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1.
Biomed Environ Sci ; 34(4): 265-271, 2021 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-33894805

RESUMEN

Objective: Airborne microbial communities include a significant number of uncultured and poorly characterized bacteria. No effective method currently exists to evaluate the health risks of such complex bacterial populations, particularly for pneumonia. Methods: We developed a method to evaluate risks from airborne microorganisms, guided by the principle that closer evolutionary relationships reflect similar biological characteristics, and thus used 16S rDNA sequences of 10 common pneumonia-related bacterial pathogens. We calculated a risk of breath-related ( Rbr) index of airborne bacterial communities and verified effectiveness with artificial flora and a clinical project. Results: We suggested applying Rbr80 to evaluate the health risks of airborne bacterial communities that comprise 80% of dominant operational taxonomic units (OTUs). The feasibility of Rbr80 was confirmed by artificial flora and by pneumonia data from a hospital. A high Rbr80 value indicated a high risk of pneumonia from airborne bacterial communities. Conclusion: Rbr80 is an effective index to evaluate the pneumonia-associated risk from airborne bacteria. Values of Rbr80 greater than 15.40 are considered high risk.


Asunto(s)
Microbiología del Aire , Bacterias/aislamiento & purificación , Neumonía Bacteriana/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Bacterias/genética , Niño , Preescolar , China/epidemiología , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Neumonía Bacteriana/microbiología , Medición de Riesgo/métodos , Adulto Joven
2.
Molecules ; 26(5)2021 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-33801538

RESUMEN

The "Saline di Tarquinia" salterns have been scarcely investigated regarding their microbiological aspects. This work studied the structure and composition of their bacterial communities along the salinity gradient (from the nearby sea through different ponds). The communities showed increasing simplification of pond bacterial diversity along the gradient (particularly if compared to those of the sea). Among the 38 assigned phyla, the most represented were Proteobacteria, Actinobacteria and Bacteroidetes. Differently to other marine salterns, where at the highest salinities Bacteroidetes dominated, preponderance of Proteobacteria was observed. At the genus level the most abundant taxa were Pontimonas, Marivita, Spiribacter, Bordetella, GpVII and Lentibacter. The α-diversity analysis showed that the communities were highly uneven, and the Canonical Correspondence Analysis indicated that they were structured by various factors (sampling site, sampling year, salinity, and sampling month). Moreover, the taxa abundance variation in relation to these significant parameters were investigated by Generalized Linear Models. This work represents the first investigation of a marine saltern, carried out by a metabarcoding approach, which permitted a broad vision of the bacterial diversity, covering both a wide temporal span (two years with monthly sampling) and the entire salinity gradient (from the nearby sea up to the crystallisation ponds).


Asunto(s)
Bacterias/clasificación , Bacterias/aislamiento & purificación , ADN Bacteriano/análisis , Filogenia , Salinidad , Agua de Mar/microbiología , Análisis Espacio-Temporal , Microbiología del Agua , Bacterias/genética , ADN Bacteriano/genética
3.
Biosensors (Basel) ; 11(4)2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33921114

RESUMEN

Given the fatal health conditions caused by emerging infectious pathogens, such as severe acute respiratory syndrome coronavirus 2, their rapid diagnosis is required for preventing secondary infections and guiding correct treatments. Although various molecular diagnostic methods based on nucleic acid amplification have been suggested as gold standards for identifying different species, these methods are not suitable for the rapid diagnosis of pathogens owing to their long result acquisition times and complexity. In this study, we developed a rapid bio-optical sensor that uses a ball-lensed optical fiber (BLOF) probe and an automatic analysis platform to precisely diagnose infectious pathogens. The BLOF probe is easy to align and has a high optical sensing sensitivity (1.5-fold) and a large detection range (1.2-fold) for an automatic optical sensing system. Automatic signal processing of up to 250 copies/reaction of DNA of Q-fever-causing Coxiella burnetii was achieved within 8 min. The clinical utility of this system was demonstrated with 18 clinical specimens (9 Q-fever and 9 other febrile disease samples) by measuring the resonant wavelength shift of positive or negative samples for Coxiella burnetii DNA. The results from the system revealed the stable and automatic optical signal measurement of DNA with 100% accuracy. We envision that this BLOF probe-based sensor would be a practical tool for the rapid, simple, and sensitive diagnosis of emerging infectious pathogens.


Asunto(s)
Técnicas Biosensibles/métodos , ADN Bacteriano/análisis , Fibras Ópticas , Fiebre Q/diagnóstico , Automatización , Técnicas Biosensibles/instrumentación , Coxiella burnetii/genética , Coxiella burnetii/aislamiento & purificación , Humanos , Fiebre Q/microbiología , Procesamiento de Señales Asistido por Computador
4.
Methods Mol Biol ; 2283: 29-36, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33765306

RESUMEN

Antimicrobial susceptibility testing (AST) for H. pylori is essential to accurately assess the prevalence of antibiotic resistance in each population. Antibiotic resistance rates form the basis of local guidelines for H. pylori treatment and AST may also be used as a personalized medicine approach to tailor therapy. This chapter provides an update on global antibiotic resistance rates and describes molecular mechanisms that confer H. pylori antibiotic resistance. An overview on the advantages and limitations of molecular AST using both invasive and noninvasive approaches is also provided.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Antibacterianos/uso terapéutico , ADN Bacteriano/análisis , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Guías de Práctica Clínica como Asunto , Medicina de Precisión
5.
Thorax ; 76(3): 281-291, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33542086

RESUMEN

BACKGROUND: Despite progress in TB control in low-burden countries like England and Wales, there are still diagnostic delays. Molecular testing and/or whole-genome sequencing (WGS) provide more rapid diagnosis but their cost-effectiveness is relatively unexplored in low-burden settings. METHODS: An integrated transmission-dynamic health economic model is used to assess the cost-effectiveness of using WGS to replace culture-based drug-sensitivity testing, versus using molecular testing versus combined use of WGS and molecular testing, for routine TB diagnosis. The model accounts for the effects of faster appropriate treatment in reducing transmission, benefiting health and reducing future treatment costs. Cost-effectiveness is assessed using incremental net benefit (INB) over a 10-year horizon with a quality-adjusted life-year valued at £20 000, and discounting at 3.5% per year. RESULTS: WGS shortens the time to drug sensitivity testing and treatment modification where necessary, reducing treatment and hospitalisation costs, with an INB of £7.1 million. Molecular testing shortens the time to TB diagnosis and treatment. Initially, this causes an increase in annual costs of treatment, but averting transmissions and future active TB disease subsequently, resulting in cost savings and health benefits to achieve an INB of £8.6 million (GeneXpert MTB/RIF) or £11.1 million (Xpert-Ultra). Combined use of Xpert-Ultra and WGS is the optimal strategy we consider, with an INB of £16.5 million. CONCLUSION: Routine use of WGS or molecular testing is cost-effective in a low-burden setting, and combined use is the most cost-effective option. Adoption of these technologies can help low-burden countries meet the WHO End TB Strategy milestones, particularly the UK, which still has relatively high TB rates.


Asunto(s)
Costo de Enfermedad , ADN Bacteriano/análisis , Modelos Económicos , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Secuenciación Completa del Genoma/métodos , Análisis Costo-Beneficio , Humanos , Tuberculosis/economía , Tuberculosis/genética
6.
Nat Commun ; 12(1): 763, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33536429

RESUMEN

Human and animal infections with bacteria of the genus Sarcina (family Clostridiaceae) are associated with gastric dilation and emphysematous gastritis. However, the potential roles of sarcinae as commensals or pathogens remain unclear. Here, we investigate a lethal disease of unknown etiology that affects sanctuary chimpanzees (Pan troglodytes verus) in Sierra Leone. The disease, which we have named "epizootic neurologic and gastroenteric syndrome" (ENGS), is characterized by neurologic and gastrointestinal signs and results in death of the animals, even after medical treatment. Using a case-control study design, we show that ENGS is strongly associated with Sarcina infection. The microorganism is distinct from Sarcina ventriculi and other known members of its genus, based on bacterial morphology and growth characteristics. Whole-genome sequencing confirms this distinction and reveals the presence of genetic features that may account for the unusual virulence of the bacterium. Therefore, we propose that this organism be considered the representative of a new species, named "Candidatus Sarcina troglodytae". Our results suggest that a heretofore unrecognized complex of related sarcinae likely exists, some of which may be highly virulent. However, the potential role of "Ca. S. troglodytae" in the etiology of ENGS, alone or in combination with other factors, remains a topic for future research.


Asunto(s)
Enfermedades del Simio Antropoideo/diagnóstico , Enfisema/diagnóstico , Gastritis/diagnóstico , Infecciones por Bacterias Grampositivas/diagnóstico , Sarcina/genética , Animales , Enfermedades del Simio Antropoideo/microbiología , ADN Bacteriano/análisis , ADN Bacteriano/genética , Enfisema/microbiología , Gastritis/microbiología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Humanos , Pan troglodytes , Sarcina/clasificación , Sarcina/patogenicidad , Sierra Leona , Virulencia/genética , Secuenciación Completa del Genoma/métodos
7.
Nature ; 590(7844): 80-84, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33536650

RESUMEN

Active matter consists of units that generate mechanical work by consuming energy1. Examples include living systems (such as assemblies of bacteria2-5 and biological tissues6,7), biopolymers driven by molecular motors8-11 and suspensions of synthetic self-propelled particles12-14. A central goal is to understand and control the self-organization of active assemblies in space and time. Most active systems exhibit either spatial order mediated by interactions that coordinate the spatial structure and the motion of active agents12,14,15 or the temporal synchronization of individual oscillatory dynamics2. The simultaneous control of spatial and temporal organization is more challenging and generally requires complex interactions, such as reaction-diffusion hierarchies16 or genetically engineered cellular circuits2. Here we report a simple technique to simultaneously control the spatial and temporal self-organization of bacterial active matter. We confine dense active suspensions of Escherichia coli cells and manipulate a single macroscopic parameter-namely, the viscoelasticity of the suspending fluid- through the addition of purified genomic DNA. This reveals self-driven spatial and temporal organization in the form of a millimetre-scale rotating vortex with periodically oscillating global chirality of tunable frequency, reminiscent of a torsional pendulum. By combining experiments with an active-matter model, we explain this behaviour in terms of the interplay between active forcing and viscoelastic stress relaxation. Our findings provide insight into the influence of bacterial motile behaviour in complex fluids, which may be of interest in health- and ecology-related research, and demonstrate experimentally that rheological properties can be harnessed to control active-matter flows17,18. We envisage that our millimetre-scale, tunable, self-oscillating bacterial vortex may be coupled to actuation systems to act a 'clock generator' capable of providing timing signals for rhythmic locomotion of soft robots and for programmed microfluidic pumping19, for example, by triggering the action of a shift register in soft-robotic logic devices20.


Asunto(s)
Escherichia coli/fisiología , Reología , Análisis Espacio-Temporal , Sustancias Viscoelásticas/química , Sustancias Viscoelásticas/metabolismo , ADN Bacteriano/análisis , ADN Bacteriano/química , Difusión , Escherichia coli/citología , Escherichia coli/aislamiento & purificación , Microfluídica , Peso Molecular , Movimiento , Robótica , Suspensiones
8.
Biomed Res Int ; 2021: 4202019, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33623780

RESUMEN

Introduction: Bloodstream infections (BSI) among patients with hematological malignancies (HM) could predispose them to higher morbidity and mortality for various underlying conditions. Several microorganisms, either pathogenic or opportunistic normal human flora, could cause severe bacteremia and septicemia. While conventional methods have their own limitations, molecular methods such as next-generation sequencing (NGS) can detect these blood infections with more reliability, specificity, and sensitivity, in addition to information on microbial population landscape. Methodology. Blood samples from HM patients (n = 50) and volunteer blood donor control individuals with no HM (n = 50) were subjected to 16S rRNA gene amplification using standard PCR protocols. A metagenomic library was prepared, and NGS was run on a MiSeq (Illumina) sequencer. Sequence reads were analyzed using MiSeq Reporter, and microbial taxa were aligned using the Green Genes library. Results: 82% of the patients showed BSI with Gram-negative bacteria as the most predominant group. E. coli comprised a major chunk of the bacterial population (19.51%), followed by K. pneumoniae (17.07%). The CoNS and Viridans Streptococci groups are 17.07% and 14.63%, respectively. Other major species were S. aureus (9.75%), P. aeruginosa (7.31%), A. baumannii (4.87%), E. cloacae (4.87%), and P. mirabilis (4.87%). 34.14% of the cases among patients showed a Gram-positive infection, while 14.63% showed polymicrobial infections. Conclusion: Most of the BSI in patients were characterized by polymicrobial infections, unlike the control samples. Molecular methods like NGS showed robust, fast, and specific identification of infectious agents in BSI in HM, indicating the possibility of its application in routine follow-up of such patients for infections.


Asunto(s)
Bacteriemia , Neoplasias Hematológicas/complicaciones , Técnicas de Diagnóstico Molecular/métodos , Análisis de Secuencia de ADN/métodos , Adolescente , Adulto , Anciano , Bacteriemia/complicaciones , Bacteriemia/diagnóstico , Bacteriemia/epidemiología , Bacteriemia/microbiología , Bacterias/clasificación , Bacterias/genética , Coinfección/complicaciones , Coinfección/diagnóstico , Coinfección/epidemiología , Coinfección/microbiología , ADN Bacteriano/análisis , ADN Bacteriano/genética , Femenino , Humanos , Masculino , Metagenoma/genética , Metagenómica/métodos , Microbiota/genética , Persona de Mediana Edad , Arabia Saudita , Adulto Joven
9.
PLoS Negl Trop Dis ; 15(2): e0009180, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33591973

RESUMEN

Outbreaks of yaws-like ulcerative skin lesions in children are frequently reported in tropical and sub-tropical countries. The origin of these lesions might be primarily traumatic or infectious; in the latter case, Treponema pallidum subspecies pertenue, the yaws agent, and Haemophilus ducreyi, the agent of chancroid, are two of the pathogens commonly associated with the aetiology of skin ulcers. In this work, we investigated the presence of T. p. pertenue and H. ducreyi DNA in skin ulcers in children living in yaws-endemic regions in Cameroon. Skin lesion swabs were collected from children presenting with yaws-suspected skin lesions during three outbreaks, two of which occurred in 2017 and one in 2019. DNA extracted from the swabs was used to amplify three target genes: the human ß2-microglobulin gene to confirm proper sample collection and DNA extraction, the polA gene, highly conserved among all subspecies of T. pallidum, and the hddA gene of H. ducreyi. A fourth target, the tprL gene was used to differentiate T. p. pertenue from the other agents of human treponematoses in polA-positive samples. A total of 112 samples were analysed in this study. One sample, negative for ß2-microglobulin, was excluded from further analysis. T. p. pertenue was only detected in the samples collected during the first 2017 outbreak (12/74, 16.2%). In contrast, H. ducreyi DNA could be amplified from samples from all three outbreaks (outbreak 1: 27/74, 36.5%; outbreak 2: 17/24, 70.8%; outbreak 3: 11/13, 84.6%). Our results show that H. ducreyi was more frequently associated to skin lesions in the examined children than T. p. pertenue, but also that yaws is still present in Cameroon. These findings strongly advocate for a continuous effort to determine the aetiology of ulcerative skin lesions during these recurring outbreaks, and to inform the planned mass treatment campaigns to eliminate yaws in Cameroon.


Asunto(s)
Chancroide/diagnóstico , Úlcera Cutánea/diagnóstico , Úlcera Cutánea/microbiología , Buba/diagnóstico , Adolescente , Camerún/epidemiología , Chancroide/epidemiología , Niño , Preescolar , ADN Bacteriano/análisis , Brotes de Enfermedades , Femenino , Haemophilus ducreyi/genética , Haemophilus ducreyi/aislamiento & purificación , Humanos , Masculino , Úlcera Cutánea/epidemiología , Treponema pallidum/genética , Treponema pallidum/aislamiento & purificación , Buba/epidemiología
10.
Food Chem ; 346: 128887, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33385916

RESUMEN

As interest in probiotics increases, the need for accurate description of probiotic compositions present in commercial products is also increasing. Since Bifidobacterium longum used as probiotics is labeled at species or subspecies levels, a detection method for distinguishing B. longum subsp. longum, infantis, and suis is needed. Thus, we designed three LAMP primer sets for B. longum subspecies. Each primer set was specific for the target subspecies. The detection level was 0.2 pg for each target DNA (about 102 CFU/mL). To apply these LAMP assays to on-site detection, a direct DNA extraction method was optimized and combined with LAMP assay. Finally, direct LAMP assays were used to monitor the presence of B. longum subspecies in 16 probiotic products. They could specifically and sensitively detect target subspecies within approximately 45 min. These rapid on-site detection methods are useful for identifying B. longum subspecies in probiotic products.


Asunto(s)
Bifidobacterium longum subspecies infantis/aislamiento & purificación , Bifidobacterium/aislamiento & purificación , ADN Bacteriano/análisis , Probióticos/análisis , Bifidobacterium/genética , Bifidobacterium longum subspecies infantis/genética , ADN Bacteriano/metabolismo , Humanos , Límite de Detección , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico
11.
J Environ Sci Health B ; 56(3): 272-281, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33400564

RESUMEN

The study's objective was to identify typical aerobic isolates from commercial, corn-soybean meal poultry diets utilizing 16S rDNA, assign them their corresponding taxonomy, and compare the data with the previously published WGS analysis of these same isolates. Ten grams of a commercial corn-soybean meal poultry diet was homogenized in 100 mL of tryptic soy broth for 2 min, serially diluted, plated onto tryptic soy agar (TSA), and incubated aerobically for 24 h at 37 °C. Subsequently, 20 unique colonies were streaked for isolation on TSA and incubated aerobically for 24 h at 37 °C. This process was repeated three consecutive times for purification of isolates until only 11 morphologically distinct colonies were obtained. DNA was extracted using Qiagen's DNeasey® Blood and Tissue Kit. The 16S rRNA V4 region was targeted using an Illumina MiSeq and analyzed via QIIME2-2020.2. Alpha diversity and Beta diversity metrics were generated, and taxa were aligned using Silva in Qiime2-2020.2. Twenty-five distinct genera were identified within the 11 different colonies. Because 16S rDNA identification can provide an understanding of pathogen associations and microbial niches within an ecosystem, the information may present a potential method to establish and characterize the hygienic indicator microorganisms associated with poultry feed.


Asunto(s)
Alimentación Animal/microbiología , Bacterias/aislamiento & purificación , Microbiota/fisiología , Aves de Corral , Aerobiosis , Animales , Bacterias/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , ADN Ribosómico/genética , ARN Ribosómico 16S/genética , Soja , Zea mays
12.
Epilepsia ; 62(2): 529-541, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33428780

RESUMEN

OBJECTIVE: A large number of studies have highlighted the important role of the gut microbiota in the pathophysiology of neurological disorders, suggesting that its manipulation might serve as a treatment strategy. We hypothesized that the gut microbiota participates in absence seizure development and maintenance in the WAG/Rij rat model and tested this hypothesis by evaluating potential gut microbiota and intestinal alterations in the model, as well as measuring the impact of microbiota manipulation using fecal microbiota transplantation (FMT). METHODS: Initially, gut microbiota composition and intestinal histology of WAG/Rij rats (a well-recognized genetic model of absence epilepsy) were studied at 1, 4, and 8 months of age in comparison to nonepileptic Wistar rats. Subsequently, in a second set of experiments, at 6 months of age, untreated Wistar or WAG/Rij rats treated with ethosuximide (ETH) were used as gut microbiota donors for FMT in WAG/Rij rats, and electroencephalographic (EEG) recordings were obtained over 4 weeks. At the end of FMT, stool and gut samples were collected, absence seizures were measured on EEG recordings, and microbiota analysis and histopathological examinations were performed. RESULTS: Gut microbiota analysis showed differences in beta diversity and specific phylotypes at all ages considered and significant variances in the Bacteroidetes/Firmicutes ratio between Wistar and WAG/Rij rats. FMT, from both Wistar and ETH-treated WAG/Rij donors to WAG/Rij rats, significantly decreased the number and duration of seizures. Histological results indicated that WAG/Rij rats were characterized by intestinal villi disruption and inflammatory infiltrates already at 1 month of age, before seizure occurrence; FMT partially restored intestinal morphology while also significantly modifying gut microbiota and concomitantly reducing absence seizures. SIGNIFICANCE: Our results demonstrate for the first time that the gut microbiota is modified and contributes to seizure occurrence in a genetic animal model of absence epilepsy and that its manipulation may be a suitable therapeutic target for absence seizure management.


Asunto(s)
Antibacterianos/farmacología , Anticonvulsivantes/farmacología , Epilepsia Tipo Ausencia/microbiología , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Animales , Bacteroidetes , Butiratos/metabolismo , Colon/patología , ADN Bacteriano/análisis , ADN Ribosómico/genética , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/fisiopatología , Epilepsia Tipo Ausencia/terapia , Etosuximida/farmacología , Ácidos Grasos Volátiles/metabolismo , Firmicutes , Motilidad Gastrointestinal , Haptoglobinas/metabolismo , Íleon/patología , Propionatos/metabolismo , Precursores de Proteínas/metabolismo , Proteobacteria , Ratas , Ratas Wistar , Convulsiones/genética , Convulsiones/microbiología , Convulsiones/fisiopatología
13.
Microbiome ; 9(1): 17, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33478576

RESUMEN

BACKGROUND: High-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities. RESULTS: PMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis. However, in realistically complex communities (computer screens, computer mice, soil, and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability (even relative to variability in amplicon sequencing), with its performance largely affected by community properties such as initial biomass, sample types, and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the "PMA-responsive" microbes, we compared our results with previous PMA-based studies and found that PMA responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies. CONCLUSIONS: This study provides a comprehensive evaluation of PMA-seq exploring its quantitative potential in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities but provided only qualitative assessments in realistically complex community samples. Video abstract.


Asunto(s)
Azidas , Escherichia coli/genética , Viabilidad Microbiana/genética , Microbiota , Propidio/análogos & derivados , Análisis de Secuencia de ADN/métodos , Streptococcus sanguis/genética , Animales , ADN Bacteriano/análisis , ADN Bacteriano/genética , Escherichia coli/aislamiento & purificación , Humanos , Ratones , Microbiota/genética , ARN Ribosómico 16S/genética , Streptococcus sanguis/aislamiento & purificación
14.
ACS Appl Mater Interfaces ; 13(2): 3098-3108, 2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33423455

RESUMEN

The digital nucleic acid assay is a precise, sensitive, and reproducible method for determining the presence of individual target molecules separated in designated partitions; thus, this technique can be used for the nucleic acid detection. Here, we propose a multifunctional micropattern array capable of isolating individual target molecules into partitions and simultaneous on-site cell lysis to achieve a direct DNA extraction and digitized quantification thereof. The multifunctional micropattern array is fabricated by the deposition of a copolymer film, poly(2-dimethylaminomethyl styrene-co-hydroxyethyl methacrylate) (pDH), directly on a microfluidic chip surface via the photoinitiated chemical vapor deposition process, followed by hydrophobic microcontact printing (µCP) to define each partition for the nucleic acid isolation. The pDH layer is a positively charged surface, which is desirable for the bacterial lysis and DNA capture, while showing exceptional water stability for more than 24 h. The hydrophobic µCP-treated pDH surface is stable under aqueous conditions at a high temperature (70 °C) for 1 h and enables the rapid and reliable formation of thousands of sessile microdroplets for the compartmentalization of an aqueous sample solution without involving bulky and costly microfluidic devices. By assembling the multifunctional micropattern array into the microfluidic chip, the isothermal amplification in each partition can detect DNA templates over a concentration range of 0.01-2 ng/µL. The untreated bacterial cells can also be directly compartmentalized via the microdroplet formation, followed by the on-site cell lysis and DNA capture on the compartmentalized pDH surface. For Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus cells, cell numbers ranging from 1.4 × 104 to 1.4 × 107 can be distinguished by using the multifunctional micropattern array, regardless of the cell type. The multifunctional micropattern array developed in this study provides a novel multifunctional compartmentalization method for rapid, simple, and accurate digital nucleic acid assays.


Asunto(s)
Bacterias/aislamiento & purificación , ADN Bacteriano/análisis , Metacrilatos/química , Análisis por Micromatrices/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Estireno/química , Bacterias/genética , Infecciones Bacterianas/microbiología , ADN Bacteriano/genética , Diseño de Equipo , Humanos , Dispositivos Laboratorio en un Chip , Impresión Tridimensional
15.
PLoS Comput Biol ; 17(1): e1008586, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33471816

RESUMEN

A streaming assembly pipeline utilising real-time Oxford Nanopore Technology (ONT) sequencing data is important for saving sequencing resources and reducing time-to-result. A previous approach implemented in npScarf provided an efficient streaming algorithm for hybrid assembly but was relatively prone to mis-assemblies compared to other graph-based methods. Here we present npGraph, a streaming hybrid assembly tool using the assembly graph instead of the separated pre-assembly contigs. It is able to produce more complete genome assembly by resolving the path finding problem on the assembly graph using long reads as the traversing guide. Application to synthetic and real data from bacterial isolate genomes show improved accuracy while still maintaining a low computational cost. npGraph also provides a graphical user interface (GUI) which provides a real-time visualisation of the progress of assembly. The tool and source code is available at https://github.com/hsnguyen/assembly.


Asunto(s)
Nanoporos , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodos , Algoritmos , Biología Computacional , ADN Bacteriano/análisis , ADN Bacteriano/genética , Genoma Bacteriano/genética , Nanotecnología , Programas Informáticos , Interfaz Usuario-Computador
16.
Artículo en Inglés | IBECS | ID: ibc-199908

RESUMEN

INTRODUCTION: Acinetobacter is a genus that comprises a group of opportunistic pathogens responsible for a variety of nosocomial infections. The Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex includes some species of clinical importance, mainly A. baumannii, A. pittii and A. nosocomialis, which share phenotypic similarities that make it very difficult to distinguish between them using a phenotypic approach. The aim of this study was to evaluate two commercial matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems for the identification of different Acinetobacter species, with a special focus among those belonging to the Acb complex. METHODS: One hundred and fifty-six Acinetobacter spp. clinical strains, identified by amplified ribosomal DNA restriction analysis (ARDRA) and rpoB gene sequencing, were analysed by two different MALDI-TOF systems. RESULTS: Considering only the 144 strains of the Acb complex evaluated in this study, the Vitek-MS(TM) and Microflex LT(TM) systems correctly identified 129 (89.6%) and 143 (99.3%) strains, respectively. CONCLUSION: After analysing 156 strains belonging to Acinetobacter spp., both Vitek-MS(TM) and Microflex LT(TM) proved to be rapid and accurate systems for the identification of Acb complex species showing a good correlation. However, both manufacturers should improve their databases to include new species in them


INTRODUCCIÓN: Acinetobacter es un género que comprende un grupo de patógenos oportunistas responsables de varias infecciones nosocomiales. El complejo Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) reúne algunas especies de importancia clínica, principalmente A. baumannii, A. pittii y A. nosocomialis, que comparten similitudes fenotípicas que hacen muy difícil poder discriminar entre ellas utilizando un enfoque fenotípico. El objetivo de este estudio fue evaluar 2 sistemas comerciales de espectrometría de masas de ionización por láser asistido con una matriz (MALDI-TOF MS) para la identificación de diferentes especies de Acinetobacter, con un enfoque especial entre los que pertenecen al complejo Acb. MÉTODOS: Analizamos 156 cepas clínicas de Acinetobacter spp., identificadas mediante análisis de restricción de ADN ribosomal amplificado (ARDRA) y secuenciación del gen rpoB, por 2 sistemas diferentes de MALDI-TOF. RESULTADOS: Teniendo en cuenta solo las 144 cepas del complejo Acb evaluadas en este estudio, los sistemas Vitek(R) MS y Microflex(R) LT identificaron correctamente 129 (89,6%) y 143 (99,3%) cepas, respectivamente. CONCLUSIÓN: Después de analizar 156 cepas pertenecientes a Acinetobacter spp., Vitek(R) MS y Microflex(R) LT demostraron ser sistemas rápidos y precisos para la identificación de especies del complejo Acb mostrando una buena correlación. Sin embargo, ambos fabricantes deberían mejorar sus bases de datos incluyendo nuevas especies en ellas


Asunto(s)
Humanos , Acinetobacter baumannii/aislamiento & purificación , Infecciones por Acinetobacter/diagnóstico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Infecciones por Acinetobacter/microbiología , ADN Bacteriano/análisis , Técnicas Bacteriológicas , ADN Ribosómico/análisis , Acinetobacter calcoaceticus/aislamiento & purificación
17.
Methods Mol Biol ; 2220: 79-88, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32975767

RESUMEN

PFGE is a valuable tool for assessing L. monocytogenes strain interrelatedness. It is based on the study of total bacterial DNA restriction patterns. Cells are embedded in agarose plugs before being lysed. The released DNA is then digested into large fragments by restriction enzymes. As DNA fragments are too large to be separated by traditional electrophoresis in an agarose gel, changes in the direction of the electrical current are periodically applied in order to allow the proper migration of large DNA fragments. Strains are characterized by the obtained DNA fragment patterns or pulsotypes which vary depending on the number and size of bands.


Asunto(s)
ADN Bacteriano/análisis , Electroforesis en Gel de Campo Pulsado/métodos , Listeria monocytogenes/aislamiento & purificación , Enzimas de Restricción del ADN/química , ADN Bacteriano/genética , Microbiología de Alimentos , Humanos , Listeria monocytogenes/genética , Listeriosis/microbiología , Sefarosa/química
18.
BMJ Case Rep ; 13(12)2020 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-33310826

RESUMEN

Tuberculosis (TB) of the breast is extremely rare and is often mistaken for benign or malignant lesions of the breast. They are rare even in countries which are endemic for TB, like India. The most common type of clinical presentation is a vague lump in the breast, but there are even other types of presentations which are documented. In olden days, there was a lot of dilemma and challenge in diagnosing TB of the breast, but thanks to improved pathological knowledge and the advent of investigations such as QuantiFERON-TB gold and GeneXpert, TB can be diagnosed early nowadays and treated accordingly. In this study series, we report 10 cases of TB of the breast with variable clinical presentations as fibroadenosis, breast abscess, duct ectasia and breast lump on evaluation, and the challenges encountered in establishing the diagnosis.


Asunto(s)
Enfermedades de la Mama/diagnóstico , Mama/microbiología , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/diagnóstico , Adulto , Mama/patología , Enfermedades de la Mama/fisiopatología , ADN Bacteriano/análisis , Diagnóstico Diferencial , Femenino , Humanos , India , Persona de Mediana Edad , Estudios Retrospectivos
19.
PLoS One ; 15(12): e0243765, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33315919

RESUMEN

BACKGROUND: Abdominal tuberculosis is a severe extrapulmonary tuberculosis, which can lead to serious complications. Early diagnosis and treatment are very important for the prognosis and the diagnosis of abdominal tuberculosis is still difficult. This study aims to evaluate the diagnostic accuracy of nucleic acid amplification tests (NAATs) for abdominal tuberculosis using meta-analysis method. METHODS: We will search PubMed, the Cochrane Library, Embase, China National Knowledge Infrastructure, and the Wanfang database for studies evaluating the diagnostic accuracy of NAATs for abdominal tuberculosis until May 2020. We will include a systematic review and meta-analysis that evaluated the accuracy of NAATs for abdominal tuberculosis. Any types of study design with full text will be sought and included. The risk of bias will be assessed using the Quality Assessment of Diagnostic Accuracy Studies tool. Stata version 15.0 with the midas command packages will be used to carry out meta-analyses. RESULTS: The results will provide clinical evidence for diagnostic accuracy of NAATs for abdominal tuberculosis, and this systematic review and meta-analysis will be submitted to a peer-reviewed journal for publication. CONCLUSION: This overview will provide evidence of NAATs for diagnosis of abdominal tuberculosis. SYSTEMATIC REVIEW REGISTRATION: INPLASY202060030.


Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , Tuberculosis/diagnóstico , Abdomen/microbiología , ADN Bacteriano/análisis , ADN Bacteriano/metabolismo , ADN Bacteriano/normas , Bases de Datos Factuales , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Estándares de Referencia , Tuberculosis/microbiología
20.
Nutrients ; 13(1)2020 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-33374116

RESUMEN

We hypothesized that the role of microbiota in breast cancer relates to its influence on gut lipid metabolism. This was tested in an in vitro model combining MCF-7 and Caco-2 cells. A total of 32 women newly diagnosed for breast cancer before any treatment and 28 healthy women provided their stools. Bacterial DNA was amplified by qPCR targeting 16s rRNA specific to Bacteroidetes and Firmicutes phyla, Lactobacillales sp., Clostridium cluster IV, Faecalibacterium prausnitzii, Clostridium cluster XIVa, Roseburia intestinalis, Blautia sp., Lactonifactor longoviformis, Bifidobacterium sp., Coriobacteriaceae, Eggertella lenta, Escherichia, and Shigella. Fecal waters (FW) were quantified for short chain fatty acids (SCFA). Caco-2 cells grown on filter inserts were incubated apically with 10% FW for 24 h, and LXR, apolipoproteins AIV, and E gene expression were estimated by real time (RT) qPCR. Then, MCF-7 cells were incubated with the whole basolateral medium for 24 h, and their viability was estimated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) test. Regression models were used to determine the correlation between MCF-7 viability and bacteria relative abundance, Caco-2 cells lipid metabolism gene expression and stool composition, as well as microbiota composition and short chain fatty acids. Logistic regression models established disease odds ratios (OR) for MCF-7 viability and Caco-2 gene expression. The OR of MCF-7 viability was 1.05 (1.01-1.10) (OR (5th-95th), p = 0.04), while that of apo AIV gene expression was 0.63 (0.39-1.01), p = 0.055). Viability correlated with % Bifidobacterium sp. (21.18 ± 7.66, p = 0.008) and valerate (-2.849 ± 1.048, p = 0.009) (ß ± s.d.). This study suggests that microbiota interacts with intestine cell lipid metabolism. Since these metabolites can reach breast cells by systemic circulation, we hypothesized that they may influence cancer disease.


Asunto(s)
Neoplasias de la Mama/microbiología , Enterocitos/metabolismo , Heces/microbiología , Microbioma Gastrointestinal/fisiología , Metabolismo de los Lípidos/fisiología , Bacterias/clasificación , Bacterias/genética , Neoplasias de la Mama/patología , Células CACO-2 , Supervivencia Celular , ADN Bacteriano/análisis , Ácidos Grasos Volátiles/análisis , Femenino , Expresión Génica , Humanos , Metabolismo de los Lípidos/genética , Células MCF-7 , Persona de Mediana Edad
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