Degradation of tetracycline, oxytetracycline & ampicillin by purified multiple copper oxidase like laccase from Stentrophomonas sp. YBX1.

Multiple copper oxidase (MCO) like laccase is widely distributed in higher plant, fungi and bacteria. This study identified MCO like laccase producing bacterium isolated from a wastewater treatment plant based on 16S rRNA sequence analysis, and they were further confirmed by phylogenetic reconstruction. Biochemical and gene characterization of MCO like laccase from Stenotrophomonas sp. YBX1 is presented. Purification of MCO like laccase was carried out by ion exchange HQ Trap column and followed by gel filtration spheracryl S-100 column. The purified MCO like laccase from Stenotrophomonas sp. YBX1 shows a total activity of 1252 units and specific activity 391.2 U/mg and protein concentration 0.32 mg/mL. In SDS PAGE, the approximate molecular mass was found at 66 kDa and further confirmed from an MS spectrum of MALDI-TOF. The purified MCO like laccase is capable of degradation of antibiotics such as tetracycline completely, whereas oxytetracycline (78%) and ampicillin (62%) degraded within 96 min without any redox mediators at pH 5 and 30 ºC. Its degradation pathway was based on identification of metabolites by LC-MS spectrum. The enzymatic degradation may be used in advanced treatment of antibiotics containing wastewater.

Identification, phylogenetic analysis, and genome mining of the tetracycline-resistant Bacillus thuringiensis strain m401 reveal its potential for biotechnological and biocontrol applications.

Bacillus thuringiensis is an entomopathogen belonging to the Bacillus cereus clade. We isolated a tetracycline-resistant strain called m401, recovered it from honey, and identified it as Bacillus thuringiensis sv. kumamotoensis based on the average nucleotide identity calculations (ANIb) comparison and the analysis of the gyrB gene sequences of different B. thuringiensis serovars. Sequences with homology to virulence factors [cytK, nheA, nheB, nheC, hblA, hblB, hblC, hblD, entFM, and inhA] and tetracycline resistance genes [tet(45), tet(V), and tet(M)/tet(W)/tet(O)/tet(S) family] were identified in the bacterial chromosome. The prediction of plasmid-coding regions revealed homolog sequences to the MarR and TetR/AcrR family of transcriptional regulators, toxins, and lantipeptides. The genome mining analysis revealed 12 regions of biosynthetic gene clusters responsible for synthesizing secondary metabolites. We identified biosynthetic gene clusters coding for bacteriocins, siderophores, ribosomally synthesized post-translationally modified peptide products, and non-ribosomal peptide synthetase clusters that provide evidence for the possible use of Bt m401 as a biocontrol agent. Furthermore, Bt m401 showed high inhibition against all Paenibacillus larvae genotypes tested in vitro. In conclusion, Bt m401 owns various genes involved in different biological processes, such as transductional regulators associated with antibiotic resistance, toxins, and antimicrobial peptides with potential biotechnological and biocontrol applications.

In-house validation for multi-residue analysis of tetracycline in cow milk by HPLC with UV detection / Validação intralaboratorial para análise múltipla dos resíduos de tetraciclina em leite bovino por cromatografia líquida de alta eficiência com detecção UV

The indiscriminate use of antibiotics in dairy cattle without complying with the waiting period results in residual contamination, whose effective control in produced milk requires validated methods toensure analytical results. The aim of this study was to optimize and validate the HPLC-UV/VIS method at 365 nm for analyzingthe tetracycline in pasteurized cow milk in accordance with the European Community (2002/657/EC). Spiked milk with analytes (oxytetracycline, tetracycline, doxycycline, and chlortetracycline) was submitted to deproteinization and cleaning by a C18 solid-phase column and analyzed by HPLC using a gradient system with 0.01 mol L?1 oxalic acid-acetonitrile-triethylamine (90:9.9:0.1) and acetonitrile on a reverse phase (C18) column. Accuracy and precision were assessed by adding analytes to levels of 0.5, 1, and 1.5 times the permissible maximum limit allowed in Brazil. The method presented selectivity with a decision limit (CC?) and detection capability (CC?) ranging from 114.2 to 143.7 and from 129.3 to 188.7 µg kg?1, respectively. The recovery of tetracyclines was higher than 82.5% with a precision of 7.1%, demonstrating theefficiency in determining tetracycline residues in cow milk.(AU)

O uso indiscriminado de antibiótico em gado leiteiro, sem cumprimento do período de carência, resulta em contaminação residual, cujo controle efetivo no leite produzido requer métodos validados que garantam os resultados analíticos. O estudo visou otimizar e validar o método de CLAE-UV/VIS a 365 nm para análise de tetraciclina em leite bovino pasteurizado em conformidade com a Comunidade Europeia (2002/657/EC). O leite fortificado com analitos (oxitetraciclina, tetraciclina, doxiciclina e clortetraciclina) foi submetido à extração por desproteinização e limpeza por coluna de fase sólida C18 e submetido à análise por CLAE empregando sistema gradiente com 0,01 mol L-1 de ácido oxálico-acetonitrila-trietilamina (90:9,9:0,1) e acetonitrila, em coluna de fase reversa (C18). A exatidão e precisão foram avaliadas adicionando o analitos em níveis de 0,5, 1 e 1,5 vezes o limite máximo permitido no Brasil. O método apresentou seletividade com limite de decisão (CC?) e capacidade de detecção (CC?) variando de 114,2 a 143,7 e 129,3 a 188,7 µg kg-1, respectivamente. A recuperação de tetraciclinas foi superior a 82,5% e a precisão de 7,1%, demonstrando eficiência para a determinação de resíduos de tetraciclina em leite bovino.(AU)

In-house validation for multi-residue analysis of tetracycline in cow milk by HPLC with UV detection / Validação intralaboratorial para análise múltipla dos resíduos de tetraciclina em leite bovino por cromatografia líquida de alta eficiência com detecção UV

The indiscriminate use of antibiotics in dairy cattle without complying with the waiting period results in residual contamination, whose effective control in produced milk requires validated methods toensure analytical results. The aim of this study was to optimize and validate the HPLC-UV/VIS method at 365 nm for analyzingthe tetracycline in pasteurized cow milk in accordance with the European Community (2002/657/EC). Spiked milk with analytes (oxytetracycline, tetracycline, doxycycline, and chlortetracycline) was submitted to deproteinization and cleaning by a C18 solid-phase column and analyzed by HPLC using a gradient system with 0.01 mol L?1 oxalic acid-acetonitrile-triethylamine (90:9.9:0.1) and acetonitrile on a reverse phase (C18) column. Accuracy and precision were assessed by adding analytes to levels of 0.5, 1, and 1.5 times the permissible maximum limit allowed in Brazil. The method presented selectivity with a decision limit (CC?) and detection capability (CC?) ranging from 114.2 to 143.7 and from 129.3 to 188.7 µg kg?1, respectively. The recovery of tetracyclines was higher than 82.5% with a precision of 7.1%, demonstrating theefficiency in determining tetracycline residues in cow milk.

O uso indiscriminado de antibiótico em gado leiteiro, sem cumprimento do período de carência, resulta em contaminação residual, cujo controle efetivo no leite produzido requer métodos validados que garantam os resultados analíticos. O estudo visou otimizar e validar o método de CLAE-UV/VIS a 365 nm para análise de tetraciclina em leite bovino pasteurizado em conformidade com a Comunidade Europeia (2002/657/EC). O leite fortificado com analitos (oxitetraciclina, tetraciclina, doxiciclina e clortetraciclina) foi submetido à extração por desproteinização e limpeza por coluna de fase sólida C18 e submetido à análise por CLAE empregando sistema gradiente com 0,01 mol L-1 de ácido oxálico-acetonitrila-trietilamina (90:9,9:0,1) e acetonitrila, em coluna de fase reversa (C18). A exatidão e precisão foram avaliadas adicionando o analitos em níveis de 0,5, 1 e 1,5 vezes o limite máximo permitido no Brasil. O método apresentou seletividade com limite de decisão (CC?) e capacidade de detecção (CC?) variando de 114,2 a 143,7 e 129,3 a 188,7 µg kg-1, respectivamente. A recuperação de tetraciclinas foi superior a 82,5% e a precisão de 7,1%, demonstrando eficiência para a determinação de resíduos de tetraciclina em leite bovino.

A simple, rapid and inexpensive screening method for the identification of Pythium insidiosum.

Growth of Pythium insidiosum mycelia around minocycline disks (30µg) did not occur within 7days of incubation at 35°C when the isolates were grown on Sabouraud, corn meal, Muller-Hinton or RPMI agar. This technique offers a simple and rapid method for the differentiation of P. insidiosum from true filamentous fungi.

NMR techniques in drug delivery: application to zein protein complexes.

Zein is a protein containing a large amount of nonpolar amino acids, which has shown the ability to form aggregates and entrap solutes, such as drugs and amino acids. NMR techniques were used to detect binding interactions and measure affinity between zein and three different drugs: tetracycline, amoxicillin and indomethacin. The release study of zein microparticle formulations containing any of these drugs was confronted with the affinity results, showing a remarkable correlation. The feasible methodology employed, focused in the functionality of the protein-drug interaction, can be very promising for the rational design of appropriate drug vehicles for drug delivery.

Global expression profile of biofilm resistance to antimicrobial compounds in the plant-pathogenic bacterium Xylella fastidiosa reveals evidence of persister cells.

Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.

Influence of Ca2+ on tetracycline adsorption on montmorillonite.

The adsorption of tetracycline (TC) on montmorillonite was studied as a function of pH and Ca(2+) concentration using a batch technique complemented with X-ray diffraction and transmission electron microscopy. In the absence of Ca(2+), TC adsorption was high at low pH and decreased as the pH increased. In the presence of Ca(2+), at least two different adsorption processes took place in the studied systems, i.e., cation exchange and Ca-bridging. Cation exchange was the prevailing process at pH<5, and thus, TC adsorption decreased by increasing total Ca(2+) concentration. On the contrary, Ca-bridging was the prevailing process at pH>5, and thus, TC adsorption increased by increasing Ca(2+) concentration. The pH 5 represents an isoadsorption pH where both adsorption processes compensate each other. TC adsorption became independent of Ca(2+) concentration at this pH. For TC adsorption on Ca(2+)-montmorillonite in 0.01 M NaCl experiments, the ratio adsorbed TC/retained Ca(2+) was close to 1 in the pH range of 5-9, indicating an important participation of Ca(2+) in the binding of TC to montmorillonite. X-ray diffraction and transmission electron microscopy showed that TC adsorption induced intercalation between montmorillonite layers forming a multiphase system with stacking of layers with and without intercalated TC.

Binding of the highly toxic tetracycline derivative, anhydrotetracycline, to bovine serum albumin.

Tetracycline (TC) derivatives are extensively used as antibiotics in human and animal medicine and, very recently, they have been screened as anti-amyloidogenic drugs. Anhydrotetracycline (AHTC) is one of the major degradation products of TC that has been linked to several side effects of the drug. We evaluated the interaction of AHTC with bovine serum albumin (BSA), one of the main carriers of amphiphilic molecules in blood, using three complementary analytical methods: fluorescence spectroscopy, isothermal titration calorimetry and differential scanning calorimetry. AHTC bound to BSA with an association constant in the order of 10(5) M(-1). Drug binding was enthalpically and entropically driven and seemed to involve hydrophobic interactions. AHTC fluorescence enhancement and hypsochromic shifts observed upon binding suggested a low-polarity location excluded from water for the bound drug. Our data are useful for evaluating the biodisponibility of the pharmacophore and the dynamic distribution of the toxic derivative.

Enhancement of europium emission band of europium tetracycline complex in the presence of cholesterol.

We report here the observation, for the first time, of the enhancement of Europium-Tetracycline complex emission in cholesterol solutions. This enhancement was initially observed with the addition of the enzyme cholesterol oxidase, which produces H(2)O(2), the agent driver of the Europium tetracycline complex, to the solution. However, it was found that the enzyme is not needed to enhance the luminescence. A calibration curve was determined, resulting in a simple method to measure the cholesterol quantity in a solution. This method shows that the complex can be used as a sensor to determine cholesterol in biological systems.

Detection of high-level tetracycline resistance in clinical isolates of Helicobacter pylori using PCR-RFLP.

Tetracycline is one of four antibiotics commonly used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing as the incidence of tetracycline resistance is increasing. In five Brazilian tetracycline-resistant (Tet(R)) H. pylori isolates, high-level tetracycline resistance is mediated by the triple-base-pair substitution AGA(926-928)-->TTC in both 16S rRNA genes, as was previously observed in two independent high-level Tet(R) H. pylori strains. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection of the AGA(926-928)-->TTC substitution, and confirmed the presence of the aforementioned triple-base-pair substitution in all five Brazilian Tet(R) isolates. This PCR-RFLP-based approach distinguishes the high-level Tet(R) isolates from low-level Tet(R) and Tet(S) H. pylori strains and thus allows the direct detection of Tet(R) H. pylori isolates.

Enhanced synchronous spectrofluorometric determination of tetracycline in blood serum by chemometric analysis. Comparison of partial least-squares and hybrid linear analysis calibrations.

Tetracycline has been determined in human serum samples by a combination of: (1) synchronous fluorescence spectra of whole sera treated with Mg2+, and (2) the multivariate calibration methods of partial least-squares (PLS-1) and a variant of the recently introduced hybrid linear analysis (HLA), which does not require the knowledge of pure-component spectra. The calibration set was designed with 50 sera spiked with concentrations of tetracycline in the range 0.0-4.0 micrograms mL-1'. Studies concerning validation, precision, accuracy and figures of merit (selectivity, sensitivity and limit of determination) were also carried out. A novel wavelength-selection procedure was applied to minimize the effect of nonmodeled interferents present in serum samples containing bilirubin, triglycerides, and salicylate. Overall, the performance of the newly developed HLA approach seems to be better than that of PLS-1.

Light emission accompanies oxygen uptake during the peroxidative metabolism of tetracyclines.

Tetracycline molecules offer several sites for peroxidative metabolism of the type known to lead to oxygen consumption and electronic excitation. Accordingly, when tetracycline and chlortetracycline were exposed to horseradish peroxidase in the presence of hydrogen peroxide, oxygen was taken up and light emission was observed. The overall quantum yield of chemiluminescence is on the order of 10(-6), but that of chemiexcitation may be orders of magnitude higher as suggested by studies of sensitized emission. Given the widespread distribution of peroxidases, the formation of highly reactive metabolites of tetracycline may have biological importance.

Estudo da resistência de amostras de Staphylococcus aureus a diferentes antibióticos / A study of multiple antibiotic resistance in samples of Staphylococcus aureus

Um total de 88 amostras de Staphylococcus aureus, das quais 44 isoladas da narina e pele de estudantes e outras 44 isoladas do ambiente hospitalar, foi testado frente a 11 antibióticos. Os resultados obtidos mostraram níveis de resistência relativamente altos, principalmente com relaçäo à penicilina, nas amostras de estudantes

Escherichia coli em produtos cárneos comercializados em Londrina-Paraná I: frequência de Escherichia coli enteropatogênica clássica (EPEC) / Escherichia coli in meat products commercialized in Londrina-Paraná I: frequency of classical enteropathogenic Escherichia coli (EPEC)

O presente estudo foi realizado para verificar a frequência de linhagens de Escherichia coli enteropatogênica clássica (EPEC) em 100 amostras de carne moída de segunda qualidade e 93 amostras de quibe cru. Das 193 amostras de alimentos analisados 93,78% continham E. coli, sendo que 8,84% apresentaram algum sorogrupo de EPEC. Em 7,2% das amostras de carne moída de segunda foram evidenciados os sorogrupos O26, O19 e O125. Em 10,71% das amostras de quibe cru foram isoladas os sorogrupos O26, O142, O55 e O127. As cepas de EPEC isoladas apresentaram resistência múltipla a no máximo três das 15 drogas testadas. A ampicilina foi a droga com maior populaçäo de EPEC resistente (68,75% das EPEC isoladas) seguida da tetraciclina (43,75%. A populaçäo de EPEC estudada mostrou sensibilidade acima de 93% para as aioutras drogas testadas, exceçäo à gentamicina (81,25%)

Changes in the predentin thickness and mineralization front configuration in developing human premolars.

The thickness of the predentin layer was studied at three different levels of developing human premolars. The results demonstrate that at the growing end next to the apex, where dentinogenesis is most active, predentin exhibits its greatest thickness (mean value 40.4 micron). However, at the coronal region, where primary dentin has been completely formed, predentin width is reduced to a mean value of 14.8 micron. Changes in the calcospheritic configuration of the mineralization front were established for each of the predentin levels studied. A comparative analysis of these calcospheritic changes and the morphology of fluorescent tetracycline lines detected in ground sections of premolars was established. Fluorescent lines observed at the coronal circumpulpal dentin showed large calcospheritic forms beneath the mantle dentin. However, lines found near the dentin-pulp border showed small calcospherites. It is concluded that the thickness of the predentin layer and the mineralization front configuration vary as a function of dentinogenic activity during development of human premolars.