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I. paraguariensis St. Hil. is a south American species of agronomic interest with studies supporting its medicinal properties. As the investigation of active ingredients with antimicrobial effect from medicinal plants is a suitable approach to the current antibacterial resistance problem, the aim of the present study was to determine the antibacterial activity of yerba mate ethanolic extracts against carbapenemase-producing gram-negative bacteria (reference strains and clinical isolates). Extracts showed antibacterial activity against Klebsiella pneumoniae ATCC® BAA-2342™ (KPC producing), Providencia rettgeri (NDM producing), Pseudomonas aeruginosa (MBL producing) and P. aeruginosa (VIM producing) at the concentrations tested. The Minimal-Inhibitory-Concentration and Minimal-Bactericidal-Concentration values ranged between 1 and 32 mg.ml-1 for the reference strains, and between 0.125 and 1 mg.ml-1 for the clinical isolates. The MBC/MIC index characterized the extracts as bactericidal. The combinations of commercial antibiotics and extracts showed a synergistic action on the reference strains studied. The lethal concentration 50 obtained using the Artemia salina toxicity assay were higher than 1 mg.ml-1 for all the extracts, indicating a low toxicity. The in vitro activity and low toxicity suggest that ethanolic I. paraguariensis leaf extracts constitute an outstanding source for new antibacterial compounds, and further studies should be carried out to understand their mechanism of action.

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RESUMEN El filtrado de secuencias es un paso esencial sin importar el tipo de tecnología aplicada para la secuenciación de un genoma, en el cual las lecturas de baja calidad o una parte son eliminadas. En un ensamblado la construcción de un genoma se realiza a partir de la unión de lecturas cortas en cóntigos. Algunos ensambladores miden la relación que existe entre secuencias de una longitud fija (k-mer) que puede verse afectada por la presencia de secuencias de baja calidad. Un enfoque común para evaluar los ensamblados se basa en el análisis del número de cóntigos, la longitud del cóntigo más largo y el valor del N50, definido como la longitud del cóntigo que representa el 50 % de la longitud del conjunto. En este contexto, el presente estudio tuvo como objetivo evaluar el efecto del uso de lecturas crudas y filtradas en los valores de los parámetros de calidad obtenidos en el ensamblado del genoma de Bacillus altitudinis 19RS3 aislada de Ilex paraguariensis. Se realizó el análisis de calidad de ambos archivos de partida con el software FastqC y se filtraron las lecturas con el softwareTrimmomatic. Para el ensamblado se utilizó el software SPAdes y para su evaluación la herramienta QUAST. El mejor ensamblado para B. altitudinis 19RS3 se obtuvo a partir de las lecturas filtradas con el valor de k-mer 79, que generó 16 cóntigos mayores a 500 pb con un N50 de 931 914 pb y el cóntigo más largo de 966 271 pb.

ABSTRACT Sequence filtering is an essential step regardless of the type of technology applied for sequencing a genome, in which low-quality readings or a portion are eliminated. In an assembly, the construction of a genome is carried out from the union of short reads in contigs. Some assemblers measure the relationship between sequences of a fixed length (k-mer) that can be affected by the presence of low-quality sequences. A common approach to evaluating assemblies is based on the analysis of the number of contigs, the length of the longest contig, and the value of N50 defined as the length of the contig representing 50 % of the length of the assembly. In this context, the objective of this study was to evaluate the effect of the use of crude and filtered reads on the values of the quality parameters obtained from the genome assembly of Bacillus altituidinis 19RS3 isolated from Ilex paraguariensis. The quality analysis of both starting files was performed with the FastqC software and the readings were filtered with the Trimmomatic software. The SPAdes software was used for the assembly and the QUAST tool for its evaluation. The best assembly for B. altitudinis 19RS3 was obtained from the filtered readings with the value of k-mer 79, which generated 16 contigs greater than 500 bp with a N50 of 931 914 bp and the longest contig of 966 271 bp.

RESUMO

Plant growth-promoting bacteria (PGPB) are a heterogeneous group of bacteria that can exert beneficial effects on plant growth directly or indirectly by different mechanisms. PGPB-based inoculant formulation has been used to replace chemical fertilizers and pesticides. In our previous studies, two endophytic endospore-forming bacteria identified as Bacillus altitudinis were isolated from roots of Ilex paraguariensis St. Hil. seedlings and selected for their plant growth-promoting (PGP) properties shown in vitro and in vivo. The purposes of this work were to assemble the genomes of B. altitudinis 19RS3 and T5S-T4, using different assemblers available for Windows and Linux and to select the best assembly for each strain. Both genomes were also automatically annotated to detect PGP genes and compare sequences with other genomes reported. Library construction and draft genome sequencing were performed by Macrogen services. Raw reads were filtered using the Trimmomatic tool. Genomes were assembled using SPAdes, ABySS, Velvet, and SOAPdenovo2 assemblers for Linux, and Geneious and CLC Genomics Workbench assemblers for Windows. Assembly evaluation was done by the QUAST tool. The parameters evaluated were the number of contigs ≥ 500 bp and ≥ 1000 bp, the length of the longest contig, and the N50 value. For genome annotation PROKKA, RAST, and KAAS tools were used. The best assembly for both genomes was obtained using Velvet. The B. altitudinis 19RS3 genome was assembled into 15 contigs with an N50 value of 1,943,801 bp. The B. altitudinis T5S-T4 genome was assembled into 24 contigs with an N50 of 344,151 bp. Both genomes comprise several genes related to PGP mechanisms, such as those for nitrogen fixation, iron metabolism, phosphate metabolism, and auxin biosynthesis. The results obtained offer the basis for a better understanding of B. altitudinis 19RS3 and T5S-T4 and make them promissory for bioinoculant development.

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BACKGROUND: In pregnant women Streptococcus agalactiae (GBS) can be transmitted to newborn causing severe infections. It is classified into 10 serotypes (Ia, Ib, II-IX). The severity of neonatal disease is determined by the capsular serotype and virulence factors such as the polysaccharide capsule, encoded by the cps gene, protein C, which includes the Cα surface proteins (bca gene), Rib (rib gene) and Cß (bac gene); the proteins Lmb (lmb gene), FbsB (fbsB gene), FbsA (fbsA gene), the cyl operon encoding a ß-hemolysin (hylB gene), the CAMP factor (cfb gene) and the C5a peptidase (scpB gene). The aim of this work was to determine the degree of GBS colonization in pregnant women, the serotypes distribution and to investigate virulence-associated genes. METHODS: We worked with 3480 samples of vagino-rectal swabs of women with 35-37 weeks of gestation. The identification of the strains was carried out using conventional biochemical tests and group confirmatory serology using a commercial latex particle agglutination kit. Two hundred GBS strains were selected. Their serotype was determined by agglutination tests. The monoplex PCR technique was used to investigate nine virulence-associated genes (cps, bca, rib, bac, lmb, fbsB, fbsA, hylB and scpB). RESULTS: The maternal colonization was 9.09%. The serotypes found were: Ia (33.50%), III (19.00%), Ib (15.50%), II (14.00%), V (7.00%) and IX (5.50%). 5.50% of strains were found to be non-serotypeable (NT). The nine virulence genes investigated were detected simultaneously in 36.50% of the strains. The genes that were most frequently detected were scpB (100.00%), fbsA (100.00%), fbsB (100.00%), cylB (95.00%), lmb (94.00%) and bca (87.50%). We found associations between serotype and genes bac (p = 0.003), cylB (p = 0.02), rib (p = 0.01) and lmb (p < 0.001). CONCLUSIONS: The frequency of vaginal-rectal colonization, serotypes distribution and associated virulence genes, varies widely among geographical areas. Therefore, epidemiological surveillance is necessary to provide data to guide decision-making and planning of prevention and control strategies.

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Yerba mate (Ilex paraguariensis St. Hil.) is a species native to the subtropical regions of South America. Despite being an important crop for the region, there are few studies on the use of microorganisms to improve the growth of seedlings in the nursery stage. The objective of this study was to isolate spore-forming endophytic bacteria with plant growth promoting properties associated with yerba mate seedlings and determine their phytobeneficial effect under controlled laboratory conditions. Isolates were selected based on their sporulation capacity and evaluated for in vitro plant growth promoting properties (nitrogen fixation, phosphate solubilization, production of siderophores and synthesis of indolic compounds). Yerba mate seedlings were inoculated with the most promising isolates, which were identified via analyses of the sequence of their 16S rDNA gene as Bacillus circulans (12RS3) and Bacillus altitudinis (19RS3, T5S-T4). After 120 days plants showed higher root dry weight when inoculated with isolate 19RS3 and higher shoot dry weight with 19RS3 and T5S-T4. In conclusion, further studies to determine the ability of these isolates to adapt to the climatic conditions and to survive amidst the native soil microflora in yerba mate cultivated native soils, will be crucial for developing such strains as biofertilizer.

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The aim of this work was to determine the susceptibility, molecular profile, and clonal relationship in Streptococcus agalactiae (group B Streptococcus [GBS]) isolated from vaginal-rectal swab samples. We worked with 200 isolates collected from pregnant women between 35 and 37 weeks of gestation. The macrolide-lincosamide-streptogramin B (MLSB) resistance phenotypes were determined using the double-disc assay. Susceptibility to erythromycin (ERI) and clindamycin (CLI) was performed with the E-test. Resistance genes ermB and ermTR were detected by polymerase chain reaction. Clonal studies were performed using the random amplification of polymorphic DNA. Twelve (6%) of the isolates were resistant to ERI and 10 (5%) of them to CLI. Fifty percent of the resistant strains corresponded to serotype III, 25% to serotype V, and the remaining 25% to serotype Ia, II, and nontypeable strains. The cMLSB phenotype was detected in eight strains (66.67%) and the iMLSB phenotype in four (33.33%). The minimum inhibitory concentration values were between 1.5 and 16 µg/mL for ERI, and between 1 and 32 µg/mL for CLI. Out of the 25 strains susceptible to ERI and CLI, the presence of the ermB gene was detected in eight of them and the ermTR gene in one strain. The ermB gene was detected in the 12 strains that initially had some macrolide resistance phenotype. The ermTR gene was detected in three out of the four strains with the iMLSB phenotype. The resistance to macrolides in the province of Misiones is due to multiclonal spread. The phenotypic and genotypic characterization of macrolide resistance in GBS strains are crucial to contribute to the correct intrapartum prophylactic antibiotic therapy of allergic pregnant women and the epidemiological surveillance of these strains.

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Streptococcus agalactiae (GBS) cause severe infections in newborns under three months. Meningitis, pneumonia and sepsis are the main infectious diseases in these children. These infections are among the most serious that an individual can suffer in his first twelve hours of life. The child acquires the infection by vertical transmission of the colonized mother. To prevent neonatal disease, penicillin is recommended as the drug of choice for intrapartum prophylaxis (PIP) in pregnant women colonized. However, strains with decreased susceptibility to penicillin have been detected so it is important to monitor the susceptibility to penicillin to ensure its usefulness during prophylaxis. The aim of this study was to determine the sensitivity to penicillin in GBS strains recovered from pregnant women with 35-37 weeks of gestation. Ninety-six isolates were studied and sensitivity was determined by the epsilometric method Etest® (LIOFILCHEM, Italy), following the recommendations of the Clinical Laboratory Standards Institute (CLSI). Minimum Inhibitory Concentration (MIC) was obtained for each bacterial isolation. 100% (96) of the strains studied were sensitive to penicillin with MIC values between 0.012 and 0.094 ?g mL-1. These results indicate that penicillin remains the antimicrobial of choice during intrapartum prophylaxis, for the prevention of neonatal disease caused by GBS in our region. The importance of epidemiological surveillance of sensitivity to penicillin and other antimicrobials is highlighted in order to alert new resistance mechanisms and to adapt treatment strategies.

Streptococcus agalactiae (SGB) es causa de infecciones severas en menores de tres meses. Meningitis, neumonía y sepsis son los principales cuadros en estos niños. Estas infecciones se encuentran entre las más graves que puede sufrir un individuo en sus primeras doce horas de vida. El niño adquiere la infección por transmisión vertical de la madre colonizada. Para prevenir la enfermedad neonatal se recomienda penicilina como droga de elección en la profilaxis intraparto (PIP) en embarazadas colonizadas. Sin embargo, actualmente se han detectado cepas con sensibilidad disminuida a penicilina por lo que resulta importante realizar la vigilancia de la sensibilidad al mismo para asegurar su utilidad durante la profilaxis. El objetivo de este trabajo fue determinar la sensibilidad a penicilina en cepas de SGB recuperados de mujeres embarazadas de 35-37 semanas de gestación. Se estudiaron 96 aislamientos y se determinó la sensibilidad por método epsilométrico Etest® (LIOFILCHEM, Italia), siguiendo las recomendaciones del Clinical Laboratory Standards Institute (CLSI). Se obtuvo la Concentración Inhibitoria Mínima (CIM) para cada aislamiento bacteriano. El 100% (96) de las cepas estudiadas fue sensible a penicilina con valores de CIM comprendidos entre 0,012 y 0,094 µg mL-1. Estos resultados indican que penicilina sigue siendo el antimicrobiano de elección durante la profilaxis intraparto, para la prevención de la enfermedad neonatal causada por SGB en nuestra región. Se destaca la importancia de la vigilancia epidemiológica de la sensibilidad a penicilina y a otros antimicrobianos para alertar sobre nuevos mecanismos de resistencia y adecuar estrategias de tratamiento.

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Introducción: Si bien Streptococcus agalactiae es comensal del tracto gastrointestinal y genitourinario, es la principal causa de enfermedades invasivas y de mortalidad en niños recién nacidos. La infección puede adquirirse a través de la aspiración de líquido amniótico infectado o durante el paso por el canal de parto. Objetivos: comparar la utilidad de diversos métodos de conservación de cepas de Streptococcus agalactiae que resulten reproducibles, accesibles a laboratorios de baja y mediana complejidad, y asegurar su estabilidad fenotípica y genotípica mediante el método de preservación llamado subcultivo continuo, para el mantenimiento del cultivo en medio adecuado con transferencias a medio fresco a intervalos variables. Métodos: Se seleccionaron al azar 40 cepas de Streptococcus agalactiae, las que se sometieron a verificación de viabilidad, pureza y caracterización fenotípica y genotípica antes y después de ser sometidas a conservación, utilizando idénticos medios, reactivos y metodología en ambas circunstancias. Se probaron diferentes medios de preservación de las cepas, que permitieran a laboratorios de baja y mediana complejidad su traslado a centros especializados para la vigilancia adecuada del organismo. Las cepas se conservaron durante nueve meses con subcultivos que mostraron las características originales. Resultados: Los medios más efectivos fueron ATS-TA y LD 4 %-SO-20 ºC, ya que garantizaron viabilidad, pureza y estabilidad fenotípica y genotípica de las cepas. Se demostró que el uso de este medio es una alternativa adecuada para la conservación de Streptococcus agalactiae en laboratorios donde la liofilización y la desecación no están disponibles y que son de bajo costo, rápidos y muy fáciles de usar en la práctica habitual. Conclusiones: los medios ATS-TA y LD 4 %-SO-20 ºC constituyen una buena alternativa para cortos períodos de preservación y transporte de cepas de Streptococcus agalactiae en laboratorios de baja y mediana complejidad.

Introduction: Although Streptococcus agalactiae is commensal of the gastrointestinal and genitourinary tract, it is the main cause of invasive diseases and mortality in newborns. The infection can be acquired through the aspiration of infected amniotic fluid or during the passage through the birth canal. Objectives: to compare the effectiveness of various methods for the conservation of Streptococcus agalactiae strains that can be reproducible and accessible to laboratories of low and medium complexity and guarantee their phenotypic and genotypic stability through a preservation method called continuous subculture, for the maintenance of the culture in an adequate environment with fresh medium transfers at variable interval periods. Methods: 40 strains of Streptococcus agalactiae were randomly selected, which were subjected to verification of viability, purity and genotypic and phenotypic characterization before and after being subjected to conservation, using identical environments, reactive and methodologies in both circumstances. Different means of preservation of strains were tested, which allowed laboratories of low and medium complexity their transfer to specialized centers for the proper surveillance of the organism. The strains were kept for nine months with subcultures that showed original characteristics. Results: The most effective environments were ATS-TA and LD-4 % -SO-20 ºC because they guaranteed viability, purity and genotypic and phenotypic stability of the strains. It was shown that the use of this environment is an adequate alternative for the conservation of Streptococcus agalactiae in laboratories where lyophilization and dessication are not available and are low cost, fast and very easy to use in regular practice. Conclusions: The ATS-TA and LD-4 % -SO-20 ºC environments are a good alternative for short periods of preservation and transportation of Streptococcus agalactiae strains in laboratories of low and medium complexity.

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The purpose of this study was to determine the susceptibilities of food-borne Aeromonas to carbapenems, as well as to investigate the presence of a metallo carbapenemase-encoding gene, named cphA. Minimum Inhibitory Concentration (MIC) was determined following NCCLS standards. All the tested microorganisms were susceptible to imipenem, meropenem and biapenem. However, a strong inoculum size effect on carbapenem MICs was observed for most of the strains. Six strains, out of seven, showed the presence of metallo--beta-lactamases but cphA gene was detected in only two strains of A. veronii bv. sobria.

O objetivo deste estudo foi determinar a suscetibilidade de aeromonas de origem alimentar a carbapenems bem como investigar a presença de um gene codificante de metalocarbapenemase, denominado "cph A". A suscetibilidade in vitro foi determinada pelo metodo de diluição em agar. Todas as cepas foram suscetíveis a Imipenem, Meropenem e Biapenem. Porém foi observado um forte efeito de tamanho do inóculo sobre as CIM das carbapenems na maioria das cepas. A detecção de metalo-beta-lactamase foi realizada pelo metodo lodometrico. Seis cepas das sete testadas demostraron a presença da enzima. A presença do gene cphA foi determinada por PCR e foi detectada em duas cepas de A veronii bv. sobria.