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1.
Science ; 368(6495): 1135-1140, 2020 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-32499444

RESUMEN

Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, a Cas13a RNA-guided nucleic acid detection assay, facilitating its implementation in a wide range of applications.


Asunto(s)
Código de Barras del ADN Taxonómico/métodos , ADN Bacteriano/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Microbiología Ambiental , Microbiota/genética , Esporas/genética , Sistemas CRISPR-Cas , ADN Bacteriano/genética , ADN de Hongos/genética , ARN Guia
2.
PLoS One ; 15(3): e0230390, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32176736

RESUMEN

The aim of the study was to detect and genetically characterize Arcobacter butzleri in pet red-footed tortoises suspected for Campylobacter spp., using molecular techniques. A written consent from tortoise owners was obtained, after explaining the advantages of the research to tortoise owners of Grenada. Fecal samples were collected from 114 tortoises from five parishes of the country and cultured for Campylobacter spp. using selective culture techniques. A. butzleri was isolated from 4.39% of pet tortoises. Total thirteen isolates were obtained; all identified as A. butzleri by a universal and a species-specific Polymerase Chain Reaction (PCR) and direct sequencing. Genetic characterization of these isolates was performed based on Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) that generated eight different genetic fingerprints with a discriminatory power of 0.91. Campylobacter species were not detected molecularly in any of the culture-positive samples. This is the first report of infection of pet tortoises in Grenada, West Indies with A. butzleri. This study emphasizes on the risk of zoonotic transmission of A. butzleri by exotic pets, which is a serious concern for public health.


Asunto(s)
Arcobacter/genética , Campylobacter/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Tortugas/microbiología , Animales , Campylobacter/aislamiento & purificación , Dermatoglifia del ADN/métodos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Heces/microbiología , Tortugas/genética
3.
An Acad Bras Cienc ; 92(suppl 1): e20180426, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32159585

RESUMEN

Effective microorganisms (EM) are inoculants formed by fungi and bacteria isolated from soil. EM are commonly used by farmers on agronomic crops to stimulate plant growth, but their composition and their benefits has been controverted. This study aimed to analyze the diversity of microorganisms growing in three EM inoculants, as well as to evaluate their efficiency in the germination of palisade grass seeds. The total DNA of the three EM inoculants was extracted, the 16S rRNA and ITS genes were amplified by PCR and sequenced on the Illumina MiSeq platform. Germination tests were conducted with three type of the EM, in three concentration and two times of the immersion. The bacterial group was the most abundant in EM, followed by fungi. Bacterial operational taxonomic units OTUs were shared by all EMs. Pre-treatments of palisade grass seeds with EMs resulted in a higher germination percentage (% G) and germination speed index (IVG) when EM was used at concentration of 1 or 2% in water. Seed immersion for 5 min was more efficient than immersion for 24 h. We can conclude that EM of different origin can share microbial groups and diversity of microorganisms, besides being an alternative to increase palisade grass seeds germination.


Asunto(s)
Inoculantes Agrícolas/genética , ADN Bacteriano/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Germinación/fisiología , Poaceae/crecimiento & desarrollo , Semillas/crecimiento & desarrollo , Biodiversidad , Productos Agrícolas/genética , Germinación/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Semillas/efectos de los fármacos , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Ácidos Sulfúricos/farmacología
4.
World J Microbiol Biotechnol ; 36(3): 49, 2020 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-32157439

RESUMEN

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.


Asunto(s)
Glicerol/metabolismo , Ingeniería Metabólica/métodos , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Acuaporinas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Capsicum/microbiología , China , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Glicerolfosfato Deshidrogenasa/genética , Redes y Vías Metabólicas/genética , Fenazinas/metabolismo , Proteínas Represoras/genética , Rizosfera
5.
World J Microbiol Biotechnol ; 36(2): 29, 2020 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-32016527

RESUMEN

Short-chain halogenated aliphatic hydrocarbons (e.g. perchloroethene, trichloroethene) are among the most toxic environmental pollutants. Perchloroethene and trichloroethene can be dechlorinated to non-toxic ethene through reductive dechlorination by Dehalococcoides sp. Bioaugmentation, applying cultures containing organohalide-respiring microorganisms, is a possible technique to remediate sites contaminated with chlorinated ethenes. Application of site specific inocula is an efficient alternative solution. Our aim was to develop site specific dechlorinating microbial inocula by enriching microbial consortia from groundwater contaminated with trichloroethene using microcosm experiments containing clay mineral as solid phase. Our main goal was to develop fast and reliable method to produce large amount (100 L) of bioactive agent with anaerobic fermentation technology. Polyphasic approach has been applied to monitor the effectiveness of dechlorination during the transfer process from bench-scale (500 mL) to industrial-scale (100 L). Gas chromatography measurement and T-RFLP (Terminal Restriction Fragment Length Polymorphism) revealed that the serial subculture of the enrichments shortened the time-course of the complete dechlorination of trichloroethene to ethene and altered the composition of bacterial communities. Complete dechlorination was observed in enrichments with significant abundance of Dehalococcoides sp. cultivated at 8 °C. Consortia incubated in fermenters at 18 °C accelerated the conversion of TCE to ethene by 7-14 days. Members of the enrichments belong to the phyla Bacteroidetes, Chloroflexi, Proteobacteria and Firmicutes. According to the operational taxonomic units, main differences between the composition of the enrichment incubated at 8 °C and 18 °C occurred with relative abundance of acetogenic and fermentative species. In addition to the temperature, the site-specific origin of the microbial communities and the solid phase applied during the fermentation technique contributed to the development of a unique microbial composition.


Asunto(s)
Anaerobiosis/fisiología , Bacterias/metabolismo , Biodegradación Ambiental , Arcilla/química , Microbiota/fisiología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteroidetes/genética , Bacteroidetes/metabolismo , Chloroflexi/genética , Chloroflexi/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Fermentación , Firmicutes/genética , Firmicutes/metabolismo , Geobacter/genética , Geobacter/metabolismo , Agua Subterránea/microbiología , Consorcios Microbianos , Polimorfismo de Longitud del Fragmento de Restricción , Proteobacteria/genética , Proteobacteria/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/aislamiento & purificación , Tricloroetileno/química , Microbiología del Agua , Contaminantes Químicos del Agua/metabolismo
6.
PLoS One ; 15(2): e0228459, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32027671

RESUMEN

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is emerging as a significant pathogen causing healthcare-associated infections. Matrix-assisted laser desorption/ionisation mass spectrometry time-of-flight mass spectrometry (MALDI-TOF MS) is used by clinical microbiology laboratories to address the need for rapid, cost-effective and accurate identification of microorganisms. We evaluated application of machine learning methods for differentiation of drug resistant bacteria from susceptible ones directly using the profile spectra of whole cells MALDI-TOF MS in 46 CRKP and 49 CSKP isolates. METHODS: We developed a two-step strategy for data preprocessing consisting of peak matching and a feature selection step before supervised machine learning analysis. Subsequently, five machine learning algorithms were used for classification. RESULTS: Random forest (RF) outperformed other four algorithms. Using RF algorithm, we correctly identified 93% of the CRKP and 100% of the CSKP isolates with an overall classification accuracy rate of 97% when 80 peaks were selected as input features. CONCLUSIONS: We conclude that CRKPs can be differentiated from CSKPs through RF analysis. We used direct colony method, and only one spectrum for an isolate for analysis, without modification of current protocol. This allows the technique to be easily incorporated into clinical practice in the future.


Asunto(s)
Algoritmos , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Infecciones por Klebsiella/diagnóstico , Klebsiella pneumoniae/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Aprendizaje Automático Supervisado , Antibacterianos/uso terapéutico , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Carbapenémicos/uso terapéutico , Infección Hospitalaria/diagnóstico , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Humanos , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Valor Predictivo de las Pruebas , Técnica del ADN Polimorfo Amplificado Aleatorio
7.
PLoS One ; 15(2): e0228133, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32023276

RESUMEN

The neonatal period, during which the initial gut microbiota is acquired, is a critical phase. The healthy development of the infant's microbiome can be interrupted by external perturbations, like antibiotics, which are associated with profound effects on the gut microbiome and various disorders later in life. The aim of this study was to investigate the development of intestinal microbiota and the effect of antibiotic exposure during the first three months of life in term infants. Fecal samples were collected from healthy infants and infants who received antibiotics in the first week of life at one week, one month, and three months after birth. Microbial composition was analyzed using IS-pro and compared between antibiotics-treated and untreated infants. In total, 98 infants, divided into four groups based on feeding type and delivery mode, were analyzed. At one week of age, samples clustered into two distinct groups, which were termed "settler types", based on their Bacteroidetes abundance. Caesarean-born infants belonged to the low-Bacteroidetes settler type, but vaginally-born infants were divided between the two groups. The antibiotics effect was assessed within a subgroup of 45 infants, vaginally-born and exclusively breastfed, to minimize the effect of other confounders. Antibiotics administration resulted in lower Bacteroidetes diversity and/or a delay in Bacteroidetes colonization, which persisted for three months, and in a differential development of the microbiota. Antibiotics resulted in pronounced effects on the Bacteroidetes composition and dynamics. Finally, we hypothesize that stratification of children's cohorts based on settler types may reveal group effects that might otherwise be masked.


Asunto(s)
Antibacterianos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Bacteroidetes/genética , Bacteroidetes/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Heces/microbiología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Análisis de Componente Principal , ARN Ribosómico 16S/aislamiento & purificación , ARN Ribosómico 16S/metabolismo
8.
Parasitol Res ; 119(4): 1423-1427, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32107621

RESUMEN

We report two cases of bovine babesiosis caused by Babesia divergens in a region of central Bosnia and Herzegovina. The cases were detected in June 2017 and July 2018 from two small backyard farms. Routine clinical assessments, including physical examination and haematology, revealed lethargy, fever, anaemia, leukopenia and haemoglobinuria in the affected animals. Serum alterations included an elevation of aspartate aminotransferase and a decrease of serum phosphate or hypophosphatemia. Thrombocytopenia was detected in the first clinical case. Microscopic examination of blood smears revealed intracytoplasmic protozoan parasites from the genus Babesia. Molecular screening of both animals confirmed the presence of Babesia divergens, the causative agent of bovine babesiosis. B. divergens DNA was also detected in two engorged female Ixodes ricinus ticks removed from these animals. In addition, Mycoplasma wenyonii DNA was identified by molecular screening in the animal examined in June 2017, and in I. ricinus ticks feeding on this animal. This study provides molecular confirmation of B. divergens as a cause of piroplasmosis in cattle in South-East Europe. The detection of M. wenyonii DNA ain I. ricinus also provides the first evidence of this bacterium in ticks in Europe.


Asunto(s)
Babesia/genética , Babesiosis/diagnóstico , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/parasitología , Infecciones por Mycoplasma/veterinaria , Mycoplasma/genética , Animales , Aspartato Aminotransferasas/sangre , Babesia/aislamiento & purificación , Babesiosis/parasitología , Bosnia y Herzegovina , Bovinos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Europa (Continente) , Granjas , Femenino , Hipofosfatemia/sangre , Ixodes/microbiología , Ixodes/parasitología , Mycoplasma/aislamiento & purificación , Infecciones por Mycoplasma/diagnóstico , Trombocitopenia/sangre
9.
PLoS One ; 15(1): e0227486, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31935223

RESUMEN

Microbiome research has experienced a surge of interest in recent years due to the advances and reduced cost of next-generation sequencing technology. The production of high quality and comparable data is dependent on proper sample collection and storage and should be standardized as far as possible. However, this becomes challenging when samples are collected in the field, especially in resource-limited settings. We investigated the impact of different stool storage methods common to the TB-CHAMP clinical trial on the microbial communities in stool. Ten stool samples were subjected to DNA extraction after 48-hour storage at -80°C, room temperature and in a cooler-box, as well as immediate DNA extraction. Three stool DNA extraction kits were evaluated based on DNA yield and quality. Quantitative PCR was performed to determine the relative abundance of the two major gut phyla Bacteroidetes and Firmicutes, and other representative microbial groups. The bacterial populations in the frozen group closely resembled the immediate extraction group, supporting previous findings that storage at -80°C is equivalent to the gold standard of immediate DNA extraction. More variation was seen in the room temperature and cooler-box groups, which may be due to the growth temperature preferences of certain bacterial populations. However, for most bacterial populations, no significant differences were found between the storage groups. As seen in other microbiome studies, the variation between participant samples was greater than that related to differences in storage. We determined that the risk of introducing bias to microbial community profiling through differences in storage will likely be minimal in our setting.


Asunto(s)
Heces/microbiología , Microbiota , Manejo de Especímenes/métodos , Preescolar , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Hongos/genética , Hongos/aislamiento & purificación , Humanos , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo
10.
PLoS One ; 15(1): e0227561, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31935259

RESUMEN

Host-parasite interactions may be modulated by host- or parasite-associated microbes, but the role of these are often overlooked. Particularly for parasites with intestinal stages (either larval or adult), the host gut microbiome may play a key role for parasite establishment; moreover, the microbiome may change in response to invading parasites. Hypothesis testing at the organismal level may be hampered, particularly in mammalian definitive hosts, by ethical, logistical, and economical restrictions. Thus, invertebrates naturally serving as intermediate hosts to parasites with complex life cycles may inform the development of mammalian models as an early-stage host-parasite model. In addition, several important pathogens are vectored by insects, and insect gut microbiome-pathogen interactions may provide essential base-line knowledge, which may be used to control vectorborne pathogens. Here, we used the grain beetle, Tenebrio molitor, a host of the tapeworm Hymenolepis diminuta, to explore interactions between infection status and resident gut microbiota at two pre-determined time points (day two and seven) post infection. Using 16S/18S microbial profiling, we measured key parameters of the composition, relative abundance, and diversity of the host gut bacteriome and mycobiome. In addition, we quantified the systemic beetle immune response to infection by Phenoloxidase activity and hemocyte abundance. We found significant changes in the gut bacteriome and mycobiome in relation to infection status and beetle age. Thus, the relative abundance of Proteobacteria was significantly higher in the gut of infected beetles and driven mostly by an increased abundance of Acinetobacter. In addition, the mycobiome was less abundant in infected beetles but maintained higher Shannon diversity in infected compared with non-infected beetles. Beetles treated with a broad-spectrum antibiotic (Tetracycline) exhibited significantly reduced parasite establishment compared with the untreated control group, indicating that the host microbiome may greatly influence hatching of eggs and subsequent establishment of H. diminuta larvae. Our results suggest that experimental work using invertebrates may provide a platform for explorative studies of host-parasite-microbe interactions and their underlying mechanisms.


Asunto(s)
Escarabajos/parasitología , Microbioma Gastrointestinal , Hymenolepis diminuta/fisiología , Animales , Antibacterianos/farmacología , Escarabajos/inmunología , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Hemolinfa/metabolismo , Interacciones Huésped-Parásitos , Monofenol Monooxigenasa/metabolismo , Micobioma/efectos de los fármacos , Análisis de Componente Principal , Proteobacteria/genética , Proteobacteria/aislamiento & purificación , Tetraciclina/farmacología
11.
PLoS One ; 15(1): e0227707, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31917801

RESUMEN

Epithelial ovarian cancer (OC) is the most deadly cancer of the female reproductive system. To date, there is no effective screening method for early detection of OC and current diagnostic armamentarium may include sonographic grading of the tumor and analyzing serum levels of tumor markers, Cancer Antigen 125 (CA-125) and Human epididymis protein 4 (HE4). Microorganisms (bacterial, archaeal, and fungal cells) residing in mucosal tissues including the gastrointestinal and urogenital tracts can be altered by different disease states, and these shifts in microbial dynamics may help to diagnose disease states. We hypothesized that the peritoneal microbial environment was altered in patients with OC and that inclusion of selected peritoneal microbial features with current clinical features into prediction analyses will improve detection accuracy of patients with OC. Blood and peritoneal fluid were collected from consented patients that had sonography confirmed adnexal masses and were being seen at SIU School of Medicine Simmons Cancer Institute. Blood was processed and serum HE4 and CA-125 were measured. Peritoneal fluid was collected at the time of surgery and processed for Next Generation Sequencing (NGS) using 16S V4 exon bacterial primers and bioinformatics analyses. We found that patients with OC had a unique peritoneal microbial profile compared to patients with a benign mass. Using ensemble modeling and machine learning pathways, we identified 18 microbial features that were highly specific to OC pathology. Prediction analyses confirmed that inclusion of microbial features with serum tumor marker levels and control features (patient age and BMI) improved diagnostic accuracy compared to currently used models. We conclude that OC pathogenesis alters the peritoneal microbial environment and that these unique microbial features are important for accurate diagnosis of OC. Our study warrants further analyses of the importance of microbial features in regards to oncological diagnostics and possible prognostic and interventional medicine.


Asunto(s)
Líquido Ascítico/microbiología , Antígeno Ca-125/sangre , Carcinoma Epitelial de Ovario/diagnóstico , Proteínas de la Membrana/sangre , Microbiota/genética , Neoplasias Ováricas/diagnóstico , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP/análisis , Anciano , Carcinoma Epitelial de Ovario/sangre , Carcinoma Epitelial de Ovario/microbiología , Carcinoma Epitelial de Ovario/cirugía , Estudios Transversales , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Femenino , Humanos , Histerectomía , Laparoscopía , Aprendizaje Automático , Persona de Mediana Edad , Modelos Biológicos , Neoplasias Ováricas/sangre , Neoplasias Ováricas/microbiología , Neoplasias Ováricas/cirugía , Ovariectomía , Proyectos Piloto , Periodo Preoperatorio , Pronóstico , ARN Ribosómico 16S/genética
12.
PLoS One ; 15(1): e0227774, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31978078

RESUMEN

The list of pharmacological agents that can modify the gut microbiome or be modified by it continues to grow at a high rate. The greatest amount of attention on drug-gut microbiome interactions has been directed primarily at pharmaceuticals used to treat infection, diabetes, cardiovascular conditions and cancer. By comparison, drugs of abuse and addiction, which can powerfully and chronically worsen human health, have received relatively little attention in this regard. Therefore, the main objective of this study was to characterize how selected synthetic psychoactive cathinones (aka "Bath Salts") and amphetamine stimulants modify the gut microbiome. Mice were treated with mephedrone (40 mg/kg), methcathinone (80 mg/kg), methamphetamine (5 mg/kg) or 4-methyl-methamphetamine (40 mg/kg), following a binge regimen consisting of 4 injections at 2h intervals. These drugs were selected for study because they are structural analogs that contain a ß-keto substituent (methcathinone), a 4-methyl group (4-methyl-methamphetamine), both substituents (mephedrone) or neither (methamphetamine). Mice were sacrificed 1, 2 or 7 days after treatment and DNA from caecum contents was subjected to 16S rRNA sequencing. We found that all drugs caused significant time- and structure-dependent alterations in the diversity and taxonomic structure of the gut microbiome. The two phyla most changed by drug treatments were Firmicutes (methcathinone, 4-methyl-methamphetamine) and Bacteriodetes (methcathinone, 4-methyl-methamphetamine, methamphetamine, mephedrone). Across time, broad microbiome changes from the phylum to genus levels were characteristic of all drugs. The present results signify that these selected psychoactive drugs, which are thought to exert their primary effects within the CNS, can have profound effects on the gut microbiome. They also suggest new avenues of investigation into the possibility that gut-derived signals could modulate drug abuse and addiction via altered communication along the gut-brain axis.


Asunto(s)
Drogas de Diseño/efectos adversos , Microbioma Gastrointestinal/efectos de los fármacos , Metanfetamina/análogos & derivados , Metanfetamina/efectos adversos , Propiofenonas/efectos adversos , Psicotrópicos/efectos adversos , Animales , ADN Bacteriano/aislamiento & purificación , Drogas de Diseño/administración & dosificación , Femenino , Microbioma Gastrointestinal/genética , Metanfetamina/administración & dosificación , Ratones , Modelos Animales , Propiofenonas/administración & dosificación , Psicotrópicos/administración & dosificación , ARN Ribosómico 16S/genética
13.
PLoS One ; 15(1): e0227285, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31940382

RESUMEN

The use of relative abundance data from next generation sequencing (NGS) can lead to misinterpretations of microbial community structures, as the increase of one taxon leads to the concurrent decrease of the other(s) in compositional data. Although different DNA- and cell-based methods as well as statistical approaches have been developed to overcome the compositionality problem, and the biological relevance of absolute bacterial abundances has been demonstrated, the human microbiome research has not yet adopted these methods, likely due to feasibility issues. Here, we describe how quantitative PCR (qPCR) done in parallel to NGS library preparation provides an accurate estimation of absolute taxon abundances from NGS data and hence provides an attainable solution to compositionality in high-throughput microbiome analyses. The advantages and potential challenges of the method are also discussed.


Asunto(s)
Bacterias/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Microbiota/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Bacterias/clasificación , Bacterias/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Estudios de Factibilidad , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Ribosómico 16S/genética , Ensayos Clínicos Controlados Aleatorios como Asunto
14.
Sci Rep ; 10(1): 1392, 2020 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-31996747

RESUMEN

Genotyping methods and genome sequencing are indispensable to reveal genomic structure of bacterial species displaying high level of genome plasticity. However, reconstruction of genome or assembly is not straightforward due to data complexity, including repeats, mobile and accessory genetic elements of bacterial genomes. Moreover, since the solution to this problem is strongly influenced by sequencing technology, bioinformatics pipelines, and selection criteria to assess assemblers, there is no systematic way to select a priori the optimal assembler and parameter settings. To assembly the genome of Pseudomonas aeruginosa strain AG1 (PaeAG1), short reads (Illumina) and long reads (Oxford Nanopore) sequencing data were used in 13 different non-hybrid and hybrid approaches. PaeAG1 is a multiresistant high-risk sequence type 111 (ST-111) clone that was isolated from a Costa Rican hospital and it was the first report of an isolate of P. aeruginosa carrying both blaVIM-2 and blaIMP-18 genes encoding for metallo-ß-lactamases (MBL) enzymes. To assess the assemblies, multiple metrics regard to contiguity, correctness and completeness (3C criterion, as we define here) were used for benchmarking the 13 approaches and select a definitive assembly. In addition, annotation was done to identify genes (coding and RNA regions) and to describe the genomic content of PaeAG1. Whereas long reads and hybrid approaches showed better performances in terms of contiguity, higher correctness and completeness metrics were obtained for short read only and hybrid approaches. A manually curated and polished hybrid assembly gave rise to a single circular sequence with 100% of core genes and known regions identified, >98% of reads mapped back, no gaps, and uniform coverage. The strategy followed to obtain this high-quality 3C assembly is detailed in the manuscript and we provide readers with an all-in-one script to replicate our results or to apply it to other troublesome cases. The final 3C assembly revealed that the PaeAG1 genome has 7,190,208 bp, a 65.7% GC content and 6,709 genes (6,620 coding sequences), many of which are included in multiple mobile genomic elements, such as 57 genomic islands, six prophages, and two complete integrons with blaVIM-2 and blaIMP-18 MBL genes. Up to 250 and 60 of the predicted genes are anticipated to play a role in virulence (adherence, quorum sensing and secretion) or antibiotic resistance (ß-lactamases, efflux pumps, etc). Altogether, the assembly and annotation of the PaeAG1 genome provide new perspectives to continue studying the genomic diversity and gene content of this important human pathogen.


Asunto(s)
Biología Computacional/métodos , Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano/genética , Pseudomonas aeruginosa/genética , Análisis de Secuencia de ADN/métodos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Técnicas de Genotipaje/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Secuencias Repetitivas Esparcidas/genética , Anotación de Secuencia Molecular , Pseudomonas aeruginosa/efectos de los fármacos
16.
J Ethnopharmacol ; 247: 112299, 2020 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-31606537

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Hua-Feng-Dan (HFD) is a traditional Chinese medicine used for neurological disorders. HFD contains cinnabar (HgS) and realgar (As4S4). The ethnopharmacological basis of cinnabar and realgar in HFD is not known. AIM OF THE STUDY: To address the role of cinnabar and realgar in HFD-produced neuroprotection against neurodegenerative diseases and disturbance of gut microbiota. MATERIALS AND METHODS: Lipopolysaccharide (LPS) plus rotenone (ROT)-elicited rat dopaminergic (DA) neuronal damage loss was performed as a Parkinson's disease animal model. Rats were given a single injection of LPS. Four months later, rats were challenged with the threshold dose of ROT. The clinical dose of HFD was administered via feed, starting from ROT administration for 46 days. Behavioral dysfunction was detected by rotarod and Y-maze tests. DA neuron loss and microglial activation were assessed via immunohistochemical staining and western bolt analysis. The colon content was collected to extract bacterial DNA followed by real-time PCR analysis with 16S rRNA primers. RESULTS: LPS plus ROT induced neurotoxicity, as evidenced by DA neuron loss in substantia nigra, impaired behavioral functions and increased microglial activation. HFD-original (containing 10% cinnabar and 10% realgar) rescued loss of DA neurons, improved behavioral dysfunction and attenuated microglial activation. Compared with HFD-original, HFD-reduced (3% cinnabar and 3% realgar) was also effective, but to be a less extent, while HFD-removed (without cinnabar and realgar) was ineffective. In analysis of gut microbiome, the increased Verrucomicrobiaceae and Lactobacteriaceae, and the decreased Enterobacteeriaceae by LPS plus ROT were ameliorated by HFD-original, and to be the less extent by HFD-reduced. CONCLUSION: Cinnabar and realgar are active ingredients in HFD to exert beneficial effects in a neurodegenerative model and gut microbiota.


Asunto(s)
Arsenicales/farmacología , Medicamentos Herbarios Chinos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Compuestos de Mercurio/farmacología , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/tratamiento farmacológico , Sulfuros/farmacología , Animales , Arsenicales/química , Arsenicales/uso terapéutico , ADN Bacteriano/aislamiento & purificación , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/uso terapéutico , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Etnofarmacología , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Lactobacillaceae/efectos de los fármacos , Lactobacillaceae/genética , Lactobacillaceae/aislamiento & purificación , Lipopolisacáridos/toxicidad , Masculino , Compuestos de Mercurio/química , Compuestos de Mercurio/uso terapéutico , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/patología , Degeneración Nerviosa , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/uso terapéutico , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/inmunología , Síndromes de Neurotoxicidad/patología , ARN Ribosómico 16S/genética , Ratas , Rotenona/toxicidad , Sulfuros/química , Sulfuros/uso terapéutico , Verrucomicrobia/efectos de los fármacos , Verrucomicrobia/genética , Verrucomicrobia/aislamiento & purificación
17.
Clin Exp Dermatol ; 45(1): 36-40, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31220362

RESUMEN

BACKGROUND: Palmoplantar pustulosis (PPP) is a distinct, chronic skin disorder characterized by intraepidermal pustules on the palms and soles. It is hypothesized that microorganisms on the skin might induce the symptoms of PPP via inflammatory cell activation. However, the microbiota has not been studied in detail because of the assumption that the pustules in PPP are sterile. AIM: To elucidate the role of microorganisms in pathogenesis of PPP. METHODS: PCR analysis was performed of microbial DNA fragments in the pustules of patients with PPP. The sequence of the D1/D2 LSU 26s rRNA gene and that of the 16S rRNA gene was used for fungal and bacterial DNA detection, respectively. RESULTS: In total, 71 samples were carefully collected from the pustules of patients with PPP. Fungal DNA bands were detected in 68 samples, and fungi including Malassezia spp. were identified in 30 of 71 samples (42.3%). Malassezia restricta was the most frequently encountered fungus (14/71; 19.7%). However, bacterial DNA was not detected by the methods used. Furthermore, identical fungal DNA was not detected in the outer lid of the pustules, suggesting that the fungi detected within the pustule did not derive from contamination via the skin surface. CONCLUSIONS: In the present study, we demonstrated for the first time that certain pustules in patients with PPP contain fungal DNA fragments, especially those of Malassezia spp. Our findings provide new insights on the role of skin microbiota in the pathogenesis of PPP.


Asunto(s)
ADN Bacteriano/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Malassezia/aislamiento & purificación , Psoriasis/microbiología , Acremonium/genética , Acremonium/aislamiento & purificación , Adulto , Anciano , Aspergillus/genética , Aspergillus/aislamiento & purificación , Cladosporium/genética , Cladosporium/aislamiento & purificación , Femenino , Humanos , Malassezia/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Saccharomycetales/genética
18.
Ann Epidemiol ; 41: 28-34, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31883841

RESUMEN

PURPOSE: Preterm birth (PTB) is a major cause of neonatal mortality. The vaginal microbiome is associated with PTB, but results vary across racial/ethnic populations. Some evidence suggests gestational age affects this association. We investigated these associations in a novel population, conducting a post hoc analysis assessing if associations differed between women swabbed at different gestational ages. METHODS: We compared vaginal microbiomes from women with PTB (n = 25) to a random sample of women with term births (n = 100) among participants in the Pregnancy Outcomes, Maternal and Infant Study, conducted in Lima, Peru. Using DADA2, we identified taxa from 16S DNA sequencing and used Dirichlet multinomial mixture models to group into community state types (CSTs). RESULTS: If gestational age at sampling was not considered, no CST (diverse, Lactobacillus-dominated or Lactobacillus iners-dominated), was associated with PTB. Among women sampled before 12 weeks' gestation, women with Lactobacillus-dominated CSTs were less likely to have a PTB than those with a diverse CST. Among those swabbed between 12 and 16 weeks' gestation, the reverse was true. CONCLUSIONS: Our study supports previous literature suggesting that what constitutes a healthy vaginal microbiome varies by race/ethnicity. Longitudinal studies are necessary to disentangle effects of vaginal microbiome differences over gestation.


Asunto(s)
Bacterias/clasificación , Lactobacillus/aislamiento & purificación , Microbiota/genética , Nacimiento Prematuro/microbiología , ARN Ribosómico 16S/genética , Vagina/microbiología , Adulto , Bacterias/genética , Bacterias/aislamiento & purificación , Estudios de Casos y Controles , ADN Bacteriano/aislamiento & purificación , Femenino , Edad Gestacional , Humanos , Recién Nacido , Lactobacillus/clasificación , Lactobacillus/genética , Masculino , Perú/epidemiología , Embarazo , Nacimiento Prematuro/epidemiología , Nacimiento Prematuro/etnología , Análisis de Secuencia de ADN
19.
PLoS One ; 14(12): e0226159, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31825981

RESUMEN

Myctophids are among the most abundant mesopelagic teleost fishes worldwide. They are dominant in the Southern Ocean, an extreme environment where they are important both as consumers of zooplankton as well as food items for larger predators. Various studies have investigated myctophids diet, but no data is yet available regarding their associated microbiota, despite that the significance of bacterial communities to fish health and adaptation is increasingly acknowledged. In order to document microbiota in key fish groups from the Southern Ocean, the bacterial communities associated with the gut, fin, gills and light organs of members of six species within the three myctophid genera Electrona, Protomyctophum and Gymnoscopelus were characterized using a 16S rRNA-based metabarcoding approach. Gut communities display limited diversity of mostly fish-specific lineages likely involved in food processing. Fin and skin communities display diversity levels and compositions resembling more those found in surrounding seawater. Community compositions are similar between genera Electrona and Protomyctophum, that differ from those found in Gymnoscopelus and in water. Low abundances of potentially light-emitting bacteria in light organs support the hypothesis of host production of light. This first description of myctophid-associated microbiota, and among the first on fish from the Southern Ocean, emphasizes the need to extend microbiome research beyond economically-important species, and start addressing ecologically-relevant species.


Asunto(s)
Bacterias/aislamiento & purificación , Peces/microbiología , Microbiota , Aletas de Animales/microbiología , Animales , Bacterias/genética , Biodiversidad , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Peces/genética , Branquias/microbiología , Intestinos/microbiología , Océanos y Mares , Análisis de Componente Principal , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Agua de Mar/microbiología
20.
PLoS One ; 14(12): e0225545, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31830061

RESUMEN

BACKGROUND: Although the significance of the human vaginal microbiome for health and disease is increasingly acknowledged, there is paucity of data on the differences in the composition of the vaginal microbiome upon infection with different sexually transmitted pathogens. METHOD: The composition of the vaginal bacterial community of women with Trichomonas vaginalis (TV, N = 18) was compared to that of women with Chlamydia trachomatis (CT, N = 14), and to that of controls (N = 21) (women negative for TV, CT and bacterial vaginosis). The vaginal bacterial composition was determined using high throughput sequencing with the Ion 16S metagenomics kit of the variable regions 2, 4 and 8 of the bacterial 16S ribosomal RNA gene from the vaginal swab DNA extract of the women. QIIME and R package "Phyloseq" were used to assess the α- and ß-diversity and absolute abundance of the 16S rRNA gene per sample in the three groups. Differences in taxa at various levels were determined using the independent T-test. RESULTS: A total of 545 operational taxonomic units (OTUs) were identified in all the three groups of which 488 occurred in all three groups (core OTUs). Bacterial α-diversity, by both Simpson's and Shannon's indices, was significantly higher, (p = 0.056) and (p = 0.001) respectively, among women with either TV or CT than among controls (mean α-diversity TV-infected > CT-infected > Controls). At the genus level, women infected with TV had a significantly (p < 0.01) higher abundance of Parvimonas and Prevotella species compared to both controls and CT-infected women, whereas women infected with CT had a significantly (p < 0.05) higher abundance of Anaerococcus, Collinsella, Corynebacterium and Dialister. CONCLUSION: The vaginal microbiomes of TV and CT-infected women were markedly different from each other and from women without TV and CT. Future studies should determine whether the altered microbiomes are merely markers of disease, or whether they actively contribute to the pathology of the two genital infections.


Asunto(s)
Infecciones por Chlamydia/microbiología , Microbiota/inmunología , Complicaciones Infecciosas del Embarazo/microbiología , Vaginitis por Trichomonas/microbiología , Vagina/microbiología , Adolescente , Adulto , Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/genética , Chlamydia trachomatis/inmunología , Chlamydia trachomatis/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Femenino , Humanos , Microbiota/genética , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , ARN Ribosómico 16S/genética , Vaginitis por Trichomonas/inmunología , Trichomonas vaginalis/genética , Trichomonas vaginalis/inmunología , Trichomonas vaginalis/aislamiento & purificación , Adulto Joven
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