined action of a 1:1 toxicity ratio. In addition, the algal cells of C. pyrenoidosa died to varying degrees, the membrane permeability of algal cells was increased, the membrane was damaged, the surface of algal cells exposed to higher concentration of pollutants was wrinkled, and their morphology was changed. The extracellular polymer of C. pyrenoidosa was affected by a change in concentration. The effect of pollutants on the reactive oxygen species (ROS) level and malondialdehyde (MDA) content of C. pyrenoidosa also had an obvious dose-effect relationship. This study contributes to the assessment of the possible ecological risks to green algae due to the presence of TC and MTZ in aquatic environments.

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

The negative impacts on the ecosystem of antibiotic residues in the environment have become a global concern. However, little is known about the transformation mechanism of antibiotics by manganese peroxidase (MnP) from microorganisms. This work investigated the transformation characteristics, the antibacterial activity of byproducts, and the degradation mechanism of tetracycline (TC) by purified MnP from Phanerochaete chrysosporium. The results show that nitrogen-limited and high level of Mn2+ medium could obtain favorable MnP activity and inhibit the expression of lignin peroxidase by Phanerochaete chrysosporium. The purified MnP could transform 80% tetracycline in 3 h, and the threshold of reaction activator (H2O2) was about 0.045 mmol L-1. After the 3rd cyclic run, the transformation rate was almost identical at the low initial concentration of TC (77.05-88.47%), while it decreased when the initial concentration was higher (49.36-60.00%). The antimicrobial potency of the TC transformation products by MnP decreased throughout reaction time. We identified seven possible degradation products and then proposed a potential TC transformation pathway, which included demethylation, oxidation of the dimethyl amino, decarbonylation, hydroxylation, and oxidative dehydrogenation. These findings provide a novel comprehension of the role of MnP on the fate of antibiotics in nature and may develop a potential technology for tetracycline removal.