New Insights for Red Propolis of Alagoas-Chemical Constituents, Topical Membrane Formulations and Their Physicochemical and Biological Properties.

The main objectives of this study were to evaluate the chemical constitution and allergenic potential of red propolis extract (RPE). They were evaluated, using high performance liquid chromatography (HPLC) and the release of ß-hexosaminidase, respectively. A plethora of biologically active polyphenols and the absence of allergic responses were evinced. RPE inhibited the release of ß-hexosaminidase, suggesting that the extract does not stimulate allergic responses. Additionally, the physicochemical properties and antibacterial activity of hydrogel membranes loaded with RPE were analyzed. Bio-polymeric hydrogel membranes (M) were obtained using 5% carboxymethylcellulose (M1 and M2), 1.0% of citric acid (M3) and 10% RPE (for all). Their characterization was performed using thermal analysis, Fourier transform infrared (FTIR), total phenolic content, phenol release test and, antioxidant activity through 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and Ferric Reducing Antioxidant Power (FRAP). The latter appointed to the similar antioxidant capacity of the M1, M2 and M3. The degradation profiles showed higher thermostability to M3, followed by M2 and M1. The incorporation of RPE into the matrices and the crosslinking of M3 were evinced by FTIR. There were differences in the release of phenolic compounds, with a higher release related to M1 and lower in the strongly crosslinked M3. The degradation profiles showed higher thermostability to M3, followed by M2 and M1. The antibacterial activity of the membranes was determined using the disc diffusion assay, in comparison with controls, obtained in the same way, without RPE. The membranes elicited antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis, with superior performance over M3. The hydrogel membranes loaded with RPE promote a physical barrier against bacterial skin infections and may be applied in the wound healing process.

First detection of metallo-β-lactamases in nosocomial isolates of Pseudomonas aeruginosa in Alagoas, Brazil / Primeira detecção de metalobetalactamases em isolados nosocomiais de Pseudomonas aeruginosa em Alagoas, Brasil

ABSTRACTIntroduction:Pseudomonas aeruginosa is a leading cause of opportunistic infections in humans, and the choice of effective antimicrobial agents to control this bacterium has been limited, mainly due to its ability to produce metallo-β-lactamases (MβL), enzymes capable of inactivating many antimicrobials through hydrolysis.Objective:This study aimed to detect the presence of multidrug-resistant (MDR) P. aeruginosa strains and the MβL-encoding genes (blaSPM, blaIMP and blaVIM) in nosocomial isolates in Maceió (AL).Methods:The isolates were collected from four public institutions/hospitals in Maceió, and cultures were identified by conventional methods. Antibiotic susceptibility was determined by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI), and polymerase chain reaction (PCR) was used to identify the presence of the MβL-encoding genes blaSPM, blaIMP and blaVIM.RESULTS:Forty-three strains of P. aeruginosa were MDR among 85 identified nosocomial isolates (50.6%), 79.1% and 20% of which were resistant to carbapenem (imipenem and meropenem) and aztreonam, respectively. PCR was performed in susceptible or resistant isolates and we identified nine (20.9%) MDR strains with blaSPM gene, whereas only one strain had blaIMP and none blaVIM positive was found.Conclusion:Production of MβL is an important mechanism of resistance to carbapenems and other β-lactams among P. aeruginosa strains in the evaluated samples. We reported the first identification of MβL-encoding genes in P. aeruginosa from nosocomial environments in Maceió, a new insight for the epidemiology of MβL in the Northeastern region of Brazil.

RESUMOIntrodução:Pseudomonas aeruginosa é a principal causa de infecções oportunistas em seres humanos, e a escolha de agentes antimicrobianos eficazes para controlar essa bactéria tem sido limitada, principalmente devido à sua capacidade de produzir metalobetalactamases (MβL), enzimas capazes de inativar muitos antimicrobianos por meio de hidrólise.Objetivo:Este estudo objetivou detectar a presença de cepas de P. aeruginosa multirresistentes e os genes codificadores de MβL (blaSPM, blaIMP e blaVIM) em isolados nosocomiais em Maceió (AL).Métodos:Os isolados foram coletados de quatro instituições públicas/hospitais em Maceió, e as culturas foram identificadas por métodos convencionais. A sensibilidade aos antibióticos foi determinada pelo método de disco-difusão de acordo com o Clinical and Laboratory Standards Institute (CLSI), e a reação em cadeia da polimerase (PCR) utilizada para identificar a presença de genes que codificam MβL - blaSPM, blaIMP e blaVIM.ResultadosQuarenta e três cepas de P. aeruginosa foram multirresistentes entre os 85 isolados nosocomiais identificados (50,6%); destes, 79,1% e 20% foram resistentes aos carbapenêmicos (imipenem e meropenen) e ao aztreonam, respectivamente. A PCR foi realizada em isolados suscetíveis ou resistentes, e nós identificamos nove (20,9%) cepas multirresistentes com gene blaSPM, enquanto apenas uma possuía blaIMP e nenhuma blaVIM positiva foi encontrada.CONCLUSÃO:A produção de MβL é um importante mecanismo de resistência aos carbapenêmicos e a outros betalactâmicos entre cepas de P. aeruginosa nas amostras avaliadas. Relatamos a primeira identificação de genes codificadores de MβL em P. aeruginosa de ambiente hospitalar de Maceió, uma nova visão para a epidemiologia de MβL na região Nordeste do Brasil.

Isolation of transcripts overexpressed in the human pathogen Trichophyton rubrum grown in lipid as carbon source.

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts overexpressed in T. rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-ß-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum.

BACKGROUND: Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. RESULTS: The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. CONCLUSION: In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.

Membrane transporter proteins are involved in Trichophyton rubrum pathogenesis.

Trichophyton rubrum is a dermatophyte responsible for the majority of human superficial mycoses. The functional expression of proteins important for the initial step and the maintenance of the infection process were identified previously in T. rubrum by subtraction suppression hybridization after growth in the presence of keratin. In this study, sequences similar to genes encoding the multidrug-resistance ATP-binding cassette (ABC) transporter, copper ATPase, the major facilitator superfamily and a permease were isolated, and used in Northern blots to monitor the expression of the genes, which were upregulated in the presence of keratin. A sequence identical to the TruMDR2 gene, encoding an ABC transporter in T. rubrum, was isolated in these experiments, and examination of a T. rubrum DeltaTruMDR2 mutant showed a reduction in infecting activity, characterized by low growth on human nails compared with the wild-type strain. The high expression levels of transporter genes by T. rubrum in mimetic infection and the reduction in virulence of the DeltaTruMDR2 mutant in a disease model in vitro suggest that transporters are involved in T. rubrum pathogenicity.

Isolation of transcripts over-expressed in human pathogen Trichophyton rubrum during growth in keratin.

Trichophyton rubrum is a cosmopolitan and anthropophilic fungus able to invade keratinized tissue, causing infection in human skin and nails. This work evaluated the changes in the extracellular pH during its growth in keratin (after 6, 12, 24, 48, 72h and 7 days) at initial pH 5.0. We observed a gradual increase of basal pH under keratin exposure when compared to glucose condition. Also, we identified 576T. rubrum transcripts differentially expressed by subtractive suppression hybridization (SSH) using conidia cultivated for 72h in keratin as tester, and cultivated in glucose as driver. The over-expression of 238 transcripts obtained under keratin condition was confirmed by macro-array dot-blot, revealing 28 unigenes. Putative proteins encoded by these genes showed similarity to fungi proteins involved in basic metabolism, growth and virulence, i.e., transporters ABC-MDR, MFS and ATPase of copper, NIMA interactive protein, Gag-Pol polyprotein, virulence factors serine-protease subtilisin and metalloprotease, cytochrome P450, GlcN-6-phosphate deaminase and Hsp30. The upregulation of T. rubrum genes encoding subtilisin, metalloprotease and Gag-Pol polyprotein was also validated by northern blot. The results of this study provide the first insight into genes differentially expressed during T. rubrum grown in keratin that may be involved in fungal pathogenesis.