Bioassay-guided In vitro Study of the Antimicrobial and Cytotoxic Properties of the Leaves from Excoecaria Lucida Sw.

BACKGROUND: Excoecaria lucida Sw. (Euphorbiaceae) is a plant conventionally used throughout the Caribbean in the treatment of infectious diseases. OBJECTIVE: To evaluate, using bioassay-guided fractionation, the in vitro cytotoxicity and antimicrobial activity of E. lucida leaves. MATERIALS AND METHODS: A 95% ethanol crude extract was dried and fractionated by solid-liquid separation in four phases (hexane, dichloromethane, ethyl acetate, and butanol). Antimicrobial activity (3 bacteria, 6 yeasts, and 2 fungi) was evaluated by the dilution method with resazurin (2048, 512, 128, 32, and 8 µg/mL). The cytotoxicity assays were evaluated in two cell lines: MRC-5 and RAW 264.7; calculating the selectivity index. Assays were performed for the total extract, the isolated compound with the highest yield, and the ethyl acetate and butanol phases. Isolated compounds were characterized by nuclear magnetic resonance and mass spectrometry techniques. RESULTS: Fractionation process led to the isolation of ellagic acid (784.29 mg), 3,3',4'-tri-O-methyl ellagic 4-O-ß-D-glucopyranoside acid (6.1 mg), and corilagin (6.91 mg). The most active were ethyl acetate phase and ellagic acid with IC50= 128 µg/mL against seven and five different species of microorganisms, respectively. The total extract (IC50=512 µg/mL) and the ethyl acetate phase (IC50=128 µg/mL) were cytotoxic in both cell lines, while butanol phase and ellagic acid both with IC50>2048 µg/mL seemed to be safer. CONCLUSIONS: The results obtained indicate that the Excoecaria leaves can be conventionally used as antimicrobial, but it should be present that some cytotoxicity could appear. In addition, the three identified compounds were reported for the first time in the species. SUMMARY: Excoecaria lucida leaves (Euphorbiaceae) are used by the Cuban population due to their antimicrobial activity. This ethnopharmacological knowledge is confirmed by the integrated antibacterial and antifungal in vitro screening developed, using the bioassay-guided fractionation method.Abbreviations Used: MRC-5-SV2: Diploid human lung fibroblasts cells, RAW 264.7: Murine macrophages cells, IC50: Inhibitory Concentration 50%, ATCC: American Type Culture Collection, CCEBI: Culture Collection of Industrial Biotechnology Center, CECT: Spanish Culture Collection Type, CFU: Colony forming units, CC50: 50% cytotoxic concentration, CO2: Carbon dioxide, SI: Selectivity index, IR: Infrared spectroscopy, 1H NMR: Nuclear Magnetic Resonance of hydrogen, 13C NMR: Nuclear Magnetic Resonance of carbon, HMQC: Heteronuclear Multiple-Quantum Correlation, HMBC: Heteronuclear Multiple Bond Correlation, COSY: Correlation Spectroscopy, NOESY: Nuclear Overhauser Effect Spectroscopy, KBr: Potassium bromide, DMSO-D6: Deuterated dimethyl sulfoxide, LC.MS: Liquid Chromatography-Mass Spectrometry, [α]D: Optical rotation, EL1: ellagic acid, EL2: 3,3',4'-tri-O-methyl ellagic 4-O-ß-D-glucopyranoside acid, EL3: corilagin, Active (+), inactive (-).

Mycobacterium tuberculosis belonging to family LAM and sublineage RD Rio: common strains in Southern Brazil for over 10 years

A sublineage of Mycobacterium tuberculosis called RD Rio was described in 2007. Although only recently described, this strain may have been present previously in the population, and its identification in clinical isolates will elucidate bacterial transmission dynamics and host-pathogen interactions. This study evaluated the clonal diversity of the RD Rio sublineage in clinical isolates from Rio Grande-RS obtained between 1998 and 2001. Among the 45 samples analyzed by the MIRU-VNTR method, there were six clusters with two samples each and 33 orphan strains with unique pattern. The strains were distributed across several different lineages including LAM (34.04%), X (14.89%), Haarlem (12.77%), UgandaI (10.64%), S (4.26%), NEW-1 (2.13%) and Cameroon (2.13%); 14.89% of the strains matched to multiple lineages. RD Rio strains were present in 28.9% of the samples and 81.25% of the identified strains belonged to the LAM family. The high clonal diversity observed in this study is a constant feature in this region. The RD Rio sublineage has been in Rio Grande-RS since 1998. The continued monitoring of RD Rio in clinical isolates will enhance the understanding of its epidemiological significance.

Mycobacterium tuberculosis belonging to family LAM and sublineage RD(Rio): common strains in Southern Brazil for over 10 years.

A sublineage of Mycobacterium tuberculosis called RD(Rio) was described in 2007. Although only recently described, this strain may have been present previously in the population, and its identification in clinical isolates will elucidate bacterial transmission dynamics and host-pathogen interactions. This study evaluated the clonal diversity of the RD(Rio) sublineage in clinical isolates from Rio Grande-RS obtained between 1998 and 2001. Among the 45 samples analyzed by the MIRU-VNTR method, there were six clusters with two samples each and 33 orphan strains with unique pattern. The strains were distributed across several different lineages including LAM (34.04%), × (14.89%), Haarlem (12.77%), UgandaI (10.64%), S (4.26%), NEW-1 (2.13%) and Cameroon (2.13%); 14.89% of the strains matched to multiple lineages. RD(Rio) strains were present in 28.9% of the samples and 81.25% of the identified strains belonged to the LAM family. The high clonal diversity observed in this study is a constant feature in this region. The RD(Rio) sublineage has been in Rio Grande-RS since 1998. The continued monitoring of RD(Rio) in clinical isolates will enhance the understanding of its epidemiological significance.

Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

Improving tuberculosis control through the partnership between university and the health system / Qualificar o controle da tuberculose mediante a parceria entre a Universidade e o Sistema de Saúde

INTRODUCTION: Tuberculosis (TB) control is linked to the availability of qualified methods for microbiological diagnostics; however, microscopy with limited sensitivity is the only method available in many locations. The objective of this study was to evaluate the introduction of culture, drug susceptibility testing (DST), and genotyping in the routine of a Municipal Program of Tuberculosis Control. METHODS: Direct microscopy of sputum and culture in Ogawa-Kudoh were performed on 1,636 samples from 787 patients. DST of positive cultures was performed by resazurin microtiter assay and genotyping by mycobacterial interspersed repetitive units-variable number tandem repeat. RESULTS: A total 91 patients with TB were identified. The culture increased case detection by 32% compared with the microscopy; acquired resistance was 3.3% and the genotyping showed high genetic diversity. CONCLUSIONS: Ogawa-Kudoh contributed significantly to the increase in case detection and is suitable for implementation in poor-resource locations. The acquired resistance rate was lower than that reported in a recent Brazilian survey. The high genetic diversity is possibly related to the high TB prevalence in the population, as well as to early detection and suitable treatment of patients. The interaction between research and health care is important for reorienting the practice, transferring technology, and improving TB control.

INTRODUÇÃO: O controle da tuberculose (TB) está relacionado com a disponibilidade de métodos de diagnóstico microbiológico qualificados, no entanto a microscopia com a sua limitada sensibilidade é o único método disponível em muitos locais.O objetivo deste estudo foi avaliar a introdução da cultura, teste de sensibilidade aos antimicrobianos (TSA) e genotipagem na rotina de um Programa Municipal de Controle da Tuberculose. MÉTODOS:A baciloscopia direta do escarro e cultura em Ogawa-Kudoh foram realizadas em 1.636 amostras de 787 pacientes. O TSA das culturas positivas foi realizado pelo método de microdiluição e a genotipagem por Mycobacterial Interspersed Repetitive Units - Variable Number Tandem Repeat (MIRU-VNTR). RESULTADOS:Foram identificados 91 pacientes com TB, com a cultura aumentando em 32% a detecção de casos em comparação com a microscopia; a resistência adquirida foi de 3,3% e a genotipagem mostrou alta diversidade genética. CONCLUSÕES: O cultivo em Ogawa-Kudoh contribuiu significativamente para o aumento na detecção de casos e é adequado para ser implementado em locais com poucos recursos. A taxa de resistência adquirida foi menor do que a relatada em recente inquérito nacional. A alta diversidade genética está possivelmente relacionada à alevada prevalência de TB na população, detecção precoce e tratamento adequado dos pacientes.A interação entre a pesquisa e serviço de saúde pública é importante para reorientar a prática, transferir tecnologia e melhorar o controle da TB.

Improving tuberculosis control through the partnership between university and the health system.

INTRODUCTION: Tuberculosis (TB) control is linked to the availability of qualified methods for microbiological diagnostics; however, microscopy with limited sensitivity is the only method available in many locations. The objective of this study was to evaluate the introduction of culture, drug susceptibility testing (DST), and genotyping in the routine of a Municipal Program of Tuberculosis Control. METHODS: Direct microscopy of sputum and culture in Ogawa-Kudoh were performed on 1,636 samples from 787 patients. DST of positive cultures was performed by resazurin microtiter assay and genotyping by mycobacterial interspersed repetitive units-variable number tandem repeat. RESULTS: A total 91 patients with TB were identified. The culture increased case detection by 32% compared with the microscopy; acquired resistance was 3.3% and the genotyping showed high genetic diversity. CONCLUSIONS: Ogawa-Kudoh contributed significantly to the increase in case detection and is suitable for implementation in poor-resource locations. The acquired resistance rate was lower than that reported in a recent Brazilian survey. The high genetic diversity is possibly related to the high TB prevalence in the population, as well as to early detection and suitable treatment of patients. The interaction between research and health care is important for reorienting the practice, transferring technology, and improving TB control.

Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis.

We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

Qualidade higiênico-sanitária de formulações ministradas a neonatos / Hygienic-sanitary quality of infant formulas given to neonates

Premature babies hospitalized at the Neonatal Intensive Care Units,particularly those with low weight and that are not allowed to be normallyfed, often need nutritional support of Infant Formulas (IF). These formulas,when contaminated, can be the source of nosocomial infections, the mostprobable way of contamination being the inadequate handling andstorage of these formulas. Evaluate the Hygienic-Sanitary quality and thesusceptibility profi le of the bacteria isolated from Ifs prepared in the lactary in the city of Rio Grande-RS to antimicrobial agents. IF samples were collected during 6 months by hospital employees working in the morning (Group A) and afternoon (group B) shifts, totalizing 72 analyzed samples. The Most Probable Number method (MPN/ml) was utilized to measure the amount of thermo-resistant and total coliforms. For mesophilic microorganisms the number of Colony-forming Units (CFU/ml) was counted. Also, samples were cultured in Blood and MacConkey Agar media and the isolates were identifi ed by 16S rRNA sequencing and submitted to antibiogram by the Kirby-Bauer method. The samples from groups A and B presented, respectively, 88.9% and 55.5% of contamination by total coliforms and 61.1% e 38.8% by thermo-resistant coliforms. Concerning the count of mesophilic microorganisms, groups A and B presented, respectively, 69.4% and 52.7% of the samples with results above the allowed limit. The identified microorganisms were Escherichia coli, Acinetobacter spp., Pseudomonas spp. and Enterobacter spp. The antimicrobial susceptibility test showed that all isolates presented resistance to at least three tested antimicrobials and that all of the isolates were resistant to tetracycline and one of them was also resistant to a carbapenem (imipinem).

Los recién nacidos prematuros hospitalizados en Unidades de Cuidados Intensivos, principalmente aquellos con bajo peso y poca habilidad para alimentarse naturalmente, necesitan el soporte nutricional de preparadospara lactantes (PL). Los PL, cuando contaminados, son fuente de infecciones hospitalaria, siendo la manipulación y el almacenamiento inadecuados las causas más frecuentes de los brotes de infección. Evaluar la calidad higiénico-sanitaria y el perfi l de susceptibilidad a los antimicrobianos de bacterias aisladas en PL. Las muestras se recogieron durante el período de 6 meses con dos grupos de trabajo: matutino (Grupo A) y vespertino (grupo B) del lactario del hospital, totalizando 72 muestras analizadas. Se realizó el recuento de número más probable (NMP / ml)de termotolerantes y coliformes totales y el recuento de unidades formadoras de colonias (UFC / ml) de los microorganismos mesófi los. Lasmuestras fueron cultivadas en Agar Sangre y MacConkey Agar y los aislados fueron identifi cados por la secuenciación del 16S rRNAy sometidos al antibiograma por el método Kirby-Bauer. Las muestras de los grupos A y B, presentaron, 88,9 y 55,5% y 61,1 y 38,8% decontaminación por coliformes totales y coliformes termotolerantes respectivamente. En cuanto a los mesófi los el recuento de microorganismos de los grupos A y B, presento, respectivamente,69,4 y 52,7% de las muestras con valores arriba del límite permitido. Los microorganismos identificados fueron Escher ichia coli , Acinetobacter spp., Pseudomonas spp. y Enterobacter spp. El antibiograma mostró quelos aislados presentaban resistencia por lo menos a tres de los antimicrobianos, todos fueron resistentes a tetraciclina y uno de ellos también fue resistente a un carbapenem (imipinem).

Prematuros que se encontram em Unidades de Tratamento Intensivo Neonatal, particularmente aqueles com baixo peso e com difi culdades parareceber a alimentação natural, necessitam frequentemente de suporte nutricional por meio de fórmulas infantis (FI). As FI, quando contaminadas, podem ser fontes de infecção hospitalar, sendo a manipulação e oarmazenamento inadequados os prováveis meios de contaminação. O estudo objetivou avaliar a qualidade higiênico-sanitária e o perfilde suscetibilidade aos antimicrobianos das bactérias isoladas em FI preparadas no lactário de um hospital da cidade de Rio Grande-RS.Amostras de FI reconstituídas por lactaristas do turno da manhã (grupo A) e da tarde (grupo B) foram coletadas durante 6 meses, totalizando72 amostras analisadas. Foi realizada a contagem do Número Mais Provável (NMP/ml) de coliformes totais e termotolerantes e a contagem das Unidades Formadoras de Colônias (UFC/ml) de microrganismos mesófi los. Além disso, amostras foram semeadas em Ágar Sangue e MacConkey e os isolados foram identificados por meio de sequenciamento do 16S rRNA e submetidos ao antibiograma através de método de Kirby-Bauer. As amostras dos grupos A e B apresentaram, respectivamente, 88,9 e 55,5% de contaminação por coliformes totais e 61,1 e 38,8% por coliformes termotolerantes. Na contagem de microrganismos mesófilos, os grupos A e B apresentaram, respectivamente, 69,4 e 52,7% das amostras com contagem acima do limite permitido. Os microrganismos identificadosforam Escherichia coli, Acinetobacter spp., Pseudomonas spp. e Enterobacter spp. O teste de suscetibilidade antimicrobiana mostrou quetodos os isolados apresentavam resistência a pelo menos três antimicrobianos testados e que todos foram resistentes à tetraciclina e um também foi resistente a um carbapenem (imipinem).