Permeation of tetracyclines through membranes of liposomes and Escherichia coli.

Uptake of tetracycline (tc), 2-tetracyclinonitrile (CN-tc), and 9-(N, N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline (DMG-DMDOT) by liposomes containing Tet repressor (TetR) and by Escherichia coli cells overexpressing TetR was examined. TetR specifically binds to tetracyclines, enhances their fluorescence and thereby allows selective detection of tetracyclines that have crossed the membranes. Analysis of the diffusion of tc and DMG-DMDOT into liposomes yielded permeation coefficients of (2.4 +/- 0.6) x 10-9 cm.s-1 and (3.3 +/- 0.8) x 10-9 cm.s-1, respectively. Similar coefficients were obtained for uptake of these tetracyclines by E. coli, indicating that diffusion through the cytoplasmic membrane is the rate-limiting step. The permeation coefficients translate into half-equilibration times of approximately 35 +/- 15 min and explain how efflux pumps can mediate resistance against tetracyclines. Furthermore, diffusion of CN-tc into liposomes was at least 400-fold slower than that of tc, indicating that the carboxamide group at position C2 is required for efficient permeation of tc through lipid membranes and thereby explaining the lack of antibiotic activity of CN-tc.     

Unusual effects of 5a,6-anhydrotetracycline and other tetracyclines. Inhibition of guanosine 5'-diphosphate 3'-diphosphate metabolism, RNA accumulation and other growth-related processes in Escherichia coli.

5a,6-Anhydrotetracycline was discovered to be unique among several tetracycline derivatives tested in its ability to inhibit RNA accumulation in vivo at low concentration (20 microgram/ml and less). In addition, in vivo protein, DNA, and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthesis were completely inhibited by 20 microgram/ml 5a,6-anhydrotetracycline. ppGpp decay in a spoT strain was inhibited by 20 microgram/ml 5a,6-anhydrotef RNA synthesis by a 5a,6-anhydrotetracycline may be due, in part, to reduced UTP and CTP synthesis. The effects of tetracyclines on in vitro ppGpp synthesis by crude stringent factor in the absence of ribosomes were investigated. It was determined that of six tetracyclines tested, four strongly inhibited the reaction (oxytetracycline, chlorotetracycline, dedimethylaminotetracycline, and tetracycline) whereas 5a,6-anhydrotetracycline gave a moderate inhibition and alpha-6-deoxyoxytetracycline resulted in only a slight reduction in ppGpp synthesis. It is proposed that tetracyclines interfere with factors involved in ppGpp metabolism and function.     

Genetic and physiological characterization of oxytetracycline-resistant bacteria from giant prawn farms

Four hundred and thirteen oxytetracycline-resistant bacteria were recovered from six freshwater giant prawn farms with a history of oxytetracycline use. Most oxytetracyclineresistant isolates were Gram-negative bacteria. Six groups of oxytetracycline-resistant bacteria were classified using cluster analysis based on a comparison of levels of oxytetracycline resistance. Complex fingerprint patterns were obtained for 71 isolates studied. In general, the band patterns of isolates from different ponds were very similar, and the data indicated that the isolates were closely related. The exploration for crossresistance found that most of the 71 oxytetracycline-resistant isolates were also resistant to tetracycline and chlortetracycline, but had a relatively low resistance to doxycycline. Many isolates showed higher chlortetracycline resistance than oxytetracycline resistance. Additionally, the oxytetracyclineresistant isolates were examined for the presence of tetracycline resistance (tet) genes. Fifty percent of the isolates carried one of the 14 known tet genes examined. The most common determinants were TetA and TetD. However, TetB, TetC, TetE, TetK, TetL, and TetM were also found with various frequencies.     

Effects of disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) and tetracyclines on drug resistant bacteria. Studies in vitro

An effect of Na2EDTA and tetracycline (oxytetracycline and doxycycline) resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa was tested. The strains were isolated from clinical specimens. The tests were performed in vitro by serial dilutions of the drugs in liquid medium. MIC for Na2EDTA, tetracyclines and a combination of Na2EDTA and tetracyclines was determined. It was shown that the combination of oxytetracycline or doxycycline with Na2EDTA caused changes in sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa resistant to these antibiotics. After an application of the mixture of various concentrations of tetracycline and Na2EDTA it was observed that, with the reduction of the effective Na2EDTA dose by about half, the lowest concentrations of tetracyclines inhibiting the growth of resistant bacteria were 2-64 times lower than MIC values of antibiotics without Na2EDTA.     

Resistance of Escherichia coli to tetracyclines. Changes in permeability to tetracyclines in Escherichia coli bearing transferable resistance factors

1. When strains of Escherichia coli, bearing transferable factors for resistance to the tetracyclines (R-factors), and previously cultured in the absence of the tetracyclines, are grown for 15–30min. in a low, subinhibitory, concentration (10μg./ml.) of oxytetracycline or tetracycline, there is a rapid and striking increase in resistance to oxytetracycline or tetracycline, this being associated with a marked fall in the absorption of the drug by the cells. 2. Very short preincubation (1min.) with oxytetracycline, followed by growth for 15–30min. in drug-free medium, produces a marked fall in the absorption of the drug by the resistant cells. Preincubation for 30min. with very low concentrations (0·05μg./ml.) of oxytetracycline produces a similar effect. 3. β-Apo-oxytetracycline, which has very little antibacterial activity, also induces a decreased absorption of oxytetracycline. 4. The ability to exclude oxytetracycline is retained by preincubated resistant cells after growth for 2hr. in drug-free medium. However, after growth for 16hr. in drug-free medium, the cells absorb oxytetracycline freely. 5. Chloramphenicol and proflavine inhibit the adaptive decrease in tetracycline absorption. 5-Fluorouracil has only a slight effect. 6. Spheroplasts prepared from resistant cells show an impaired response to preincubation with tetracycline, compared with intact cells. 7. The relevance of these results to the probable mechanism of tetracycline resistance in R-factor-bearing E. coli is discussed.     

Purification of the Tn 10-specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein

The bacterial tetracycline-resistance determinant from Tn 10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/ac operator in a plasmid that provided fusion of TetA to a polyhis-tidine-carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10–40% that of the wild-type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3–13-fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an α-helix content of 54–64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane-spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13-(cyclopentylthio)-5-hydroxy-6-α-deoxyte-tracycline. These findings suggested that the purified protein was in a native state.     

Role of oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B.

Photoillumination of tetracycline derivatives with low-intensity (320- to 400-nm) light and visible light generated superoxide, observed as the reduction of ferricytochrome c. The rate of reduction was dependent on the tetracycline concentration and on the derivative being examined, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. Tetracycline-mediated cytochrome c reduction was oxygen dependent and inhibited up to 70% by superoxide dismutase. Illuminated tetracyclines were lethal to Escherichia coli B incubated in a glucose minimal medium containing chloramphenicol. This lethality was light dependent, oxygen dependent, and dependent on the concentration of tetracycline. Kill rates also varied according to the derivative under study, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. The addition of superoxide dismutase and catalase to the incubation medium partially protected E. coli B against the light-dependent lethality. Preinduction of intracellular superoxide dismutase and catalase substantially protected E. coli B against the phototoxicity of tetracyclines. Iron EDTA augmented the phototoxicity of tetracyclines, while diethylenetriaminepentaacetic acid protected against their lethality. Hydroxyl radical scavengers also conferred protection against tetracycline phototoxicity. The extent of protection was in order of the in vitro reactivity of the scavengers with the hydroxyl radical. These results indicate that superoxide, hydrogen peroxide, and the hydroxyl radical are generated by illuminated tetracyclines and are molecular agents of tetracycline phototoxicity in E. coli B.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein

Interaction of tetracycline (TC) derivatives viz. oxytetracycline, doxycycline, demeclocycline and chlorotetracycline with bovine serum albumin (BSA) and concomitant changes in protein conformation were studied using fluorescence quenching and circular dichroism measurements. Fluorescence data revealed the presence of one to three binding sites on BSA for different TC derivatives. Binding studies with the marker ligands, warfarin and bilirubin, elucidated site-I as a primary binding site for TCs on albumin. Scatchard analysis revealed the binding affinity (K_a) and capacity (n) for these derivatives vary in the range from 0.8 to 3.2×10⁶ l/mole and 1.3–3.4, respectively. Significant reduction (60–45%) in secondary structure (-helical content) of BSA was noticed upon interaction with different TC derivatives in presence of Cu (II) ions. High affinity binding of TCs with BSA signifies drug stability. However, excessive binding at higher TC concentrations in combination with Cu (II) induces conformational change in protein structure, which may exert detrimental effect on cellular protein.     

Metal ion-tetracycline interactions in biological fluids. 2. Potentiometric study of magnesium complexes with tetracycline, oxytetracycline, doxycycline, and minocycline, and discussion of their possible influence on the bioavailability of these antibiotics in blood plasma

The formation constants of the various complexes formed by magnesium with four tetracycline derivatives, namely, tetracycline itself, oxytetracycline, doxycycline, and minocycline, were determined by potentiometry over large pH ranges under experimental conditions pertaining to blood plasma (37 degrees C, NaCl 0.15 mol dm-3). The results were used, together with those previously obtained on the complexation of these tetracyclines with proton and calcium, to assess the influence of the two alkali earth metal ions on the bioavailability of these drugs in blood plasma. Accordingly, simulations of the distribution of the four tetracyclines into their different proton and metal complex species were calculated. The distributions confirm that, in combination with the protein-bound fraction of the tetracyclines, the metal-bound fraction represents more than 99% of these drugs in plasma, the extent of their free fraction commonly being less than 1%.     

O2- photogenerated from aqueous solutions of tetracycline antibiotics (pH 7.3) as evidenced by DMPO spin trapping and cytochrome c reduction

UV-irradiation of several tetracycline antibiotics in aqueous buffer (pH 7.3) resulted in the generation of the superoxide anion radical (O2-) which was detected by cytochrome c reduction and by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide and was inhibited by superoxide dismutase. A comparison of the O2- yields from the tetracyclines examined showed the trend chlortetracycline (CTC) greater than oxytetracycline (OXY) greater than demeclocycline (DEM) much greater than (doxycycline (DOXY) = tetracycline (TC) = minocycline (MINO) = 0). This trend is in reasonable agreement with clinical reports that CTC, OXY and DEM are potent photosensitizers, TC is only weakly phototoxic whereas MINO is not. These findings suggest that the O2- production may be involved in tetracycline-induced phototoxicity. While the two methods for O2- detection gave comparable results for most of the tetracyclines, the spin trapping technique was clearly superior for DOXY which reduced cytochrome c in the dark.     

Effects of orally administered tetracycline on the intestinal community structure of chickens and on tet determinant carriage by commensal bacteria and Campylobacter jejuni

There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli, and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 microg/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at <2 microg/ml for Campylobacter isolates before and after tetracycline treatment of the chickens, even though isolates expressing MICs of >16 mug/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment.     

Simultaneous determination of fluoroquinolones and tetracyclines in chicken muscle using HPLC with fluorescence detection

A multiresidue method has been developed which allows for the simultaneous determination of both fluoroquinolones and tetracyclines in chicken muscle. Samples were extracted with a mix of acetonitrile and 0.1 M citrate, 150 mM MgCl(2), pH 5.0. After centrifugation and evaporation, the extracts could be analyzed by liquid chromatography with fluorescence detection. Good recoveries (63-95%) were obtained from samples fortified with a mix of five fluoroquinolones and three tetracyclines, with satisfactory relative standard deviations. Limits of detection were 0.5 ng/g (danofloxacin), 1 ng/g (oxytetracycline, ciprofloxacin, enrofloxacin), 1.5 ng/g (tetracycline), 2 ng/g (difloxacin) and 5 ng/g (sarafloxacin, chlortetracycline). Enrofloxacin and its metabolite ciprofloxacin, as well as oxytetracycline were determined in enrofloxacin and oxytetracycline incurred chicken muscle using this method.     

Effects of fused-ring antibiotics on metallic corrosion

The effect on metallic corrosion of antibiotics containing the fused-ring structure of tetracycline (doxycycline and the dihydrate and hydrochloride salts of oxytetracycline) when present in 1% KCl solution was investigated. Corrosion potential and zero resistance ammetry studies were carried out; the effects observed were variable and depended upon the nature of the metal and its surface condition. All three antibiotics appeared to stimulate the corrosion of Vitallium (cobalt-chromium alloy), but corrosion inhibition was found for as-received titanium with all three antibiotics, for abraded titanium with doxycycline and for stainless steel with oxytetracycline dihydrate.     

TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems

The complete nucleotide sequence of the tetracycline resistance plasmid pAG1 from the gram-positive soil bacterium Corynebacterium glutamicum 22243 (formerly Corynebacterium melassecola 22243) was determined. The R-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. The resistance determinant of pAG1 revealed homology to gram-negative tetracycline efflux and repressor systems of Tet classes A through J. The highest levels of amino acid sequence similarity were observed to the transmembrane tetracycline efflux protein TetA(A) and to the tetracycline repressor TetR(A) of transposon Tn1721 with 64 and 56% similarity, respectively. This is the first time a repressor-regulated tet gene has been found in gram-positive bacteria. A new class of tetracycline resistance and repressor proteins, termed TetA(Z) and TetR(Z), is proposed.     

The efficacy of tetracyclines in peripheral and intracerebral prion infection

We have previously shown that tetracyclines interact with and reverse the protease resistance of pathological prion protein extracted from scrapie-infected animals and patients with all forms of Creutzfeldt-Jakob disease, lowering the prion titre and prolonging survival of cerebrally infected animals. To investigate the effectiveness of these drugs as anti-prion agents Syrian hamsters were inoculated intramuscularly or subcutaneously with 263K scrapie strain at a 10(-4) dilution. Tetracyclines were injected intramuscularly or intraperitoneally at the dose of 10 mg/kg. A single intramuscular dose of doxycycline one hour after infection in the same site of inoculation prolonged median survival by 64%. Intraperitoneal doses of tetracyclines every two days for 40 or 44 days increased survival time by 25% (doxycycline), 32% (tetracycline); and 81% (minocycline) after intramuscular infection, and 35% (doxycycline) after subcutaneous infection. To extend the therapeutic potential of tetracyclines, we investigated the efficacy of direct infusion of tetracyclines in advanced infection. Since intracerebroventricular infusion of tetracycline solutions can cause overt acute toxicity in animals, we entrapped the drugs in liposomes. Animals were inoculated intracerebrally with a 10(-4) dilution of the 263K scrapie strain. A single intracerebroventricular infusion of 25 microg/20 microl of doxycycline or minocycline entrapped in liposomes was administered 60 days after inoculation, when 50% of animals showed initial symptoms of the disease. Median survival increased of 8.1% with doxycycline and 10% with minocycline. These data suggest that tetracyclines might have therapeutic potential for humans.     

Prevalence of Antimicrobial Resistance and Transfer of Tetracycline Resistance Genes in Escherichia coli Isolates from Beef Cattle

The aim of this study was to investigate the prevalence and transferability of resistance in tetracycline-resistant Escherichia coli isolates recovered from beef cattle in South Korea. A total of 155 E. coli isolates were collected from feces in South Korea, and 146 were confirmed to be resistant to tetracycline. The tetracycline resistance gene tet(A) (46.5%) was the most prevalent, followed by tet(B) (45.1%) and tet(C) (5.8%). Strains carrying tet(A) plus tet(B) and tet(B) plus tet(C) were detected in two isolates each. In terms of phylogenetic grouping, 101 (65.2%) isolates were classified as phylogenetic group B1, followed in decreasing order by D (17.4%), A (14.2%), and B2 (3.2%). Ninety-one (62.3%) isolates were determined to be multidrug resistant by the disk diffusion method. MIC testing using the principal tetracyclines, namely, tetracycline, chlortetracycline, oxytetracycline, doxycycline, and minocycline, revealed that isolates carrying tet(B) had higher MIC values than isolates carrying tet(A). Conjugation assays showed that 121 (82.9%) isolates could transfer a tetracycline resistance gene to a recipient via the IncFIB replicon (65.1%). This study suggests that the high prevalence of tetracycline-resistant E. coli isolates in beef cattle is due to the transferability of tetracycline resistance genes between E. coli populations which have survived the selective pressure caused by the use of antimicrobial agents.     

Genetic analysis of the tetA(C) gene on plasmid pBR322.

The TetA(C) protein, encoded by the tetA(C) gene of plasmid pBR322, is a member of a family of membrane-bound proteins that mediate energy-dependent efflux of tetracycline from the bacterial cell. The tetA(C) gene was mutagenized with hydroxylamine, and missense mutations causing the loss of tetracycline resistance were identified at 30 distinct codons. Mutations that encoded substitutions within putative membrane-spanning alpha-helical regions were scattered throughout the gene. In contrast, mutations outside the alpha-helical regions were clustered in two cytoplasmic loops, between helices 2 and 3 and helices 10 and 11, suggesting that these regions play a critical role in the recognition of tetracycline and/or energy transduction. All of the missense mutations encoded a protein that retained the ability to rescue an Escherichia coli strain defective in potassium uptake, suggesting that the loss of tetracycline resistance was not due to an unstable TetA(C) protein or to the failure of the protein to be inserted in the membrane. We postulate that the mutations encode residues that are critical for the active efflux of tetracycline, except for mutations that result in the introduction of charged residues within hydrophobic regions of the TetA(C) protein.     

Structure-based design of Tet repressor to optimize a new inducer specificity

We constructed a mutant of the tetracycline-inducible repressor protein TetR with specificity for the tc analogue 4-de(dimethylamino)anhydrotetracycline (4-ddma-atc), which is neither an antibiotic nor an inducer for the wild-type protein. The previously published relaxed specificity mutant TetR H64K S135L displays reduced induction by tc but full induction by doxycycline (dox), anhydrotetracycline (atc), and 4-de(dimethylamino)-6-demethyl-6-deoxytetracycline (cmt3). To create induction specificity for tc derivatives lacking the 4-dimethylamino grouping such as cmt3 and 4-ddma-atc, the residues at positions 82 and 138, which are located close to that moiety in the crystal structure of the TetR-[tc-Mg](+)(2) complex, were randomized. We anticipated that a residue with increased size may lead to sterical hindrance, and screening for 4-ddma-atc-specific induction indeed revealed the mutant TetR H64K S135L S138I. Out of 24 exchanges only the addition of S138I to TetR H64K S135L yielded a mutant with a pronounced reduction of affinity for atc and dox, while the one for 4-ddma-atc is not affected. The ratio of binding constants revealed a 200-fold specificity increase for 4-ddma-atc over atc. The contributions of each single mutant to specificity indicate that the tc variants bind slightly different positions in the TetR tc binding pocket.     

Use of metal chelate affinity chromatography and membrane-based ion-exchange as clean-up procedure for trace residue analysis of tetracyclines in animal tissues and egg

A new and efficient procedure for the clean-up of tetracycline residues in animal tissues and egg prior to reversed-phase high-performance liquid chromatography is described. The principal steps involve homogenization of the tissues in sodium succinate buffer and methanol, followed by centrifugation and clean-up with metal chelate affinity chromatography (MCAC). After further concentration on an Empore extraction membrane with cation-exchange properties, the sample is analysed by HPLC with fluorescence detection. The method was tested on porcine kidney and muscle, bovine liver and whole chicken's egg. The recoveries were determined from spiked tissues for oxytetracycline, tetracycline, chlortetracycline and doxycycline and ranged from 40 to 70%, with repeatabilities below 10% R.S.D.. The analytical responses were linear in the range up to at least 1000 ng/g. The detection limits of the method were estimated at 0.42 ng/g of oxytetracycline, 0.49 ng/g of tetracycline, 0.66 ng/g of chlortetracycline and 1.38 ng/g of doxycycline in porcine muscle, using signal-to-noise ratios of 4:1. Similar detection limits were estimated for kidney, liver and egg. The measured limits of quantification were 2 ng/g for oxytetracycline, 3 ng/g for tetracycline, 4 ng/g for chlortetracycline and 5 ng/g for doxycycline in porcine kidney. The advantage of this method over existing methods is its increased limit of detection.