RESUMO
Chromoblastomycosis is a fungal chronic disease, which affects humans, especially in cutaneous and subcutaneous tissues. There is no standard treatment for Chromoblastomycosis, and it is a therapeutic challenge, due natural resistance of their causative agents, inadequate response of patients and common cases of relapse. Protocols for determination of antifungal drugs susceptibility are not standardized for chromoblastomycosis agents and endpoint definition is usually based on visual inspection, which depends on the analyst, making it sometimes inaccurate. We presented a colorimetric and quantitative methodology based on resazurin reduction to resofurin to determine the metabolic status of viable cells of Fonsecaea sp. Performing antifungal susceptibility assay by a modified EUCAST protocol allied to resazurin, we validated the method to identify the minimum inhibitory concentrations of itraconazole, fluconazole, amphotericin B, and terbinafine for eight Fonsecaea clinical isolates. According to our data, resazurin is a good indicator of metabolic status of viable cells, including those exposed to antifungal drugs. This work aimed to test resazurin as an indicator of the metabolic activity of Fonsecaea species in susceptibility assays to antifungal drugs. Species of this genus are the main causative agents of Chromoblastomycosis, which affects humans.
Assuntos
Antifúngicos , Cromoblastomicose , Fonsecaea , Testes de Sensibilidade Microbiana , Oxazinas , Xantenos , Xantenos/metabolismo , Oxazinas/metabolismo , Antifúngicos/farmacologia , Humanos , Fonsecaea/efeitos dos fármacos , Fonsecaea/genética , Fonsecaea/metabolismo , Cromoblastomicose/microbiologia , Cromoblastomicose/tratamento farmacológico , Colorimetria/métodosRESUMO
Citrus Canker disease is one of the most important disease in citrus production worldwide caused by gram-negative bacterial pathogen Xanthomonas citri subsp. citri, leading to great economic losses. Currently, a spray of copper-based bactericides is the primary measure for citrus canker management. However, these measures can lead to the contamination of soil by metal contamination, but also the development of copper-resistant Xanthomonas populations. Considering the need to discovery new alternatives to control the citrus canker disease, actinomycetes isolated from the Brazilian Caatinga biome and their crude extracts were tested against different strains of Xanthomonas citri subsp. citri. Streptomyces sp. Caat 1-54 crude extract showed the highest antibiotic activity against Xcc. The crude extract dereplication was performed by LC-MS/MS. Through bioassay-guided fractionation strategy, the antimicrobial activity was assigned to Lysolipins, showing a MIC around 0.4-0.8 µg/mL. Growth media optimization using statistical experimental design increased the Lysolipins production in three-fold production. The preventive and curative effects of the optimized crude extract obtained by experimental design of Caat-1-54 against citrus canker were evaluated in potted 'Pera' sweet orange nursery trees. Caat 1-54 extract was effective in preventing new infections by Xcc on leaves but was not able to reduce Xcc population in pre-established citrus canker lesions. Streptomyces sp. Caat 1-54 extract is a promising, environmentally-friendly source of antimicrobial compound to protect citrus trees against citrus canker.
Assuntos
Actinobacteria/metabolismo , Xantenos/isolamento & purificação , Xanthomonas/efeitos dos fármacos , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Anti-Infecciosos , Cromatografia Líquida/métodos , Citrus/efeitos dos fármacos , Doenças das Plantas , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Espectrometria de Massas em Tandem/métodos , Xantenos/metabolismo , Xanthomonas/patogenicidadeRESUMO
The discovery of new secondary metabolites is a challenge to biotechnologists due to the emergence of superbugs and drug resistance. Knowledge about biodiversity and the discovery of new microorganisms have become major objectives; thus, new habitats like extreme ecosystems have become places of interest to research. In this context, caatinga is an unexplored biome. The ecosystem caatinga is a rich habitat for thermophilic microbes. Its high temperature and dry climate cause selective microbes to flourish and become established. Actinobacteria (Caat 1-54 genus Streptomyces sp.) isolated from the soil of caatinga was investigated to characterize and map its secondary metabolites by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). With this technique, the production of bioactive metabolites was detected and associated with the different morphological differentiation stages within a typical Streptomyces sp. life cycle. High-resolution mass spectrometry, tandem mass spectrometry, UV-Vis profiling and NMR analysis were also performed to characterize the metabolite ions detected by DESI-MS. A novel compound, which is presumed to be an analogue of the antifungal agent lienomycin, along with the antimicrobial compound lysolipin I were identified in this study to be produced by the bacterium. The potency of these bioactive compounds was further studied by disc diffusion assays and their minimum inhibitory concentrations (MIC) against Bacillus and Penicillium were determined. These bioactive metabolites could be useful to the pharmaceutical industry as candidate compounds, especially given growing concern about increasing resistance to available drugs with the emergence of superbugs. Consequently, the unexplored habitat caatinga affords new possibilities for novel bioactive compound discovery. Graphical Abstract á .
Assuntos
Metabolismo Secundário , Espectrometria de Massas por Ionização por Electrospray/métodos , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Bacillus/efeitos dos fármacos , Humanos , Metabolômica/métodos , Micoses/tratamento farmacológico , Penicillium/efeitos dos fármacos , Polienos/química , Polienos/metabolismo , Polienos/farmacologia , Streptomyces/química , Espectrometria de Massas em Tandem/métodos , Xantenos/química , Xantenos/metabolismo , Xantenos/farmacologiaRESUMO
Drug resistance in tuberculosis is a major threat to public health and control of the disease worldwide. Given the need of a rapid and accurate detection of Mycobacterium tuberculosis resistance to second-line drugs, this study evaluated the performance of the BACTEC MGIT 960 for second-line, drug susceptibility testing in comparison with the resazurin microtiter assay (REMA), in order to implement the automated methodology in the diagnostic routine of a reference laboratory. Drug susceptibility testing (DST) for second-line drugs of 151 MDR M. tuberculosis clinical isolates was performed by both BACTEC MGIT 960 and REMA, and a panel of 26 M. tuberculosis reference isolates from a proficiency test was tested by the BACTEC MGIT 960. DST for second-line drugs by the BACTEC MGIT 960 system was more rapid, highly reproducible and showed 100% of proficiency. After these results, this methodology was successfully implemented in our diagnostic routine for all MDR-TB patients.
Assuntos
Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Oxazinas/metabolismo , Xantenos/metabolismo , Automação Laboratorial , Humanos , Testes de Sensibilidade Microbiana/instrumentação , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
BACKGROUND: Mycobacterium abscessus group has heterogeneous susceptibility pattern among species. The species is most common cause of nosocomial infections. Macrolides minimum Inhibitory concentration (MIC) determination is essential for the treatment. METHODS: Thirty-six strains were randomly selected for performing Resazurin Microtiter Assay (REMA) for clarithromycin testing in comparison to MIC test according to Clinical and Laboratory Standards Institute (2011) recommendation. REMA has been used for detection of drug resistance in M. tuberculosis. Extended incubation was performed to detect induced resistance. RESULTS: Thirty microliters of resazurin (0.01%) was added after visually taking MIC reading. Resistance was observed in 11.1% of M. bolletti and 4.8% of M. abscessus strains; and induced resistance was detected in 77.8% and 95.2% of M. bolletti and M. abscessus strains, respectively. All strains of M. massiliense were susceptible. The samples presented same MIC value both by visual reading and through resazurin. CONCLUSION: The present study showed 100% concordance between both readings, with REMA providing easier to read and report results benefit. This change in reading can also reflect on the MIC determination and report, improving the test.
Assuntos
Claritromicina/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium/isolamento & purificação , Oxazinas/metabolismo , Xantenos/metabolismo , HumanosRESUMO
The susceptibility of rapidly growing mycobacteria (RGM) to linezolid and ciprofloxacin was evaluated by using resazurin as a growth indicator. The assay with resazurin supplemented medium performed as well as its addition to the medium at reading time and was efficient for the determination for the susceptibility profile in RGM.
Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Micobactérias não Tuberculosas/efeitos dos fármacos , Micobactérias não Tuberculosas/crescimento & desenvolvimento , Oxazinas/metabolismo , Xantenos/metabolismo , Ciprofloxacina/farmacologia , Linezolida/farmacologia , Micobactérias não Tuberculosas/metabolismoAssuntos
Xylopia/química , Xylopia/metabolismo , Alcaloides/análise , Alcaloides/metabolismo , Benzoatos/análise , Benzoatos/metabolismo , Metabolismo dos Carboidratos , Carboidratos/análise , Clorofila/análise , Clorofila/metabolismo , Flavonoides/análise , Flavonoides/metabolismo , Lignanas/análise , Lignanas/metabolismo , Terpenos/análise , Terpenos/metabolismo , Xantenos/análise , Xantenos/metabolismoRESUMO
CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs.
Assuntos
Antifúngicos/farmacologia , Oxazinas/metabolismo , Paracoccidioides/efeitos dos fármacos , Coloração e Rotulagem/métodos , Xantenos/metabolismo , Anfotericina B/farmacologia , Humanos , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana/métodos , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/metabolismoRESUMO
The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Nitrato Redutase/metabolismo , Oxazinas/metabolismo , Valor Preditivo dos Testes , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Xantenos/metabolismoRESUMO
The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.