A Preliminary Study on the Concentration of Oxytetracycline and 4-Epi-Oxytetracycline in Sow Milk.

Even though modern analytical chemistry has developed a methodology enabling evaluation of the presence of OTC in milk, data regarding its concentration in the material collected from lactating sows are missing. Therefore, this paper was intended to provide new data on the transmission of OTC and its epimer, 4-epi-oxytetracycline (4-epi-OTC), in the milk of lactating sows after a singular intramuscular administration of a long-acting form of the antibiotic. The determination of OTC and 4-epi-OTC was carried out using ultrahigh-performance liquid chromatography with mass spectrometry (UHPLC-MS/MS). The highest average concentration of antibiotic (1132.2 µgL-1) was observed in samples collected 1 day after the administration of the drug. The average OTC level at day 3 was 358 µgL-1. The average concentration of the antibiotic found on the 21st day was 12.3 µgL-1. The highest average concentration of 4-epi-OTC-i.e., 54 µgL-1-was noted 1 day after the administration. Amongst samples collected at day 3, the average level of the substance in question was 26.4 µgL-1. The average value observed at day 21 was 1.5 µgL-1. Our results indicated considerable OTC and 4-epi-OTC transmission into the milk of lactating sows.

Diagnosis of EGFR exon21 L858R point mutation as lung cancer biomarker by electrochemical DNA biosensor based on reduced graphene oxide /functionalized ordered mesoporous carbon/Ni-oxytetracycline metallopolymer nanoparticles modified pencil graphite electrode.

In this present work we made a novel, fast, selective and sensitive electrochemical genobiosensor to detection of EGFR exon 21 point mutation based on two step electropolymerization of Ni(II)-oxytetracycline conducting metallopolymer nanoparticles (Ni-OTC NPs) on the surface of pencil graphite electrode (PGE) which was modified by reduced graphene oxide/carboxyl functionalized ordered mesoporous carbon (rGO/f-OMC) nanocomposite. ssDNA capture probe with amine groups at the5' end which applied as recognition element was immobilized on the rGO/f-OMC/PGE surface via the strong amide bond. Ni-OTC metallopolymer NPs were electropolymerized to rGO/ssDNA-OMC/PGE surface and then hybridization fallows through the peak current change in differential pulse voltammetry (DPV) using Ni-OTC NPs as a redox label. The biosensor was characterized by field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), FT-IR spectroscopy, energy dispersive X-ray spectroscopy (EDX), cyclic voltammetry and Nitrogen adsorption-desorption analysis. The Ni-OTC current response verified only the complementary sequence indicating a significant reduction current signal in comparison to single point mismatched and non-complementary and sequences. Under optimal conditions, the prepared biosensor showed long-term stability (21 days) with a wide linear range from 0.1 µM to 3 µM with high sensitivity (0.0188 mA/µM) and low detection limit (120 nM).

Development of a quantum dot-based immunochromatography test strip for rapid screening of oxytetracycline and 4-epi-oxytetracycline in edible animal tissues.

A rapid and sensitive immunochromatographic test strip (ICTS) was developed in a competitive format with the quantum dot-conjugated monoclonal antibody specifically to determine the residues of oxytetracycline (OTC) and its metabolite 4-epi-oxytetracycline (4-epi-OTC). Using an ICTS reader, the 50% inhibition concentration (IC50) was found to be 3.41 ± 0.29 ng ml-1 of OTC in phosphate-buffered saline samples and the detection limit was 0.44 ± 0.05 ng ml-1. The visual cut-off level of the ICTS is 25 ng ml-1. The recoveries from tissue samples spiked with OTC of 50-600 µg kg-1 were 74.2-107.2%, with coefficients of variation below 15%. The method was used to analyse incurred tissue samples, and there was good correlation (R2 = 0.999) between the ICTS and high-performance liquid chromatography. These results indicate that ICTS is suitable for the rapid and quantitative detection of OTC and 4-epi-OTC residues in edible animal tissues.

Residue depletion of oxytetracycline (OTC) and 4-epi-oxytetracycline (4-epi-OTC) in broiler chicken's claws by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Antibiotics are widely used in poultry production for the treatment of bacterial diseases. However, residues may remain in products and by-products destined for human consumption or animal feeding. The claws of chickens, which are a by-product of the poultry industry, can directly or indirectly enter the food chain as meals destined to feed other productive animals. Thus, it becomes necessary to determine and quantify antimicrobial residues present in this matrix. The objective of the study was to assess the depletion of oxytetracycline (OTC) and its metabolite 4-epi-OTC in broiler chicken's claws. Claws of 32 broilers treated with a therapeutic dosage of 10% OTC during 7 days were analysed. Samples were taken at days 3, 9, 15 and 19 post-treatment. As for the control group, eight broiler chickens were raised under the same conditions. Extraction was carried out through EDTA-McIlvaine buffer, and clean-up employed a SPE C-18 Sep-Pak®. Instrumental analysis was performed through LC-MS/MS. The concentrations of both analytes were determined in claw samples until day 19 post-treatment. Average concentrations were within the LOD (20 µg kg-1) and LOQ (22 µg kg-1) for OTC and 84 µg kg-1 for 4-epi-OTC. Withdrawal times (WDTs) of 39 days for OTC and 54 days for 4-epi-OTC were established in claws based on 95% confidence. These findings demonstrate that claws can be a source of antimicrobial residue entry into the food chain, since the results showed that OTC and its metabolite can be found in chicken's claws for long periods, even exceeding the average lifespan of a broiler chicken.

Impact of 4-epi-oxytetracycline on the gut microbiota and blood metabolomics of Wistar rats.

The impact of 4-epi-oxytetracycline (4-EOTC), one of the main oxytetracycline (OTC) metabolites, on the gut microbiota and physiological metabolism of Wistar rats was analyzed to explore the dynamic alterations apparent after repeated oral exposure (0.5, 5.0 or 50.0 mg/kg bw) for 15 days as shown by 16S rRNA pyrosequencing and UPLC-Q-TOF/MS analysis. Both principal component analysis and cluster analysis showed consistently altered patterns with distinct differences in the treated groups versus the control groups. 4-EOTC treatment at 5.0 or 50.0 mg/kg increased the relative abundance of the Actinobacteria, specifically Bifidobacteriaceae, and improved the synthesis of lysophosphatidylcholine (LysoPC), as shown by the lipid biomarkers LysoPC(16:0), LysoPC(18:3), LysoPC(20:3), and LysoPC(20:4). The metabolomic analysis of urine samples also identified four other decreased metabolites: diacylglycerol, sphingomyelin, triacylglycerol, and phosphatidylglycerol. Notably, the significant changes observed in these biomarkers demonstrated the ongoing disorder induced by 4-EOTC. Blood and urine analysis revealed that residual 4-EOTC accumulated in the rats, even two weeks after oral 4-EOTC administration, ceased. Thus, through thorough analysis, it can be concluded that the alteration of the gut microbiota and disorders in blood metabolomics are correlated with 4-EOTC treatment.

High-throughput analysis of tetracycline antibiotics and their epimers in liquid hog manure using Ultra Performance Liquid Chromatography with UV detection.

Antibiotics contained in animal manure can contaminate soil, groundwater and eventually surface and drinking water. To reduce the usage of antibiotics in livestock industry the EU banned their application as growth promoters in 2006. Even though the antibiotics are still used for this purpose and therefore it is necessary to control their applications. An Ultra Performance Liquid Chromatography method (UPLC) with UV detection for determination of tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX) including their epimers in the liquid hog manure was developed. The antibiotics were extracted with ethyl acetate and separated on UPLC BEH Shield RP18 column. The validated method was selective for all analytes and system suitability was assessed. Calibration curves ranged from 7.8 to 250.0mugmL(-1) with determination coefficient of 0.9999. The method limits of quantification ranged from 0.9 to 1.6mgkg(-1). Recoveries were 52.4+/-3.8%, 72.4+/-5.0%, 83.8+/-5.7% and 95.9+/-4.7% for TC, OTC, CT, and DOX, respectively. The method was used for the determination of TC, OTC, CT, and DOX in liquid hog manure samples.

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.

C(18) columns for the simultaneous determination of oxytetracycline and its related substances by reversed-phase high performance liquid chromatography and UV detection.

Simultaneous determination of oxytetracycline, 4-epioxytetracycline, alpha-apooxytetracycline, tetracycline and beta-apooxytetracycline on C(18) columns has been accomplished using a high performance liquid chromatographic method with UV detection. Separation was achieved on a Hypersil BDS RP-C(18) column (250 mm x 4.6 mm) and on a Waters C(18) Symmetry column (150 mm x 3.9mm), 5 microm particle size each. These columns were equilibrated with mobile phases consisted of methanol-acetonitrile-0.1M phosphate buffer pH 8.0 (12.5:12.5:75, v/v/v) and (15:15:70, v/v/v), respectively. The flow rate was 1.0 ml/min and the total elution time was 15 and 5 min, respectively. Both methods were applied to oxytetracycline raw material, human and veterinary formulations, where the excipients did not interfere. External standard calibration curves were linear for 4-epioxytetracycline, oxytetracycline, alpha-apooxytetracycline, tetracycline and beta-apooxytetracycline in the concentration range of 0.27-200 microM, 0.05-200 microM, 0.03-200 microM, 0.35-200 microM and 0.20-200 microM on column A and 0.08-200 microM, 0.15-200 microM, 0.09-200 microM, 0.25-200 microM and 0.47-200 microM on column B, respectively. Day-to-day relative standard deviation of the determination for every component was less than 3%. Concerning the first column, limits of detection and quantification of the above compounds were in the concentration ranges of 10-106 nM and 30-352 nM, respectively, whereas on the second column these ranges became 27-144 nM and 81-475 nM, respectively. Recovery of the separated compounds was 95-105%.

Topical kanamycin: an effective therapeutic option in aerobic vaginitis.

Eighty-one patients with clinical diagnosis of aerobic vaginitis (AV) were included in the study. The patients were randomized for treatment, 45 with kanamycin (100 mg vaginal ovules for 6 days, consecutively) and 36 with meclocycline (35 mg vaginal ovules for 6 days, consecutively). The patients were examined before starting the study, 1-2 days after treatment and 30 days after the end of the study. At the first follow-up the patients showed different levels of symptom reduction. Reduction in the presence of leukocytes, vaginal mucosa burning and itching were statistically significant in the group treated with kanamycin with respect to the group treated with meclocycline. Moreover, there was also reduced isolation of Enterobacteriaeae (97%) in the group treated with kanamycin versus those treated with meclocycline (76%). At the second follow-up, vaginal homeostasis (normalization of pH and presence of lactobacilli) was more evident in the kanamycin-treated group. In conclusion, our data suggest that the topical use of kanamycin could be considered a specific antibiotic for the therapy of this new pathology.

Disruption of an aromatase/cyclase from the oxytetracycline gene cluster of Streptomyces rimosus results in production of novel polyketides with shorter chain lengths.

Oxytetracycline is a polyketide antibiotic made by Streptomyces rimosus. From DNA sequencing, the gene product of otcD1 is deduced to function as a bifunctional cyclase/aromatase involved in ring closure of the polyketide backbone. Although otcD1 is contiguous with the ketoreductase gene, they are located an unusually large distance from the genes encoding the "minimal polyketide synthase" of the oxytetracycline gene cluster. A recombinant, disrupted in the genomic copy of otcD1, made four novel polyketides, all of shorter chain length (by up to 10 carbons) than oxytetracycline. All four novel structures contained the unusual carboxamido group, typical of oxytetracycline. This implies that the carboxamido group is present at the start of biosynthesis of oxytetracycline, a topic that has been debated in the literature. Loss of the cyclase protein has a profound influence on the length of polyketide chain assembled, implying that OtcD1 plays a greater role in the overall integrity of the quaternary structure of the polyketide complex than hitherto imagined.

Application of reversed-phase liquid chromatography and prepacked C18 cartridges for the analysis of oxytetracycline and related compounds.

The reversed-phase (RP) chromatographic separation of oxytetracycline (OTC) 4-epioxytetracycline (4-epiOTC), alpha-apooxytetracycline (alpha-apoOTC), and beta-apooxytetracycline (beta-apoOTC) has been accomplished on an Inertsil C8 column at ambient temperature. Using the simplex method of solvent optimization, a 0.1 M ammonium acetate buffer (pH 3.0)-acetonitrile-tetrahydrofuran (72.5:12.5:15, v/v/v) mobile phase was found to give excellent separation of the compounds. OTC, 4-epiOTC, alpha-apoOTC and beta-apoOTC were resolved in 35 min with calculated detection limits of 40, 20, 50 and 140 ng/ml, respectively. Solid-phase extraction (using RP C18 cartridges) of OTC and OTC degradation compounds from distilled water and porcine muscle was tested at four concentration levels ranging from 200 to 2000 ng/ml (g); overall mean recovery of OTC from distilled water and porcine tissue was greater than 90% and 70%, respectively.

Topical meclocycline sulfosalicylate, benzoyl peroxide, and a combination of the two in the treatment of acne vulgaris.

One hundred and six patients with acne vulgaris of the face were treated for 10 weeks with either topical meclocycline sulfosalicylate, topical benzoyl peroxide or both preparations. A randomized, double-blind parallel group study was used. Benzoyl peroxide proved more effective than meclocycline in reducing acne lesion counts, while local side effects were more common in the benzoyl peroxide-treated patients. The combined treatment was of intermediate efficacy with fewer local side effects.

[Activity of topical flunoxaprofen in nonspecific vaginitis. Comparison with meclocycline sulfosalicylate]. / Attività di flunoxaprofene per uso topico nelle vaginiti aspecifiche. Confronto con meclociclina solfosalicilato.

A trial was performed in 30 patients affected by non-specific vaginitis. The results show that the topical application (by vaginal washings) of flunoxaprofen produces a high therapeutic activity like that of meclocycline. Contrary to meclocycline, flunoxaprofen does not possess bactericidal and bacteriostatic properties. Therefore, the normalization of vaginal flora, with a remarkable increase of Döderlein bacillus, is exclusively due to local antiphlogistic activity of flunoxaprofen. Contrary to meclocycline, flunoxaprofen induces a precocious increase of Döderlein bacillus, necessary for a definitive normalization and to limit the possibility of relapses.

[Double-blind group comparison of topical meclocycline, erythromycin and placebo in the treatment of papulo-pustulosa acne]. / Doppelblinder Gruppenvergleich von topischem Meclocyclin, Erythromycin und Placebo in der Behandlung der Akne papulo-pustulosa.

Ten institutions participated in a controlled clinical trial in order to evaluate the efficacy of topical meclocycline and erythromycin in comparison to placebo with regard to papulopustular acne. Both drugs had been incorporated in the same galenic formulation that served as placebo. The vehicle employed in this study guaranteed equally favorable drug relies for both preparations. At the end of the trial, 419 patients could be evaluated for efficacy. As impartial criterion for evaluation, the number of inflammatory lesions on the right side of the face was counted before and after three months of treatment. In addition, we recorded the patients' and physicians' overall judgment at the end of the study. As compared with placebo, meclocycline as well as erythromycin brought about statistically significant improvement already after two months of treatment. After three months, the results were statistically very highly significant (p less than 0.001). At any time of the study, there could not be demonstrated any difference between the two groups treated with meclocycline and erythromycin.

Determination of meclocycline, a tetracycline analogue, in cream formulations by liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic quantitation of meclocycline (I) in a cream formulation is described. The acidified methanolic extract of the sample was diluted with mobile phase and analyzed on a reverse-phase column by using a mobile phase consisting of EDTA buffer (pH 6.6)-tetrahydrofuran (85:15 v/v). The method gave linear, quantitative, and reproducible results with a detection limit of 0.4 ppm for meclocycline.

Meclosorb, a new topical antibiotic agent in the treatment of acne vulgaris: a double-blind clinical study.

The clinical effect on acne vulgaris of topical treatment with meclocycline sulfosalicylate and systemic treatment with peroral tetracycline (500 mg daily) was compared in a double-blind study of 60 patients treated for 8 weeks. The reducing effect of Meclosorb cream and tetracycline tablets on the number of closed comedones, pustules, papules and cysts was marked and not significantly different. The effect of Meclosorb on open comedones was weak and of slow onset. No side effects were registered. Topical treatment with Meclosorb is an effective and safe alternative to systemic tetracycline treatment of acne vulgaris.

[Effect of propyl gallate on the antibacterial activity of meclocycline sulfosalicylate]. / Influenza del propile gallato sull'attività antibatterica della meclociclina solfosalicilato.

Propyl gallate shows little antibacterial activity however it markedly potentiates the activity of meclocycline against Pseudomonas, Proteus, Serratia, Escherichia coli and Klebsiella strains. The potentiating effect of propyl gallate is seen especially with resistant strains whereas sensitive strains of Salmonella do not manifest a potentiating effect on meclocycline by propyl gallate. The mechanism of action of propyl gallate is discussed.