Electrochemistry of chloramphenicol on laser-induced graphene electrodes and its voltammetric determination in honey.

Laser scribing is a promising technology for the rapid and large-scale production of low-cost electrochemical sensors from diverse substrates. Polyimide has been the most popular so far because of its low cost, flexibility and capability of generating high-quality porous graphene films, known as laser-induced graphene (LIG). Herein we report the electrochemistry of chloramphenicol (CAP) on LIG electrodes and its determination in honey samples. LIG electrodes were fabricated by the photothermal conversion of sp3 carbon within the polymeric matrix into sp2 carbon using a CO2 laser cutter. The LIG electrode associated with differential pulse voltammetry (DPV) showed good linearity (R2 > 0.99) in the range from 10 to 160 µmol L-1 with a limit of detection of 1.0 µmol L-1 and good precision (RSD < 5%) for the electrochemical reduction of CAP species. Detection was possible free from the interference of other antibiotics, such as amoxicillin, tetracycline, sulfanilamide, and sulfamethoxazole. Spiked honey samples were analyzed by the standard-addition method and recovery values between 86 and 109% were obtained, which confirmed the absence of sample matrix effects. Therefore, the proposed sensor is an alternative, feasible, low-cost, and powerful analytical tool for the determination of CAP in honey.

Electro-, photo-, and photoelectrochemical degradation of chloramphenicol on self-doping Ti nanotubes.

In this work, the photo-, electro-, and photo-electro-oxidation of chloramphenicol was investigated. The photo-experiments were carried out with different irradiation sources (an ultraviolet and a simulated solar source) using self-doped titanium nanotubes (SDTNT), a very promising and innovative material that deserves further investigations in the degradation of different pollutants. The photo-electrooxidation (j = 15 mA cm-2) under simulated solar irradiation presented the best efficiency, with ca. 100% degradation and kinetic constant of k = 0.04427 min-1. The FTIR analysis demonstrated a structural modification of the standard molecule occurred for all conditions used, suggesting a modification in functional groups responsible for the biological activity. Furthermore, the TOC analysis showed a significant mineralization of the pollutant (66% from the initial concentration). In addition, both photo-electrooxidation approaches have demonstrated a positive value of S, where the simulated solar irradiation reached the highest value S = 0.6960. The experimental results pointed out evidence that the methodology employed herein for chloramphenicol degradation is greatly interesting and the photo-electrooxidation under simulated solar irradiation is a promising approach for this purpose.

Comparative Genomics Determines Strain-Dependent Secondary Metabolite Production in Streptomyces venezuelae Strains.

Streptomyces venezuelae is well known to produce various secondary metabolites, including chloramphenicol, jadomycin, and pikromycin. Although many strains have been classified as S. venezuelae species, only a limited number of strains have been explored extensively for their genomic contents. Moreover, genomic differences and diversity in secondary metabolite production between the strains have never been compared. Here, we report complete genome sequences of three S. venezuelae strains (ATCC 10712, ATCC 10595, and ATCC 21113) harboring chloramphenicol and jadomycin biosynthetic gene clusters (BGC). With these high-quality genome sequences, we revealed that the three strains share more than 85% of total genes and most of the secondary metabolite biosynthetic gene clusters (smBGC). Despite such conservation, the strains produced different amounts of chloramphenicol and jadomycin, indicating differential regulation of secondary metabolite production at the strain level. Interestingly, antagonistic production of chloramphenicol and jadomycin was observed in these strains. Through comparison of the chloramphenicol and jadomycin BGCs among the three strains, we found sequence variations in many genes, the non-coding RNA coding regions, and binding sites of regulators, which affect the production of the secondary metabolites. We anticipate that these genome sequences of closely related strains would serve as useful resources for understanding the complex secondary metabolism and for designing an optimal production process using Streptomyces strains.

Characterisation of OXA-258 enzymes and AxyABM efflux pump in Achromobacter ruhlandii.

OBJECTIVES: The aim of this study was to characterise OXA-258 variants and other features that may contribute to carbapenem resistance in Achromobacter ruhlandii. METHODS: Kinetic parameters for purified OXA-258a and OXA-258b were determined measuring the rate of hydrolysis of a representative group of antimicrobial agents. Whole-genome shotgun sequencing was performed on A. ruhlandii 38 (producing OXA-258a) and A. ruhlandii 319 (producing OXA-258b), and in silico analysis of antimicrobial resistance determinants was conducted. Substrates of the AxyABM efflux pump were investigated by inhibition assays using phenylalanine-arginine ß-naphthylamide (PAßN). Outer membrane protein profiles were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Kinetic measurements of purified OXA-258 variants displayed an overall weak catalytic efficiency toward ß-lactams. A detectable hydrolysis of imipenem was observed. In silico genomic analysis confirmed the presence of 32 and 35 putative efflux pump-encoding genes in A. ruhlandii strains 38 and 319, respectively. Complete sequences for AxyABM and AxyXY efflux pumps, previously described in Achromobacter xylosoxidans, were detected. Decreases in the MICs for chloramphenicol, nalidixic acid and trimethoprim/sulfamethoxazole were observed in the presence of the inhibitor PAßN, suggesting that these antibiotics are substrates of AxyABM. AxyXY-encoding genes of A. ruhlandii 38 and A. ruhlandii 319 displayed 99% identity. No differences were observed in the outer membrane protein profiles. CONCLUSIONS: The contribution of OXA-258 enzymes to the final ß-lactam resistance profile may be secondary. Further studies on other putative resistance markers identified in the whole-genome analysis should be conducted to understand the carbapenem resistance observed in A. ruhlandii.

Preparation of Chloramphenicol/Amino Acid Combinations Exhibiting Enhanced Dissolution Rates and Reduced Drug-Induced Oxidative Stress.

Chloramphenicol is an old antibiotic agent that is re-emerging as a valuable alternative for the treatment of multidrug-resistant pathogens. However, it exhibits suboptimal biopharmaceutical properties and toxicity profiles. In this work, chloramphenicol was combined with essential amino acids (arginine, cysteine, glycine, and leucine) with the aim of improving its dissolution rate and reduce its toxicity towards leukocytes. The chloramphenicol/amino acid solid samples were prepared by freeze-drying method and characterized in the solid state by using Fourier transform infrared spectroscopy, powder X-ray diffraction, differential scanning calorimetry, scanning electron microscopy, and solid-state nuclear magnetic resonance. The dissolution properties, antimicrobial activity, reactive oxygen species production, and stability of the different samples were studied. The dissolution rate of all combinations was significantly increased in comparison to that of the pure active pharmaceutical ingredient. Additionally, oxidative stress production in human leukocytes caused by chloramphenicol was decreased in the chloramphenicol/amino acid combinations, while the antimicrobial activity of the antibiotic was maintained. The CAP:Leu binary combination resulted in the most outstanding solid system makes it suitable candidate for the development of pharmaceutical formulations of this antimicrobial agent with an improved safety profile.

Activation of chloramphenicol biosynthesis in Streptomyces venezuelae ATCC 10712 by ethanol shock: insights from the promoter fusion studies.

BACKGROUND: Streptomyces venezuelae ATCC 10712 produces antibiotics chloramphenicol (Cml) and jadomycin (Jad) in response to nutrient limitation and ethanol shock (ES), respectively. Biosynthesis of Cml and Jad was shown to be reciprocally regulated via the action of regulatory proteins JadR1 and JadR2 encoded by the jad cluster, and mechanism of such regulation has been characterized. However, detailed analysis of the regulatory mechanism controlling Cml biosynthesis is still lacking. RESULTS: In the present study, several promoters from the cml cluster were fused to the reporter gene gusA. Reporter protein activity and Cml production were assayed in the wild-type strain with and without ES, followed by similar experiments with the jadR1 deletion mutant. The latter gene was earlier reported to negatively control Cml biosynthesis, while serving as a positive regulator for the jad cluster. A double deletion mutant deficient in both jadR1 and the cml cluster was also constructed and used in promoter fusion studies. Analyses of the results revealed that ES activates Cml biosynthesis in both wild-type and jadR1 deletion mutant, while Cml production by the latter was ca 80% lower. CONCLUSIONS: These results contradict earlier reports regarding the function of JadR1, but correlate well with the reporter activity data for some promoters, while reaction of others to the ES is genotype-dependent. Remarkably, the absence of Cml production in the double mutant has a profound effect on the way certain cml promoters react to ES. The latter suggests direct involvement of Cml in this complex regulatory mechanism.

Optimization of bioassay for tetracycline detection in milk by means of chemometric techniques.

AIMS: In this study, a microbiological method of dichotomous response using Bacillus cereus was designed and optimized to detect tetracyclines (TCs) at concentrations near to the maximum residue limits (MRLs). METHODS AND RESULTS: In a first stage, the response time of bioassay was reduced to 5 h when the logarithm of spore concentration (log S) was increased. Later, a Plackett Burman design (2(6-3)) was analysed using logistic regression model. This design indicates significant effects of log S and chloramphenicol (CAP) on the detection limit (DL) of TC. Then, the response surfaces (RS) of the TCs DTs as a function of log S and CAP were plotted using a Dohlert design and the logistic regression model. These RS show a linear decrease with the raise of CAP and a quadratic effect of log S. Finally, the DTs of TC (109 µg l(-1)) and oxytetracycline (100 µg l(-1)) were adjusted to their MRLs through the desirability function. CONCLUSIONS: By successive application of experimental design techniques could be optimized a bioassay for the detection of TC residues in milk. The best conditions have been achieved when the assay was made with log S = 5·12 and CAP = 470 µg l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: Experimental design techniques together with the logistic regression model and the desirability function represent an adequate tool for the optimization of a bioassay with binary response.

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation.

Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1H-NMR established that the principal metabolite in the disrupted strains was the alpha-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH2-dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

Structural basis for chloramphenicol tolerance in Streptomyces venezuelae by chloramphenicol phosphotransferase activity.

Streptomyces venezuelae synthesizes chloramphenicol (Cm), an inhibitor of ribosomal peptidyl transferase activity, thereby inhibiting bacterial growth. The producer escapes autoinhibition by its own secondary metabolite through phosphorylation of Cm by chloramphenicol phosphotransferase (CPT). In addition to active site binding, CPT binds its product 3-phosphoryl-Cm, in an alternate product binding site. To address the mechanisms of Cm tolerance of the producer, the crystal structures of CPT were determined in complex with either the nonchlorinated Cm (2-N-Ac-Cm) at 3.1 A resolution or the antibiotic's immediate precursor, the p-amino analog p-NH(2)-Cm, at 2.9 A resolution. Surprisingly, p-NH(2)-Cm binds CPT in a novel fashion. Additionally, neither 2-N-Ac-Cm nor p-NH(2)-Cm binds to the secondary product binding site.

Metodologia para determinaçäo de resíduos de clorafenicol em músculo bovino por cromatografia líquida de alta eficiência / Method for Determination of Clorampfenicol Residus In Bovine Muscle by High Performance Liquid Chromatography

Uma metodologia analítica par determinaão de resíduos de clorafenicol em tecido muscular foi padronizada e validada empregando extração com acetado de etila, purificação com cartuchos de sílica e detecção e quantificação por CLAE/UV. A faixa de linearidade da resposta, sem inteferência da matriz, foi de 10 a 400 ng/mL. Nos ensaios com amostras fortificadas entre 5 e 40µg/kg

Cubic crystals of chloramphenicol phosphotransferase from Streptomyces venezuelae in complex with chloramphenicol.

Chloramphenicol 3-O-phosphotransferase (CPT) from Streptomyces venezuelae ISP5230, a novel chloramphenicol-inactivating kinase, has been overexpressed and purified using Escherichia coli as the heterologous host. Crystals of CPT in complex with its substrate chloramphenicol (Cm) were obtained which were suitable for X-ray diffraction. The crystals belong to the cubic space group I4132 with unit-cell dimension a = 200.0 A. The initial CPT crystals diffracted to 3.5 A and the diffraction was improved significantly upon adding acetonitrile and Cm to the crystallization drop. The CPT-Cm crystals diffract to at least 2.8 A resolution.

[Drug combinations in the care of bed sores]. / Atenção nos cuidados de enfermagem das escaras quanto às associações terapêuticas.

The present study aims at discussing the types of prescriptions and caring bedsores related to IRUXOL ointment use, as well as bandages practice reports performed by the nursing team in care units. Nineteen patient files from three School Hospitals ins Rio de Janeiro city have been analyzed. The results presented eight different kinds of drug prescription for bedsore treatment in which four of them were related to IRUXOL. The most frequent formulation was: cleaning with PVP-I + Physiologic Solution 0.9% and IRUXOL. It has also been observed that the nursing professional had used seventeen different combinations with several substances in which ten of them were associated to IRUXOL. They have also reported the use of 24 different products/substances when performing bedsores bandages. The indiscriminated use of products which alters injury pH (acid-base contents) such a factor which interferes debridant action of IRUXOL, has been verified.

Inactivation of chloramphenicol by O-phosphorylation. A novel resistance mechanism in Streptomyces venezuelae ISP5230, a chloramphenicol producer.

Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic DNA from the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, confers resistance when introduced by transformation into the Cm-sensitive host Streptomyces lividans M252 (Mosher, R. H. Ranade, N. P., Schrempf, H., and Vining, L. C. (1990) J. Gen. Microbiol. 136, 293-301). Transformants rapidly metabolized Cm to one major product, which was isolated and purified by reversed phase chromatography. The metabolite was identified by nuclear magnetic resonance spectroscopy and mass spectrometry as 3'-O-phospho-Cm, and was shown to have negligible inhibitory activity against Cm-sensitive Micrococcus luteus. The nucleotide sequence of the S. venezuelae DNA insert in pJV4 contains an open reading frame (ORF) that encodes a polypeptide (19 kDa) with a consensus motif at its NH2 terminus corresponding to a nucleotide-binding amino acid sequence (motif A or P-loop; Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). When a recombinant vector containing this ORF as a 1.6-kilobase pair SmaI-SmaI fragment was used to transform S. lividans M252, uniformly Cm-resistant transformants were obtained. A strain of S. lividans transformed by a vector in which the ORF had been disrupted by an internal deletion yielded clones that were unable to phosphorylate Cm, and exhibited normal susceptibility to the antibiotic. The results implicate the product of the ORF from S. venezuelae as an enzymic effector of Cm resistance in the producing organism by 3'-O-phosphorylation. We suggest the trivial name chloramphenicol 3'-O-phosphotransferase for the enzyme.

X-ray studies on phospholipid bilayers. XI. Interactions with chloramphenicol.

Chloramphenicol is a widely used antibiotic with low levels of toxicity. However, scanning electron microscopy revealed morphological changes in human erythrocytes when they interacted in vitro with therapeutical concentrations of chloramphenicol. To explain this shape change, a study concerned with the possible interactions of this antibiotic with bilayers built-up of phospholipids located in either side of the red cell membrane was performed by X-ray diffraction. Results indicated that chloramphenicol was unable to perturb in any significant extent the structure of the phospholipids under study. The only noticeable effects were phase transitions produced to dimyristoylphosphatidylethanolamine bilayers.